The efficient packaging of Venezuelan equine encephalitis virus-specific RNAs into viral particles is determined by nsP1–3 synthesis

PubMed Central

Volkova, Eugenia; Gorchakov, Rodion; Frolov, Ilya

2008-01-01

Alphaviruses are regarded as attractive systems for expression of heterologous genes and development of recombinant vaccines. Venezuelan equine encephalitis virus (VEE)-based vectors are particularly promising because of their specificity to lymphoid tissues and strong resistance to interferon. To improve understanding of the VEE genome packaging and optimize application of this virus as a vector, we analyzed in more detail the mechanism of packaging of the VEE-specific RNAs. The presence of the RNAs in the VEE particles during serial passaging in tissue culture was found to depend not only on the presence of packaging signal(s), but also on the ability of these RNAs to express in cis nsP1, nsP2 and nsP3 in the form of a P123 precursor. Packaging of VEE genomes into infectious virions was also found to be more efficient compared to that of Sindbis virus, in spite of lower levels of RNA replication and structural protein production. PMID:16239019

Influence of sequence and size of DNA on packaging efficiency of parvovirus MVM-based vectors.

PubMed

Brandenburger, A; Coessens, E; El Bakkouri, K; Velu, T

1999-05-01

We have derived a vector from the autonomous parvovirus MVM(p), which expresses human IL-2 specifically in transformed cells (Russell et al., J. Virol 1992;66:2821-2828). Testing the therapeutic potential of these vectors in vivo requires high-titer stocks. Stocks with a titer of 10(9) can be obtained after concentration and purification (Avalosse et al., J. Virol. Methods 1996;62:179-183), but this method requires large culture volumes and cannot easily be scaled up. We wanted to increase the production of recombinant virus at the initial transfection step. Poor vector titers could be due to inadequate genome amplification or to inefficient packaging. Here we show that intracellular amplification of MVM vector genomes is not the limiting factor for vector production. Several vector genomes of different size and/or structure were amplified to an equal extent. Their amplification was also equivalent to that of a cotransfected wild-type genome. We did not observe any interference between vector and wild-type genomes at the level of DNA amplification. Despite equivalent genome amplification, vector titers varied greatly between the different genomes, presumably owing to differences in packaging efficiency. Genomes with a size close to 100% that of wild type were packaged most efficiently with loss of efficiency at lower and higher sizes. However, certain genomes of identical size showed different packaging efficiencies, illustrating the importance of the DNA sequence, and probably its structure.

Retroviral packaging cells encapsulated in TheraCyte immunoisolation devices enable long-term in vivo gene delivery.

PubMed

Krupetsky, Anna; Parveen, Zahida; Marusich, Elena; Goodrich, Adrienne; Dornburg, Ralph

2003-05-01

The method of delivering a therapeutic gene into a patient is still one of the major obstacles towards successful human gene therapy. Here we describe a novel gene delivery approach using TheraCyte immunoisolation devices. Retroviral vector producing cells, derived from the avian retrovirus spleen necrosis virus, SNV, were encapsulated in TheraCyte devices and tested for the release of retroviral vectors. In vitro experiments show that such devices release infectious retroviral vectors into the tissue culture medium for up to 4 months. When such devices were implanted subcutaneously in SCID mice, infectious virus was released into the blood stream. There, the vectors were transported to and infected tumors, which had been induced by subcutaneous injection of tissue culture cells. Thus, this novel concept of a continuous, long-term gene delivery may constitute an attractive approach for future in vivo human gene therapy.

Use of the piggyBac transposon to create stable packaging cell lines for the production of clinical-grade self-inactivating γ-retroviral vectors.

PubMed

Feldman, Steven A; Xu, Hui; Black, Mary A; Park, Tristen S; Robbins, Paul F; Kochenderfer, James N; Morgan, Richard A; Rosenberg, Steven A

2014-08-01

Efforts to improve the biosafety of γ-retroviral-mediated gene therapy have resulted in a shift toward the use of self-inactivating (SIN) γ-retroviral vectors. However, scale-up and manufacturing of such vectors requires significant optimization of transient transfection-based processes or development of novel platforms for the generation of stable producer cell clones. To that end, we describe the use of the piggybac transposon to generate stable producer cell clones for the production of SIN γ-retroviral vectors. The piggybac transposon is a universal tool allowing for the stable integration of SIN γ-retroviral constructs into murine (PG13) and human 293-based Phoenix (GALV and RD114, respectively) packaging cell lines without reverse transcription. Following transposition, a high-titer clone is selected for manufacture of a master cell bank and subsequent γ-retroviral vector supernatant production. Packaging cell clones created using the piggybac transposon have comparable titers to non-SIN vectors generated via conventional methods. We describe herein the use of the piggybac transposon for the production of stable packaging cell clones for the manufacture of clinical-grade SIN γ-retroviral vectors for ex vivo gene therapy clinical trials.

Rational plasmid design and bioprocess optimization to enhance recombinant adeno-associated virus (AAV) productivity in mammalian cells.

PubMed

Emmerling, Verena V; Pegel, Antje; Milian, Ernest G; Venereo-Sanchez, Alina; Kunz, Marion; Wegele, Jessica; Kamen, Amine A; Kochanek, Stefan; Hoerer, Markus

2016-02-01

Viral vectors used for gene and oncolytic therapy belong to the most promising biological products for future therapeutics. Clinical success of recombinant adeno-associated virus (rAAV) based therapies raises considerable demand for viral vectors, which cannot be met by current manufacturing strategies. Addressing existing bottlenecks, we improved a plasmid system termed rep/cap split packaging and designed a minimal plasmid encoding adenoviral helper function. Plasmid modifications led to a 12-fold increase in rAAV vector titers compared to the widely used pDG standard system. Evaluation of different production approaches revealed superiority of processes based on anchorage- and serum-dependent HEK293T cells, exhibiting about 15-fold higher specific and volumetric productivity compared to well-established suspension cells cultivated in serum-free medium. As for most other viral vectors, classical stirred-tank bioreactor production is thus still not capable of providing drug product of sufficient amount. We show that manufacturing strategies employing classical surface-providing culture systems can be successfully transferred to the new fully-controlled, single-use bioreactor system Integrity(TM) iCELLis(TM) . In summary, we demonstrate substantial bioprocess optimizations leading to more efficient and scalable production processes suggesting a promising way for flexible large-scale rAAV manufacturing. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Partial E3 Deletion in Replication-Defective Adenoviral Vectors Allows for Stable Expression of Potentially Toxic Transgene Products.

PubMed

Haut, Larissa H; Gill, Amanda L; Kurupati, Raj K; Bian, Ang; Li, Yan; Giles-Davis, Wynetta; Xiang, Zhiquan; Zhou, Xiang Yang; Ertl, Hildegund C J

2016-10-01

Adenovirus (Ad) is used extensively for construction of viral vectors, most commonly with deletion in its E1 and/or E3 genomic regions. Previously, our attempts to insert envelope proteins (Env) of HIV-1 into such vectors based on chimpanzee-derived Ad (AdC) viruses were thwarted. Here, we describe that genetic instability of an E1- and E3-deleted AdC vector of serotype C6 expressing Env of HIV-1 can be overcome by reinsertion of E3 sequences with anti-apoptotic activities. This partial E3 deletion presumably delays premature death of HEK-293 packaging cell lines due to Env-induced cell apoptosis. The same partial E3 deletion also allows for the generation of stable glycoprotein 140 (gp140)- and gp160-expressing Ad vectors based on AdC7, a distinct AdC serotype. Env-expressing AdC vectors containing the partial E3 deletion are genetically stable upon serial cell culture passaging, produce yields comparable to those of other AdC vectors, and induce transgene product-specific antibody responses in mice. A partial E3 deletion thereby allows expansion of the repertoire of transgenes that can be expressed by Ad vectors.

Expressing Transgenes That Exceed the Packaging Capacity of Adeno-Associated Virus Capsids

PubMed Central

Chamberlain, Kyle; Riyad, Jalish Mahmud; Weber, Thomas

2016-01-01

Recombinant adeno-associated virus vectors (rAAV) are being explored as gene delivery vehicles for the treatment of various inherited and acquired disorders. rAAVs are attractive vectors for several reasons: wild-type AAVs are nonpathogenic, and rAAVs can trigger long-term transgene expression even in the absence of genome integration—at least in postmitotic tissues. Moreover, rAAVs have a low immunogenic profile, and the various AAV serotypes and variants display broad but distinct tropisms. One limitation of rAAVs is that their genome-packaging capacity is only ∼5 kb. For most applications this is not of major concern because the median human protein size is 375 amino acids. Excluding the ITRs, for a protein of typical length, this allows the incorporation of ∼3.5 kb of DNA for the promoter, polyadenylation sequence, and other regulatory elements into a single AAV vector. Nonetheless, for certain diseases the packaging limit of AAV does not allow the delivery of a full-length therapeutic protein by a single AAV vector. Hence, approaches to overcome this limitation have become an important area of research for AAV gene therapy. Among the most promising approaches to overcome the limitation imposed by the packaging capacity of AAV is the use of dual-vector approaches, whereby a transgene is split across two separate AAV vectors. Coinfection of a cell with these two rAAVs will then—through a variety of mechanisms—result in the transcription of an assembled mRNA that could not be encoded by a single AAV vector because of the DNA packaging limits of AAV. The main purpose of this review is to assess the current literature with respect to dual-AAV-vector design, to highlight the effectiveness of the different methodologies and to briefly discuss future areas of research to improve the efficiency of dual-AAV-vector transduction. PMID:26757051

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

PubMed Central

Podsakoff, G; Wong, K K; Chatterjee, S

1994-01-01

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells. Images PMID:8057446

Efficient gene transfer into nondividing cells by adeno-associated virus-based vectors.

PubMed

Podsakoff, G; Wong, K K; Chatterjee, S

1994-09-01

Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.

Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

DOE Office of Scientific and Technical Information (OSTI.GOV)

Huang, Shuohao; Kawabe, Yoshinori; Ito, Akira

2012-01-06

Highlights: Black-Right-Pointing-Pointer Adeno-associated virus (AAV) is capable of targeted integration in human cells. Black-Right-Pointing-Pointer Integrase-defective retroviral vector (IDRV) enables a circular DNA delivery. Black-Right-Pointing-Pointer A targeted integration system of IDRV DNA using the AAV integration mechanism. Black-Right-Pointing-Pointer Targeted IDRV integration ameliorates the safety concerns for retroviral vectors. -- Abstract: Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors,more » therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.« less

Characteristics of Minimally Oversized Adeno-Associated Virus Vectors Encoding Human Factor VIII Generated Using Producer Cell Lines and Triple Transfection.

PubMed

Nambiar, Bindu; Cornell Sookdeo, Cathleen; Berthelette, Patricia; Jackson, Robert; Piraino, Susan; Burnham, Brenda; Nass, Shelley; Souza, David; O'Riordan, Catherine R; Vincent, Karen A; Cheng, Seng H; Armentano, Donna; Kyostio-Moore, Sirkka

2017-02-01

Several ongoing clinical studies are evaluating recombinant adeno-associated virus (rAAV) vectors as gene delivery vehicles for a variety of diseases. However, the production of vectors with genomes >4.7 kb is challenging, with vector preparations frequently containing truncated genomes. To determine whether the generation of oversized rAAVs can be improved using a producer cell-line (PCL) process, HeLaS3-cell lines harboring either a 5.1 or 5.4 kb rAAV vector genome encoding codon-optimized cDNA for human B-domain deleted Factor VIII (FVIII) were isolated. High-producing "masterwells" (MWs), defined as producing >50,000 vg/cell, were identified for each oversized vector. These MWs provided stable vector production for >20 passages. The quality and potency of the AAVrh8R/FVIII-5.1 and AAVrh8R/FVIII-5.4 vectors generated by the PCL method were then compared to those prepared via transient transfection (TXN). Southern and dot blot analyses demonstrated that both production methods resulted in packaging of heterogeneously sized genomes. However, the PCL-derived rAAV vector preparations contained some genomes >4.7 kb, whereas the majority of genomes generated by the TXN method were ≤4.7 kb. The PCL process reduced packaging of non-vector DNA for both the AAVrh8R/FVIII-5.1 and the AAVrh8R/FVIII-5.4 kb vector preparations. Furthermore, more DNA-containing viral particles were obtained for the AAVrh8R/FVIII-5.1 vector. In a mouse model of hemophilia A, animals administered a PCL-derived rAAV vector exhibited twofold higher plasma FVIII activity and increased levels of vector genomes in the liver than mice treated with vector produced via TXN did. Hence, the quality of oversized vectors prepared using the PCL method is greater than that of vectors generated using the TXN process, and importantly this improvement translates to enhanced performance in vivo.

Vectors expressing chimeric Japanese encephalitis dengue 2 viruses.

PubMed

Wei, Y; Wang, S; Wang, X

2014-01-01

Vectors based on self-replicating RNAs (replicons) of flaviviruses are becoming powerful tool for expression of heterologous genes in mammalian cells and development of novel antiviral and anticancer vaccines. We constructed two vectors expressing chimeric viruses consisting of attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) in which the PrM/M-E genes were replaced fully or partially with those of dengue 2 virus (DENV-2). These vectors, named pJED2 and pJED2-1770 were transfected to BHK-21 cells and produced chimeric viruses JED2V and JED2-1770V, respectively. The chimeric viruses could be passaged in C6/36 but not BHK-21 cells. The chimeric viruses produced in C6/36 cells CPE 4-5 days after infection and RT-PCR, sequencing, immunofluorescence assay (IFA) and Western blot analysis confirmed the chimeric nature of produced viruses. The immunogenicity of chimeric viruses in mice was proved by detecting DENV-2 E protein-specific serum IgG antibodies with neutralization titer of 10. Successful preparation of infectious clones of chimeric JEV-DENV-2 viruses showed that JEV-based expression vectors are fully functional.

Packaging of HCV-RNA into lentiviral vector

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caval, Vincent; Piver, Eric; Service de Biochimie et Biologie Moleculaire, CHRU de Tours

2011-11-04

Highlights: Black-Right-Pointing-Pointer Description of HCV-RNA Core-D1 interactions. Black-Right-Pointing-Pointer In vivo evaluation of the packaging of HCV genome. Black-Right-Pointing-Pointer Determination of the role of the three basic sub-domains of D1. Black-Right-Pointing-Pointer Heterologous system involving HIV-1 vector particles to mobilise HCV genome. Black-Right-Pointing-Pointer Full length mobilisation of HCV genome and HCV-receptor-independent entry. -- Abstract: The advent of infectious molecular clones of Hepatitis C virus (HCV) has unlocked the understanding of HCV life cycle. However, packaging of the genomic RNA, which is crucial to generate infectious viral particles, remains poorly understood. Molecular interactions of the domain 1 (D1) of HCV Core protein andmore » HCV RNA have been described in vitro. Since compaction of genetic information within HCV genome has hampered conventional mutational approach to study packaging in vivo, we developed a novel heterologous system to evaluate the interactions between HCV RNA and Core D1. For this, we took advantage of the recruitment of Vpr fusion-proteins into HIV-1 particles. By fusing HCV Core D1 to Vpr we were able to package and transfer a HCV subgenomic replicon into a HIV-1 based lentiviral vector. We next examined how deletion mutants of basic sub-domains of Core D1 influenced HCV RNA recruitment. The results emphasized the crucial role of the first and third basic regions of D1 in packaging. Interestingly, the system described here allowed us to mobilise full-length JFH1 genome in CD81 defective cells, which are normally refractory to HCV infection. This finding paves the way to an evaluation of the replication capability of HCV in various cell types.« less

Reverse Transcription Quantitative Polymerase Chain Reaction for Detection of and Differentiation Between RNA and DNA of HIV-1-Based Lentiviral Vectors.

PubMed

Pavlovic, Melanie; Koehler, Nina; Anton, Martina; Dinkelmeier, Anna; Haase, Maren; Stellberger, Thorsten; Busch, Ulrich; Baiker, Armin E

2017-08-01

The purpose of the described method is the detection of and differentiation between RNA and DNA of human immunodeficiency virus (HIV)-derived lentiviral vectors (LV) in cell culture supernatants and swab samples. For the analytical surveillance of genetic engineering, operations methods for the detection of the HIV-1-based LV generations are required. Furthermore, for research issues, it is important to prove the absence of LV particles for downgrading experimental settings in terms of the biosafety level. Here, a quantitative polymerase chain reaction method targeting the long terminal repeat U5 subunit and the start sequence of the packaging signal ψ is described. Numerous controls are included in order to monitor the technical procedure.

APOBEC3-mediated hypermutation of retroviral vectors produced from some retrovirus packaging cell lines.

PubMed

Miller, A D; Metzger, M J

2011-05-01

APOBEC3 proteins are packaged into retrovirus virions and can hypermutate retroviruses during reverse transcription. We found that HT-1080 human fibrosarcoma cells hypermutate retroviruses, and that the HT-1080 cell-derived FLYA13 retrovirus packaging cells also hypermutate a retrovirus vector produced using these cells. We found no hypermutation of the same vector produced by the mouse cell-derived packaging line PT67 or by human 293 cells transfected with the vector and retrovirus packaging plasmids. We expect that avoidance of vector hypermutation will be particularly important for vectors used in gene therapy, wherein mutant proteins might stimulate deleterious immune responses.

Adaptation of retrovirus producer cells to serum deprivation: Implications in lipid biosynthesis and vector production.

PubMed

Rodrigues, A F; Amaral, A I; Veríssimo, V; Alves, P M; Coroadinha, A S

2012-05-01

The manufacture of enveloped virus, particularly retroviral (RV) and lentiviral (LV) vectors, faces the challenge of low titers that are aggravated under serum deprivation culture conditions. Also, the scarce knowledge on the biochemical pathways related with virus production is still limiting the design of rational strategies for improved production yields. This work describes the adaptation to serum deprivation of two human RV packaging cell lines, 293 FLEX and Te Fly and its effects on lipid biosynthetic pathways and infectious vector production. Total lipid content as well as cellular cholesterol were quantified and lipid biosynthesis was assessed by (13)C-NMR spectroscopy; changes in gene expression of lipid biosynthetic enzymes were also evaluated. The effects of adaptation to serum deprivation in lipid biosynthesis were cell line specific and directly correlated with infectious virus titers: 293 FLEX cells faced severe lipid starvation-up to 50% reduction in total lipid content-along with a 68-fold reduction in infectious vector titers; contrarily, Te Fly cells were able to maintain identical levels of total lipid content by rising de novo lipid biosynthesis, particularly for cholesterol-50-fold increase-with the consequent recovery of infectious vector productivities. Gene expression analysis of lipid biosynthetic enzymes further confirmed cholesterol production pathway to be prominently up-regulated under serum deprivation conditions for Te Fly cells, providing a genotype-phenotype validation for enhanced cholesterol synthesis. These results highlight lipid metabolism dynamics and the ability to activate lipid biosynthesis under serum deprivation as an important feature for high retroviral titers. Mechanisms underlying virus production and its relationship with lipid biosynthesis, with special focus on cholesterol, are discussed as potential targets for cellular metabolic engineering. Copyright © 2011 Wiley Periodicals, Inc.

cis-Acting elements important for retroviral RNA packaging specificity.

PubMed

Beasley, Benjamin E; Hu, Wei-Shau

2002-05-01

Spleen necrosis virus (SNV) proteins can package RNA from distantly related murine leukemia virus (MLV), whereas MLV proteins cannot package SNV RNA efficiently. We used this nonreciprocal recognition to investigate regions of packaging signals that influence viral RNA encapsidation specificity. Although the MLV and SNV packaging signals (Psi and E, respectively) do not contain significant sequence homology, they both contain a pair of hairpins. This hairpin pair was previously proposed to be the core element in MLV Psi. In the present study, MLV-based vectors were generated to contain chimeric SNV/MLV packaging signals in which the hairpins were replaced with the heterologous counterpart. The interactions between these chimeras and MLV or SNV proteins were examined by virus replication and RNA analyses. SNV proteins recognized all of the chimeras, indicating that these chimeras were functional. We found that replacing the hairpin pair did not drastically alter the ability of MLV proteins to package these chimeras. These results indicate that, despite the important role of the hairpin pair in RNA packaging, it is not the major motif responsible for the ability of MLV proteins to discriminate between the MLV and SNV packaging signals. To determine the role of sequences flanking the hairpins in RNA packaging specificity, vectors with swapped flanking regions were generated and evaluated. SNV proteins packaged all of these chimeras efficiently. In contrast, MLV proteins strongly favored chimeras with the MLV 5'-flanking regions. These data indicated that MLV Gag recognizes multiple elements in the viral packaging signal, including the hairpin structure and flanking regions.

Psi- vectors: murine leukemia virus-based self-inactivating and self-activating retroviral vectors.

PubMed Central

Delviks, K A; Hu, W S; Pathak, V K

1997-01-01

We have developed murine leukemia virus (MLV)-based self-inactivating and self-activating vectors to show that the previously demonstrated high-frequency direct repeat deletions are not unique to spleen necrosis virus (SNV) or the neomycin drug resistance gene. Retroviral vectors pKD-HTTK and pKD-HTpTK containing direct repeats composed of segments of the herpes simplex virus type 1 thymidine kinase (HTK) gene were constructed; in pKD-HTpTK, the direct repeat flanked the MLV packaging signal. The generation of hypoxanthine-aminopterin-thymidine-resistant colonies after one cycle of retroviral replication demonstrated functional reconstitution of the HTK gene. Quantitative Southern analysis indicated that direct repeat deletions occurred in 57 and 91% of the KD-HTTK and KD-HTpTK proviruses, respectively. These results demonstrate that (i) deletion of direct repeats occurs at similar high frequencies in SNV and MLV vectors, (ii) MLV psi can be efficiently deleted by using direct repeats, (iii) suicide genes can be functionally reconstituted during reverse transcription, and (iv) the psi region may be a hot spot for reverse transcriptase template switching events. PMID:9223521

Jaagsiekte Sheep Retrovirus Envelope Efficiently Pseudotypes Human Immunodeficiency Virus Type 1-Based Lentiviral Vectors

PubMed Central

Liu, Shan-Lu; Halbert, Christine L.; Miller, A. Dusty

2004-01-01

Jaagsiekte sheep retrovirus (JSRV) infects lung epithelial cells in sheep, and oncoretroviral vectors bearing JSRV Env can mediate transduction of human cells, suggesting that such vectors might be useful for lung-directed gene therapy. Here we show that JSRV Env can also efficiently pseudotype a human immunodeficiency virus type 1-based lentiviral vector, a more suitable vector for transduction of slowly dividing lung epithelial cells. We created several chimeric Env proteins that, unlike the parental Env, do not transform rodent fibroblasts but are still capable of pseudotyping lentiviral and oncoretroviral vectors. PMID:14963173

Attenuated Human Parainfluenza Virus Type 1 Expressing the Respiratory Syncytial Virus (RSV) Fusion (F) Glycoprotein from an Added Gene: Effects of Prefusion Stabilization and Packaging of RSV F

PubMed Central

Liu, Xiang; Liang, Bo; Ngwuta, Joan; Liu, Xueqiao; Surman, Sonja; Lingemann, Matthias; Kwong, Peter D.; Graham, Barney S.; Collins, Peter L.

2017-01-01

ABSTRACT Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the prefusion (pre-F) conformation by previously described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane and cytoplasmic tail (TMCT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild-type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo. In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine. IMPORTANCE RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a stabilized attenuating mutation, to express the RSV F glycoprotein bearing amino acid substitutions that increase its stability in the pre-F form, the most immunogenic form that elicits highly functional virus-neutralizing antibodies. RSV F was expressed from the pre-N or N-P gene position of the rHPIV1 vector as a full-length protein or as a chimeric form with its TMCT domain derived from HPIV1 F. TMCT modification greatly increased packaging of RSV F into the vector particles but also increased vector attenuation in vivo, resulting in reduced immunogenicity. In contrast, full-length RSV F expressed from the pre-N position was immunogenic, eliciting complement-independent RSV-neutralizing antibodies and providing protection against RSV challenge. PMID:28835504

Control of cucurbit viruses.

PubMed

Lecoq, Hervé; Katis, Nikolaos

2014-01-01

More than 70 well-characterized virus species transmitted by a diversity of vectors may infect cucurbit crops worldwide. Twenty of those cause severe epidemics in major production areas, occasionally leading to complete crop failures. Cucurbit viruses' control is based on three major axes: (i) planting healthy seeds or seedlings in a clean environment, (ii) interfering with vectors activity, and (iii) using resistant cultivars. Seed disinfection and seed or seedling quality controls guarantee growers on the sanitary status of their planting material. Removal of virus or vector sources in the crop environment can significantly delay the onset of viral epidemics. Insecticide or oil application may reduce virus spread in some situations. Diverse cultural practices interfere with or prevent vector reaching the crop. Resistance can be obtained by grafting for soil-borne viruses, by cross-protection, or generally by conventional breeding or genetic engineering. The diversity of the actions that may be taken to limit virus spread in cucurbit crops and their limits will be discussed. The ultimate goal is to provide farmers with technical packages that combine these methods within an integrated disease management program and are adapted to different countries and cropping systems.

Detection of osteoclastic cell-cell fusion through retroviral vector packaging.

PubMed

Kondo, Takako; Ikeda, Kyoji; Matsuo, Koichi

2004-11-01

Cell-cell fusion generates multinucleated cells such as osteoclasts in bone, myotubes in muscle, and trophoblasts in placenta. Molecular details governing these fusion processes are still largely unknown. As a step toward identification of fusogenic genes, we tested the concept that retroviral vectors can be packaged as a result of cell-cell fusion. First, we introduced replication-deficient retroviral vectors expressing mCAT-1, which mediates fusogenic interaction with the retroviral envelope protein Env, into Chinese hamster ovary (CHO) cells to generate vector cells. Plasmids expressing virion proteins Gag, Pol, and Env were introduced into a separate culture of CHO cells to generate packaging cells. Co-culturing vector and packaging cells resulted in production of infectious retroviruses carrying the mCAT-1 gene as a consequence of cell-cell fusion. Second, we introduced a retroviral vector into primary osteoclast precursors and co-cultured them with established osteoclast precursor RAW264.7 cells, which turned out to harbor packaging activity. Packaged retroviral vector was detected in culture supernatants only where the osteoclast differentiation factor receptor activator for NF-kappaB ligand (RANKL) induced fusion between these two cell types. These data suggest that retrovirus production can occur as a result of cell-cell fusion. This provides a novel approach for isolating and characterizing fusogenic genes using retroviral expression vectors.

Murine Stem Cell-Based Retrovirus Production for Marking Primary Mouse Mammary Cells for Metastasis Studies.

PubMed

Beverly, Levi J; Podsypanina, Katrina

2016-02-01

Since the introduction of retroviral vector technology, permanent genetic marking of cells has considerably contributed to the understanding of different physiological and disease processes in vivo. Recent marking strategies aim to elucidate the contribution of cells on the clonal level, and the advent of fluorescent proteins has opened new avenues for the in vivo analysis of gene-marked cells. Gene-modified cells are easily identifiable (e.g., via the introduced fluorescent protein) within whole organ structures, allowing one to measure the contribution of transduced cells to malignant outgrowth. In our laboratory, we use the tetracycline-inducible system to study oncogene cooperation in metastatic progression. We use bicistronic retroviruses expressing the tetracycline transactivator (tTA) and the candidate gene (MIT-gene) or the tTA alone (MIT-Rx) to infect primary mammary cells from mice harboring tetracycline-inducible transgenes. This allows for constitutive expression of the candidate gene and tTA-dependent expression of the inducible oncogene. We also use MIG-based vectors, which allow for constitutive expression of the candidate gene and a green fluorescent protein. Here we describe how to produce retroviral particles carrying both MIT- and MIG-based vectors. Because of the fragility of the retroviral envelope, we do not attempt to concentrate the virus, and we directly use packaging cell media to infect primary epithelial cells (either normal or tumor). Infected cells can be transplanted into recipient mice to investigate metastatic colonization. © 2016 Cold Spring Harbor Laboratory Press.

Production and purification of lentiviral vectors generated in 293T suspension cells with baculoviral vectors.

PubMed

Lesch, H P; Laitinen, A; Peixoto, C; Vicente, T; Makkonen, K-E; Laitinen, L; Pikkarainen, J T; Samaranayake, H; Alves, P M; Carrondo, M J T; Ylä-Herttuala, S; Airenne, K J

2011-06-01

Lentivirus can be engineered to be a highly potent vector for gene therapy applications. However, generation of clinical grade vectors in enough quantities for therapeutic use is still troublesome and limits the preclinical and clinical experiments. As a first step to solve this unmet need we recently introduced a baculovirus-based production system for lentiviral vector (LV) production using adherent cells. Herein, we have adapted and optimized the production of these vectors to a suspension cell culture system using recombinant baculoviruses delivering all elements required for a safe latest generation LV preparation. High-titer LV stocks were achieved in 293T cells grown in suspension. Produced viruses were accurately characterized and the functionality was also tested in vivo. Produced viruses were compared with viruses produced by calcium phosphate transfection method in adherent cells and polyethylenimine transfection method in suspension cells. Furthermore, a scalable and cost-effective capture purification step was developed based on a diethylaminoethyl monolithic column capable of removing most of the baculoviruses from the LV pool with 65% recovery.

Attenuated Human Parainfluenza Virus Type 1 Expressing the Respiratory Syncytial Virus (RSV) Fusion (F) Glycoprotein from an Added Gene: Effects of Prefusion Stabilization and Packaging of RSV F.

PubMed

Liu, Xiang; Liang, Bo; Ngwuta, Joan; Liu, Xueqiao; Surman, Sonja; Lingemann, Matthias; Kwong, Peter D; Graham, Barney S; Collins, Peter L; Munir, Shirin

2017-11-15

Human respiratory syncytial virus (RSV) is the most prevalent worldwide cause of severe respiratory tract infection in infants and young children. Human parainfluenza virus type 1 (HPIV1) also causes severe pediatric respiratory illness, especially croup. Both viruses lack vaccines. Here, we describe the preclinical development of a bivalent RSV/HPIV1 vaccine based on a recombinant HPIV1 vector, attenuated by a stabilized mutation, that expresses RSV F protein modified for increased stability in the prefusion (pre-F) conformation by previously described disulfide bond (DS) and hydrophobic cavity-filling (Cav1) mutations. RSV F was expressed from the first or second gene position as the full-length protein or as a chimeric protein with its transmembrane and cytoplasmic tail (TMCT) domains substituted with those of HPIV1 F in an effort to direct packaging in the vector particles. All constructs were recovered by reverse genetics. The TMCT versions of RSV F were packaged in the rHPIV1 particles much more efficiently than their full-length counterparts. In hamsters, the presence of the RSV F gene, and in particular the TMCT versions, was attenuating and resulted in reduced immunogenicity. However, the vector expressing full-length RSV F from the pre-N position was immunogenic for RSV and HPIV1. It conferred complement-independent high-quality RSV-neutralizing antibodies at titers similar to those of wild-type RSV and provided protection against RSV challenge. The vectors exhibited stable RSV F expression in vitro and in vivo In conclusion, an attenuated rHPIV1 vector expressing a pre-F-stabilized form of RSV F demonstrated promising immunogenicity and should be further developed as an intranasal pediatric vaccine. IMPORTANCE RSV and HPIV1 are major viral causes of acute pediatric respiratory illness for which no vaccines or suitable antiviral drugs are available. The RSV F glycoprotein is the major RSV neutralization antigen. We used a rHPIV1 vector, bearing a stabilized attenuating mutation, to express the RSV F glycoprotein bearing amino acid substitutions that increase its stability in the pre-F form, the most immunogenic form that elicits highly functional virus-neutralizing antibodies. RSV F was expressed from the pre-N or N-P gene position of the rHPIV1 vector as a full-length protein or as a chimeric form with its TMCT domain derived from HPIV1 F. TMCT modification greatly increased packaging of RSV F into the vector particles but also increased vector attenuation in vivo , resulting in reduced immunogenicity. In contrast, full-length RSV F expressed from the pre-N position was immunogenic, eliciting complement-independent RSV-neutralizing antibodies and providing protection against RSV challenge. Copyright © 2017 American Society for Microbiology.

9 CFR 112.10 - Special packaging and labeling.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Special packaging and labeling. 112.10... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING § 112.10 Special packaging and labeling. A biological product, which requires special packaging and/or...

[Recombinant hFOXA2 and hPDX1 lentivirus induced dental pulp stem cells from deciduous teeth reprogramming for insulin-producing cells].

PubMed

Shi, Jian-feng; Zhu, Chun-hui; Liu, Jin; Sun, Jun-yi; Rao, Guo-zhou; Li, Ang

2013-12-01

The purpose of this study was to culture and identify dental pulp stem cells(DPSCs) from deciduous teeth in vitro and construct the recombinant hFOXA2 and hPDX1 lentivirus vectors and transfect the DPSCs to induce insulin-producing cells (IPCs). DPSCs were separated and cultured by enzyme digest method, and purified by limited dilution method. Flow cytometry was used to determine the surface marker expression of the DPSCs, and the ability of multiple differentiations was determined by specific staining. hFOXA2 and hPDX1 genes were amplified by PCR, and the recombinant hFOXA2 and hPDX1 lentivirus vectors were reconstructed and transfected into 293T cells by lipofectamine2000 for virus packaging. The viral infection efficiency and titer were determined through fluorescence cell count. The recombinant virus was used to infect the DPSCs cells via multiplicity of infection (MOI) and induce the DPSCs reprogramming for IPCs. Immunofluorescence staining was used to measure the expression of proinsulin, FOXA2 and PDX1. ELISA method was used to detect the insulin secretion. The data was analyzed Using SPSS13.0 software package. DPSCs were isolated and cultured successfully. Cell surface highly expressed STRO-1 (98.01%), CDl46 (98.51%), CD34 (99.54%) and CD45 (24.08%). The multi-lineage differentiation capacity into osteoblasts, chondrocytes, and adipose was achieved. The recombinant hFOXA2 and hPDX1 lentivirus vectors were successfully constructed. Double enzyme digestion and sequencing appraisal showed that the sequence was fully consistent with GenBank retrieval. Virus packing efficiency was (96.15±0.17) % and (95.49±0.21) % respectively, and the infection titer was about 1.80±108 GTU/mL. The best MOI of the virus was 20. After inducing the cells to express proinsulin, FOXA2 and PDX1, insulin secretion volume was about 1.92 μmol/L. Compared with the uninduced group and control group, insulin secretion increased significantly (P<0.01). The recombinant transcription factor virus can activate cell reprogramming mechanism, form insulin-producing cells, and can be used for gene therapy of diabetes seed cells. Supported by Science and Technology Research Program of Shaanxi Province (2009K17-06) and Science and Technology Innovation as a Whole Plan Resources Leading Industry Key Technology (Chain) Project of Shaanxi Province (2011KTCL03-24).

Comparison of HIV- and EIAV-based vectors on their efficiency in transducing murine and human hematopoietic repopulating cells.

PubMed

Siapati, Elena K; Bigger, Brian W; Miskin, James; Chipchase, Daniel; Parsley, Kathryn L; Mitrophanous, Kyriacos; Themis, Mike; Thrasher, Adrian J; Bonnet, Dominique

2005-09-01

The use of lentiviral vectors for gene transfer into hematopoietic stem cells has raised considerable interest as these vectors can permanently integrate their genome into quiescent cells. Vectors based on alternative lentiviruses would theoretically be safer than HIV-1-based vectors and could also be used in HIV-positive patients, minimizing the risk of generating replication-competent virus. Here we report the use of third-generation equine infectious anemia virus (EIAV)- and HIV-1-based vectors with minimal viral sequences and absence of accessory proteins. We have compared their efficiency in transducing mouse and human hematopoietic stem cells both in vitro and in vivo to that of a previously documented second-generation HIV-1 vector. The third-generation EIAV- and HIV-based vectors gave comparable levels of transduction and transgene expression in both mouse and human NOD/SCID repopulating cells but were less efficient than the second-generation HIV-1 vector in human HSCs. For the EIAV vector this is possibly a reflection of the lower protein expression levels achieved in human cells, as vector copy number analysis revealed that this vector exhibited a trend to integrate equally efficiently compared to the third-generation HIV-1 vector in both mouse and human HSCs. Interestingly, the presence or absence of Tat in viral preparations did not influence the transduction efficiency of HIV-1 vectors in human HSCs.

Characterization of replication-competent retroviruses from nonhuman primates with virus-induced T-cell lymphomas and observations regarding the mechanism of oncogenesis.

PubMed Central

Vanin, E F; Kaloss, M; Broscius, C; Nienhuis, A W

1994-01-01

Rapidly progressive T-cell lymphomas were observed in 3 of 10 rhesus monkeys several months after autologous transplantation of enriched bone marrow stem cells that had been transduced with a retroviral vector preparation containing replication-competent virus (R. E. Donahue, S. W. Kessler, D. Bodice, K. McDonagh, C. Dunbar, S. Goodman, B. Agricola, E. Byrne, M. Raffeld, R. Moen, J. Bacher, K. M. Zsebo, and A. W. Nienhuis, J. Exp. Med. 176:1124-1135, 1992). The animals with lymphoma appeared to be tolerant to retroviral antigens in that their sera lacked antibodies reactive with viral proteins and contained 10(4) to 10(5) infectious virus particles per ml. By molecular cloning and DNA sequencing, we have now demonstrated that the serum from one of the monkeys contained a replication-competent retrovirus that arose by recombination between vector and packaging encoding sequences (vector/helper [V/H] recombinant) in the producer clone used for transduction of bone marrow stem cells. Southern blot analysis demonstrated 14 or 25 copies of this genome per cell where present in two animals. The genome of a second replication-competent virus was also recovered by molecular cloning; it arose by recombination involving the genome of the V/H recombinant and endogenous murine retroviral genomes in the producer clone. Twelve copies of this amphotropic virus/mink cell focus-forming virus genome were present in tumor DNA of one animal, but it was not found in tumor DNA of the other two animals with lymphoma. Southern blot analysis of DNA from various tissues demonstrated common insertion site bands in several samples of tumor DNA from one animal, suggesting clonal origin of the lymphoma. Our data are most consistent with a pathogenic mechanism in which chronic productive retroviral infection allowed insertional mutagenesis of critical growth control genes, leading to cell transformation and clonal tumor evolution. Images PMID:8207799

A novel intranuclear RNA vector system for long-term stem cell modification

PubMed Central

Ikeda, Yasuhiro; Makino, Akiko; Matchett, William E.; Holditch, Sara J.; Lu, Brian; Dietz, Allan B.; Tomonaga, Keizo

2015-01-01

Genetically modified stem and progenitor cells have emerged as a promising regenerative platform in the treatment of genetic and degenerative disorders, highlighted by their successful therapeutic use in inherent immunodeficiencies. However, biosafety concerns over insertional mutagenesis resulting from integrating recombinant viral vectors have overshadowed the widespread clinical applications of genetically modified stem cells. Here, we report an RNA-based episomal vector system, amenable for long-term transgene expression in stem cells. Specifically, we used a unique intranuclear RNA virus, Borna disease virus (BDV), as the gene transfer vehicle, capable of persistent infections in various cell types. BDV-based vectors allowed for long-term transgene expression in mesenchymal stem cells (MSCs) without affecting cellular morphology, cell surface CD105 expression, or the adipogenicity of MSCs. Similarly, replication-defective BDV vectors achieved long-term transduction of human induced pluripotent stem cells (iPSCs), while maintaining the ability to differentiate into three embryonic germ layers. Thus, the BDV-based vectors offer a genomic modification-free, episomal RNA delivery system for sustained stem cell transduction. PMID:26632671

Distribution of AAV8 particles in cell lysates and culture media changes with time and is dependent on the recombinant vector

PubMed Central

Piras, Bryan A; Drury, Jason E; Morton, Christopher L; Spence, Yunyu; Lockey, Timothy D; Nathwani, Amit C; Davidoff, Andrew M; Meagher, Michael M

2016-01-01

With clinical trials ongoing, efficient clinical production of adeno-associated virus (AAV) to treat large numbers of patients remains a challenge. We compared distribution of AAV8 packaged with Factor VIII (FVIII) in cell culture media and lysates on days 3, 5, 6, and 7 post-transfection and found increasing viral production through day 6, with the proportion of viral particles in the media increasing from 76% at day 3 to 94% by day 7. Compared to FVIII, AAV8 packaged with Factor IX and Protective Protein/Cathepsin A vectors demonstrated a greater shift from lysate towards media from day 3 to 6, implying that particle distribution is dependent on recombinant vector. Larger-scale productions showed that the ratio of full-to-empty AAV particles is similar in media and lysate, and that AAV harvested on day 6 post-transfection provides equivalent function in mice compared to AAV harvested on day 3. This demonstrates that AAV8 production can be optimized by prolonging the duration of culture post-transfection, and simplified by allowing harvest of media only, with disposal of cells that contain 10% or less of total vector yield. Additionally, the difference in particle distribution with different expression cassettes implies a recombinant vector-dependent processing mechanism which should be taken into account during process development. PMID:27069949

9 CFR 112.6 - Packaging biological products.

Code of Federal Regulations, 2014 CFR

2014-01-01

... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING... Disease Vaccine. (2) Poultry vaccines administered to individual birds using automatic vaccinating..., unless each final container bears, or is packaged in a carton with, complete and approved labeling which...

9 CFR 112.6 - Packaging biological products.

Code of Federal Regulations, 2013 CFR

2013-01-01

... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING... Disease Vaccine. (2) Poultry vaccines administered to individual birds using automatic vaccinating..., unless each final container bears, or is packaged in a carton with, complete and approved labeling which...

9 CFR 112.6 - Packaging biological products.

Code of Federal Regulations, 2012 CFR

2012-01-01

... AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING... Disease Vaccine. (2) Poultry vaccines administered to individual birds using automatic vaccinating..., unless each final container bears, or is packaged in a carton with, complete and approved labeling which...

Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors.

PubMed

Gray, Steven J; Foti, Stacey B; Schwartz, Joel W; Bachaboina, Lavanya; Taylor-Blake, Bonnie; Coleman, Jennifer; Ehlers, Michael D; Zylka, Mark J; McCown, Thomas J; Samulski, R Jude

2011-09-01

With the increased use of small self-complementary adeno-associated viral (AAV) vectors, the design of compact promoters becomes critical for packaging and expressing larger transgenes under ubiquitous or cell-specific control. In a comparative study of commonly used 800-bp cytomegalovirus (CMV) and chicken β-actin (CBA) promoters, we report significant differences in the patterns of cell-specific gene expression in the central and peripheral nervous systems. The CMV promoter provides high initial neural expression that diminishes over time. The CBA promoter displayed mostly ubiquitous and high neural expression, but substantially lower expression in motor neurons (MNs). We report the creation of a novel hybrid form of the CBA promoter (CBh) that provides robust long-term expression in all cells observed with CMV or CBA, including MNs. To develop a short neuronal promoter to package larger transgenes into AAV vectors, we also found that a 229-bp fragment of the mouse methyl-CpG-binding protein-2 (MeCP2) promoter was able to drive neuron-specific expression within the CNS. Thus the 800-bp CBh promoter provides strong, long-term, and ubiquitous CNS expression whereas the MeCP2 promoter allows an extra 570-bp packaging capacity, with low and mostly neuronal expression within the CNS, similar to the MeCP2 transcription factor.

An avian cell line designed for production of highly attenuated viruses.

PubMed

Jordan, Ingo; Vos, Ad; Beilfuss, Stefanie; Neubert, Andreas; Breul, Sabine; Sandig, Volker

2009-01-29

Several viral vaccines, including highly promising vectors such as modified vaccinia Ankara (MVA), are produced on chicken embryo fibroblasts. Dependence on primary cells complicates production especially in large vaccination programs. With primary cells it is also not possible to create packaging lines for replication-deficient vectors that are adapted to proliferation in an avian host. To obviate requirement for primary cells permanent lines from specific tissues of muscovy duck were derived (AGE1.CR, CS, and CA) and further modified: we demonstrate that stable expression of the structural gene pIX from human adenovirus increases titers for unrelated poxvirus in the avian cells. This augmentation appears to be mediated via induction of heat shock and thus provides a novel cellular substrate that may allow further attenuation of vaccine strains.

[Overexpression of SEPP1 inhibits the proliferation and induces cell cycle G2/M arrest of 786-O and 769-P human renal carcinoma cells].

PubMed

Liu, Kan; Zhao, Chaofei; Chen, Jianwen; Wu, Shengpan; Yao, Yuanxin; Wu, Chong; Luo, Guoxiong; Zhang, Xu

2016-06-01

Objective To establish selenoprotein P, plasma 1 (SEPP1) gene recombinant lentiviral vector and investigate the effect of SEPP1 on the proliferation of human clear cell renal cell carcinoma (ccRCC) cells. Methods cDNA sequence of SEPP1 was cloned from the total cDNA of HEK293T cells by PCR. Then, the cDNA fragment was combined with the pLV-EGFP(2A)Puro vector and the constructed plasmid pLV-EGFP(2A)Puro-SEPP1 was transfected into HEK293T cells for packaging the virus. Forty-eight hours after transfected with the virus supernatant, the level of SEPP1 protein in 769-P and 786-O cells were tested by Western blotting. Cells were divided into recombinant lentivirus-infected cells, empty vector lentivirus-infected cells and the blank control cells. Cell proliferation rate was detected by MTS assay, colony forming ability was evaluated by plate clony formation assay and cell cycle change was assayed by flow cytometry after transfected with pLV-EGFP(2A)Puro-SEPP1 or empty pLV-EGFP(2A)Puro vector. Results Enzyme digestion analysis and DNA sequencing showed that the recombinant plasmid pLV-EGFP(2A)Puro-SEPP1 was constructed successfully. After being infected by the virus supernatant, the 786-O and 769-P cells expressed EGFP. Compared with the empty vector group and the blank control group, expression level of SEPP1 in the experimental group was much higher. The cell proliferative ability was inhibited in the cells overexpressing SEPP1, and the colony forming ability of SEPP1-overexpressed cells evidently decreased. Cell cycle was arrested in G2/M phase in 786-O cells overexpressing SEPP1. Conclusion The recombinant plasmid pLV-EGFP(2A)Puro-SEPP1 has been constructed successfully. Overexpression of SEPP1 could significantly reduce the proliferation rate of 786-O and 769P cells, and cause G2/M phase arrest of 786-O cells.

Viral vector-based influenza vaccines

PubMed Central

de Vries, Rory D.; Rimmelzwaan, Guus F.

2016-01-01

ABSTRACT Antigenic drift of seasonal influenza viruses and the occasional introduction of influenza viruses of novel subtypes into the human population complicate the timely production of effective vaccines that antigenically match the virus strains that cause epidemic or pandemic outbreaks. The development of game-changing vaccines that induce broadly protective immunity against a wide variety of influenza viruses is an unmet need, in which recombinant viral vectors may provide. Use of viral vectors allows the delivery of any influenza virus antigen, or derivative thereof, to the immune system, resulting in the optimal induction of virus-specific B- and T-cell responses against this antigen of choice. This systematic review discusses results obtained with vectored influenza virus vaccines and advantages and disadvantages of the currently available viral vectors. PMID:27455345

Viral vector-based influenza vaccines.

PubMed

de Vries, Rory D; Rimmelzwaan, Guus F

2016-11-01

Antigenic drift of seasonal influenza viruses and the occasional introduction of influenza viruses of novel subtypes into the human population complicate the timely production of effective vaccines that antigenically match the virus strains that cause epidemic or pandemic outbreaks. The development of game-changing vaccines that induce broadly protective immunity against a wide variety of influenza viruses is an unmet need, in which recombinant viral vectors may provide. Use of viral vectors allows the delivery of any influenza virus antigen, or derivative thereof, to the immune system, resulting in the optimal induction of virus-specific B- and T-cell responses against this antigen of choice. This systematic review discusses results obtained with vectored influenza virus vaccines and advantages and disadvantages of the currently available viral vectors.

Mouse mammary tumor virus-based vector transduces non-dividing cells, enters the nucleus via a TNPO3-independent pathway and integrates in a less biased fashion than other retroviruses.

PubMed

Konstantoulas, Constantine James; Indik, Stanislav

2014-04-30

Mouse mammary tumor virus (MMTV) is a complex, milk-born betaretrovirus, which preferentially infects dendritic cells (DC) in the gastrointestinal tract and then spreads to T and B lymphocytes and finally to the mammary gland. It is not clear how the prototypic betaretrovirus infects mucosal DCs and naïve lymphocytes as these cells are considered to be non-proliferative. Studies of MMTV biology have been hampered by the difficulty of obtaining sufficient virus/vector titers after transfection of a molecular clone in cultured cells. To surmount this barrier we developed a novel MMTV-based vector system with a split genome design containing potent posttranscriptional regulatory functions. Using this system, vector particles were produced to markedly greater titers (>1000-fold) than those obtained previously. The titers (>106 transduction units /ml) were comparable to those achieved with lentiviral or gammaretroviral vectors. Importantly, the vector transduced the enhanced green fluorescence protein gene into the chromosomes of non-dividing cells, such as cells arrested at the G2/M phase of the cell cycle and unstimulated hematopoietic progenitor cells, at an efficiency similar to that obtained with the HIV-1-based vector. In contrast to HIV-1, MMTV transductions were not affected by knocking down the expression of a factor involved in nuclear import of the HIV-1 pre-integration complexes, TNPO3. In contrast to HIV-1, the MMTV-based vector did not preferentially integrate in transcription units. Additionally, no preference for integration near transcription start sites, the regions preferentially targeted by gammaretroviral vectors, was observed. The vector derived from MMTV exhibits a random integration pattern. Overall, the betaretroviral vector system should facilitate molecular virology studies of the prototypic betaretrovirus as well as studies attempting to elucidate fundamental cellular processes such as nuclear import pathways. Random integration in cycling and non-cycling cells may be applicable in unbiased gene delivery.

A multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors for functional gene analysis.

PubMed

Weber, Kristoffer; Bartsch, Udo; Stocking, Carol; Fehse, Boris

2008-04-01

Functional gene analysis requires the possibility of overexpression, as well as downregulation of one, or ideally several, potentially interacting genes. Lentiviral vectors are well suited for this purpose as they ensure stable expression of complementary DNAs (cDNAs), as well as short-hairpin RNAs (shRNAs), and can efficiently transduce a wide spectrum of cell targets when packaged within the coat proteins of other viruses. Here we introduce a multicolor panel of novel lentiviral "gene ontology" (LeGO) vectors designed according to the "building blocks" principle. Using a wide spectrum of different fluorescent markers, including drug-selectable enhanced green fluorescent protein (eGFP)- and dTomato-blasticidin-S resistance fusion proteins, LeGO vectors allow simultaneous analysis of multiple genes and shRNAs of interest within single, easily identifiable cells. Furthermore, each functional module is flanked by unique cloning sites, ensuring flexibility and individual optimization. The efficacy of these vectors for analyzing multiple genes in a single cell was demonstrated in several different cell types, including hematopoietic, endothelial, and neural stem and progenitor cells, as well as hepatocytes. LeGO vectors thus represent a valuable tool for investigating gene networks using conditional ectopic expression and knock-down approaches simultaneously.

Packaging of Human Chromosome 19-Specific Adeno-Associated Virus (AAV) Integration Sites in AAV Virions during AAV Wild-Type and Recombinant AAV Vector Production

PubMed Central

Hüser, Daniela; Weger, Stefan; Heilbronn, Regine

2003-01-01

Adeno-associated virus type 2 (AAV-2) establishes latency by site-specific integration into a unique locus on human chromosome 19, called AAVS1. During the development of a sensitive real-time PCR assay for site-specific integration, AAV-AAVS1 junctions were reproducibly detected in highly purified AAV wild-type and recombinant AAV vector stocks. A series of controls documented that the junctions were packaged in AAV capsids and were newly generated during a single round of AAV production. Cloned junctions displayed variable AAV sequences fused to AAVS1. These data suggest that packaged junctions represent footprints of AAV integration during productive infection. Apparently, AAV latency established by site-specific integration and the helper virus-dependent, productive AAV cycle are more closely related than previously thought. PMID:12663794

Assessment of different virus-mediated approaches for retinal gene therapy of Usher 1B.

PubMed

Lopes, Vanda S; Diemer, Tanja; Williams, David S

2014-01-01

Usher syndrome type 1B, which is characterized by congenital deafness and progressive retinal degeneration, is caused by the loss of the function of MYO7A. Prevention of the retinal degeneration should be possible by delivering functional MYO7A to retinal cells. Although this approach has been used successfully in clinical trials for Leber congenital amaurosis (LCA2), it remains a challenge for Usher 1B because of the large size of the MYO7A cDNA. Different viral vectors have been tested for use in MYO7A gene therapy. Here, we review approaches with lentiviruses, which can accommodate larger genes, as well as attempts to use adeno-associated virus (AAV), which has a smaller packaging capacity. In conclusion, both types of viral vector appear to be effective. Despite concerns about the ability of lentiviruses to access the photoreceptor cells, a phenotype of the photoreceptors of Myo7a-mutant mice can be corrected. And although MYO7A cDNA is significantly larger than the nominal carrying capacity of AAV, AAV-MYO7A in single vectors also corrected Myo7a-mutant phenotypes in photoreceptor and RPE cells. Interestingly, however, a dual AAV vector approach was found to be much less effective.

Potential of Equine Herpesvirus 1 as a Vector for Immunization

PubMed Central

Trapp, Sascha; von Einem, Jens; Hofmann, Helga; Köstler, Josef; Wild, Jens; Wagner, Ralf; Beer, Martin; Osterrieder, Nikolaus

2005-01-01

Key problems using viral vectors for vaccination and gene therapy are antivector immunity, low transduction efficiencies, acute toxicity, and limited capacity to package foreign genetic information. It could be demonstrated that animal and human cells were efficiently transduced with equine herpesvirus 1 (EHV-1) reconstituted from viral DNA maintained and manipulated in Escherichia coli. Between 13 and 23% of primary human CD3+, CD4+, CD8+, CD11b+, and CD19+ cells and more than 70% of CD4+ MT4 cells or various human tumor cell lines (MeWo, Huh7, HeLa, 293T, or H1299) could be transduced with one infectious unit of EHV-1 per cell. After intranasal instillation of EHV-1 into mice, efficient transgene expression in lungs was detectable. Successful immunization using EHV-1 was shown after delivery of the human immunodeficiency virus type 1 Pr55gag precursor by the induction of a Gag-specific CD8+ immune response in mice. Because EHV-1 was not neutralized by human sera containing high titers of antibodies directed against human herpesviruses 1 to 5, it is concluded that this animal herpesvirus has enormous potential as a vaccine vector, because it is able to efficiently transduce a variety of animal and human cells, has high DNA packaging capacity, and can conveniently be maintained and manipulated in prokaryotic cells. PMID:15827159

Improved Prefusion Stability, Optimized Codon Usage, and Augmented Virion Packaging Enhance the Immunogenicity of Respiratory Syncytial Virus Fusion Protein in a Vectored-Vaccine Candidate

PubMed Central

Liang, Bo; Ngwuta, Joan O.; Surman, Sonja; Kabatova, Barbora; Liu, Xiang; Lingemann, Matthias; Liu, Xueqiao; Yang, Lijuan; Herbert, Richard; Swerczek, Joanna; Chen, Man; Moin, Syed M.; Kumar, Azad; McLellan, Jason S.; Kwong, Peter D.; Graham, Barney S.; Collins, Peter L.

2017-01-01

ABSTRACT Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory tract disease worldwide, but it lacks a licensed vaccine or suitable antiviral drug. A live attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) was developed previously as a vector expressing RSV fusion (F) protein to confer bivalent protection against RSV and HPIV3. In a previous clinical trial in virus-naive children, rB/HPIV3 was well tolerated but the immunogenicity of wild-type RSV F was unsatisfactory. We previously modified RSV F with a designed disulfide bond (DS) to increase stability in the prefusion (pre-F) conformation and to be efficiently packaged in the vector virion. Here, we further stabilized pre-F by adding both disulfide and cavity-filling mutations (DS-Cav1), and we also modified RSV F codon usage to have a lower CpG content and a higher level of expression. This RSV F open reading frame was evaluated in rB/HPIV3 in three forms: (i) pre-F without vector-packaging signal, (ii) pre-F with vector-packaging signal, and (iii) secreted pre-F ectodomain trimer. Despite being efficiently expressed, the secreted pre-F was poorly immunogenic. DS-Cav1 stabilized pre-F, with or without packaging, induced higher titers of pre-F specific antibodies in hamsters, and improved the quality of RSV-neutralizing serum antibodies. Codon-optimized RSV F containing fewer CpG dinucleotides had higher F expression, replicated more efficiently in vivo, and was more immunogenic. The combination of DS-Cav1 pre-F stabilization, optimized codon usage, reduced CpG content, and vector packaging significantly improved vector immunogenicity and protective efficacy against RSV. This provides an improved vectored RSV vaccine candidate suitable for pediatric clinical evaluation. IMPORTANCE RSV and HPIV3 are the first and second leading viral causes of severe pediatric respiratory disease worldwide. Licensed vaccines or suitable antiviral drugs are not available. We are developing a chimeric rB/HPIV3 vector expressing RSV F as a bivalent RSV/HPIV3 vaccine and have been evaluating means to increase RSV F immunogenicity. In this study, we evaluated the effects of improved stabilization of F in the pre-F conformation and of codon optimization resulting in reduced CpG content and greater pre-F expression. Reduced CpG content dampened the interferon response to infection, promoting higher replication and increased F expression. We demonstrate that improved pre-F stabilization and strategic manipulation of codon usage, together with efficient pre-F packaging into vector virions, significantly increased F immunogenicity in the bivalent RSV/HPIV3 vaccine. The improved immunogenicity included induction of increased titers of high-quality complement-independent antibodies with greater pre-F site Ø binding and greater protection against RSV challenge. PMID:28539444

A nonproliferating parvovirus vaccine vector elicits sustained, protective humoral immunity following a single intravenous or intranasal inoculation.

PubMed

Palmer, Gene A; Brogdon, Jennifer L; Constant, Stephanie L; Tattersall, Peter

2004-02-01

An ideal vaccine delivery system would elicit persistent protection following a single administration, preferably by a noninvasive route, and be safe even in the face of immunosuppression, either inherited or acquired, of the recipient. We have exploited the unique life cycle of the autonomous parvoviruses to develop a nonproliferating vaccine platform that appears to both induce priming and continually boost a protective immune response following a single inoculation. A crippled parvovirus vector was constructed, based on a chimera between minute virus of mice (MVM) and LuIII, which expresses Borrelia burgdorferi outer surface protein A (OspA) instead of its coat protein. The vector was packaged into an MVM lymphotropic capsid and inoculated into naive C3H/HeNcr mice. Vaccination with a single vector dose, either intravenously or intranasally, elicited high-titer anti-OspA-specific antibody that provided protection from live spirochete challenge and was sustained over the lifetime of the animal. Both humoral and cell-mediated Th(1) immunity was induced, as shown by anti-OspA immunoglobulin G2a antibody and preferential gamma interferon production by OspA-specific CD4(+) T cells.

A Nonproliferating Parvovirus Vaccine Vector Elicits Sustained, Protective Humoral Immunity following a Single Intravenous or Intranasal Inoculation

PubMed Central

Palmer, Gene A.; Brogdon, Jennifer L.; Constant, Stephanie L.; Tattersall, Peter

2004-01-01

An ideal vaccine delivery system would elicit persistent protection following a single administration, preferably by a noninvasive route, and be safe even in the face of immunosuppression, either inherited or acquired, of the recipient. We have exploited the unique life cycle of the autonomous parvoviruses to develop a nonproliferating vaccine platform that appears to both induce priming and continually boost a protective immune response following a single inoculation. A crippled parvovirus vector was constructed, based on a chimera between minute virus of mice (MVM) and LuIII, which expresses Borrelia burgdorferi outer surface protein A (OspA) instead of its coat protein. The vector was packaged into an MVM lymphotropic capsid and inoculated into naive C3H/HeNcr mice. Vaccination with a single vector dose, either intravenously or intranasally, elicited high-titer anti-OspA-specific antibody that provided protection from live spirochete challenge and was sustained over the lifetime of the animal. Both humoral and cell-mediated Th1 immunity was induced, as shown by anti-OspA immunoglobulin G2a antibody and preferential gamma interferon production by OspA-specific CD4+ T cells. PMID:14722265

Expression of human argininosuccinate synthetase after retroviral-mediated gene transfer.

PubMed

Wood, P A; Partridge, C A; O'Brien, W E; Beaudet, A L

1986-09-01

The cDNA sequence for human argininosuccinate synthetase (AS) was introduced into plasmid expression vectors with an SV40 promoter or Rous sarcoma virus promoter to construct pSV2-AS and pRSV-AS, respectively, and human enzyme was synthesized after gene transfer into Chinese hamster cells. The functional cDNA was inserted into the retroviral vectors pZIP-NeoSV(X) and pZIP-NeoSV(B). Ecotropic AS retrovirus was produced after calcium-phosphate-mediated gene transfer of these constructions into the packaging cell line psi-2, and viral titers up to 10(5) CFU/ml were obtained. Recombinant AS retrovirus was evaluated by detecting G-418-resistant colonies after infection of the rodent cells, XC, NRK, and 3T3. Colonies were also obtained when infected XC cells were selected in citrulline medium for expression of AS activity. Southern blot analysis of infected cells demonstrated that the recombinant retroviral genome was not altered grossly after infecting some rodent cells, while other cells showed evidence of rearrangement. A rapid assay for detecting AS retrovirus was developed based on the incorporation of [14C]citrulline into protein by intact 3T3 cells or XC cells.

Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function.

PubMed

Stanton, Richard J; McSharry, Brian P; Armstrong, Melanie; Tomasec, Peter; Wilkinson, Gavin W G

2008-12-01

With the enhanced capacity of bioinformatics to interrogate extensive banks of sequence data, more efficient technologies are needed to test gene function predictions. Replication-deficient recombinant adenovirus (Ad) vectors are widely used in expression analysis since they provide for extremely efficient expression of transgenes in a wide range of cell types. To facilitate rapid, high-throughput generation of recombinant viruses, we have re-engineered an adenovirus vector (designated AdZ) to allow single-step, directional gene insertion using recombineering technology. Recombineering allows for direct insertion into the Ad vector of PCR products, synthesized sequences, or oligonucleotides encoding shRNAs without requirement for a transfer vector Vectors were optimized for high-throughput applications by making them "self-excising" through incorporating the I-SceI homing endonuclease into the vector removing the need to linearize vectors prior to transfection into packaging cells. AdZ vectors allow genes to be expressed in their native form or with strep, V5, or GFP tags. Insertion of tetracycline operators downstream of the human cytomegalovirus major immediate early (HCMV MIE) promoter permits silencing of transgenes in helper cells expressing the tet repressor thus making the vector compatible with the cloning of toxic gene products. The AdZ vector system is robust, straightforward, and suited to both sporadic and high-throughput applications.

Construction of Various γ34.5 Deleted Fluorescent-Expressing Oncolytic herpes Simplex type 1 (oHSV) for Generation and Isolation of HSV-Based Vectors

PubMed

Abdoli, Shahriyar; Roohvand, Farzin; Teimoori-Toolabi, Ladan; Shokrgozar, Mohammad Ali; Bahrololoumi, Mina; Azadmanesh, Kayhan

2017-07-01

Oncolytic herpes simplex virus (oHSV)-based vectors lacking γ34.5 gene, are considered as ideal templates to construct efficient vectors for (targeted) cancer gene therapy. Herein, we reported the construction of three single/dually-flourescence labeled and γ34.5-deleted, recombinant HSV-1 vectors for rapid generation and easy selection/isolation of different HSV-Based vectors. Generation of recombinant viruses was performed with conventional homologous recombination methods using green fluorescent protein (GFP) and BleCherry harboring shuttle vectors. Viruses were isolated by direct fluorescence observation and standard plaque purifying methods and confirmed by PCR and sequencing and flow cytometry. XTT and plaque assay titration were performed on Vero, U87MG, and T98 GBM cell lines. We generated three recombinant viruses, HSV-GFP, HSV-GR (Green-Red), and HSV-Red. The HSV-GFP showed two log higher titer (1010 PFU) than wild type (108 PFU). In contrast, HSV-GR and HSV-Red showed one log lower titer (107 PFU) than parental HSV. Cytotoxicity analysis showed that HSV-GR and HSV-Red can lyse target tumor cells at multiplicity of infection of 10 and 1 (P<0.001). Moreover, HSV-GFP showed higher infection potency (98%) in comparison with HSV-GR (82%). Our oHSVs provide a simple and an efficient platform for construction and rapid isolation of 2nd and 3rd generation oHSVs by replacing the inserted dyes with transgenes and also for rapid identification via fluorescence activated cell sorting. These vectors can also be used for tracing the efficacy of therapeutic agents on target cells, imaging of neural or tumoral cells in vitro/in vivo and as oncolytic agents in cancer therapy.

Development of a functional cell-based assay that probes the specific interaction between influenza A virus NP and its packaging signal sequence RNA.

PubMed

Woo, Jiwon; Yu, Kyung Lee; Lee, Sun Hee; You, Ji Chang

2015-02-06

Although cis-acting packaging signal RNA sequences for the influenza virus NP encoding vRNA have been identified recently though genetic studies, little is known about the interaction between NP and the vRNA packaging signals either in vivo or in vitro. Here, we provide evidence that NP is able to interact specifically with the vRNA packaging sequence RNA within living cells and that the specific RNA binding activity of NP in vivo requires both the N-terminal and central region of the protein. This assay established would be a valuable tool for further detailed studies of the NP-packaging signal RNA interaction in living cells. Copyright © 2014 Elsevier Inc. All rights reserved.

Undetectable Transcription of cap in a Clinical AAV Vector: Implications for Preformed Capsid in Immune Responses

PubMed Central

Hauck, Bernd; Murphy, Samuel L; Smith, Peter H; Qu, Guang; Liu, Xingge; Zelenaia, Olga; Mingozzi, Federico; Sommer, Jürg M; High, Katherine A; Wright, J. Fraser

2008-01-01

In a gene therapy clinical trial for hemophilia B, adeno-associated virus 2 (AAV2) capsid–specific CD8+ T cells were previously implicated in the elimination of vector-transduced hepatocytes, resulting in loss of human factor IX (hFIX) transgene expression. To test the hypothesis that expression of AAV2 cap DNA impurities in the AAV2-hFIX vector was the source of epitopes presented on transduced cells, transcription of cap was assessed by quantitative reverse transcription–PCR (Q-RT-PCR) following transduction of target cells with the vector used in the clinical trial. Transcriptional profiling was also performed for residual AmpR, and adenovirus E2A and E4. Although trace amounts of DNA impurities were present in the clinical vector, transcription of these sequences was not detected after transduction of human hepatocytes, nor in mice administered a dose 26-fold above the highest dose administered in the clinical study. Two methods used to minimize encapsidated DNA impurities in the clinical vector were: (i) a vector (cis) production plasmid with a backbone exceeding the packaging limit of AAV; and (ii) a vector purification step that achieved separation of the vector from vector-related impurities (e.g., empty capsids). In conclusion, residual cap expression was undetectable following transduction with AAV2-hFIX clinical vectors. Preformed capsid protein is implicated as the source of epitopes recognized by CD8+ T cells that eliminated vector-transduced cells in the clinical study. PMID:18941440

Parainfluenza Virus Infection Sensitizes Cancer Cells to DNA-Damaging Agents: Implications for Oncolytic Virus Therapy.

PubMed

Fox, Candace R; Parks, Griffith D

2018-04-01

A parainfluenza virus 5 (PIV5) with mutations in the P/V gene (P/V-CPI - ) is restricted for spread in normal cells but not in cancer cells in vitro and is effective at reducing tumor burdens in mouse model systems. Here we show that P/V-CPI - infection of HEp-2 human laryngeal cancer cells results in the majority of the cells dying, but unexpectedly, over time, there is an emergence of a population of cells that survive as P/V-CPI - persistently infected (PI) cells. P/V-CPI - PI cells had elevated levels of basal caspase activation, and viability was highly dependent on the activity of cellular inhibitor-of-apoptosis proteins (IAPs) such as Survivin and XIAP. In challenge experiments with external inducers of apoptosis, PI cells were more sensitive to cisplatin-induced DNA damage and cell death. This increased cisplatin sensitivity correlated with defects in DNA damage signaling pathways such as phosphorylation of Chk1 and translocation of damage-specific DNA binding protein 1 (DDB1) to the nucleus. Cisplatin-induced killing of PI cells was sensitive to the inhibition of wild-type (WT) p53-inducible protein 1 (WIP1), a phosphatase which acts to terminate DNA damage signaling pathways. A similar sensitivity to cisplatin was seen with cells during acute infection with P/V-CPI - as well as during acute infections with WT PIV5 and the related virus human parainfluenza virus type 2 (hPIV2). Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish PI as well as the potential for combining chemotherapy with oncolytic RNA virus vectors. IMPORTANCE There is intense interest in developing oncolytic viral vectors with increased potency against cancer cells, particularly those cancer cells that have gained resistance to chemotherapies. We have found that infection with cytoplasmically replicating parainfluenza virus can result in increases in the killing of cancer cells by agents that induce DNA damage, and this is linked to alterations to DNA damage signaling pathways that balance cell survival versus death. Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish persistent infection, the repurposing of drugs that target cellular IAPs as antivirals, and the combined use of DNA-damaging chemotherapy agents in conjunction with oncolytic RNA virus vectors. Copyright © 2018 American Society for Microbiology.

Noninfectious virus-like particles produced by Moloney murine leukemia virus-based retrovirus packaging cells deficient in viral envelope become infectious in the presence of lipofection reagents

PubMed Central

Sharma, Sanjai; Murai, Fukashi; Miyanohara, Atsushi; Friedmann, Theodore

1997-01-01

Retrovirus packaging cell lines expressing the Moloney murine leukemia virus gag and pol genes but lacking virus envelope genes produce virus-like particles constitutively, whether or not they express a transcript from an integrated retroviral provirus. In the absence of a proviral transcript, the assembled particles contain processed gag and reverse transcriptase, and particles made by cells expressing an integrated lacZ provirus also contain viral RNA. The virus-like particles from both cell types are enveloped and are secreted/budded into the extracellular space but are noninfectious. Their physicochemical properties are similar to those of mature retroviral particles. The noninfectious gag pol RNA particles can readily be made infectious by the addition of lipofection reagents to produce preparations with titers of up to 105 colony-forming units per ml. PMID:9380714

9 CFR 113.35 - Detection of viricidal activity.

Code of Federal Regulations, 2011 CFR

2011-01-01

... OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD... used as diluent for a desiccated live virus vaccine in a combination package. (a) Bulk or final... with each virus fraction for which it is to be used as a diluent. If the vaccine to be rehydrated...

[Construction and Function Verification of a Novel Shuttle Vector Containing a Marker Gene Self-deletion System].

PubMed

Li, Lili; Wang, Zhan; Zhou, Yubai; Zhang, Fang; Shen, Sisi; Li, Zelin; Zeng, Yi

2015-09-01

For rapid and accurate screening of recombinant modified vaccinia virus Ankara (rMVA) that satisfied the quality standards of clinical trials, a novel shuttle vector that can delete the marker gene automatically during virus propagation was construted: pZL-EGFP. To construct the pZL-EGFP, the original shuttle vector pSC11 was modified by replacing the LacZ marker gene with enhanced green fluorescent protein (EGFP) and then inserting homologous sequences of TKL into the flank regions of EGFP. Baby hamster kidney (BHK)-21 cells were cotransfected with pZL-EGFP and MVA, and underwent ten passages and one plaque screening to obtain the EGFP-free rMVA carrying the exogenous gene. Resulting rMVA was tested by polymerase chain reaction and western blotting to verify pZL-EGFP function. A novel shuttle vector pZL-EGFP containing an EGFP marker gene which could be deleted automatically was constructed. This gene deletion had no effect on the activities of rMVA, and the exogenous gene could be expressed stably. These results suggest that rMVA can be packaged efficiently by homologous recombination between pZL-EGFP and MVA in BHK-21 cells, and that the carried EGFP gene can be removed automatically by intramolecular homologous recombination during virus passage. Meanwhile, the gene deletion had no influence on the activities of rMVA and the expression of exogenous target gene. This study lays a solid foundation for the future research.

Determination of Anti-Adeno-Associated Virus Vector Neutralizing Antibody Titer with an In Vitro Reporter System

PubMed Central

Meliani, Amine; Leborgne, Christian; Triffault, Sabrina; Jeanson-Leh, Laurence; Veron, Philippe

2015-01-01

Abstract Adeno-associated virus (AAV) vectors are a platform of choice for in vivo gene transfer applications. However, neutralizing antibodies (NAb) to AAV can be found in humans and some animal species as a result of exposure to the wild-type virus, and high-titer NAb develop following AAV vector administration. In some conditions, anti-AAV NAb can block transduction with AAV vectors even when present at low titers, thus requiring prescreening before vector administration. Here we describe an improved in vitro, cell-based assay for the determination of NAb titer in serum or plasma samples. The assay is easy to setup and sensitive and, depending on the purpose, can be validated to support clinical development of gene therapy products based on AAV vectors. PMID:25819687

A New Genetic Vaccine Platform Based on an Adeno-Associated Virus Isolated from a Rhesus Macaque ▿

PubMed Central

Lin, Jianping; Calcedo, Roberto; Vandenberghe, Luk H.; Bell, Peter; Somanathan, Suryanarayan; Wilson, James M.

2009-01-01

We created a hybrid adeno-associated virus (AAV) from two related rhesus macaque isolates, called AAVrh32.33, and evaluated it as a vaccine carrier for human immunodeficiency virus type 1 (HIV-1) and type A influenza virus antigens. The goal was to overcome the limitations of vaccines based on other AAVs, which generate dysfunctional T-cell responses and are inhibited by antibodies found in human sera. Injection of a Gag-expressing AAVrh32.33 vector into mice resulted in a high-quality CD8+ T-cell response. The resulting Gag-specific T cells express multiple cytokines at high levels, including interleukin-2, with many having memory phenotypes; a subsequent boost with an adenovirus vector yielded a brisk expansion of Gag-specific T cells. A priming dose of AAVrh32.33 led to high levels of Gag antibodies, which exceed levels found after injection of adenovirus vectors. Importantly, passive transfer of pooled human immunoglobulin into mice does not interfere with the efficacy of AAVrh32.33 expressing nucleoproteins from influenza virus, as measured by protection to a lethal dose of influenza virus, which is consistent with the very low seroprevalence to this virus in humans. Studies of macaques with vectors expressing gp140 from HIV-1 (i.e., with AAVrh32.33 as the prime and simian adenovirus type 24 as the boost) demonstrated results similar to those for mice with high-level and high-quality CD8+ T-cell responses to gp140 and high-titered neutralizing antibodies to homologous HIV-1. The biology of this novel AAV hybrid suggests that it should be a preferred genetic vaccine carrier, capable of generating robust T- and B-cell responses. PMID:19812149

Adeno-associated virus (AAV)-3-based vectors transduce haematopoietic cells not susceptible to transduction with AAV-2-based vectors.

PubMed

Handa, A; Muramatsu, S; Qiu, J; Mizukami, H; Brown, K E

2000-08-01

Although adeno-associated virus (AAV)-2 has a broad tissue-host range and can transduce a wide variety of tissue types, some cells, such as erythro-megakaryoblastoid cells, are non-permissive and appear to lack the AAV-2 receptor. However, limited studies have been reported with the related dependovirus AAV-3. We have previously cloned this virus, characterized its genome and produced an infectious clone. In this study, the gene for green fluorescent protein (GFP) was inserted into AAV-2- and AAV-3-based plasmids and recombinant viruses were produced. These viruses were then used to transduce haematopoietic cells and the transduction efficiencies were compared. In contrast to recombinant (r) AAV-2, rAAV-3 successfully transduced erythroid and megakaryoblastoid cells, although rAAV-2 was superior in transduction of lymphocyte-derived cell lines. Recently, it was reported that heparan sulphate can act as a receptor of AAV-2. The infectivity of rAAV-2 and rAAV-3 was tested with mutant cell lines of Chinese hamster ovary cells that were defective for heparin or heparan sulphate expression on the cell surface. There was no correlation between the ability of rAAV-2 or rAAV-3 to infect cells and the cell surface expression of heparan sulphate and, although heparin blocked both rAAV-2 and rAAV-3 transduction, the ID(50) of rAAV-3 was higher than that of rAAV-2. In addition, virus-binding overlay assays indicated that AAV-2 and AAV-3 bound different membrane proteins. These results suggest not only that there are different cellular receptors for AAV-2 and AAV-3, but that rAAV-3 vectors may be preferred for transduction of some haematopoietic cell types.

Nucleic acid delivery using magnetic nanoparticles: the Magnetofection technology.

PubMed

Laurentt, Nicolas; Sapet, Cédric; Le Gourrierec, Loic; Bertosio, Elodie; Zelphati, Olivier

2011-04-01

In recent years, gene therapy has received considerable interest as a potential method for the treatment of numerous inherited and acquired diseases. However, successes have so far been hampered by several limitations, including safety issues of viral-based nucleic acid vectors and poor in vivo efficiency of nonviral vectors. Magnetofection has been introduced as a novel and powerful tool to deliver genetic material into cells. This technology is defined as the delivery of nucleic acids, either 'naked' or packaged (as complexes with lipids or polymers, and viruses) using magnetic nanoparticles under the guidance of an external magnetic field. This article first discusses the principles of the Magnetofection technology and its benefits as compared with standard transfection methods. A number of relevant examples of its use, both in vitro and in vivo, will then be highlighted. Future trends in the development of new magnetic nanoparticle formulations will also be outlined.

Quantitative and qualitative features of heterologous virus-vector-induced antigen-specific CD8+ T cells against Trypanosoma cruzi infection.

PubMed

Takayama, Eiji; Ono, Takeshi; Carnero, Elena; Umemoto, Saori; Yamaguchi, Yoko; Kanayama, Atsuhiro; Oguma, Takemi; Takashima, Yasuhiro; Tadakuma, Takushi; García-Sastre, Adolfo; Miyahira, Yasushi

2010-11-01

We studied some aspects of the quantitative and qualitative features of heterologous recombinant (re) virus-vector-induced, antigen-specific CD8(+) T cells against Trypanosoma cruzi. We used three different, highly attenuated re-viruses, i.e., influenza virus, adenovirus and vaccinia virus, which all expressed a single, T. cruzi antigen-derived CD8(+) T-cell epitope. The use of two out of three vectors or the triple virus-vector vaccination regimen not only confirmed that the re-vaccinia virus, which was placed last in order for sequential immunisation, was an effective booster for the CD8(+) T-cell immunity in terms of the number of antigen-specific CD8(+) T cells, but also demonstrated that (i) the majority of cells exhibit the effector memory (T(EM)) phenotype, (ii) robustly secrete IFN-γ, (iii) express higher intensity of the CD122 molecule and (iv) present protective activity against T. cruzi infection. In contrast, placing the re-influenza virus last in sequential immunisation had a detrimental effect on the quantitative and qualitative features of CD8(+) T cells. The triple virus-vector vaccination was more effective at inducing a stronger CD8(+) T-cell immunity than using two re-viruses. The different quantitative and qualitative features of CD8(+) T cells induced by different immunisation regimens support the notion that the refinement of the best choice of multiple virus-vector combinations is indispensable for the induction of a maximum number of CD8(+) T cells of high quality. Copyright © 2010 Australian Society for Parasitology Inc. All rights reserved.

The Function of Herpes Simplex Virus Genes: A Primer for Genetic Engineering of Novel Vectors

NASA Astrophysics Data System (ADS)

Roizman, Bernard

1996-10-01

Herpes simplex virus vectors are being developed for delivery and expression of human genes to the central nervous system, selective destruction of cancer cells, and as carriers for genes encoding antigens that induce protective immunity against infectious agents. Vectors constructed to meet these objectives must differ from wild-type virus with respect to host range, reactivation from latency, and expression of viral genes. The vectors currently being developed are (i) helper free amplicons, (ii) replication defective viruses, and (iii) genetically engineered replication competent viruses with restricted host range. Whereas the former two types of vectors require stable, continuous cell lines expressing viral genes for their replication, the replication competent viruses will replicate on approved primary human cell strains.

Engineering T7 bacteriophage as a potential DNA vaccine targeting delivery vector.

PubMed

Xu, Hai; Bao, Xi; Wang, Yiwei; Xu, Yue; Deng, Bihua; Lu, Yu; Hou, Jibo

2018-03-20

DNA delivery with bacteriophage by surface-displayed mammalian cell penetrating peptides has been reported. Although, various phages have been used to facilitate DNA transfer by surface displaying the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide), no similar study has been conducted using T7 phage. In this study, we engineeredT7 phage as a DNA targeting delivery vector to facilitate cellular internalization. We constructed recombinant T7 phages that displayed Tat peptide on their surface and carried eukaryotic expression box (EEB) as a part of their genomes (T7-EEB-Tat). We demonstrated that T7 phage harboring foreign gene insertion had packaged into infective progeny phage particles. Moreover, when mammalian cells that were briefly exposed to T7-EEB-Tat, expressed a significant higher level of the marker gene with the control cells infected with the wide type phage without displaying Tat peptides. These data suggested that the potential of T7 phage as an effective delivery vector for DNA vaccine transfer.

Single-Step Conversion of Cells to Retrovirus Vector Producers with Herpes Simplex Virus–Epstein-Barr Virus Hybrid Amplicons

PubMed Central

Sena-Esteves, Miguel; Saeki, Yoshinaga; Camp, Sara M.; Chiocca, E. Antonio; Breakefield, Xandra O.

1999-01-01

We report here on the development and characterization of a novel herpes simplex virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV–Epstein-Barr virus (EBV) hybrid amplicons to efficiently convert cells to retrovirus vector producer cells after single-step transduction. The retrovirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV hybrid amplicon. Retrovirus expression cassettes were used to generate the HSV-EBV-retrovirus hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. Retrovirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retrovirus titers between 106 and 107 transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. Retrovirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retrovirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retrovirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery. PMID:10559361

Expression of Rous sarcoma virus-derived retroviral vectors in the avian blastoderm: Potential as stable genetic markers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Reddy, S.T.; Stoker, A.W.; Bissell, M.J.

1991-12-01

Retroviruses are valuable tools in studies of embryonic development, both as gene expression vectors and as cell lineage markers. In this study early chicken blastoderm cells are shown to be permissive for infection by Rous sarcoma virus and derivative replication-defective by Rous sarcoma virus and derivative replication-defective vectors, and, in contrast to previously published data, these cells will readily express viral genes. In cultured blastoderm cells, Rous sarcoma virus stably integrates and is transcribed efficiently, producing infectious virus particles. Using replication-defective vectors encoding the bacterial lacZ gene, the authors further show that blastoderms can be infected in culture and inmore » ovo. In ovo, lacZ expression is seen within 24 hours of virus inoculation, and by 96 hours stably expressing clones of cells are observed in diverse tissues throughout the embryo, including epidermis, somites, and heart, as well as in extraembryonic membranes. Given the rapid onset of vector expression and the broad range of permissive cell types, it should be feasible to use Rous sarcoma virus-derived retroviruses as early lineage markers and expression vectors beginning at the blastoderm stage of avian embryogenesis.« less

RD2-MolPack-Chim3, a packaging cell line for stable production of lentiviral vectors for anti-HIV gene therapy.

PubMed

Stornaiuolo, Anna; Piovani, Bianca Maria; Bossi, Sergio; Zucchelli, Eleonora; Corna, Stefano; Salvatori, Francesca; Mavilio, Fulvio; Bordignon, Claudio; Rizzardi, Gian Paolo; Bovolenta, Chiara

2013-08-01

Over the last two decades, several attempts to generate packaging cells for lentiviral vectors (LV) have been made. Despite different technologies, no packaging clone is currently employed in clinical trials. We developed a new strategy for LV stable production based on the HEK-293T progenitor cells; the sequential insertion of the viral genes by integrating vectors; the constitutive expression of the viral components; and the RD114-TR envelope pseudotyping. We generated the intermediate clone PK-7 expressing constitutively gag/pol and rev genes and, by adding tat and rd114-tr genes, the stable packaging cell line RD2-MolPack, which can produce LV carrying any transfer vector (TV). Finally, we obtained the RD2-MolPack-Chim3 producer clone by transducing RD2-MolPack cells with the TV expressing the anti-HIV transgene Chim3. Remarkably, RD114-TR pseudovirions have much higher potency when produced by stable compared with transient technology. Most importantly, comparable transduction efficiency in hematopoietic stem cells (HSC) is obtained with 2-logs less physical particles respect to VSV-G pseudovirions produced by transient transfection. Altogether, RD2-MolPack technology should be considered a valid option for large-scale production of LV to be used in gene therapy protocols employing HSC, resulting in the possibility of downsizing the manufacturing scale by about 10-fold in respect to transient technology.

Effects of vector backbone and pseudotype on lentiviral vector-mediated gene transfer: studies in infant ADA-deficient mice and rhesus monkeys.

PubMed

Carbonaro Sarracino, Denise; Tarantal, Alice F; Lee, C Chang I; Martinez, Michele; Jin, Xiangyang; Wang, Xiaoyan; Hardee, Cinnamon L; Geiger, Sabine; Kahl, Christoph A; Kohn, Donald B

2014-10-01

Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options.

Virus-mimetic polyplex particles for systemic and inflammation-specific targeted delivery of large genetic contents.

PubMed

Kang, S; Lu, K; Leelawattanachai, J; Hu, X; Park, S; Park, T; Min, I M; Jin, M M

2013-11-01

Systemic and target-specific delivery of large genetic contents has been difficult to achieve. Although viruses effortlessly deliver kilobase-long genome into cells, its clinical use has been hindered by serious safety concerns and the mismatch between native tropisms and desired targets. Nonviral vectors, in contrast, are limited by low gene transfer efficiency and inherent cytotoxicity. Here we devised virus-mimetic polyplex particles (VMPs) based on electrostatic self-assembly among polyanionic peptide (PAP), cationic polymer polyethyleneimine (PEI) and nucleic acids. We fused PAP to the engineered ligand-binding domain of integrin αLβ2 to target intercellular adhesion molecule-1 (ICAM-1), an inducible marker of inflammation. Fully assembled VMPs packaged large genetic contents, bound specifically to target molecules, elicited receptor-mediated endocytosis and escaped endosomal pathway, resembling intracellular delivery processes of viruses. Unlike conventional PEI-mediated transfection, molecular interaction-dependent gene delivery of VMPs was unaffected by the presence of serum and achieved higher efficiency without toxicity. By targeting overexpressed ICAM-1, VMPs delivered genes specifically to inflamed endothelial cells and macrophages both in vitro and in vivo. Simplicity and versatility of the platform and inflammation-specific delivery may open up opportunities for multifaceted gene therapy that can be translated into the clinic and treat a broad range of debilitating immune and inflammatory diseases.

Efficient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection

PubMed Central

Throm, Robert E.; Ouma, Annastasia A.; Zhou, Sheng; Chandrasekaran, Anantharaman; Lockey, Timothy; Greene, Michael; De Ravin, Suk See; Moayeri, Morvarid; Malech, Harry L.; Sorrentino, Brian P.

2009-01-01

Retroviral vectors containing internal promoters, chromatin insulators, and self-inactivating (SIN) long terminal repeats (LTRs) may have significantly reduced genotoxicity relative to the conventional retroviral vectors used in recent, otherwise successful clinical trials. Large-scale production of such vectors is problematic, however, as the introduction of SIN vectors into packaging cells cannot be accomplished with the traditional method of viral transduction. We have derived a set of packaging cell lines for HIV-based lentiviral vectors and developed a novel concatemeric array transfection technique for the introduction of SIN vector genomes devoid of enhancer and promoter sequences in the LTR. We used this method to derive a producer cell clone for a SIN lentiviral vector expressing green fluorescent protein, which when grown in a bioreactor generated more than 20 L of supernatant with titers above 107 transducing units (TU) per milliliter. Further refinement of our technique enabled the rapid generation of whole populations of stably transformed cells that produced similar titers. Finally, we describe the construction of an insulated, SIN lentiviral vector encoding the human interleukin 2 receptor common γ chain (IL2RG) gene and the efficient derivation of cloned producer cells that generate supernatants with titers greater than 5 × 107 TU/mL and that are suitable for use in a clinical trial for X-linked severe combined immunodeficiency (SCID-X1). PMID:19286997

Highly efficient gene transfer in naive human T cells with a murine leukemia virus-based vector.

PubMed

Dardalhon, V; Jaleco, S; Rebouissou, C; Ferrand, C; Skander, N; Swainson, L; Tiberghien, P; Spits, H; Noraz, N; Taylor, N

2000-08-01

Retroviral vectors based on the Moloney murine leukemia virus (MuLV) have become the primary tool for gene delivery into hematopoietic cells, but clinical trials have been hampered by low transduction efficiencies. Recently, we and others have shown that gene transfer of MuLV-based vectors into T cells can be significantly augmented using a fibronectin-facilitated protocol. Nevertheless, the relative abilities of naive (CD45RA(+)) and memory (CD45RO(+)) lymphocyte subsets to be transduced has not been assessed. Although naive T cells demonstrate a restricted cytokine profile following antigen stimulation and a decreased susceptibility to infection with human immunodeficiency virus, it was not clear whether they could be efficiently infected with a MuLV vector. This study describes conditions that permitted gene transfer of an enhanced green fluorescent protein-expressing retroviral vector in more than 50% of naive umbilical cord (UC) blood and peripheral blood (PB) T cells following CD3/CD28 ligation. Moreover, treatment of naive T cells with interleukin-7 resulted in the maintenance of a CD45RA phenotype and gene transfer levels approached 20%. Finally, it was determined that parameters for optimal transduction of CD45RA(+) T cells isolated from PB and UC blood differed: transduction of the UC cells was significantly increased by the presence of autologous mononuclear cells (24.5% versus 56.5%). Because naive T cells harbor a receptor repertoire that allows them to respond to novel antigens, the development of protocols targeting their transduction is crucial for gene therapy applications. This approach will also allow the functions of exogenous genes to be evaluated in primary nontransformed naive T cells.

A novel system for the production of high levels of functional human therapeutic proteins in stable cells with a Semliki Forest virus noncytopathic vector.

PubMed

Casales, Erkuden; Aranda, Alejandro; Quetglas, Jose I; Ruiz-Guillen, Marta; Rodriguez-Madoz, Juan R; Prieto, Jesus; Smerdou, Cristian

2010-05-31

Semliki Forest virus (SFV) vectors lead to high protein expression in mammalian cells, but expression is transient due to vector cytopathic effects, inhibition of host cell proteins and RNA-based expression. We have used a noncytopathic SFV mutant (ncSFV) RNA vector to generate stable cell lines expressing two human therapeutic proteins: insulin-like growth factor I (IGF-I) and cardiotrophin-1 (CT-1). Therapeutic genes were fused at the carboxy-terminal end of Puromycin N-acetyl-transferase gene by using as a linker the sequence coding for foot-and-mouth disease virus (FMDV) 2A autoprotease. These cassettes were cloned into the ncSFV vector. Recombinant ncSFV vectors allowed rapid and efficient selection of stable BHK cell lines with puromycin. These cells expressed IGF-I and CT-1 in supernatants at levels reaching 1.4 and 8.6 microg/10(6)cells/24 hours, respectively. Two cell lines generated with each vector were passaged ten times during 30 days, showing constant levels of protein expression. Recombinant proteins expressed at different passages were functional by in vitro signaling assays. Stability at RNA level was unexpectedly high, showing a very low mutation rate in the CT-1 sequence, which did not increase at high passages. CT-1 was efficiently purified from supernatants of ncSFV cell lines, obtaining a yield of approximately 2mg/L/24 hours. These results indicate that the ncSFV vector has a great potential for the production of recombinant proteins in mammalian cells. 2010 Elsevier B.V. All rights reserved.

A human parvovirus, adeno-associated virus, as a eucaryotic vector: Transient expression and encapsidation of the procaryotic gene for chloramphenicol acetyltransferase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tratschin, J.D.; West, M.H.P.; Sandbank, T.

1984-10-01

The authors have used the defective human parvovirus adeno-associated virus (AAV) as a novel eurocaryotic vector (parvector) for the expression of a foreign gene in human cells. The recombinant, pAV2, contains the AAV genome in a pBR322-derived bacterial plasmid. When pAV2 is transfected into human cells together with helper adenovirus particles, the AAV genome is rescued from the recombinant plasmid and replicated to produce infectious AAV particles at high efficiency. To create a vector, we inserted a procaryotic sequence coding for chloramphenicol acetyltransferase (CAT) into derivatives of pAV2 following either of the AAV promoters p/sub 40/ (pAVHiCAT) and p/sub 19/more » (pAVBcCAT). When transfected into human 293 cells or HeLa cells, pAVHiCAT expressed CAT activity in the absence of adenovirus. In the presence of adenovirus, this vector produced increased amounts of CAT activity and the recombinant AAV-CAT genome was replicated. In 293 cells, pAVBcCAT expressed a similar amount of CAT activity in the absence or presence of adenovirus and the recombinant AAV-CAT genome was not replicated. In HeLa cells, pAVBcCAT expressed low levels of CAT activity, but this level was elevated by coinfection with adenovirus particles or by cotransfection with a plasmid which expressed the adenovirus early region 1A (E1A) product. The E1A product is a transcriptional activator and is expressed in 293 cells. Thus, expression from two AAV promoters is differentially regulated: expression from p/sub 19/ is increased by E1A, whereas p/sub 40/ yields high levels of constitutive expression in the absence of E1A. Both AAV vectors were packaged into AAV particles by complementation with wild-type AAV and yielded CAT activity when subsequently infected into cells in the presence of adenovirus.« less

Comparative Characterization of the Sindbis Virus Proteome from Mammalian and Invertebrate Hosts Identifies nsP2 as a Component of the Virus Nucleocapsid and Sorting Nexin 5 as a Significant Host Factor for Alphavirus Replication.

PubMed

Schuchman, Ryan; Kilianski, Andy; Piper, Amanda; Vancini, Ricardo; Ribeiro, José M C; Sprague, Thomas R; Nasar, Farooq; Boyd, Gabrielle; Hernandez, Raquel; Glaros, Trevor

2018-05-09

Recent advances in mass spectrometry methods and instrumentation now allow for more accurate identification of proteins in low abundance. This technology was applied to Sindbis virus, the prototypical alphavirus to investigate the viral proteome. To determine if host proteins are specifically packaged into alphavirus virions, Sindbis virus (SINV) was grown in multiple host cells representing vertebrate and mosquito hosts and total protein content of purified virions was determined. This analysis identified host factors not previously associated with alphavirus entry, replication, or egress. One host protein, sorting nexin 5 (SNX5), was shown to be critical for the replication of three different alphaviruses, Sindbis, Mayaro and Chikungunya virus. The most significant finding was that in addition to the host proteins, SINV non-structural protein 2 (nsP2) was detected within virions grown in all host cells examined. The protein and RNA-interacting capabilities of nsP2 coupled with its presence in the virion support a role for nsP2 during packaging and/or entry of progeny virus. This function has not been identified for this protein. Taken together, this strategy identified at least one host factor integrally involved in alphavirus replication. Identification of other host proteins provides insight into alphavirus-host interactions during viral replication in both vertebrate and invertebrate hosts. This method of virus proteome analysis may also be useful for the identification of protein candidates for host-based therapeutics. IMPORTANCE Pathogenic Alphaviruses, such as Chikungunya and Mayaro virus, continue to plague public health in developing and developed countries alike. Alphaviruses belong to a group of viruses vectored in nature by hematophagous (blood-feeding) insects and are termed Arboviruses (arthropod-borne viruses). This group of viruses contains many human pathogens such as dengue fever, West Nile and Yellow fever viruses. With few exceptions there are no vaccines or prophylactics for these agents leaving one third of the world population at risk of infection. Identifying effective antivirals has been a long term goal for combating these diseases not only because of the lack of vaccines but also because they are effective during an ongoing epidemic. Mass spectrometry-based analysis of the Sindbis virus proteome can be effective in identifying host genes involved in virus replication and novel functions for virus proteins. Identification of these factors is invaluable for the prophylaxis of this group of viruses. Copyright © 2018 Schuchman et al.

Large Animal Models for Foamy Virus Vector Gene Therapy

PubMed Central

Trobridge, Grant D.; Horn, Peter A.; Beard, Brian C.; Kiem, Hans-Peter

2012-01-01

Foamy virus (FV) vectors have shown great promise for hematopoietic stem cell (HSC) gene therapy. Their ability to efficiently deliver transgenes to multi-lineage long-term repopulating cells in large animal models suggests they will be effective for several human hematopoietic diseases. Here, we review FV vector studies in large animal models, including the use of FV vectors with the mutant O6-methylguanine-DNA methyltransferase, MGMTP140K to increase the number of genetically modified cells after transplantation. In these studies, FV vectors have mediated efficient gene transfer to polyclonal repopulating cells using short ex vivo transduction protocols designed to minimize the negative effects of ex vivo culture on stem cell engraftment. In this regard, FV vectors appear superior to gammaretroviral vectors, which require longer ex vivo culture to effect efficient transduction. FV vectors have also compared favorably with lentiviral vectors when directly compared in the dog model. FV vectors have corrected leukocyte adhesion deficiency and pyruvate kinase deficiency in the dog large animal model. FV vectors also appear safer than gammaretroviral vectors based on a reduced frequency of integrants near promoters and also near proto-oncogenes in canine repopulating cells. Together, these studies suggest that FV vectors should be highly effective for several human hematopoietic diseases, including those that will require relatively high percentages of gene-modified cells to achieve clinical benefit. PMID:23223198

Induction of pluripotent stem cells from a cynomolgus monkey using a polycistronic simian immunodeficiency virus-based vector, differentiation toward functional cardiomyocytes, and generation of stably expressing reporter lines.

PubMed

Wunderlich, Stephanie; Haase, Alexandra; Merkert, Sylvia; Beier, Jennifer; Schwanke, Kristin; Schambach, Axel; Glage, Silke; Göhring, Gudrun; Curnow, Eliza C; Martin, Ulrich

2012-12-01

Induced pluripotent stem cells (iPSCs) represent a novel cell source for regenerative therapies. Many emerging iPSC-based therapeutic concepts will require preclinical evaluation in suitable large animal models. Among the large animal species frequently used in preclinical efficacy and safety studies, macaques show the highest similarities to humans at physiological, cellular, and molecular levels. We have generated iPSCs from cynomolgus monkeys (Macaca fascicularis) as a segue to regenerative therapy model development in this species. Because typical human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors show poor transduction of simian cells, a simian immunodeficiency virus (SIV)-based vector was chosen for efficient transduction of cynomolgus skin fibroblasts. A corresponding polycistronic vector with codon-optimized reprogramming factors was constructed for reprogramming. Growth characteristics as well as cell and colony morphology of the resulting cynomolgus iPSCs (cyiPSCs) were demonstrated to be almost identical to cynomolgus embryonic stem cells (cyESCs), and cyiPSCs expressed typical pluripotency markers including OCT4, SOX2, and NANOG. Furthermore, differentiation in vivo and in vitro into derivatives of all three germ layers, as well as generation of functional cardiomyocytes, could be demonstrated. Finally, a highly efficient technique for generation of transgenic cyiPSC clones with stable reporter expression in undifferentiated cells as well as differentiated transgenic cyiPSC progeny was developed to enable cell tracking in recipient animals. In conclusion, our data indicate that cyiPSCs represent a valuable cell source for establishment of macaque-based allogeneic and autologous preclinical cell transplantation models for various fields of regenerative medicine.

Characterization of an internal ribosomal entry segment within the 5' leader of avian reticuloendotheliosis virus type A RNA and development of novel MLV-REV-based retroviral vectors.

PubMed

López-Lastra, M; Gabus, C; Darlix, J L

1997-11-01

The murine leukemia virus (MLV)-related type C viruses constitute a major class of retroviruses that includes numerous endogenous and exogenous mammalian viruses and the related avian spleen necrosis virus (SNV). The MLV-related viruses possess a long and multifunctional 5' untranslated leader involved in key steps of the viral life cycle--splicing, translation, RNA dimerization, encapsidation, and reverse transcription. Recent studies have shown that the 5' leader of Friend murine leukemia virus and Moloney murine leukemia virus can direct cap independent translation of gag precursor proteins (Berlioz et al., 1995; Vagner et al., 1995b). These data, together with structural homology studies (Koning et al., 1992), prompted us to undertake a search for new internal ribosome entry segment (IRES) of retroviral origin. Here we describe an IRES element within the 5' leader of avian reticuloendotheliosis virus type A (REV-A) genomic RNA. Data show that the REV-A 5' IRES element maps downstream of the packaging/dimerization (E/DLS) sequence (Watanabe and Temin, 1982; Darlix et al., 1992) and the minimal IRES sequence appears to be within a 129 nt fragment (nucleotides 452-580) of the 5' leader, immediately upstream of the gag AUG codon. The REV-A IRES has been successfully utilized in the construction of novel high titer MLV-based retroviral vectors, containing one or more IRES elements of retroviral origin. These retroviral constructs, which represent a starting point for the design of novel vectors suitable for gene therapy, are also of interest as a model system of internal translation initiation and its possible regulation during development, cancer, or virus infection.

Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors

PubMed Central

Ban, Hiroshi; Nishishita, Naoki; Fusaki, Noemi; Tabata, Toshiaki; Saeki, Koichi; Shikamura, Masayuki; Takada, Nozomi; Inoue, Makoto; Hasegawa, Mamoru; Kawamata, Shin; Nishikawa, Shin-Ichi

2011-01-01

After the first report of induced pluripotent stem cells (iPSCs), considerable efforts have been made to develop more efficient methods for generating iPSCs without foreign gene insertions. Here we show that Sendai virus vector, an RNA virus vector that carries no risk of integrating into the host genome, is a practical solution for the efficient generation of safer iPSCs. We improved the Sendai virus vectors by introducing temperature-sensitive mutations so that the vectors could be easily removed at nonpermissive temperatures. Using these vectors enabled the efficient production of viral/factor-free iPSCs from both human fibroblasts and CD34+ cord blood cells. Temperature-shift treatment was more effective in eliminating remaining viral vector-related genes. The resulting iPSCs expressed human embryonic stem cell markers and exhibited pluripotency. We suggest that generation of transgene-free iPSCs from cord blood cells should be an important step in providing allogeneic iPSC-derived therapy in the future. PMID:21821793

Reprogramming Methods Do Not Affect Gene Expression Profile of Human Induced Pluripotent Stem Cells.

PubMed

Trevisan, Marta; Desole, Giovanna; Costanzi, Giulia; Lavezzo, Enrico; Palù, Giorgio; Barzon, Luisa

2017-01-20

Induced pluripotent stem cells (iPSCs) are pluripotent cells derived from adult somatic cells. After the pioneering work by Yamanaka, who first generated iPSCs by retroviral transduction of four reprogramming factors, several alternative methods to obtain iPSCs have been developed in order to increase the yield and safety of the process. However, the question remains open on whether the different reprogramming methods can influence the pluripotency features of the derived lines. In this study, three different strategies, based on retroviral vectors, episomal vectors, and Sendai virus vectors, were applied to derive iPSCs from human fibroblasts. The reprogramming efficiency of the methods based on episomal and Sendai virus vectors was higher than that of the retroviral vector-based approach. All human iPSC clones derived with the different methods showed the typical features of pluripotent stem cells, including the expression of alkaline phosphatase and stemness maker genes, and could give rise to the three germ layer derivatives upon embryoid bodies assay. Microarray analysis confirmed the presence of typical stem cell gene expression profiles in all iPSC clones and did not identify any significant difference among reprogramming methods. In conclusion, the use of different reprogramming methods is equivalent and does not affect gene expression profile of the derived human iPSCs.

Effects of Vector Backbone and Pseudotype on Lentiviral Vector-mediated Gene Transfer: Studies in Infant ADA-Deficient Mice and Rhesus Monkeys

PubMed Central

Carbonaro Sarracino, Denise; Tarantal, Alice F; Lee, C Chang I.; Martinez, Michele; Jin, Xiangyang; Wang, Xiaoyan; Hardee, Cinnamon L; Geiger, Sabine; Kahl, Christoph A; Kohn, Donald B

2014-01-01

Systemic delivery of a lentiviral vector carrying a therapeutic gene represents a new treatment for monogenic disease. Previously, we have shown that transfer of the adenosine deaminase (ADA) cDNA in vivo rescues the lethal phenotype and reconstitutes immune function in ADA-deficient mice. In order to translate this approach to ADA-deficient severe combined immune deficiency patients, neonatal ADA-deficient mice and newborn rhesus monkeys were treated with species-matched and mismatched vectors and pseudotypes. We compared gene delivery by the HIV-1-based vector to murine γ-retroviral vectors pseudotyped with vesicular stomatitis virus-glycoprotein or murine retroviral envelopes in ADA-deficient mice. The vesicular stomatitis virus-glycoprotein pseudotyped lentiviral vectors had the highest titer and resulted in the highest vector copy number in multiple tissues, particularly liver and lung. In monkeys, HIV-1 or simian immunodeficiency virus vectors resulted in similar biodistribution in most tissues including bone marrow, spleen, liver, and lung. Simian immunodeficiency virus pseudotyped with the gibbon ape leukemia virus envelope produced 10- to 30-fold lower titers than the vesicular stomatitis virus-glycoprotein pseudotype, but had a similar tissue biodistribution and similar copy number in blood cells. The relative copy numbers achieved in mice and monkeys were similar when adjusted to the administered dose per kg. These results suggest that this approach can be scaled-up to clinical levels for treatment of ADA-deficient severe combined immune deficiency subjects with suboptimal hematopoietic stem cell transplantation options. PMID:24925206

Development of Defective and Persistent Sendai Virus Vector

PubMed Central

Nishimura, Ken; Sano, Masayuki; Ohtaka, Manami; Furuta, Birei; Umemura, Yoko; Nakajima, Yoshiro; Ikehara, Yuzuru; Kobayashi, Toshihiro; Segawa, Hiroaki; Takayasu, Satoko; Sato, Hideyuki; Motomura, Kaori; Uchida, Eriko; Kanayasu-Toyoda, Toshie; Asashima, Makoto; Nakauchi, Hiromitsu; Yamaguchi, Teruhide; Nakanishi, Mahito

2011-01-01

The ectopic expression of transcription factors can reprogram differentiated tissue cells into induced pluripotent stem cells. However, this is a slow and inefficient process, depending on the simultaneous delivery of multiple genes encoding essential reprogramming factors and on their sustained expression in target cells. Moreover, once cell reprogramming is accomplished, these exogenous reprogramming factors should be replaced with their endogenous counterparts for establishing autoregulated pluripotency. Complete and designed removal of the exogenous genes from the reprogrammed cells would be an ideal option for satisfying this latter requisite as well as for minimizing the risk of malignant cell transformation. However, no single gene delivery/expression system has ever been equipped with these contradictory characteristics. Here we report the development of a novel replication-defective and persistent Sendai virus (SeVdp) vector based on a noncytopathic variant virus, which fulfills all of these requirements for cell reprogramming. The SeVdp vector could accommodate up to four exogenous genes, deliver them efficiently into various mammalian cells (including primary tissue cells and human hematopoietic stem cells) and express them stably in the cytoplasm at a prefixed balance. Furthermore, interfering with viral transcription/replication using siRNA could erase the genomic RNA of SeVdp vector from the target cells quickly and thoroughly. A SeVdp vector installed with Oct4/Sox2/Klf4/c-Myc could reprogram mouse primary fibroblasts quite efficiently; ∼1% of the cells were reprogrammed to Nanog-positive induced pluripotent stem cells without chromosomal gene integration. Thus, this SeVdp vector has potential as a tool for advanced cell reprogramming and for stem cell research. PMID:21138846

A single EBV-based vector for stable episomal maintenance and expression of GFP in human embryonic stem cells.

PubMed

Thyagarajan, Bhaskar; Scheyhing, Kelly; Xue, Haipeng; Fontes, Andrew; Chesnut, Jon; Rao, Mahendra; Lakshmipathy, Uma

2009-03-01

Stable expression of transgenes in stem cells has been a challenge due to the nonavailability of efficient transfection methods and the inability of transgenes to support sustained gene expression. Several methods have been reported to stably modify both embryonic and adult stem cells. These methods rely on integration of the transgene into the genome of the host cell, which could result in an expression pattern dependent on the number of integrations and the genomic locus of integration. To overcome this issue, site-specific integration methods mediated by integrase, adeno-associated virus or via homologous recombination have been used to generate stable human embryonic stem cell (hESC) lines. In this study, we describe a vector that is maintained episomally in hESCs. The vector used in this study is based on components derived from the Epstein-Barr virus, containing the Epstein-Barr virus nuclear antigen 1 expression cassette and the OriP origin of replication. The vector also expresses the drug-resistance marker gene hygromycin, which allows for selection and long-term maintenance of cells harboring the plasmid. Using this vector system, we show sustained expression of green fluorescent protein in undifferentiated hESCs and their differentiating embryoid bodies. In addition, the stable hESC clones show comparable expression with and without drug selection. Consistent with this observation, bulk-transfected adipose tissue-derived mesenchymal stem cells showed persistent marker gene expression as they differentiate into adipocytes, osteoblasts and chondroblasts. Episomal vectors offer a fast and efficient method to create hESC reporter lines, which in turn allows one to test the effect of overexpression of various genes on stem cell growth, proliferation and differentiation.

Matrix-M™ adjuvant enhances immunogenicity of both protein- and modified vaccinia virus Ankara-based influenza vaccines in mice.

PubMed

Magnusson, Sofia E; Altenburg, Arwen F; Bengtsson, Karin Lövgren; Bosman, Fons; de Vries, Rory D; Rimmelzwaan, Guus F; Stertman, Linda

2018-04-01

Influenza viruses continuously circulate in the human population and escape recognition by virus neutralizing antibodies induced by prior infection or vaccination through accumulation of mutations in the surface proteins hemagglutinin (HA) and neuraminidase (NA). Various strategies to develop a vaccine that provides broad protection against different influenza A viruses are under investigation, including use of recombinant (r) viral vectors and adjuvants. The replication-deficient modified vaccinia virus Ankara (MVA) is a promising vaccine vector that efficiently induces B and T cell responses specific for the antigen of interest. It is assumed that live vaccine vectors do not require an adjuvant to be immunogenic as the vector already mediates recruitment and activation of immune cells. To address this topic, BALB/c mice were vaccinated with either protein- or rMVA-based HA influenza vaccines, formulated with or without the saponin-based Matrix-M™ adjuvant. Co-formulation with Matrix-M significantly increased HA vaccine immunogenicity, resulting in antigen-specific humoral and cellular immune responses comparable to those induced by unadjuvanted rMVA-HA. Of special interest, rMVA-HA immunogenicity was also enhanced by addition of Matrix-M, demonstrated by enhanced HA inhibition antibody titres and cellular immune responses. Matrix-M added to either protein- or rMVA-based HA vaccines mediated recruitment and activation of antigen-presenting cells and lymphocytes to the draining lymph node 24 and 48 h post-vaccination. Taken together, these results suggest that adjuvants can be used not only with protein-based vaccines but also in combination with rMVA to increase vaccine immunogenicity, which may be a step forward to generate new and more effective influenza vaccines.

Differential CD4+ versus CD8+ T-cell responses elicited by different poxvirus-based human immunodeficiency virus type 1 vaccine candidates provide comparable efficacies in primates.

PubMed

Mooij, Petra; Balla-Jhagjhoorsingh, Sunita S; Koopman, Gerrit; Beenhakker, Niels; van Haaften, Patricia; Baak, Ilona; Nieuwenhuis, Ivonne G; Kondova, Ivanela; Wagner, Ralf; Wolf, Hans; Gómez, Carmen E; Nájera, José L; Jiménez, Victoria; Esteban, Mariano; Heeney, Jonathan L

2008-03-01

Poxvirus vectors have proven to be highly effective for boosting immune responses in diverse vaccine settings. Recent reports reveal marked differences in the gene expression of human dendritic cells infected with two leading poxvirus-based human immunodeficiency virus (HIV) vaccine candidates, New York vaccinia virus (NYVAC) and modified vaccinia virus Ankara (MVA). To understand how complex genomic changes in these two vaccine vectors translate into antigen-specific systemic immune responses, we undertook a head-to-head vaccine immunogenicity and efficacy study in the pathogenic HIV type 1 (HIV-1) model of AIDS in Indian rhesus macaques. Differences in the immune responses in outbred animals were not distinguished by enzyme-linked immunospot assays, but differences were distinguished by multiparameter fluorescence-activated cell sorter analysis, revealing a difference between the number of animals with both CD4(+) and CD8(+) T-cell responses to vaccine inserts (MVA) and those that elicit a dominant CD4(+) T-cell response (NYVAC). Remarkably, vector-induced differences in CD4(+)/CD8(+) T-cell immune responses persisted for more than a year after challenge and even accompanied antigenic modulation throughout the control of chronic infection. Importantly, strong preexposure HIV-1/simian immunodeficiency virus-specific CD4(+) T-cell responses did not prove deleterious with respect to accelerated disease progression. In contrast, in this setting, animals with strong vaccine-induced polyfunctional CD4(+) T-cell responses showed efficacies similar to those with stronger CD8(+) T-cell responses.

Large-scale Clinical-grade Retroviral Vector Production in a Fixed-Bed Bioreactor

PubMed Central

Wang, Xiuyan; Olszewska, Malgorzata; Qu, Jinrong; Wasielewska, Teresa; Bartido, Shirley; Hermetet, Gregory; Sadelain, Michel

2015-01-01

The successful genetic engineering of patient T cells with γ-retroviral vectors expressing chimeric antigen receptors or T-cell receptors for phase II clinical trials and beyond requires the large-scale manufacture of high-titer vector stocks. The production of retroviral vectors from stable packaging cell lines using roller bottles or 10- to 40-layer cell factories is limited by a narrow harvest window, labor intensity, open-system operations, and the requirement for significant incubator space. To circumvent these shortcomings, we optimized the production of vector stocks in a disposable fixed-bed bioreactor using good manufacturing practice–grade packaging cell lines. High-titer vector stocks were harvested over 10 days, representing a much broader harvest window than the 3-day harvest afforded by cell factories. For PG13 and 293Vec packaging cells, the average vector titer and the vector stocks’ yield in the bioreactor were higher by 3.2- to 7.3-fold, and 5.6- to 13.1-fold, respectively, than those obtained in cell factories. The vector production was 10.4 and 18.6 times more efficient than in cell factories for PG13 and 293Vec cells, respectively. Furthermore, the vectors produced from the fixed-bed bioreactors passed the release test assays for clinical applications. Therefore, a single vector lot derived from 293Vec is suitable to transduce up to 500 patients cell doses in the context of large clinical trials using chimeric antigen receptors or T-cell receptors. These findings demonstrate for the first time that a robust fixed-bed bioreactor process can be used to produce γ-retroviral vector stocks scalable up to the commercialization phase. PMID:25751502

Versatile Cosmid Vectors for the Isolation, Expression, and Rescue of Gene Sequences: Studies with the Human α -globin Gene Cluster

NASA Astrophysics Data System (ADS)

Lau, Yun-Fai; Kan, Yuet Wai

1983-09-01

We have developed a series of cosmids that can be used as vectors for genomic recombinant DNA library preparations, as expression vectors in mammalian cells for both transient and stable transformations, and as shuttle vectors between bacteria and mammalian cells. These cosmids were constructed by inserting one of the SV2-derived selectable gene markers-SV2-gpt, SV2-DHFR, and SV2-neo-in cosmid pJB8. High efficiency of genomic cloning was obtained with these cosmids and the size of the inserts was 30-42 kilobases. We isolated recombinant cosmids containing the human α -globin gene cluster from these genomic libraries. The simian virus 40 DNA in these selectable gene markers provides the origin of replication and enhancer sequences necessary for replication in permissive cells such as COS 7 cells and thereby allows transient expression of α -globin genes in these cells. These cosmids and their recombinants could also be stably transformed into mammalian cells by using the respective selection systems. Both of the adult α -globin genes were more actively expressed than the embryonic zeta -globin genes in these transformed cell lines. Because of the presence of the cohesive ends of the Charon 4A phage in the cosmids, the transforming DNA sequences could readily be rescued from these stably transformed cells into bacteria by in vitro packaging of total cellular DNA. Thus, these cosmid vectors are potentially useful for direct isolation of structural genes.

Recombinant modified vaccinia virus Ankara-based malaria vaccines.

PubMed

Sebastian, Sarah; Gilbert, Sarah C

2016-01-01

A safe and effective malaria vaccine is a crucial part of the roadmap to malaria elimination/eradication by the year 2050. Viral-vectored vaccines based on adenoviruses and modified vaccinia virus Ankara (MVA) expressing malaria immunogens are currently being used in heterologous prime-boost regimes in clinical trials for induction of strong antigen-specific T-cell responses and high-titer antibodies. Recombinant MVA is a safe and well-tolerated attenuated vector that has consistently shown significant boosting potential. Advances have been made in large-scale MVA manufacture as high-yield producer cell lines and high-throughput purification processes have recently been developed. This review describes the use of MVA as malaria vaccine vector in both preclinical and clinical studies in the past 5 years.

[Use of a novel baculovirus vector to express nucleoprotein gene of Crimean-Congo hemorrhagic fever virus in both insect and mammalian cells].

PubMed

Ma, Benjiang; Hang, Changshou; Zhao, Yun; Wang, Shiwen; Xie, Yanxiang

2002-09-01

To construct a novel baculovirus vector which is capable of promoting the high-yield expression of foreign gene in mammalian cells and to express by this vector the nucleoprotein (NP) gene of Crimean-Congo hemorrhagic fever virus (CCHFV) Chinese isolate (Xinjiang hemorrhagic fever virus, XHFV) BA88166 in insect and Vero cells. Human cytomegalovirus (CMV) immediate early (IE) promoter was ligated to the baculovirus vector pFastBac1 downstream of the polyhedrin promoter to give rise to the novel vector pCB1. XHFV NP gene was cloned to this vector and was well expressed in COS-7 cells and Vero cells by means of recombinant plasmid transfection and baculovirus infection. The XHFV NP gene in vector pCB1 could be well expressed in mammalian cells. Vero cells infected with recombinant baculovirus harboring NP gene could be employed as antigens to detect XHF serum specimens whose results were in good correlation with those of ELISA and in parallel with clinical diagnoses. This novel baculovirus vector is able to express the foreign gene efficiently in both insect and mammalian cells, which provides not only the convenient diagnostic antigens but also the potential for developing recombinant virus vaccines and gene therapies.

The expression of full length Gp91-phox protein is associated with reduced amphotropic retroviral production.

PubMed

Bellantuono, I; Lashford, L S; Rafferty, J A; Fairbairn, L J

2000-05-01

As a single gene defect in mature bone marrow cells, chronic granulomatous disease (X-CGD) represents a disorder which may be amenable to gene therapy by the transfer of the missing subunit into hemopoietic stem cells. In the majority of cases lack of Gp91-phox causes the disease. So far, studies involving transfer of Gp91-phox cDNA, including a phase I clinical trial, have yielded disappointing results. Most often, low titers of virus have been reported. In the present study we investigated the possible reasons for low titer amphotropic viral production. To investigate the effect of Gp91 cDNA on the efficiency of retroviral production from the packaging cell line, GP+envAm12, we constructed vectors containing either the native cDNA, truncated versions of the cDNA or a mutated form (LATG) in which the natural translational start codon was changed to a stop codon. Following derivation of clonal packaging cell lines, these were assessed for viral titer by RNA slot blot and analyzed by non-parametrical statistical analysis (Whitney-Mann U-test). An improvement in viral titer of just over two-fold was found in packaging cells containing the start-codon mutant of Gp91 and no evidence of truncated viral RNA was seen in these cells. Further analysis revealed the presence of rearranged forms of the provirus in Gp91-expressing cells, and the production of truncated, unpackaged viral RNA. Protein analysis revealed that LATG-transduced cells did not express full-length Gp91-phox, whereas those containing the wild-type cDNA did. However, a truncated protein was seen in ATG-transduced cells which was also present in wild type cells. No evidence for the presence of a negative transcriptional regulatory element was found from studies with the deletion mutants. A statistically significant effect of protein production on the production of virus from Gp91-expressing cells was found. Our data point to a need to restrict expression of the Gp91-phox protein and its derivatives in order to enhance retroviral production and suggest that improvements in current vectors for CGD gene therapy may need to include controlled, directed expression only in mature neutrophils.

From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging

PubMed Central

Ferrer, Mireia; Henriet, Simon; Chamontin, Célia; Lainé, Sébastien; Mougel, Marylène

2016-01-01

In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV) and complex human immunodeficiency virus type 1 (HIV-1) in mind, the techniques were described in order to benefit to a larger community. PMID:27556480

Applications and challenges of multivalent recombinant vaccines

PubMed Central

Naim, Hussein Y.

2013-01-01

The exceptional discoveries of antigen/gene delivery systems have allowed the development of novel prophylactic and therapeutic vaccine candidates. The vaccine candidates employ various antigen-delivery systems, particularly recombinant viral vectors. Recombinant viral vectors are experimental vaccines similar to DNA vaccines, but they use attenuated viruses or bacterium as a carrier “vector” to introduce microbial DNA to cells of the body. They closely mimic a natural infection and therefore can efficiently stimulate the immune system. Although such recombinant vectors may face extensive preclinical testing and will possibly have to meet stringent regulatory requirements, some of these vectors (e.g. measles virus vectors) may benefit from the profound industrial and clinical experience of the parent vaccine. Most notably, novel vaccines based on live attenuated viruses combine the induction of broad, strong and persistent immune responses with acceptable safety profiles. We assess certain technologies in light of their use against human immunodeficiency virus (HIV). PMID:23249651

Virus-Like-Vaccines against HIV

PubMed Central

Andersson, Anne-Marie C.; Schwerdtfeger, Melanie; Holst, Peter J.

2018-01-01

Protection against chronic infections has necessitated the development of ever-more potent vaccination tools. HIV seems to be the most challenging foe, with a remarkable, poorly immunogenic and fragile surface glycoprotein and the ability to overpower the cell immune system. Virus-like-particle (VLP) vaccines have emerged as potent inducers of antibody and helper T cell responses, while replication-deficient viral vectors have yielded potent cytotoxic T cell responses. Here, we review the emerging concept of merging these two technologies into virus-like-vaccines (VLVs) for the targeting of HIV. Such vaccines are immunologically perceived as viruses, as they infect cells and produce VLPs in situ, but they only resemble viruses, as the replication defective vectors and VLPs cannot propagate an infection. The inherent safety of such a platform, despite robust particle production, is a distinct advantage over live-attenuated vaccines that must balance safety and immunogenicity. Previous studies have delivered VLVs encoded in modified Vaccinia Ankara vectors and we have developed the concept into a single-reading adenovirus-based technology capable of eliciting robust CD8+ and CD4+ T cells responses and trimer binding antibody responses. Such vaccines offer the potential to display the naturally produced immunogen directly and induce an integrated humoral and cellular immune response. PMID:29439476

Virus-Like-Vaccines against HIV.

PubMed

Andersson, Anne-Marie C; Schwerdtfeger, Melanie; Holst, Peter J

2018-02-11

Protection against chronic infections has necessitated the development of ever-more potent vaccination tools. HIV seems to be the most challenging foe, with a remarkable, poorly immunogenic and fragile surface glycoprotein and the ability to overpower the cell immune system. Virus-like-particle (VLP) vaccines have emerged as potent inducers of antibody and helper T cell responses, while replication-deficient viral vectors have yielded potent cytotoxic T cell responses. Here, we review the emerging concept of merging these two technologies into virus-like-vaccines (VLVs) for the targeting of HIV. Such vaccines are immunologically perceived as viruses, as they infect cells and produce VLPs in situ, but they only resemble viruses, as the replication defective vectors and VLPs cannot propagate an infection. The inherent safety of such a platform, despite robust particle production, is a distinct advantage over live-attenuated vaccines that must balance safety and immunogenicity. Previous studies have delivered VLVs encoded in modified Vaccinia Ankara vectors and we have developed the concept into a single-reading adenovirus-based technology capable of eliciting robust CD8⁺ and CD4⁺ T cells responses and trimer binding antibody responses. Such vaccines offer the potential to display the naturally produced immunogen directly and induce an integrated humoral and cellular immune response.

Induction of complex immune responses and strong protection against retrovirus challenge by adenovirus-based immunization depends on the order of vaccine delivery.

PubMed

Kaulfuß, Meike; Wensing, Ina; Windmann, Sonja; Hrycak, Camilla Patrizia; Bayer, Wibke

2017-02-06

In the Friend retrovirus mouse model we developed potent adenovirus-based vaccines that were designed to induce either strong Friend virus GagL 85-93 -specific CD8 + T cell or antibody responses, respectively. To optimize the immunization outcome we evaluated vaccination strategies using combinations of these vaccines. While the vaccines on their own confer strong protection from a subsequent Friend virus challenge, the simple combination of the vaccines for the establishment of an optimized immunization protocol did not result in a further improvement of vaccine effectivity. We demonstrate that the co-immunization with GagL 85-93 /leader-gag encoding vectors together with envelope-encoding vectors abrogates the induction of GagL 85-93 -specific CD8 + T cells, and in successive immunization protocols the immunization with the GagL 85-93 /leader-gag encoding vector had to precede the immunization with an envelope encoding vector for the efficient induction of GagL 85-93 -specific CD8 + T cells. Importantly, the antibody response to envelope was in fact enhanced when the mice were adenovirus-experienced from a prior immunization, highlighting the expedience of this approach. To circumvent the immunosuppressive effect of envelope on immune responses to simultaneously or subsequently administered immunogens, we developed a two immunizations-based vaccination protocol that induces strong immune responses and confers robust protection of highly Friend virus-susceptible mice from a lethal Friend virus challenge.

Targeted systemic gene therapy and molecular imaging of cancer contribution of the vascular-targeted AAVP vector.

PubMed

Hajitou, Amin

2010-01-01

Gene therapy and molecular-genetic imaging have faced a major problem: the lack of an efficient systemic gene delivery vector. Unquestionably, eukaryotic viruses have been the vectors of choice for gene delivery to mammalian cells; however, they have had limited success in systemic gene therapy. This is mainly due to undesired uptake by the liver and reticuloendothelial system, broad tropism for mammalian cells causing toxicity, and their immunogenicity. On the other hand, prokaryotic viruses such as bacteriophage (phage) have no tropism for mammalian cells, but can be engineered to deliver genes to these cells. However, phage-based vectors have inherently been considered poor vectors for mammalian cells. We have reported a new generation of vascular-targeted systemic hybrid prokaryotic-eukaryotic vectors as chimeras between an adeno-associated virus (AAV) and targeted bacteriophage (termed AAV/phage; AAVP). In this hybrid vector, the targeted bacteriophage serves as a shuttle to deliver the AAV transgene cassette inserted in an intergenomic region of the phage DNA genome. As a proof of concept, we assessed the in vivo efficacy of vector in animal models of cancer by displaying on the phage capsid the cyclic Arg-Gly-Asp (RGD-4C) ligand that binds to alphav integrin receptors specifically expressed on the angiogenic blood vessels of tumors. The ligand-directed vector was able to specifically deliver imaging and therapeutic transgenes to tumors in mice, rats, and dogs while sparing the normal organs. This chapter reviews some gene transfer strategies and the potential of the vascular-targeted AAVP vector for enhancing the effectiveness of existing systemic gene delivery and genetic-imaging technologies. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Generation of a non-transmissive Borna disease virus vector lacking both matrix and glycoprotein genes.

PubMed

Fujino, Kan; Yamamoto, Yusuke; Daito, Takuji; Makino, Akiko; Honda, Tomoyuki; Tomonaga, Keizo

2017-09-01

Borna disease virus (BoDV), a prototype of mammalian bornavirus, is a non-segmented, negative strand RNA virus that often causes severe neurological disorders in infected animals, including horses and sheep. Unique among animal RNA viruses, BoDV transcribes and replicates non-cytopathically in the cell nucleus, leading to establishment of long-lasting persistent infection. This striking feature of BoDV indicates its potential as an RNA virus vector system. It has previously been demonstrated by our team that recombinant BoDV (rBoDV) lacking an envelope glycoprotein (G) gene develops persistent infections in transduced cells without loss of the viral genome. In this study, a novel non-transmissive rBoDV, rBoDV ΔMG, which lacks both matrix (M) and G genes in the genome, is reported. rBoDV-ΔMG expressing green fluorescence protein (GFP), rBoDV ΔMG-GFP, was efficiently generated in Vero/MG cells stably expressing both BoDV M and G proteins. Infection with rBoDV ΔMG-GFP was persistently maintained in the parent Vero cells without propagation within cell culture. The optimal ratio of M and G for efficient viral particle production by transient transfection of M and G expression plasmids into cells persistently infected with rBoDV ΔMG-GFP was also demonstrated. These findings indicate that the rBoDV ΔMG-based BoDV vector may provide an extremely safe virus vector system and could be a novel strategy for investigating the function of M and G proteins and the host range of bornaviruses. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

[Development of viral vectors and the application for viral entry mechanisms].

PubMed

Tani, Hideki

2011-06-01

Virus is identified as one of the obligate intracellular parasites, which only amplify in cells of specific living things. Viral vectors, which are developed by utilizing these properties, are available in the various fields such as basic research of medical biology or application of gene therapy. Our research group has studied development of viral vectors using properties of baculovirus or vesicular stomatitis virus (VSV). Due to the development of new baculoviral vectors for mammalian cells, it is possible to be more efficient transduction of foreign gene in mammalian cells and animals. Furthermore, pseudotype or recombinant VSV possessing the envelope proteins of hepatitis C virus, Japanese encephalitis virus or baculovirus were constructed, and characteristics of the envelope proteins or entry mechanisms of these viruses were analyzed.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Claus, Claudia; Tzeng, W.-P.; Liebert, Uwe Gerd

During serial passaging of rubella virus (RUB) in cell culture, the dominant species of defective-interfering RNA (DI) generated contains an in-frame deletion between the capsid protein (C) gene and E1 glycoprotein gene resulting in production of a C-E1 fusion protein that is necessary for the maintenance of the DI [Tzeng, W.P., Frey, T.K. (2006). C-E1 fusion protein synthesized by rubella virus DI RNAs maintained during serial passage. Virology 356 198-207.]. A BHK cell line stably expressing the RUB structural proteins was established which was used to package DIs into virus particles following transfection with in vitro transcripts from DI infectiousmore » cDNA constructs. Packaging of a DI encoding an in-frame C-GFP-E1 reporter fusion protein corresponding to the C-E1 fusion protein expressed in a native DI was only marginally more efficient than packaging of a DI encoding GFP, indicating that the C-E1 fusion protein did not function by enhancing packaging. However, infection with the DI encoding the C-GFP-E1 fusion protein (in the absence of wt RUB helper virus) resulted in formation of clusters of GFP-positive cells and the percentage of GFP-positive cells in the culture following infection remained relatively constant. In contrast, a DI encoding GFP did not form GFP-positive clusters and the percentage of GFP-positive cells declined by roughly half from 2 to 4 days post-infection. Cluster formation and sustaining the percentage of infected (GFP-positive) cells required the C part of the fusion protein, including the downstream but not the upstream of two arginine clusters (both of which are associated with RNA binding and association with mitochondrial p32 protein) and the E1 part through the transmembrane sequence, but not the C-terminal cytoplasmic tail. Among a collection of mutant DI constructs, cluster formation and sustaining infected cell percentage correlated with maintenance during serial passage with wt RUB. We hypothesize that cluster formation and sustaining infected cell percentage increase the likelihood of co-infection by a DI and wt RUB during serial passage thus enhancing maintenance of the DI. Cluster formation and sustaining infected cell percentage were found to be due to a combination of attenuated cytopathogenicity of DIs that express the C-E1 fusion protein and cell-to-cell movement of the DI. In infected cells, the C-GFP-E1 fusion protein was localized to potentially novel vesicular structures that appear to originate from ER-Golgi transport vacuoles. This species of DI expressing a C-E1 fusion protein that exhibits attenuated cytopathogenicity and the ability to increase the number of infected cells through cell-to-cell movement could be the basis for development of an attractive vaccine vector.« less

Vector Design Tour de Force: Integrating Combinatorial and Rational Approaches to Derive Novel Adeno-associated Virus Variants

PubMed Central

Marsic, Damien; Govindasamy, Lakshmanan; Currlin, Seth; Markusic, David M; Tseng, Yu-Shan; Herzog, Roland W; Agbandje-McKenna, Mavis; Zolotukhin, Sergei

2014-01-01

Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of “virtual family shuffling” to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 105 complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8. PMID:25048217

Perspective on Adeno-Associated Virus Capsid Modification for Duchenne Muscular Dystrophy Gene Therapy.

PubMed

Nance, Michael E; Duan, Dongsheng

2015-12-01

Duchenne muscular dystrophy (DMD) is a X-linked, progressive childhood myopathy caused by mutations in the dystrophin gene, one of the largest genes in the genome. It is characterized by skeletal and cardiac muscle degeneration and dysfunction leading to cardiac and/or respiratory failure. Adeno-associated virus (AAV) is a highly promising gene therapy vector. AAV gene therapy has resulted in unprecedented clinical success for treating several inherited diseases. However, AAV gene therapy for DMD remains a significant challenge. Hurdles for AAV-mediated DMD gene therapy include the difficulty to package the full-length dystrophin coding sequence in an AAV vector, the necessity for whole-body gene delivery, the immune response to dystrophin and AAV capsid, and the species-specific barriers to translate from animal models to human patients. Capsid engineering aims at improving viral vector properties by rational design and/or forced evolution. In this review, we discuss how to use the state-of-the-art AAV capsid engineering technologies to overcome hurdles in AAV-based DMD gene therapy.

Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys.

PubMed

Lingemann, Matthias; Liu, Xueqiao; Surman, Sonja; Liang, Bo; Herbert, Richard; Hackenberg, Ashley D; Buchholz, Ursula J; Collins, Peter L; Munir, Shirin

2017-05-15

The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (i.n.) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were replaced with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (C Δ170 ). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic i.n. vaccine candidate expressing GP from the pre-N position. It is expected to be well tolerated in humans and is available for clinical evaluation. IMPORTANCE EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect against mucosal as well as systemic inoculation are needed. We evaluated a version of human parainfluenza virus type 1 (HPIV1) bearing a stabilized attenuating mutation in the P/C gene (C Δ170 ) as an intranasal vaccine vector to express the EBOV glycoprotein GP. We evaluated expression from two different genome positions (pre-N and N-P) and investigated the use of vector packaging signals. African green monkeys immunized with two doses of the vector expressing GP from the pre-N position developed high titers of GP neutralizing serum antibodies. The attenuated vaccine candidate is expected to be safe and immunogenic and is available for clinical development. Copyright © 2017 American Society for Microbiology.

Geminiviruses for biotechnology: the art of parasite taming.

PubMed

Lozano-Durán, Rosa

2016-04-01

Viruses are intracellular pathogens that have evolved efficient strategies for replication and expression of their proteins in the host cells. Geminiviruses - plant viruses with small circular single-stranded DNA genomes - effectively manipulate plant cell processes for viral functions, entailing great potential for biotechnological applications. This potentiality has been realized in the form of protein expression and gene-silencing vectors, and, more recently, vectors for genome editing - a technology that these viruses seem particularly well-suited to facilitate. This insight offers an overview of the biological properties of geminiviruses, with emphasis on those leveraging development of geminivirus-based replicons. It illustrates the basis for engineering geminivirus-based replicons and their applications. Furthermore, it discusses the reported use and future perspectives of geminivirus-based replicons for genome editing. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

Randomization and In Vivo Selection Reveal a GGRG Motif Essential for Packaging Human Immunodeficiency Virus Type 2 RNA ▿ †

PubMed Central

Baig, Tayyba T.; Lanchy, Jean-Marc; Lodmell, J. Stephen

2009-01-01

The packaging signal (ψ) of human immunodeficiency virus type 2 (HIV-2) is present in the 5′ noncoding region of RNA and contains a 10-nucleotide palindrome (pal; 5′-392-GGAGUGCUCC) located upstream of the dimerization signal stem-loop 1 (SL1). pal has been shown to be functionally important in vitro and in vivo. We previously showed that the 3′ side of pal (GCUCC-3′) is involved in base-pairing interactions with a sequence downstream of SL1 to make an extended SL1, which is important for replication in vivo and the regulation of dimerization in vitro. However, the role of the 5′ side of pal (5′-GGAGU) was less clear. Here, we characterized this role using an in vivo SELEX approach. We produced a population of HIV-2 DNA genomes with random sequences within the 5′ side of pal and transfected these into COS-7 cells. Viruses from COS-7 cells were used to infect C8166 permissive cells. After several weeks of serial passage in C8166 cells, surviving viruses were sequenced. On the 5′ side of pal there was a striking convergence toward a GGRGN consensus sequence. Individual clones with consensus and nonconsensus sequences were tested in infectivity and packaging assays. Analysis of individuals that diverged from the consensus sequence showed normal viral RNA and protein synthesis but had replication defects and impaired RNA packaging. These findings clearly indicate that the GGRG motif is essential for viral replication and genomic RNA packaging. PMID:18971263

A Promising IFN-Deficient System to Manufacture IFN-Sensitive Influenza Vaccine Virus.

PubMed

Chen, Can; Fan, Wenhui; Li, Jing; Zheng, Weinan; Zhang, Shuang; Yang, Limin; Liu, Di; Liu, Wenjun; Sun, Lei

2018-01-01

Interferon (IFN)-sensitive and replication-incompetent influenza viruses are likely to be the alternatives to inactivated and attenuated virus vaccines. Some IFN-sensitive influenza vaccine candidates with modified non-structural protein 1 (NS1) are highly attenuated in IFN-competent hosts but induce robust antiviral immune responses. However, little research has been done on the manufacturability of these IFN-sensitive vaccine viruses. Here, RIG-I-knockout 293T cells were used to package the IFN-sensitive influenza A/WSN/33 (H1N1) virus expressing the mutant NS1 R38A/K41A. We found that the packaging efficiency of the NS1 R38A/K41A virus in RIG-I-knockout 293T cells was much higher than that in 293T cells. Moreover, the NS1 R38A/K41A virus almost lost its IFN antagonist activity and could no longer replicate in A549, MDCK, and Vero cells after 3-6 passages. This indicated that the replication of NS1 R38A/K41A virus is limited in conventional cells. Therefore, we further established a stable Vero cell line expressing the wild-type (WT) NS1 of the WSN virus, based on the Tet-On 3G system. The NS1 R38A/K41A virus was able to steadily propagate in this IFN-deficient cell line for at least 20 passages. In a mouse model, the NS1 R38A/K41A virus showed more than a 4-log reduction in lung virus titers compared to the WT virus at 3 and 5 days post infection. Furthermore, we observed that the NS1 R38A/K41A virus triggered high-level of IFN-α/β production in lung tissues and was eliminated from the host in a relatively short period of time. Additionally, this virus induced high-titer neutralizing antibodies against the WT WSN, A/Puerto Rico/8/1934 (PR8), or A/California/04/2009 (CA04) viruses and provided 100% protection against the WT WSN virus. Thus, we found that the replication of the NS1 R38A/K41A virus was limited in IFN-competent cells and mice. We also presented a promising IFN-deficient system, involving a RIG-I-knockout 293T cell line to package the IFN-sensitive vaccine virus and a stable Vero cell line expressing NS1 to propagate the IFN-sensitive vaccine virus. The IFN-deficient system is applicable for the manufacture of IFN-sensitive vaccine virus.

[Effects of SREBP-1 over-expression on fatty acid metabolism related genes expression in goats].

PubMed

Xu, Huifen; Luo, Jun; Li, Fang; Yu, Kang; Shi, Hengbo; Li, Jun; Lin, Xianzi; Zhu, Jiangjiang

2012-11-01

The aim of the study was to construct a recombinant adenovirus overexpression vector for Sterol Regulatory Element Binding Protein-1 (SREBP-1) of Xinong Saanen dairy goat, and to detect its effect on genes related to fatty acid metabolism in goat mammary epithelial cells, to establish foundation for further study of its roles in metabolism of fatty acid synthesis and lactation. First, we designed primers based on the SREBP-1 gene sequence in GenBank for PCR amplification and inserted the sequence into shuttle vector pAdTrack-CMV. The recombinant plasmid pAdTrack-CMV-SREBP-1 linearized by Pme I was transformed into E. coli BJ5183 competence cell containing the backbone vector pAdEasy-1 to obtain recombinant vector pAd-SREBP-1 by homologous recombination. pAd-SREBP-1 was linearized by Pac I and transfected into HEK 293 cell. Then we infected goat mammary epithelial cells with recombinant adenovirus which was packaged in HEK 293 cell line. The results showed that the recombinant adenovirus vector containing SREBP-1 was successfully constructed, and the titer of virus was 10(9) U/mL. Compared with the control group, mRNA level of SREBP-1 increased by about 15 times after infected for 48 h and 30 times after infected for 72 h. Fatty acid synthase (FASN) and Acetyl-CoA carboxylase (ACC) was upregulated by almost 2 times. The expression level of Peroxisome proliferator activated receptorgamma (PPARgamma) increased by 1.5 times. Liver X receptoralpha (LXRalpha) and Adipose triglyceride lipase (ATGL) upregulated by 1.2 times compared with that of control. But Stearoyl-coenzyme A desaturase (SCD) had no obvious change. In conclusion, SREBP-1 can activate the expression of genes related to fatty acid synthesis in mammary epithelial cells of Xinong Saanen dairy goat, demonstrated a regulatory function on the fatty acid metabolism in goat mammary gland.

Transduction of skin-migrating dendritic cells by human adenovirus 5 occurs via an actin-dependent phagocytic pathway.

PubMed

Guzman, Efrain; Taylor, Geraldine; Hope, Jayne; Herbert, Rebecca; Cubillos-Zapata, Carolina; Charleston, Bryan

2016-10-01

Dendritic cells (DC) are central to the initiation of immune responses, and various approaches have been used to target vaccines to DC in order to improve immunogenicity. Cannulation of lymphatic vessels allows for the collection of DC that migrate from the skin. These migrating DC are involved in antigen uptake and presentation following vaccination. Human replication-deficient adenovirus (AdV) 5 is a promising vaccine vector for delivery of recombinant antigens. Although the mechanism of AdV attachment and penetration has been extensively studied in permissive cell lines, few studies have addressed the interaction of AdV with DC. In this study, we investigated the interaction of bovine skin-migrating DC and replication-deficient AdV-based vaccine vectors. We found that, despite lack of expression of Coxsackie B-Adenovirus Receptor and other known adenovirus receptors, AdV readily enters skin-draining DC via an actin-dependent endocytosis. Virus exit from endosomes was pH independent, and neutralizing antibodies did not prevent virus entry but did prevent virus translocation to the nucleus. We also show that combining adenovirus with adjuvant increases the absolute number of intracellular virus particles per DC but not the number of DC containing intracellular virus. This results in increased trans-gene expression and antigen presentation. We propose that, in the absence of Coxsackie B-Adenovirus Receptor and other known receptors, AdV5-based vectors enter skin-migrating DC using actin-dependent endocytosis which occurs in skin-migrating DC, and its relevance to vaccination strategies and vaccine vector targeting is discussed.

Protective immunity against influenza in HLA-A2 transgenic mice by modified vaccinia virus Ankara vectored vaccines containing internal influenza proteins.

PubMed

Di Mario, Giuseppina; Sciaraffia, Ester; Facchini, Marzia; Gubinelli, Francesco; Soprana, Elisa; Panigada, Maddalena; Bernasconi, Valentina; Garulli, Bruno; Siccardi, Antonio; Donatelli, Isabella; Castrucci, Maria R

2017-03-01

The emergence of novel strains of influenza A viruses with hemagglutinins (HAs) that are antigenically distinct from those circulating in humans, and thus have pandemic potential, pose concerns and call for the development of more broadly protective influenza vaccines. In the present study, modified vaccinia virus Ankara (MVA) encoding internal influenza antigens were evaluated for their immunogenicity and ability to protect HLA-A2.1 transgenic (AAD) mice from infection with influenza viruses. MVAs expressing NP (MVA-NP), M1 (MVA-M1) or polymerase PB1 (MVA-PB1) of A/California/4/09 (CA/09) virus were generated and used to immunize AAD mice. Antibodies and CD8+T cell responses were assessed by ELISA and ELISPOT, respectively, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus. CD8+T cells specific to immunodominant and subdominant epitopes on the internal influenza proteins were elicited by MVA-based vectors in AAD mice, whereas influenza-specific antibodies were detected only in MVA-NP-immunized mice. Both M1- and NP-based MVA vaccines, regardless of whether they were applied individually or in combination, conferred protection against lethal influenza virus challenge. Our data further emphasize the promising potential of MVA vector expressing internal antigens toward the development of a universal influenza vaccine.

Production and first-in-man use of T cells engineered to express a HSVTK-CD34 sort-suicide gene.

PubMed

Zhan, Hong; Gilmour, Kimberly; Chan, Lucas; Farzaneh, Farzin; McNicol, Anne Marie; Xu, Jin-Hua; Adams, Stuart; Fehse, Boris; Veys, Paul; Thrasher, Adrian; Gaspar, Hubert; Qasim, Waseem

2013-01-01

Suicide gene modified donor T cells can improve immune reconstitution after allogeneic haematopoietic stem cell transplantation (SCT), but can be eliminated in the event of graft versus host disease (GVHD) through the administration of prodrug. Here we report the production and first-in-man use of mismatched donor T cells modified with a gamma-retroviral vector expressing a herpes simplex thymidine kinase (HSVTK):truncated CD34 (tCD34) suicide gene/magnetic selection marker protein. A stable packaging cell line was established to produce clinical grade vector pseudotyped with the Gibbon Ape Leukaemia Virus (GALV). T cells were transduced in a closed bag system following activation with anti-CD3/CD28 beads, and enriched on the basis of CD34 expression. Engineered cells were administered in two escalating doses to three children receiving T-depleted, CD34 stem cell selected, mismatched allogeneic grafts. All patients had pre-existing viral infections and received chemotherapy conditioning without serotherapy. In all three subjects cell therapy was tolerated without acute toxicity or the development of acute GVHD. Circulating gene modified T cells were detectable by flow cytometry and by molecular tracking in all three subjects. There was resolution of virus infections, concordant with detectable antigen-specific T cell responses and gene modified cells persisted for over 12 months. These findings highlight the suitability of tCD34 as a GMP compliant selection marker and demonstrate the feasibility, safety and immunological potential of HSVTK-tCD34 suicide gene modified donor T cells. ClinicalTrials.gov NCT01204502

RNA interference targets arbovirus replication in Culicoides cells.

PubMed

Schnettler, Esther; Ratinier, Maxime; Watson, Mick; Shaw, Andrew E; McFarlane, Melanie; Varela, Mariana; Elliott, Richard M; Palmarini, Massimo; Kohl, Alain

2013-03-01

Arboviruses are transmitted to vertebrate hosts by biting arthropod vectors such as mosquitoes, ticks, and midges. These viruses replicate in both arthropods and vertebrates and are thus exposed to different antiviral responses in these organisms. RNA interference (RNAi) is a sequence-specific RNA degradation mechanism that has been shown to play a major role in the antiviral response against arboviruses in mosquitoes. Culicoides midges are important vectors of arboviruses, known to transmit pathogens of humans and livestock such as bluetongue virus (BTV) (Reoviridae), Oropouche virus (Bunyaviridae), and likely the recently discovered Schmallenberg virus (Bunyaviridae). In this study, we investigated whether Culicoides cells possess an antiviral RNAi response and whether this is effective against arboviruses, including those with double-stranded RNA (dsRNA) genomes, such as BTV. Using reporter gene-based assays, we established the presence of a functional RNAi response in Culicoides sonorensis-derived KC cells which is effective in inhibiting BTV infection. Sequencing of small RNAs from KC and Aedes aegypti-derived Aag2 cells infected with BTV or the unrelated Schmallenberg virus resulted in the production of virus-derived small interfering RNAs (viRNAs) of 21 nucleotides, similar to the viRNAs produced during arbovirus infections of mosquitoes. In addition, viRNA profiles strongly suggest that the BTV dsRNA genome is accessible to a Dicer-type nuclease. Thus, we show for the first time that midge cells target arbovirus replication by mounting an antiviral RNAi response mainly resembling that of other insect vectors of arboviruses.

Integration of adeno-associated virus vectors in CD34+ human hematopoietic progenitor cells after transduction.

PubMed

Fisher-Adams, G; Wong, K K; Podsakoff, G; Forman, S J; Chatterjee, S

1996-07-15

Gene transfer vectors based on adeno-associated virus (AAV) appear promising because of their high transduction frequencies regardless of cell cycle status and ability to integrate into chromosomal DNA. We tested AAV-mediated gene transfer into a panel of human bone marrow or umbilical cord-derived CD34+ hematopoietic progenitor cells, using vectors encoding several transgenes under the control of viral and cellular promoters. Gene transfer was evaluated by (1) chromosomal integration of vector sequences and (2) analysis of transgene expression. Southern hybridization and fluorescence in situ hybridization analysis of transduced CD34 genomic DNA showed the presence of integrated vector sequences in chromosomal DNA in a portion of transduced cells and showed that integrated vector sequences were replicated along with cellular DNA during mitosis. Transgene expression in transduced CD34 cells in suspension cultures and in myeloid colonies differentiating in vitro from transduced CD34 cells approximated that predicted by the multiplicity of transduction. This was true in CD34 cells from different donors, regardless of the transgene or selective pressure. Comparisons of CD34 cell transduction either before or after cytokine stimulation showed similar gene transfer frequencies. Our findings suggest that AAV transduction of CD34+ hematopoietic progenitor cells is efficient, can lead to stable integration in a population of transduced cells, and may therefore provide the basis for safe and efficient ex vivo gene therapy of the hematopoietic system.

Characterization of a third generation lentiviral vector pseudotyped with Nipah virus envelope proteins for endothelial cell transduction.

PubMed

Witting, S R; Vallanda, P; Gamble, A L

2013-10-01

Lentiviruses are becoming progressively more popular as gene therapy vectors due to their ability to integrate into quiescent cells and recent clinical trial successes. Directing these vectors to specific cell types and limiting off-target transduction in vivo remains a challenge. Replacing the viral envelope proteins responsible for cellular binding, or pseudotyping, remains a common method to improve lentiviral targeting. Here, we describe the development of a high titer, third generation lentiviral vector pseudotyped with Nipah virus fusion protein (NiV-F) and attachment protein (NiV-G). Critical to high titers was truncation of the cytoplasmic domains of both NiV-F and NiV-G. As known targets of wild-type Nipah virus, primary endothelial cells are shown to be effectively transduced by the Nipah pseudotype. In contrast, human CD34+ hematopoietic progenitors were not significantly transduced. Additionally, the Nipah pseudotype has increased stability in human serum compared with vesicular stomatitis virus pseudotyped lentivirus. These findings suggest that the use of Nipah virus envelope proteins in third generation lentiviral vectors would be a valuable tool for gene delivery targeted to endothelial cells.

Dual Insect specific virus infection limits Arbovirus replication in Aedes mosquito cells.

PubMed

Schultz, Michaela J; Frydman, Horacio M; Connor, John H

2018-05-01

Aedes mosquitoes are vectors for many pathogenic viruses. Cell culture systems facilitate the investigation of virus growth in the mosquito vector. We found Zika virus (ZIKV) growth to be consistent in A. albopictus cells but hypervariable in A. aegypti cell lines. As a potential explanation of this variability, we tested the hypothesis that our cells harbored opportunistic viruses. We screened Aedes cell lines for the presence of insect specific viruses (ISVs), Cell-fusing agent virus (CFAV) and Phasi charoen-like virus (PCLV). PCLV was present in the ZIKV-growth-variable A. aegypti cell lines but absent in A. albopictus lines, suggesting that these ISVs may interfere with ZIKV growth. In support of this hypothesis, PCLV infection of CFAV-positive A. albopictus cells inhibited the growth of ZIKV, dengue virus and La Crosse virus. These data suggest ISV infection of cell lines can impact arbovirus growth leading to significant changes in cell permissivity to arbovirus infection. Copyright © 2018 Elsevier Inc. All rights reserved.

Overexpression of stearoyl-CoA desaturase 1 in bone marrow mesenchymal stem cells enhance the expression of induced endothelial cells

PubMed Central

2014-01-01

Background Bone marrow mesenchymal stem cells (BM-MSCs) are capable of differentiating into endothelial cells in vitro and acquire major characteristics of mature endothelial-like expression of vWF and CD31. SFAs and lipid oxidation products have been linked with postprandial endothelial dysfunction. Consumption of SFAs impairs arterial endothelial function, while a Mediterranean-type MUFA-diet has a beneficial effect on endothelial function by producing a decrease in levels of vWF, TFPI and PAI-1. Stearoyl-CoA desaturase 1 (SCD1), which converts SFA to MUFA, is involved in the cellular biosynthesis of MUFAs from SFA substrates. High expression of SCD1 is corresponded with low rates of fatty acid oxidation, therefore it might reduce inflammatory responses and be beneficial for the growth of induced endothelial cells. Overexpression of SCD1 in BM-MSCs might increase the growth of induced endothelial cells. The goal of this research is to study the relationship between overexpression of SCD1 and the expression of induced endothelial cells in BM-MSCs in vitro. Methods The gene SCD1 was integrated into a lentiviral vector, and then 293 T cells were transfected by the connected product to produce a packaged virus. BM-MSCs were infected by the packaged virus. Cell culture and endothelial induction were performed. Fluorescent quantitative PCR of CD31, vWF and VE-cad was performed after 1 week and 2 weeks to test the growth of induced endothelial cells. Results The mRNA amount of CD31, vWF and VE-cad of the SCD1 overexpressed group was statistically higher than that of the empty vector (EV) group and that of the normal group after 1 week and 2 weeks, respectively (p < 0.05). Immunocytochemical staining of CD31 or vWF was detected by visualizing red color. Conclusions This study suggested that overexpression of SCD1 in BM-MSCs could increase the expression of induced endothelial cells in vitro. PMID:24650127

Parainfluenza virus 5-based vaccine vectors expressing vaccinia virus (VACV) antigens provide long-term protection in mice from lethal intranasal VACV challenge.

PubMed

Clark, Kimberly M; Johnson, John B; Kock, Nancy D; Mizel, Steven B; Parks, Griffith D

2011-10-25

To test the potential for parainfluenza virus 5 (PIV5)-based vectors to provide protection from vaccinia virus (VACV) infection, PIV5 was engineered to express secreted VACV L1R and B5R proteins, two important antigens for neutralization of intracellular mature (IMV) and extracellular enveloped (EEV) virions, respectively. Protection of mice from lethal intranasal VACV challenge required intranasal immunization with PIV5-L1R/B5R in a prime-boost protocol, and correlated with low VACV-induced pathology in the respiratory tract and anti-VACV neutralizing antibody. Mice immunized with PIV5-L1R/B5R showed some disease symptoms following VACV challenge such as loss of weight and hunching, but these symptoms were delayed and less severe than with unimmunized control mice. While immunization with PIV5 expressing B5R alone conferred at least some protection, the most effective immunization included the PIV5 vector expressing L1R alone or in combination with PIV5-B5R. PIV5-L1R/B5R vectors elicited protection from VACV challenge even when CD8+ cells were depleted, but not in the case of mice that were defective in B cell production. Mice were protected from VACV challenge out to at least 1.5 years after immunization with PIV5-L1R/B5R vectors, and showed significant levels of anti-VACV neutralizing antibodies. These results demonstrate the potential for PIV5-based vectors to provide long lasting protection against complex human respiratory pathogens such as VACV, but also highlight the need to understand mechanisms for the generation of strong immune responses against poorly immunogenic viral proteins. Copyright © 2011 Elsevier Inc. All rights reserved.

Inhibition of hepatitis B virus replication via HBV DNA cleavage by Cas9 from Staphylococcus aureus.

PubMed

Liu, Yu; Zhao, Miaoxian; Gong, Mingxing; Xu, Ying; Xie, Cantao; Deng, Haohui; Li, Xueying; Wu, Hongkai; Wang, Zhanhui

2018-04-01

Chronic hepatitis B virus (HBV) infection is difficult to cure due to the presence of covalently closed circular DNA (cccDNA). Accumulating evidence indicates that the CRISPR/Cas9 system effectively disrupts HBV genome, including cccDNA, in vitro and in vivo. However, efficient delivery of CRISPR/Cas9 system to the liver or hepatocytes using an adeno-associated virus (AAV) vector remains challenging due to the large size of Cas9 from Streptococcus pyogenes (Sp). The recently identified Cas9 protein from Staphylococcus aureus (Sa) is smaller than SpCas9 and thus is able to be packaged into the AAV vector. To examine the efficacy of SaCas9 system on HBV genome destruction, we designed 5 guide RNAs (gRNAs) that targeted different HBV genotypes, 3 of which were shown to be effective. The SaCas9 system significantly reduced HBV antigen expression, as well as pgRNA and cccDNA levels, in Huh7, HepG2.2.15 and HepAD38 cells. The dual expression of gRNAs/SaCas9 in these cell lines resulted in more efficient HBV genome cleavage. In the mouse model, hydrodynamic injection of gRNA/SaCas9 plasmids resulted in significantly lower levels of HBV protein expression. We also delivered the SaCas9 system into mice with persistent HBV replication using an AAV vector. Both the AAV vector and the mRNA of Cas9 could be detected in the C3H mouse liver cells. Decreased hepatitis B surface antigen (HBsAg), HBV DNA and pgRNA levels were observed when a higher titer of AAV was injected, although this decrease was not significantly different from the control. In summary, the SaCas9 system accurately and efficiently targeted the HBV genome and inhibited HBV replication both in vitro and in vivo. The system was delivered by an AAV vector and maybe used as a novel therapeutic strategy against chronic HBV infection. Copyright © 2018 Elsevier B.V. All rights reserved.

Helper-Free Foamy Virus Vectors

PubMed Central

TROBRIDGE, GRANT D.; RUSSELL, DAVID W.

2010-01-01

Retroviral vectors based on human foamy virus (HFV) have been developed and show promise as gene therapy vehicles. Here we describe a method for the production of HFV vector stocks free of detectable helper virus. The helper and vector plasmid constructs used both lack the HFV bel genes, so recombination between these constructs cannot create a wild-type virus. A fusion promoter that combines portions of the cytomegalovirus (CMV) immediate-early and HFV long terminal repeat (LTR) promoters was used to drive expression of both the helper and vector constructs. The CMV–LTR fusion promoter allows for HFV vector production in the absence of the Bel-1 trans-activator protein, which would otherwise be necessary for efficient transcription from the HFV LTR. Vector stocks containing either neomycin phosphotransferase or alkaline phosphatase reporter genes were produced by transient transfection at titers greater than 105 transducing units/ml. G418-resistant BHK-21 cells obtained by transduction with neo vectors contained randomly integrated HFV vector proviruses without detectable deletions or rearrangements. The vector stocks generated were free of replication-competent retrovirus (RCR), as determined by assays for LTR trans-activation and a marker rescue assay developed here for the detection of Bel-independent RCR. OVERVIEW SUMMARY Vectors based on human foamy virus have been developed but low titers and the presence of replication-competent retrovirus (RCR) in vector stocks have prevented their use in preclinical animal experiments. We have developed a transient transfection method that can be used to produce replication-incompetent HFV vector stocks at titers greater than 105/ml, and that does not produce contaminating RCR. The use of CMV-HFV LTR fusion promoters in the helper and vector constructs has circumvented the requirement for the HFV Bel-1 trans-activator protein. Consequently, the potential for generating wild-type HFV by recombination between helper and vector constructs during vector production has been eliminated. Here we describe HFV vector production using this Bel-independent system. PMID:9853518

Adeno-Associated Virus Vectors and Stem Cells: Friends or Foes?

PubMed

Brown, Nolan; Song, Liujiang; Kollu, Nageswara R; Hirsch, Matthew L

2017-06-01

The infusion of healthy stem cells into a patient-termed "stem-cell therapy"-has shown great promise for the treatment of genetic and non-genetic diseases, including mucopolysaccharidosis type 1, Parkinson's disease, multiple sclerosis, numerous immunodeficiency disorders, and aplastic anemia. Stem cells for cell therapy can be collected from the patient (autologous) or collected from another "healthy" individual (allogeneic). The use of allogenic stem cells is accompanied with the potentially fatal risk that the transplanted donor T cells will reject the patient's cells-a process termed "graft-versus-host disease." Therefore, the use of autologous stem cells is preferred, at least from the immunological perspective. However, an obvious drawback is that inherently as "self," they contain the disease mutation. As such, autologous cells for use in cell therapies often require genetic "correction" (i.e., gene addition or editing) prior to cell infusion and therefore the requirement for some form of nucleic acid delivery, which sets the stage for the AAV controversy discussed herein. Despite being the most clinically applied gene delivery context to date, unlike other more concerning integrating and non-integrating vectors such as retroviruses and adenovirus, those based on adeno-associated virus (AAV) have not been employed in the clinic. Furthermore, published data regarding AAV vector transduction of stem cells are inconsistent in regards to vector transduction efficiency, while the pendulum swings far in the other direction with demonstrations of AAV vector-induced toxicity in undifferentiated cells. The variation present in the literature examining the transduction efficiency of AAV vectors in stem cells may be due to numerous factors, including inconsistencies in stem-cell collection, cell culture, vector preparation, and/or transduction conditions. This review summarizes the controversy surrounding AAV vector transduction of stem cells, hopefully setting the stage for future elucidation and eventual therapeutic applications.

Inducing indel mutation in the SOX6 gene by zinc finger nuclease for gamma reactivation: An approach towards gene therapy of beta thalassemia.

PubMed

Modares Sadeghi, Mehran; Shariati, Laleh; Hejazi, Zahra; Shahbazi, Mansoureh; Tabatabaiefar, Mohammad Amin; Khanahmad, Hossein

2018-03-01

β-thalassemia is a common autosomal recessive disorder characterized by a deficiency in the synthesis of β-chains. Evidences show that increased HbF levels improve the symptoms in patients with β-thalassemia or sickle cell anemia. In this study, ZFN technology was applied to induce a mutation in the binding domain region of SOX6 to reactivate γ-globin expression. The sequences coding for ZFP arrays were designed and sub cloned in TDH plus as a transfer vector. The ZFN expression was confirmed using Western blot analysis. In the next step, using the site-directed mutagenesis strategy through the overlap PCR, a missense mutation (D64V) was induced in the catalytic domain of the integrase gene in the packaging plasmid and verified using DNA sequencing. Then, the integrase minus lentivirus containing ZFN cassette was packaged. Transduction of K562 cells with this virus was performed. Mutation detection assay was performed. The indel percentage of the cells transducted with lenti virus containing ZFN was 31%. After 5 days of erythroid differentiation with 15 μg/mL cisplatin, the levels of γ-globin mRNA were sixfold in the cells treated with ZFN compared to untreated cells. In the meantime, the measurement of HbF expression levels was carried out using hemoglobin electrophoresis and showed the same results. Integrase minus lentivirus can provide a useful tool for efficient transient gene expression and helps avoid disadvantages of gene targeting using the native virus. The ZFN strategy applied here to induce indel on SOX6 gene in adult erythroid progenitors may provide a method to activate fetal hemoglobin expression in individuals with β-thalassemia. © 2017 Wiley Periodicals, Inc.

Friendly fire: redirecting herpes simplex virus-1 for therapeutic applications.

PubMed

Advani, S J; Weichselbaum, R R; Whitley, R J; Roizman, B

2002-09-01

Herpes simplex virus-1 (HSV-1) is a relatively large double-stranded DNA virus encoding at least 89 proteins with well characterized disease pathology. An understanding of the functions of viral proteins together with the ability to genetically engineer specific viral mutants has led to the development of attenuated HSV-1 for gene therapy. This review highlights the progress in creating attenuated genetically engineered HSV-1 mutants that are either replication competent (viral non-essential gene deleted) or replication defective (viral essential gene deleted). The choice between a replication-competent or -defective virus is based on the end-goal of the therapeutic intervention. Replication-competent HSV-1 mutants have primarily been employed as antitumor oncolytic viruses, with the lytic nature of the virus harnessed to destroy tumor cells selectively. In replacement gene therapy, replication-defective viruses have been utilized as delivery vectors. The advantages of HSV-1 vectors are that they infect quiescent and dividing cells efficiently and can encode for relatively large transgenes.

Interactions of Prototype Foamy Virus Capsids with Host Cell Polo-Like Kinases Are Important for Efficient Viral DNA Integration

PubMed Central

Zurnic, Irena; Hütter, Sylvia; Rzeha, Ute; Stanke, Nicole; Reh, Juliane; Müllers, Erik; Hamann, Martin V.; Kern, Tobias; Gerresheim, Gesche K.; Serrao, Erik; Lesbats, Paul; Engelman, Alan N.; Cherepanov, Peter; Lindemann, Dirk

2016-01-01

Unlike for other retroviruses, only a few host cell factors that aid the replication of foamy viruses (FVs) via interaction with viral structural components are known. Using a yeast-two-hybrid (Y2H) screen with prototype FV (PFV) Gag protein as bait we identified human polo-like kinase 2 (hPLK2), a member of cell cycle regulatory kinases, as a new interactor of PFV capsids. Further Y2H studies confirmed interaction of PFV Gag with several PLKs of both human and rat origin. A consensus Ser-Thr/Ser-Pro (S-T/S-P) motif in Gag, which is conserved among primate FVs and phosphorylated in PFV virions, was essential for recognition by PLKs. In the case of rat PLK2, functional kinase and polo-box domains were required for interaction with PFV Gag. Fluorescently-tagged PFV Gag, through its chromatin tethering function, selectively relocalized ectopically expressed eGFP-tagged PLK proteins to mitotic chromosomes in a Gag STP motif-dependent manner, confirming a specific and dominant nature of the Gag-PLK interaction in mammalian cells. The functional relevance of the Gag-PLK interaction was examined in the context of replication-competent FVs and single-round PFV vectors. Although STP motif mutated viruses displayed wild type (wt) particle release, RNA packaging and intra-particle reverse transcription, their replication capacity was decreased 3-fold in single-cycle infections, and up to 20-fold in spreading infections over an extended time period. Strikingly similar defects were observed when cells infected with single-round wt Gag PFV vectors were treated with a pan PLK inhibitor. Analysis of entry kinetics of the mutant viruses indicated a post-fusion defect resulting in delayed and reduced integration, which was accompanied with an enhanced preference to integrate into heterochromatin. We conclude that interaction between PFV Gag and cellular PLK proteins is important for early replication steps of PFV within host cells. PMID:27579920

Tissue-Specific Expression of Herpes Simplex Virus Thymidine Kinase Gene Delivered by Adeno-Associated Virus Inhibits the Growth of Human Hepatocellular Carcinoma in Athymic Mice

NASA Astrophysics Data System (ADS)

Su, Hua; Lu, Ronghua; Chang, Judy C.; Kan, Yuet Wai

1997-12-01

About 70% of hepatocellular carcinomas are known to express α -fetoprotein, which is normally expressed in fetal but not in adult livers. To induce herpes simplex virus-thymidine kinase expression in these cancer cells, we constructed an adeno-associated viral vector containing the HSV-TK gene under the control of the α -fetoprotein enhancer and albumin promoter. We previously demonstrated in vitro that although this vector can transduce a variety of human cells, only transduced AFP and albumin-expressing hepatocellular carcinoma cell lines were sensitive to killing by ganciclovir (GCV). In the present study, we explored the effect of this vector on hepatocellular carcinoma cells in vivo. Subcutaneous tumors generated in nude mice by implanting hepatocellular carcinoma cells previously transduced with this vector shrank dramatically after treatment with GCV. Bystander effect was also observed on the tumors generated by mixing transduced and untransduced cells. To test whether the tumor cells can be transduced by the virus in vivo, we injected the recombinant adeno-associated virus into tumors generated by untransduced hepatocarcinoma cell line. Tumor growth were retarded after treatment with GCV. These experiments demonstrate the feasibility of in vivo transduction of tumor cell with rAAV.

Properties of the tick-borne encephalitis virus population during persistent infection of ixodid ticks and tick cell lines.

PubMed

Belova, Oxana A; Litov, Alexander G; Kholodilov, Ivan S; Kozlovskaya, Liubov I; Bell-Sakyi, Lesley; Romanova, Lidiya Iu; Karganova, Galina G

2017-10-01

Tick-borne encephalitis virus (TBEV) is the causative agent of tick-borne encephalitis (TBE), a vector-borne zoonotic neuroinfection. For successful circulation in natural foci the virus has to survive in the vector for a long period of time. Information about the effect of long-term infection of ticks on properties of the viral population is of great importance. In recent years, changes in the eco-epidemiology of TBEV due to changes in distribution of ixodid ticks have been observed. These changes in TBEV-endemic areas could result in a shift of the main tick vector species, which in turn may lead to changes in properties of the virus. In the present study we evaluated the selective pressure on the TBEV population during persistent infection of various species of ticks and tick cell lines. TBEV effectively replicated and formed persistent infection in ticks and tick cell lines of the vector species (Ixodes spp.), potential vectors (Dermacentor spp.) and non-vector ticks (Hyalomma spp.). During TBEV persistence in Ixodes and Dermacentor ticks, properties of the viral population remained virtually unchanged. In contrast, persistent TBEV infection of tick cell lines from both vector and non-vector ticks favoured selection of viral variants with low neuroinvasiveness for laboratory mice and substitutions in the E protein that increased local positive charge of the virion. Thus, selective pressure on viral population may differ in ticks and tick cell lines during persistent infection. Nevertheless, virus variants with properties of the original strain adapted to mouse CNS were not eliminated from the viral population during long-term persistence of TBEV in ticks and tick cell lines. Copyright © 2017 Elsevier GmbH. All rights reserved.

Investigation of viral vectors using atomic force microscopy and microfluidic devices

NASA Astrophysics Data System (ADS)

Negishi, Atsuko

Researchers are modifying viruses into gene delivery vehicles in hope to cure diseases such as muscular dystrophy, hemophilia and cancer. Significant progress has been made toward this end, but further development and success of viral vectors depend on a deeper understanding of viral structure and physiology. Recent advances in microscopy have allowed new approaches to studying viruses that complement existing methodologies. Presented in this dissertation are novel viral studies using the atomic force microscope (AFM), a microscope that provides topographic information at the nanometer scale. As well microfluidic channels were used to study the effect of fluid flow properties on infection. A number of viruses are currently under study as potential vectors. We focus our studies on the adenovirus (Ad) and the adeno-associated virus (AAV) which have numerous attractive properties as vectors. The AFM is used to probe first, the structural aspects of the Ad and second, the virus-receptor interactions between AAV and its cell surface receptor, heparan sulfate proteoglycan (HSPG). The AFM was capable of imaging the capsid facets of intact Ad and DNA strands released from disrupted Ad capsids. In addition, we found that the stability of the capsid depended on the surface chemistry. An AFM-based binding assay was developed to study the binding between AAV and HSPG. The advantage of using the AFM for this purpose is its ability to simultaneously provide structural and quantitative information at the single molecule level. We measured a binding constant of 3.4 +/- 0.3 nM which is consistent with published reports. Microfluidic devices were used to study the dependence of fluid flow on infection. Cells were cultured in microfluidic channels and exposed to AAV vectors at various shear stresses. We found that a lower percentage of the cells were infected at higher shear stress. We also found that fluid forces can indirectly play a role in viral infection by influencing the cell state. A significantly lower percentage of cells that were treated with shear stress prior to vector exposure were infected compared to cells which were not exposed to shear stress.

Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector.

PubMed

Chen, Yong; Chen, Qian; Li, Manman; Mao, Qianzhuo; Chen, Hongyan; Wu, Wei; Jia, Dongsheng; Wei, Taiyun

2017-11-01

Many viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. Generally, an insect vector can use autophagy as an intrinsic antiviral defense mechanism against viral infection. Whether viruses can evolve to exploit autophagy to promote their transmission by insect vectors is still unknown. Here, we show that the autophagic process is triggered by the persistent replication of a plant reovirus, rice gall dwarf virus (RGDV) in cultured leafhopper vector cells and in intact insects, as demonstrated by the appearance of obvious virus-containing double-membrane autophagosomes, conversion of ATG8-I to ATG8-II and increased level of autophagic flux. Such virus-containing autophagosomes seem able to mediate nonlytic viral release from cultured cells or facilitate viral spread in the leafhopper intestine. Applying the autophagy inhibitor 3-methyladenine or silencing the expression of Atg5 significantly decrease viral spread in vitro and in vivo, whereas applying the autophagy inducer rapamycin or silencing the expression of Torc1 facilitate such viral spread. Furthermore, we find that activation of autophagy facilitates efficient viral transmission, whereas inhibiting autophagy blocks viral transmission by its insect vector. Together, these results indicate a plant virus can induce the formation of autophagosomes for carrying virions, thus facilitating viral spread and transmission by its insect vector. We believe that such a role for virus-induced autophagy is common for vector-borne persistent viruses during their transmission by insect vectors.

Autophagy pathway induced by a plant virus facilitates viral spread and transmission by its insect vector

PubMed Central

Mao, Qianzhuo; Chen, Hongyan; Wu, Wei

2017-01-01

Many viral pathogens are persistently transmitted by insect vectors and cause agricultural or health problems. Generally, an insect vector can use autophagy as an intrinsic antiviral defense mechanism against viral infection. Whether viruses can evolve to exploit autophagy to promote their transmission by insect vectors is still unknown. Here, we show that the autophagic process is triggered by the persistent replication of a plant reovirus, rice gall dwarf virus (RGDV) in cultured leafhopper vector cells and in intact insects, as demonstrated by the appearance of obvious virus-containing double-membrane autophagosomes, conversion of ATG8-I to ATG8-II and increased level of autophagic flux. Such virus-containing autophagosomes seem able to mediate nonlytic viral release from cultured cells or facilitate viral spread in the leafhopper intestine. Applying the autophagy inhibitor 3-methyladenine or silencing the expression of Atg5 significantly decrease viral spread in vitro and in vivo, whereas applying the autophagy inducer rapamycin or silencing the expression of Torc1 facilitate such viral spread. Furthermore, we find that activation of autophagy facilitates efficient viral transmission, whereas inhibiting autophagy blocks viral transmission by its insect vector. Together, these results indicate a plant virus can induce the formation of autophagosomes for carrying virions, thus facilitating viral spread and transmission by its insect vector. We believe that such a role for virus-induced autophagy is common for vector-borne persistent viruses during their transmission by insect vectors. PMID:29125860

Lack of dependence on p53 for DNA double strand break repair of episomal vectors in human lymphoblasts

NASA Technical Reports Server (NTRS)

Kohli, M.; Jorgensen, T. J.

1999-01-01

The p53 tumor suppressor gene has been shown to be involved in a variety of repair processes, and recent findings have suggested that p53 may be involved in DNA double strand break repair in irradiated cells. The role of p53 in DNA double strand break repair, however, has not been fully investigated. In this study, we have constructed a novel Epstein-Barr virus (EBV)-based shuttle vector, designated as pZEBNA, to explore the influence of p53 on DNA strand break repair in human lymphoblasts, since EBV-based vectors do not inactivate the p53 pathway. We have compared plasmid survival of irradiated, restriction enzyme linearized, and calf intestinal alkaline phosphatase (CIP)-treated pZEBNA with a Simian virus 40 (SV40)-based shuttle vector, pZ189, in TK6 (wild-type p53) and WTK1 (mutant p53) lymphoblasts and determined that p53 does not modulate DNA double strand break repair in these cell lines. Copyright 1999 Academic Press.

Properties of a herpes simplex virus multiple immediate-early gene-deleted recombinant as a vaccine vector

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Daisuke; Brockman, Mark A.; Ndung'u, Thumbi

2007-01-20

Herpes simplex virus (HSV) recombinants induce durable immune responses in rhesus macaques and mice and have induced partial protection in rhesus macaques against mucosal challenge with virulent simian immunodeficiency virus (SIV). In this study, we evaluated the properties of a new generation HSV vaccine vector, an HSV-1 multiple immediate-early (IE) gene deletion mutant virus, d106, which contains deletions in the ICP4, ICP27, ICP22, and ICP47 genes. Because several of the HSV IE genes have been implicated in immune evasion, inactivation of the genes encoding these proteins was expected to result in enhanced immunogenicity. The d106 virus expresses few HSV genemore » products and shows minimal cytopathic effect in cultured cells. When d106 was inoculated into mice, viral DNA accumulated at high levels in draining lymph nodes, consistent with an ability to transduce dendritic cells and activate their maturation and movement to lymph nodes. A d106 recombinant expressing Escherichia coli {beta}-galactosidase induced durable {beta}-gal-specific IgG and CD8{sup +} T cell responses in naive and HSV-immune mice. Finally, d106-based recombinants have been constructed that express simian immunodeficiency virus (SIV) gag, env, or a rev-tat-nef fusion protein for several days in cultured cells. Thus, d106 shows many of the properties desirable in a vaccine vector: limited expression of HSV gene products and cytopathogenicity, high level expression of transgenes, ability to induce durable immune responses, and an ability to transduce dendritic cells and induce their maturation and migration to lymph nodes.« less

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

PubMed Central

Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

1997-01-01

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population. PMID:9032306

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

PubMed

Malik, P; McQuiston, S A; Yu, X J; Pepper, K A; Krall, W J; Podsakoff, G M; Kurtzman, G J; Kohn, D B

1997-03-01

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population.

Targeted Entry via Somatostatin Receptors Using a Novel Modified Retrovirus Glycoprotein That Delivers Genes at Levels Comparable to Those of Wild-Type Viral Glycoproteins

PubMed Central

Li, Fang; Ryu, Byoung Y.; Krueger, Robin L.; Heldt, Scott A.

2012-01-01

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 106 transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins. PMID:22013043

Recombinant yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 gag induces SIV-specific CD8+ T-cell responses in rhesus macaques.

PubMed

Bonaldo, Myrna C; Martins, Mauricio A; Rudersdorf, Richard; Mudd, Philip A; Sacha, Jonah B; Piaskowski, Shari M; Costa Neves, Patrícia C; Veloso de Santana, Marlon G; Vojnov, Lara; Capuano, Saverio; Rakasz, Eva G; Wilson, Nancy A; Fulkerson, John; Sadoff, Jerald C; Watkins, David I; Galler, Ricardo

2010-04-01

Here we describe a novel vaccine vector for expressing human immunodeficiency virus (HIV) antigens. We show that recombinant attenuated yellow fever vaccine virus 17D expressing simian immunodeficiency virus SIVmac239 Gag sequences can be used as a vector to generate SIV-specific CD8(+) T-cell responses in the rhesus macaque. Priming with recombinant BCG expressing SIV antigens increased the frequency of these SIV-specific CD8(+) T-cell responses after recombinant YF17D boosting. These recombinant YF17D-induced SIV-specific CD8(+) T cells secreted several cytokines, were largely effector memory T cells, and suppressed viral replication in CD4(+) T cells.

West Nile virus infectious replicon particles generated using a packaging-restricted cell line is a safe reporter system.

PubMed

Li, Wei; Ma, Le; Guo, Li-Ping; Wang, Xiao-Lei; Zhang, Jing-Wei; Bu, Zhi-Gao; Hua, Rong-Hong

2017-06-12

West Nile virus (WNV) is a neurotropic pathogen which causes zoonotic disease in humans. Recently, there have been an increasing number of infected cases and there are no clinically approved vaccines or effective drugs to treat WNV infections in humans. The purpose of this study was to facilitate vaccine and antiviral drug discovery by developing a packaging cell line-restricted WNV infectious replicon particle system. We constructed a DNA-based WNV replicon lacking the C-prM-E coding region and replaced it with a GFP coding sequence. To produce WNV replicon particles, cell lines stably-expressing prM-E and C-prM-E were constructed. When the WNV replicon plasmid was co-transfected with a WNV C-expressing plasmid into the prM-E-expressing cell line or directly transfected the C-prM-E expressing cell line, the replicon particle was able to replicate, form green fluorescence foci, and exhibit cytopathic plaques similar to that induced by the wild type virus. The infectious capacity of the replicon particles was restricted to the packaging cell line as the replicons demonstrated only one round of infection in other permissive cells. Thus, this system provides a safe and convenient reporter WNV manipulating tool which can be used to study WNV viral invasion mechanisms, neutralizing antibodies and antiviral efficacy.

[Sendai virus vector: vector development and its application to health care and biotechnology].

PubMed

Iida, Akihiro

2007-06-01

Sendai virus (SeV) is an enveloped virus with a nonsegmented negative-strand RNA genome and a member of the paramyxovirus family. We have developed SeV vector which has shown a high efficiently of gene transfer and expression of foreign genes to a wide range of dividing and non-dividing mammalian cells and tissues. One of the characteristics of the vector is that the genome is located exclusively in the cytoplasm of infected cells and does not go through a DNA phase; thus there is no concern about unwanted integration of foreign sequences into chromosomal DNA. Therefore, this new class of "cytoplasmic RNA vector", an RNA vector with cytoplasmic expression, is expected to be a safer and more efficient viral vector than existing vectors for application to human therapy in various fields including gene therapy and vaccination. In this review, I describe development of Sendai virus vector, its application in the field of biotechnology and clinical application aiming to treat for a large number of diseases including cancer, cardiovascular disease, infectious diseases and neurologic disorders.

Retrovirus-specific differences in matrix and nucleocapsid protein-nucleic acid interactions: implications for genomic RNA packaging.

PubMed

Sun, Meng; Grigsby, Iwen F; Gorelick, Robert J; Mansky, Louis M; Musier-Forsyth, Karin

2014-01-01

Retroviral RNA encapsidation involves a recognition event between genomic RNA (gRNA) and one or more domains in Gag. In HIV-1, the nucleocapsid (NC) domain is involved in gRNA packaging and displays robust nucleic acid (NA) binding and chaperone functions. In comparison, NC of human T-cell leukemia virus type 1 (HTLV-1), a deltaretrovirus, displays weaker NA binding and chaperone activity. Mutation of conserved charged residues in the deltaretrovirus bovine leukemia virus (BLV) matrix (MA) and NC domains affects virus replication and gRNA packaging efficiency. Based on these observations, we hypothesized that the MA domain may generally contribute to NA binding and genome encapsidation in deltaretroviruses. Here, we examined the interaction between HTLV-2 and HIV-1 MA proteins and various NAs in vitro. HTLV-2 MA displays higher NA binding affinity and better chaperone activity than HIV-1 MA. HTLV-2 MA also binds NAs with higher affinity than HTLV-2 NC and displays more robust chaperone function. Mutation of two basic residues in HTLV-2 MA α-helix II, previously implicated in BLV gRNA packaging, reduces NA binding affinity. HTLV-2 MA binds with high affinity and specificity to RNA derived from the putative packaging signal of HTLV-2 relative to nonspecific NA. Furthermore, an HIV-1 MA triple mutant designed to mimic the basic character of HTLV-2 MA α-helix II dramatically improves binding affinity and chaperone activity of HIV-1 MA in vitro and restores RNA packaging to a ΔNC HIV-1 variant in cell-based assays. Taken together, these results are consistent with a role for deltaretrovirus MA proteins in viral RNA packaging.

Characterization of a 3rd Generation Lentiviral Vector Pseudotyped With Nipah Virus Envelope Proteins For Endothelial Cell Transduction

PubMed Central

Witting, Scott R.; Vallanda, Priya; Gamble, Aisha L.

2013-01-01

Lentiviruses are becoming progressively more popular as gene therapy vectors due to their ability to integrate into quiescent cells and recent clinical trial successes. Directing these vectors to specific cell types and limiting off-target transduction in vivo remains a challenge. Replacing the viral envelope proteins responsible for cellular binding, or pseudotyping, remains a common method to improve lentiviral targeting. Here, we describe the development of a high titer, 3rd generation lentiviral vector pseudotyped with Nipah virus fusion protein (NiV-F) and attachment protein (NiV-G). Critical to high titers was truncation of the cytoplasmic domains of both NiV-F and NiV-G. As known targets of wild-type Nipah virus, primary endothelial cells are shown to be effectively transduced by the Nipah pseudotype. In contrast, human CD34+ hematopoietic progenitors were not significantly transduced. Additionally, the Nipah pseudotype has increased stability in human serum compared to VSV pseudotyped lentivirus. These findings suggest that the use of Nipah virus envelope proteins in 3rd generation lentiviral vectors would be a valuable tool for gene delivery targeted to endothelial cells. PMID:23698741

Virus induced gene silencing (VIGS) for functional analysis of wheat genes involved in Zymoseptoria tritici susceptibility and resistance.

PubMed

Lee, Wing-Sham; Rudd, Jason J; Kanyuka, Kostya

2015-06-01

Virus-induced gene silencing (VIGS) has emerged as a powerful reverse genetic technology in plants supplementary to stable transgenic RNAi and, in certain species, as a viable alternative approach for gene functional analysis. The RNA virus Barley stripe mosaic virus (BSMV) was developed as a VIGS vector in the early 2000s and since then it has been used to study the function of wheat genes. Several variants of BSMV vectors are available, with some requiring in vitro transcription of infectious viral RNA, while others rely on in planta production of viral RNA from DNA-based vectors delivered to plant cells either by particle bombardment or Agrobacterium tumefaciens. We adapted the latest generation of binary BSMV VIGS vectors for the identification and study of wheat genes of interest involved in interactions with Zymoseptoria tritici and here present detailed and the most up-to-date protocols. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Establishment of MicroRNA delivery system by PP7 bacteriophage-like particles carrying cell-penetrating peptide.

PubMed

Sun, Yanli; Sun, Yanhua; Zhao, Ronglan

2017-08-01

MicroRNAs have great therapeutic potential in cancer and other diseases. However, their instability and low in vivo delivery efficiency limits their application. Recombinant PP7 bacteriophage-based virus-like particles (VLPs) could protect microRNAs against rapid degradation by RNase by packaging specific exogenous pre-microRNAs using the pac site. Insertion of a cell-penetrating peptide (CPP) into the AB-loop of VLPs could significantly improve the delivery efficiency of microRNAs into mammalian cells. Unlike other microRNA delivery methods (viral or non-viral vectors), recombinant PP7 VLPs carrying a CPP and microRNA could be efficiently expressed in Escherichia coli using the one-plasmid double expression system. Here we showed that PP7 VLPs carrying a CPP penetrated hepatoma SK-HEP-1 cells and delivered the pre-microRNA-23b, which was processed into a mature product within 24 h; a concentration of 10 nM was sufficient for the inhibition of hepatoma cell migration via the downregulation of liver-intestine cadherin expression. Furthermore, PP7 VLPs carrying a CPP and a pre-microRNA were not infectious, replicative, or cytotoxic. Therefore, recombinant PP7 VLPs can be used for simultaneous and targeted delivery of both microRNAs and peptides because of their ability to package specific exogenous RNA using the pac site and to display peptides. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Immunogenicity of modified vaccinia virus Ankara expressing the hemagglutinin stalk domain of pandemic (H1N1) 2009 influenza virus.

PubMed

Di Mario, Giuseppina; Soprana, Elisa; Gubinelli, Francesco; Panigada, Maddalena; Facchini, Marzia; Fabiani, Concetta; Garulli, Bruno; Basileo, Michela; Cassone, Antonio; Siccardi, Antonio; Donatelli, Isabella; Castrucci, Maria R

2017-03-01

Vaccination offers protection against influenza, although current vaccines need to be reformulated each year. The development of a broadly protective influenza vaccine would guarantee the induction of heterosubtypic immunity also against emerging influenza viruses of a novel subtype. Vaccine candidates based on the stalk region of the hemagglutinin (HA) have the potential to induce broad and persistent protection against diverse influenza A viruses. Modified vaccinia virus Ankara (MVA) expressing a headless HA (hlHA) of A/California/4/09 (CA/09) virus was used as a vaccine to immunize C57BL/6 mice. Specific antibody and cell-mediated immune responses were determined, and challenge experiments were performed by infecting vaccinated mice with CA/09 virus. Immunization of mice with CA/09-derived hlHA, vectored by MVA, was able to elicit influenza-specific broad cross-reactive antibodies and cell-mediated immune responses, but failed to induce neutralizing antibodies and did not protect mice against virus challenge. Although highly immunogenic, our vaccine was unable to induce a protective immunity against influenza. A misfolded and unstable conformation of the hlHA molecule may have affected its capacity of inducing neutralizing antiviral, conformational antibodies. Design of stable hlHA-based immunogens and their delivery by recombinant MVA-based vectors has the potential of improving this promising approach for a universal influenza vaccine.

Expression of CD154 by a Simian Immunodeficiency Virus Vector Induces Only Transitory Changes in Rhesus Macaques

PubMed Central

Hodara, Vida L.; Velasquillo, M. Cristina; Parodi, Laura M.; Giavedoni, Luis D.

2005-01-01

Human immunodeficiency virus infection is characterized by dysregulation of antigen-presenting cell function and defects in cell-mediated immunity. Recent evidence suggests that impaired ability of CD4+ T cells to upregulate the costimulatory molecule CD154 is at the core of this dysregulation. To test the hypothesis that increased expression of CD154 on infected CD4+ T cells could modulate immune function, we constructed a replication-competent simian immunodeficiency virus (SIV) vector that expressed CD154. We found that this recombinant vector directed the expression of CD154 on the surface of infected CD4+ T cells and that expression of CD154 resulted in activation of B cells present in the same cultures. Experimental infection of rhesus macaques resulted in very low viral loads for the CD154-expressing virus and the control virus, indicating that expression of CD154 did not result in increased viral replication. Analyses of the anti-SIV immune responses and the phenotype of lymphocytes in blood and lymphoid tissues showed changes that occurred during the acute phase of infection only in animals infected with the CD154-expressing SIV, but that became indistinguishable from those seen in animals infected with the control virus at later time points. We conclude that the level of expression of CD154 in itself is not responsible for affecting the immune response to an attenuated virus. Considering that the CD154-expressing SIV vector and the virus control did not carry an active nef gene, our results suggest that, in CD4+ T cells infected with wild-type virus, Nef is the viral factor that interferes with the immune mechanisms that regulate expression of CD154. PMID:15795254

Adeno-associated virus-mediated gene transfer

PubMed Central

Srivastava, Arun

2008-01-01

Although the remarkable versatility and efficacy of recombinant adeno-associated virus 2 (AAV2) vectors in transducing a wide variety of cells and tissues in vitro, and in numerous pre-clinical animal models of human diseases in vivo, have been well established, the published literature is replete with controversies with regard to the efficacy of AAV2 vectors in hematopoietic stem cell (HSC) transduction. A number of factors have contributed to these controversies, the molecular bases of which have begun to come to light in recent years. With the availability of several novel serotypes (AAV1 through AAV12), rational design of AAV capsid mutants, and strategies (self-complementary vector genomes, hematopoietic cell-specific promoters), it is indeed becoming feasible to achieve efficient transduction of HSC by AAV vectors in a murine serial bone marrow transplantation model in vivo, where stable integration of the proviral AAV genome does not lead to any overt hematological abnormalities. Thus, a better understanding of the AAV-HSC interactions, and the availability of a vast repertoire of novel serotype vectors, are likely to have significant implications in the use of AAV vectors in high-efficiency transduction of HSCs as well as in gene therapy applications involving the hematopoietic system. PMID:18500727

Cytoplasmic utilization of human immunodeficiency virus type 1 genomic RNA is not dependent on a nuclear interaction with gag.

PubMed

Grewe, Bastian; Hoffmann, Bianca; Ohs, Inga; Blissenbach, Maik; Brandt, Sabine; Tippler, Bettina; Grunwald, Thomas; Uberla, Klaus

2012-03-01

In some retroviruses, such as Rous sarcoma virus and prototype foamy virus, Gag proteins are known to shuttle between the nucleus and the cytoplasm and are implicated in nuclear export of the viral genomic unspliced RNA (gRNA) for subsequent encapsidation. A similar function has been proposed for human immunodeficiency virus type 1 (HIV-1) Gag based on the identification of nuclear localization and export signals. However, the ability of HIV-1 Gag to transit through the nucleus has never been confirmed. In addition, the lentiviral Rev protein promotes efficient nuclear gRNA export, and previous reports indicate a cytoplasmic interaction between Gag and gRNA. Therefore, functional effects of HIV-1 Gag on gRNA and its usage were explored. Expression of gag in the absence of Rev was not able to increase cytoplasmic gRNA levels of subgenomic, proviral, or lentiviral vector constructs, and gene expression from genomic reporter plasmids could not be induced by Gag provided in trans. Furthermore, Gag lacking the reported nuclear localization and export signals was still able to mediate an efficient packaging process. Although small amounts of Gag were detectable in the nuclei of transfected cells, a Crm1-dependent nuclear export signal in Gag could not be confirmed. Thus, our study does not provide any evidence for a nuclear function of HIV-1 Gag. The encapsidation process of HIV-1 therefore clearly differs from that of Rous sarcoma virus and prototype foamy virus.

Virus-Like Particles Derived from HIV-1 for Delivery of Nuclear Proteins: Improvement of Production and Activity by Protein Engineering.

PubMed

Robert, Marc-André; Lytvyn, Viktoria; Deforet, Francis; Gilbert, Rénald; Gaillet, Bruno

2017-01-01

Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4). The sizes of the VLPs were 120-150 nm in diameter and they were resistant to freeze/thaw cycles. Protein delivery by the VLPs reached up to 100% efficacy in human cells and was well tolerated. Gag-cTA triggered up to 1100-fold gene activation of the reporter gene in comparison to the negative control. Protein engineering was required to detect Gag-KLF4 activity. Thus, insertion of the VP16 transactivation domain increased the activity of the VLPs by eightfold. An additional 2.4-fold enhancement was obtained by inserting nuclear export signal. In conclusion, our platform produced VLPs capable of efficient protein transfer, and it was shown that protein engineering can be used to improve the activity of the delivered proteins as well as VLP production.

A novel small animal model to study the replication of simian foamy virus in vivo.

PubMed

Blochmann, Rico; Curths, Christoph; Coulibaly, Cheick; Cichutek, Klaus; Kurth, Reinhard; Norley, Stephen; Bannert, Norbert; Fiebig, Uwe

2014-01-05

Preclinical evaluation in a small animal model would help the development of gene therapies and vaccines based on foamy virus vectors. The establishment of persistent, non-pathogenic infection with the prototype foamy virus in mice and rabbits has been described previously. To extend this spectrum of available animal models, hamsters were inoculated with infectious cell supernatant or bioballistically with a foamy virus plasmid. In addition, a novel foamy virus from a rhesus macaque was isolated and characterised genetically. Hamsters and mice were infected with this new SFVmac isolate to evaluate whether hamsters are also susceptible to infection. Both hamsters and mice developed humoral responses to either virus subtype. Virus integration and replication in different animal tissues were analysed by PCR and co-cultivation. The results strongly indicate establishment of a persistent infection in hamsters. These studies provide a further small animal model for studying FV-based vectors in addition to the established models. © 2013 Elsevier Inc. All rights reserved.

Vaccination with an adenoviral vector that encodes and displays a retroviral antigen induces improved neutralizing antibody and CD4+ T-cell responses and confers enhanced protection.

PubMed

Bayer, Wibke; Tenbusch, Matthias; Lietz, Ruth; Johrden, Lena; Schimmer, Simone; Uberla, Klaus; Dittmer, Ulf; Wildner, Oliver

2010-02-01

We present a new type of adenoviral vector that both encodes and displays a vaccine antigen on the capsid, thus combining in itself gene-based and protein vaccination; this vector resulted in an improved vaccination outcome in the Friend virus (FV) model. For presentation of the envelope protein gp70 of Friend murine leukemia virus on the adenoviral capsid, gp70 was fused to the adenovirus capsid protein IX. When compared to vaccination with conventional FV Env- and Gag-encoding adenoviral vectors, vaccination with the adenoviral vector that encodes and displays pIX-gp70 combined with an FV Gag-encoding vector resulted in significantly improved protection against systemic FV challenge infection, with highly controlled viral loads in plasma and spleen. This improved protection correlated with improved neutralizing antibody titers and stronger CD4(+) T-cell responses. Using a vector that displays gp70 without encoding it, we found that while the antigen display on the capsid alone was sufficient to induce high levels of binding antibodies, in vivo expression was necessary for the induction of neutralizing antibodies. This new type of adenovirus-based vaccine could be a valuable tool for vaccination.

HIV/AIDS Vaccine Candidates Based on Replication-Competent Recombinant Poxvirus NYVAC-C-KC Expressing Trimeric gp140 and Gag-Derived Virus-Like Particles or Lacking the Viral Molecule B19 That Inhibits Type I Interferon Activate Relevant HIV-1-Specific B and T Cell Immune Functions in Nonhuman Primates

PubMed Central

García-Arriaza, Juan; Perdiguero, Beatriz; Heeney, Jonathan L.; Seaman, Michael S.; Montefiori, David C.; Yates, Nicole L.; Tomaras, Georgia D.; Ferrari, Guido; Foulds, Kathryn E.; Roederer, Mario; Self, Steven G.; Borate, Bhavesh; Gottardo, Raphael; Phogat, Sanjay; Tartaglia, Jim; Barnett, Susan W.; Burke, Brian; Cristillo, Anthony D.; Weiss, Deborah E.; Lee, Carter; Kibler, Karen V.; Jacobs, Bertram L.; Wagner, Ralf; Ding, Song; Pantaleo, Giuseppe

2017-01-01

ABSTRACT The nonreplicating attenuated poxvirus vector NYVAC expressing clade C(CN54) HIV-1 Env(gp120) and Gag-Pol-Nef antigens (NYVAC-C) showed limited immunogenicity in phase I clinical trials. To enhance the capacity of the NYVAC vector to trigger broad humoral responses and a more balanced activation of CD4+ and CD8+ T cells, here we compared the HIV-1-specific immunogenicity elicited in nonhuman primates immunized with two replicating NYVAC vectors that have been modified by the insertion of the K1L and C7L vaccinia virus host range genes and express the clade C(ZM96) trimeric HIV-1 gp140 protein or a Gag(ZM96)-Pol-Nef(CN54) polyprotein as Gag-derived virus-like particles (termed NYVAC-C-KC). Additionally, one NYVAC-C-KC vector was generated by deleting the viral gene B19R, an inhibitor of the type I interferon response (NYVAC-C-KC-ΔB19R). An immunization protocol mimicking that of the RV144 phase III clinical trial was used. Two groups of macaques received two doses of the corresponding NYVAC-C-KC vectors (weeks 0 and 4) and booster doses with NYVAC-C-KC vectors plus the clade C HIV-1 gp120 protein (weeks 12 and 24). The two replicating NYVAC-C-KC vectors induced enhanced and similar HIV-1-specific CD4+ and CD8+ T cell responses, similar levels of binding IgG antibodies, low levels of IgA antibodies, and high levels of antibody-dependent cellular cytotoxicity responses and HIV-1-neutralizing antibodies. Small differences within the NYVAC-C-KC-ΔB19R group were seen in the magnitude of CD4+ and CD8+ T cells, the induction of some cytokines, and the neutralization of some HIV-1 isolates. Thus, replication-competent NYVAC-C-KC vectors acquired relevant immunological properties as vaccine candidates against HIV/AIDS, and the viral B19 molecule exerts some control of immune functions. IMPORTANCE It is of special importance to find a safe and effective HIV/AIDS vaccine that can induce strong and broad T cell and humoral immune responses correlating with HIV-1 protection. Here we developed novel replicating poxvirus NYVAC-based HIV/AIDS vaccine candidates expressing clade C HIV-1 antigens, with one of them lacking the vaccinia virus B19 protein, an inhibitor of the type I interferon response. Immunization of nonhuman primates with these novel NYVAC-C-KC vectors and the protein component gp120 elicited high levels of T cell and humoral immune responses, with the vector containing a deletion in B19R inducing a trend toward a higher magnitude of CD4+ and CD8+ T cell responses and neutralization of some HIV-1 strains. These poxvirus vectors could be considered HIV/AIDS vaccine candidates based on their activation of potential immune correlates of protection. PMID:28179536

Antibody-mediated targeting of replication-competent retroviral vectors.

PubMed

Tai, Chien-Kuo; Logg, Christopher R; Park, Jinha M; Anderson, W French; Press, Michael F; Kasahara, Noriyuki

2003-05-20

Replication-competent murine leukemia virus (MLV) vectors can be engineered to achieve high efficiency gene transfer to solid tumors in vivo and tumor-restricted replication, however their safety can be further enhanced by redirecting tropism of the virus envelope. We have therefore tested the targeting capability and replicative stability of ecotropic and amphotropic replication-competent retrovirus (RCR) vectors containing two tandem repeats from the immunoglobulin G-binding domain of Staphylococcal protein A inserted into the proline-rich "hinge" region of the envelope, which enables modular use of antibodies of various specificities for vector targeting. The modified envelopes were efficiently expressed and incorporated into virions, were capable of capturing monoclonal anti-HER2 antibodies, and mediated efficient binding of the virus-antibody complex to HER2-positive target cells. While infectivity was markedly reduced by pseudotyping with targeted envelopes alone, coexpression of wild-type envelope rescued efficient cellular entry. Both ecotropic and amphotropic RCR vector/anti-HER2 antibody complexes achieved significant enhancement of transduction on murine target cells overexpressing HER2, which could be competed by preincubation with excess free antibodies. Interestingly, HER2-expressing human breast cancer cells did not show enhancement of transduction despite efficient antibody-mediated cell surface binding, suggesting that target cell-specific parameters markedly affect the efficiency of post-binding entry processes. Serial replication of targeted vectors resulted in selection of Z domain deletion variants, but reduction of the overall size of the vector genome enhanced its stability. Application of antibody-mediated targeting to the initial localization of replication-competent virus vectors to tumor sites will thus require optimized target selection and vector design.

CRISPR/Cas9-Advancing Orthopoxvirus Genome Editing for Vaccine and Vector Development.

PubMed

Okoli, Arinze; Okeke, Malachy I; Tryland, Morten; Moens, Ugo

2018-01-22

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them.

[Ubiquitination of recombinant adeno-associated viral vector and its application].

PubMed

Wang, Qi-zhao; Lu, Ying-hui; Diao, Yong; Xu, Rui-an

2012-09-01

Recombinant adeno-associated virus (rAAV) has been widely used as vector for gene therapy. However, the effectiveness of gene therapy based on rAAV needs to be further improved. Enhancement of the transduction efficiency is one of the most important fields for rAAV-based gene therapy. Recent results have showed that the ubiquitin-proteasome system plays an important role in the trafficking of rAAV vector in cytoplasm, and regulation of its function may significantly improve the transduction efficiency of rAAV vector in various types of cells and tissues.

Large-scale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application.

PubMed

Merten, Otto-Wilhelm; Charrier, Sabine; Laroudie, Nicolas; Fauchille, Sylvain; Dugué, Céline; Jenny, Christine; Audit, Muriel; Zanta-Boussif, Maria-Antonietta; Chautard, Hélène; Radrizzani, Marina; Vallanti, Giuliana; Naldini, Luigi; Noguiez-Hellin, Patricia; Galy, Anne

2011-03-01

From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 × 10(9) infectious particles per milliliter were obtained, generating up to 6 × 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.

Magnetic nanoparticles for efficient cell transduction with Semliki Forest virus.

PubMed

Kurena, Baiba; Vežāne, Aleksandra; Skrastiņa, Dace; Trofimova, Olga; Zajakina, Anna

2017-07-01

Semliki Forest virus (SFV) is a potential cancer gene therapy vector capable of providing high and transient expression of heterologous proteins in mammalian cells. However, SFV has shown suboptimal transduction levels in several cancer cell types as well as wide biodistribution of SFV has been observed after in vivo applications. Magnetic nanoparticles (MNPs) have been shown to increase cell transduction with several viral vectors in vitro under an external magnetic field and enhance magnetically guided viral vector delivery. Here, we examined a panel of MNPs for enhanced cancer cell transduction with SFV vector. Magneto-transduction using positively charged MNPs increased Semliki Forest virus transduction in TS/A mouse mammary carcinoma cells in vitro in the presence of fetal bovine serum. Positively charged MNPs efficiently captured SFV particles independently of capturing medium, and MNPs-SFV complexes were successfully separated from suspension by magnetic precipitation. These results reveal the potential application of MNPs for enhanced gene delivery by SFV vector as well as proposes magnetic precipitation for efficient concentration of SFV particles from different media. Copyright © 2017 Elsevier B.V. All rights reserved.

[Construction of lentiviral mediated CyPA siRNA and its functions in non-small cell lung cancer].

PubMed

FENG, Yan-ming; WU, Yi-ming; TU, Xin-ming; XU, Zheng-shun; WU, Wei-dong

2010-02-01

To construct a lentiviral-vector-mediated CyPA small interference RNA (siRNA) and study its function in non-small cell lung cancer. First, four target sequences were selected according to CyPA mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed, synthesized and cloned into the pGCL-GFP vector, which contained U6 promoter and green fluorescent protein (GFP). The resulting lentiviral vector containing CyPA shRNA was named Lv-shCyPA, and it was confirmed by PCR and sequencing. Next, it was cotransfected by Lipofectamine 2000 along with pHelper1.0 and pHelper 2.0 into 293T cells to package lentivirus particles. At the same time, the packed virus infected non-small cell lung cancer cell (A549), the level of CyPA protein at 5 d after infection was detected by Western Blot to screen the target of CyPA. A549 were infected with Lv-shCyPA and grown as xenografts in severe combined immunodeficient mice. Cell cycle and apoptosis were measured by FCM. It was confirmed by PCR and DNA sequencing that lentiviral-vector-mediated CyPA siRNA (Lv-shCyPA) producing CyPA shRNA was constructed successfully. The titer of concentrated virus were 1 x 10(7) TU/ml. Flow cytometric analysis demonstrated G2-M phase (11.40% +/- 0.68%) was decreased relatively in A549/LvshCyPA compared with control groups (14.52% +/- 1.19%) (P<0.05). The apoptosis rate of A549/Lv-shCyPA (5.01% +/- 0.5%) was higher than control groups (0.35% +/- 0.17%) (P<0.05). Visible tumors were only detectable at 6th day after inoculated by A549/Lv-shCyPA. The xenograft tumors of A549/Lv-shCyPA remarkably delayed tumor growth and remained at a similarly small average size at 38th days after inoculation compared with the control group (P < 0.05). Lentiviral-vector-mediated siRNA technique effectively inhibits the expression of CyPA, induces the NSCLC cell apoptosis, inhibits the tumor growth. Elucidation of the precise role of CypA in these pathways may lead to new targeted therapies for non-small cell lung cancer.

A Mutation in UL15 of Herpes Simplex Virus 1 That Reduces Packaging of Cleaved Genomes▿

PubMed Central

Yang, Kui; Wills, Elizabeth G.; Baines, Joel D.

2011-01-01

Herpesvirus genomic DNA is cleaved from concatemers that accumulate in infected cell nuclei. Genomic DNA is inserted into preassembled capsids through a unique portal vertex. Extensive analyses of viral mutants have indicated that intact capsids, the portal vertex, and all components of a tripartite terminase enzyme are required to both cleave and package viral DNA, suggesting that DNA cleavage and packaging are inextricably linked. Because the processes have not been functionally separable, it has been difficult to parse the roles of individual proteins in the DNA cleavage/packaging reaction. In the present study, a virus bearing the deletion of codons 400 to 420 of UL15, encoding a terminase component, was analyzed. This virus, designated vJB27, failed to replicate on noncomplementing cells but cleaved concatemeric DNA to ca. 35 to 98% of wild-type levels. No DNA cleavage was detected in cells infected with a UL15-null virus or a virus lacking UL15 codons 383 to 385, comprising a motif proposed to couple ATP hydrolysis to DNA translocation. The amount of vJB27 DNA protected from DNase I digestion was reduced compared to the wild-type virus by 6.5- to 200-fold, depending on the DNA fragment analyzed, thus indicating a profound defect in DNA packaging. Capsids containing viral DNA were not detected in vJB27-infected cells, as determined by electron microscopy. These data suggest that pUL15 plays an essential role in DNA translocation into the capsid and indicate that this function is separable from its role in DNA cleavage. PMID:21880766

Sodium Chloride Enhances Recombinant Adeno-Associated Virus Production in a Serum-Free Suspension Manufacturing Platform Using the Herpes Simplex Virus System

PubMed Central

Adamson-Small, Laura; Potter, Mark; Byrne, Barry J.; Clément, Nathalie

2017-01-01

The increase in effective treatments using recombinant adeno-associated viral (rAAV) vectors has underscored the importance of scalable, high-yield manufacturing methods. Previous work from this group reported the use of recombinant herpes simplex virus type 1 (rHSV) vectors to produce rAAV in adherent HEK293 cells, demonstrating the capacity of this system and quality of the product generated. Here we report production and optimization of rAAV using the rHSV system in suspension HEK293 cells (Expi293F) grown in serum and animal component-free medium. Through adjustment of salt concentration in the medium and optimization of infection conditions, titers greater than 1 × 1014 vector genomes per liter (VG/liter) were observed in purified rAAV stocks produced in Expi293F cells. Furthermore, this system allowed for high-titer production of multiple rAAV serotypes (2, 5, and 9) as well as multiple transgenes (green fluorescent protein and acid α-glucosidase). A proportional increase in vector production was observed as this method was scaled, with a final 3-liter shaker flask production yielding an excess of 1 × 1015 VG in crude cell harvests and an average of 3.5 × 1014 total VG of purified rAAV9 material, resulting in greater than 1 × 105 VG/cell. These results support the use of this rHSV-based rAAV production method for large-scale preclinical and clinical vector production. PMID:28117600

A virus responds instantly to the presence of the vector on the host and forms transmission morphs

PubMed Central

Martinière, Alexandre; Bak, Aurélie; Macia, Jean-Luc; Lautredou, Nicole; Gargani, Daniel; Doumayrou, Juliette; Garzo, Elisa; Moreno, Aranzazu; Fereres, Alberto; Blanc, Stéphane; Drucker, Martin

2013-01-01

Many plant and animal viruses are spread by insect vectors. Cauliflower mosaic virus (CaMV) is aphid-transmitted, with the virus being taken up from specialized transmission bodies (TB) formed within infected plant cells. However, the precise events during TB-mediated virus acquisition by aphids are unknown. Here, we show that TBs react instantly to the presence of the vector by ultra-rapid and reversible redistribution of their key components onto microtubules throughout the cell. Enhancing or inhibiting this TB reaction pharmacologically or by using a mutant virus enhanced or inhibited transmission, respectively, confirming its requirement for efficient virus-acquisition. Our results suggest that CaMV can perceive aphid vectors, either directly or indirectly by sharing the host perception. This novel concept in virology, where viruses respond directly or via the host to the outside world, opens new research horizons, that is, investigating the impact of ‘perceptive behaviors’ on other steps of the infection cycle. DOI: http://dx.doi.org/10.7554/eLife.00183.001 PMID:23358702

Identification of Essential Genetic Baculoviral Elements for Recombinant Protein Expression by Transactivation in Sf21 Insect Cells.

PubMed

Bleckmann, Maren; Schürig, Margitta; Chen, Fang-Fang; Yen, Zen-Zen; Lindemann, Nils; Meyer, Steffen; Spehr, Johannes; van den Heuvel, Joop

2016-01-01

The Baculovirus Expression Vector System (BEVS) is widely used to produce high amounts of recombinant proteins. Nevertheless, generating recombinant baculovirus in high quality is rather time-consuming and labor-intensive. Alternatively, virus-free expression in insect cells did not achieve similar expression levels for most proteins so far. The transactivation method is a promising approach for protein expression in Sf21 cells. It combines advantages of BEVS and plasmid-based expression by activating strong virus-dependent promoters on a transfected plasmid by baculoviral coinfection. Here, we identified expression elements required for transactivation. Therefore, we designed several vectors comprising different viral promoters or promoter combinations and tested them for eGFP expression using the automated BioLector microcultivation system. Remarkably, only the combination of the very late promoter p10 together with the homologous region 5 (hr5) could boost expression during transactivation. Other elements, like p10 alone or the late viral promoter polH, did not respond to transactivation. A new combination of hr5 and p10 with the strongest immediate early OpMNPV viral promoter OpIE2 improved the yield of eGFP by ~25% in comparison to the previous applied hr5-IE1-p10 expression cassette. Furthermore, we observed a strong influence of the transcription termination sequence and vector backbone on the level of expression. Finally, the expression levels for transactivation, BEVS and solely plasmid-based expression were compared for the marker protein eGFP, underlining the potential of transactivation for fast recombinant protein expression in Sf21 cells. In conclusion, essential elements for transactivation could be identified. The optimal elements were applied to generate an improved vector applicable in virus-free plasmid-based expression, transactivation and BEVS.

Novel Nonreplicating Vaccinia Virus Vector Enhances Expression of Heterologous Genes and Suppresses Synthesis of Endogenous Viral Proteins.

PubMed

Wyatt, Linda S; Xiao, Wei; Americo, Jeffrey L; Earl, Patricia L; Moss, Bernard

2017-06-06

Viruses are used as expression vectors for protein synthesis, immunology research, vaccines, and therapeutics. Advantages of poxvirus vectors include the accommodation of large amounts of heterologous DNA, the presence of a cytoplasmic site of transcription, and high expression levels. On the other hand, competition of approximately 200 viral genes with the target gene for expression and immune recognition may be disadvantageous. We describe a vaccinia virus (VACV) vector that uses an early promoter to express the bacteriophage T7 RNA polymerase; has the A23R intermediate transcription factor gene deleted, thereby restricting virus replication to complementing cells; and has a heterologous gene regulated by a T7 promoter. In noncomplementing cells, viral early gene expression and DNA replication occurred normally but synthesis of intermediate and late proteins was prevented. Nevertheless, the progeny viral DNA provided templates for abundant expression of heterologous genes regulated by a T7 promoter. Selective expression of the Escherichia coli lac repressor gene from an intermediate promoter reduced transcription of the heterologous gene specifically in complementing cells, where large amounts might adversely impact VACV replication. Expression of heterologous proteins mediated by the A23R deletion vector equaled that of a replicating VACV, was higher than that of a nonreplicating modified vaccinia virus Ankara (MVA) vector used for candidate vaccines in vitro and in vivo , and was similarly immunogenic in mice. Unlike the MVA vector, the A23R deletion vector still expresses numerous early genes that can restrict immunogenicity as demonstrated here by the failure of the prototype vector to induce interferon alpha. By deleting immunomodulatory genes, we anticipate further improvements in the system. IMPORTANCE Vaccines provide an efficient and effective way of preventing infectious diseases. Nevertheless, new and better vaccines are needed. Vaccinia virus, which was used successfully as a live vaccine to eradicate smallpox, has been further attenuated and adapted as a recombinant vector for immunization against other pathogens. However, since the initial description of this vector system, only incremental improvements largely related to safety have been implemented. Here we described novel modifications of the platform that increased expression of the heterologous target gene and decreased expression of endogenous vaccinia virus genes while providing safety by preventing replication of the candidate vaccine except in complementing cells used for vector propagation. Copyright © 2017 Wyatt et al.

Molecular interactions and immune responses between maize fine streak virus and the leafhopper vector G. nigrifrons through differential expression and RNA interference

USDA-ARS?s Scientific Manuscript database

Maize fine streak virus (MFSV) is an emerging virus of maize that is transmitted by an insect vector, the leafhopper called Graminella nigrifrons. Virus transmission by the leafhopper requires that the virus enter into and multiply in insect cells, tissues and organs before being transmitted to a ne...

Infection studies of nontarget mammalian cell lines with Bombyx mori macula-like virus.

PubMed

Innami, Katsuhisa; Aizawa, Takahiro; Tsukui, Toshihiro; Katsuma, Susumu; Imanishi, Shigeo; Kawasaki, Hideki; Iwanaga, Masashi

2016-03-01

Bombyx mori-derived cell lines are generally used for Bombyx mori nucleopolyhedrovirus (BmNPV)-based baculovirus expression vector system (BEVS). However, almost all of the B. mori-derived cell lines are persistently infected with Bombyx mori macula-like virus (BmMLV). In this study, nontarget mammalian cell lines were exposed to BmMLV, and their susceptibility was investigated. Real-time PCR showed that viral RNA in virus-inoculated nine mammalian cell lines decreased sharply at 7 days postinfection. Also, there was no significant effect on cell viability of mammalian cells after inoculation with BmMLV. These findings indicate that mammalian cell lines used in this study are not permissive to BmMLV, and BmMLV contamination might not affect the safety aspect of BmNPV-based BEVS. Copyright © 2015 Elsevier B.V. All rights reserved.

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity

PubMed Central

Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

2015-01-01

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3’untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3’ UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3’UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3’UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3’ UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity. PMID:26646790

Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

PubMed

Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

2015-12-01

Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs suggests this that interaction is a bona fide target for the design of compounds with antiviral activity.

Conditional Cytotoxic Anti-HIV Gene Therapy for Selectable Cell Modification

PubMed Central

Garg, Himanshu; Joshi, Anjali

2016-01-01

Gene therapy remains one of the potential strategies to achieve a cure for HIV infection. One of the major limitations of anti-HIV gene therapy concerns recovering an adequate number of modified cells to generate an HIV-proof immune system. Our study addresses this issue by developing a methodology that can mark conditional vector-transformed cells for selection and subsequently target HIV-infected cells for elimination by treatment with ganciclovir (GCV). We used the herpes simplex virus thymidine kinase (TK) mutant SR39, which is highly potent at killing cells at low GCV concentrations. This gene was cloned into a conditional HIV vector, pNL-GFPRRESA, which expresses the gene of interest as well as green fluorescent protein (GFP) in the presence of HIV Tat protein. We show here that TK-SR39 was more potent that wild-type TK (TK-WT) at eliminating infected cells at lower concentrations of GCV. As the vector expresses GFP in the presence of Tat, transient expression of Tat either by Tat RNA transfection or transduction by a nonintegrating lentiviral (NIL) vector marked the cells with GFP for selection. In cells selected by this strategy, TK-SR39 was more potent at limiting virus replication than TK-WT. Finally, in Jurkat cells modified and selected by this approach, infection with CXCR4-tropic Lai virus could be suppressed by treatment with GCV. GCV treatment limited the number of HIV-infected cells, virus production, as well as virus-induced cytopathic effects in this model. We provide proof of principle that TK-SR39 in a conditional HIV vector can provide a safe and effective anti-HIV strategy. PMID:26800572

Conditional Cytotoxic Anti-HIV Gene Therapy for Selectable Cell Modification.

PubMed

Garg, Himanshu; Joshi, Anjali

2016-05-01

Gene therapy remains one of the potential strategies to achieve a cure for HIV infection. One of the major limitations of anti-HIV gene therapy concerns recovering an adequate number of modified cells to generate an HIV-proof immune system. Our study addresses this issue by developing a methodology that can mark conditional vector-transformed cells for selection and subsequently target HIV-infected cells for elimination by treatment with ganciclovir (GCV). We used the herpes simplex virus thymidine kinase (TK) mutant SR39, which is highly potent at killing cells at low GCV concentrations. This gene was cloned into a conditional HIV vector, pNL-GFPRRESA, which expresses the gene of interest as well as green fluorescent protein (GFP) in the presence of HIV Tat protein. We show here that TK-SR39 was more potent that wild-type TK (TK-WT) at eliminating infected cells at lower concentrations of GCV. As the vector expresses GFP in the presence of Tat, transient expression of Tat either by Tat RNA transfection or transduction by a nonintegrating lentiviral (NIL) vector marked the cells with GFP for selection. In cells selected by this strategy, TK-SR39 was more potent at limiting virus replication than TK-WT. Finally, in Jurkat cells modified and selected by this approach, infection with CXCR4-tropic Lai virus could be suppressed by treatment with GCV. GCV treatment limited the number of HIV-infected cells, virus production, as well as virus-induced cytopathic effects in this model. We provide proof of principle that TK-SR39 in a conditional HIV vector can provide a safe and effective anti-HIV strategy.

HSV as a vector in vaccine development and gene therapy.

PubMed

Marconi, Peggy; Argnani, Rafaela; Epstein, Alberto L; Manservigi, Roberto

2009-01-01

The very deep knowledge acquired on the genetics and molecular biology of herpes simplex virus (HSV), major human pathogen whose lifestyle is based on a long-term dual interaction with the infected host characterized by the existence of lytic and latent infections, has allowed the development of potential vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous system, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases and targeted infection of specific tissues or organs. Three different classes of vectors can be derived from HSV-1: replication-competent attenuated vectors, replication-incompetent recombinant vectors and defective helper-dependent vectors known as amplicons. This chapter highlights the current knowledge concerning design, construction and recent applications, as well as the potential and current limitations of the three different classes of HSV-1-based vectors.

Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging

PubMed Central

Drouin, Lauren M.; Lins, Bridget; Janssen, Maria; Bennett, Antonette; Chipman, Paul; McKenna, Robert; Chen, Weijun; Muzyczka, Nicholas; Cardone, Giovanni

2016-01-01

ABSTRACT The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency. IMPORTANCE The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ∼1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production. PMID:27440903

Deep sequencing of foot-and-mouth disease virus reveals RNA sequences involved in genome packaging.

PubMed

Logan, Grace; Newman, Joseph; Wright, Caroline F; Lasecka-Dykes, Lidia; Haydon, Daniel T; Cottam, Eleanor M; Tuthill, Tobias J

2017-10-18

Non-enveloped viruses protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. Packaging and capsid assembly in RNA viruses can involve interactions between capsid proteins and secondary structures in the viral genome as exemplified by the RNA bacteriophage MS2 and as proposed for other RNA viruses of plants, animals and human. In the picornavirus family of non-enveloped RNA viruses, the requirements for genome packaging remain poorly understood. Here we show a novel and simple approach to identify predicted RNA secondary structures involved in genome packaging in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data generated from both packaged and unpackaged populations of RNA we have determined multiple regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. This study provides evidence for the involvement of predicted RNA structures in picornavirus packaging and offers a readily transferable methodology for identifying packaging requirements in many other viruses. Importance In order to transmit their genetic material to a new host, non-enveloped viruses must protect their genomes by packaging them into an outer shell or capsid of virus-encoded proteins. For many non-enveloped RNA viruses the requirements for this critical part of the viral life cycle remain poorly understood. We have identified RNA sequences involved in genome packaging of the picornavirus foot-and-mouth disease virus. This virus causes an economically devastating disease of livestock affecting both the developed and developing world. The experimental methods developed to carry out this work are novel, simple and transferable to the study of packaging signals in other RNA viruses. Improved understanding of RNA packaging may lead to novel vaccine approaches or targets for antiviral drugs with broad spectrum activity. Copyright © 2017 Logan et al.

Design of retrovirus vectors for transfer and expression of the human. beta. -globin gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, A.D.; Bender, M.A.; Harris, E.A.S.

1988-11-01

Regulated expression of the human ..beta..-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective ..beta..-globin genes. The authors found regions that interfered with virus production within intron 2 of the ..beta..-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for ..beta..-globin expression. Inclusion of ..beta..-globin introns necessitatesmore » an antisense orientation of the gene within the retrovirus vector. However, they found no effect of the antisense ..beta..-globin transcription on virus production. A region downstream of the ..beta..-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10/sup 6/ CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human ..beta..-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human ..beta..-globin gene in hematopoietic cells (CFU-S cells) in mice.« less

Structural gene (prME) chimeras of St Louis encephalitis virus and West Nile virus exhibit altered in vitro cytopathic and growth phenotypes

PubMed Central

Maharaj, Payal D.; Anishchenko, Michael; Langevin, Stanley A.; Fang, Ying; Reisen, William K.

2012-01-01

Despite utilizing the same avian hosts and mosquito vectors, St Louis encephalitis virus (SLEV) and West Nile virus (WNV) display dissimilar vector-infectivity and vertebrate-pathogenic phenotypes. SLEV exhibits a low oral infection threshold for Culex mosquito vectors and is avirulent in avian hosts, producing low-magnitude viraemias. In contrast, WNV is less orally infective to mosquitoes and elicits high-magnitude viraemias in a wide range of avian species. In order to identify the genetic determinants of these different phenotypes and to assess the utility of mosquito and vertebrate cell lines for recapitulating in vivo differences observed between these viruses, reciprocal WNV and SLEV pre-membrane and envelope protein (prME) chimeric viruses were generated and growth of these mutant viruses was characterized in mammalian (Vero), avian (duck) and mosquito [Aedes (C6/36) and Culex (CT)] cells. In both vertebrate lines, WNV grew to 100-fold higher titres than SLEV, and growth and cytopathogenicity phenotypes, determined by chimeric phenotypes, were modulated by genetic elements outside the prME gene region. Both chimeras exhibited distinctive growth patterns from those of SLEV in C6/36 cells, indicating the role of both structural and non-structural gene regions for growth in this cell line. In contrast, growth of chimeric viruses was indistinguishable from that of virus containing homologous prME genes in CT cells, indicating that structural genetic elements could specifically dictate growth differences of these viruses in relevant vectors. These data provide genetic insight into divergent enzootic maintenance strategies that could also be useful for the assessment of emergence mechanisms of closely related flaviviruses. PMID:21940408

Glyceraldehyde 3-phosphate dehydrogenase negatively regulates human immunodeficiency virus type 1 infection

PubMed Central

2012-01-01

Background Host proteins are incorporated inside human immunodeficiency virus type 1 (HIV-1) virions during assembly and can either positively or negatively regulate HIV-1 infection. Although the identification efficiency of host proteins is improved by mass spectrometry, how those host proteins affect HIV-1 replication has not yet been fully clarified. Results In this study, we show that virion-associated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) does not allosterically inactivate HIV-1 reverse transcriptase (RT) but decreases the efficiency of reverse transcription reactions by decreasing the packaging efficiency of lysyl-tRNA synthetase (LysRS) and tRNALys3 into HIV-1 virions. Two-dimensional (2D) gel electrophoresis demonstrated that some isozymes of GAPDH with different isoelectric points were expressed in HIV-1-producing CEM/LAV-1 cells, and a proportion of GAPDH was selectively incorporated into the virions. Suppression of GAPDH expression by RNA interference in CEM/LAV-1 cells resulted in decreased GAPDH packaging inside the virions, and the GAPDH-packaging-defective virus maintained at least control levels of viral production but increased the infectivity. Quantitative analysis of reverse transcription products indicated that the levels of early cDNA products of the GAPDH-packaging-defective virus were higher than those of the control virus owing to the higher packaging efficiencies of LysRS and tRNALys3 into the virions rather than the GAPDH-dependent negative allosteric modulation for RT. Furthermore, immunoprecipitation assay using an anti-GAPDH antibody showed that GAPDH directly interacted with Pr55gag and p160gag-pol and the overexpression of LysRS in HIV-1-producing cells resulted in a decrease in the efficiency of GAPDH packaging in HIV particles. In contrast, the viruses produced from cells expressing a high level of GAPDH showed decreased infectivity in TZM-bl cells and reverse transcription efficiency in TZM-bl cells and peripheral blood mononuclear cells (PBMCs). Conclusions These findings indicate that GAPDH negatively regulates HIV-1 infection and provide insights into a novel function of GAPDH in the HIV-1 life cycle and a new host defense mechanism against HIV-1 infection. PMID:23237566

Influenza virus-specific TCR-transduced T cells as a model for adoptive immunotherapy

PubMed Central

Berdien, Belinda; Reinhard, Henrike; Meyer, Sabrina; Spöck, Stefanie; Kröger, Nicolaus; Atanackovic, Djordje; Fehse, Boris

2013-01-01

Adoptive transfer of T lymphocytes equipped with tumor-antigen specific T-cell receptors (TCRs) represents a promising strategy in cancer immunotherapy, but the approach remains technically demanding. Using influenza virus (Flu)-specific T-cell responses as a model system we compared different methods for the generation of T-cell clones and isolation of antigen-specific TCRs. Altogether, we generated 12 CD8+ T-cell clones reacting to the Flu matrix protein (Flu-M) and 6 CD4+ T-cell clones reacting to the Flu nucleoprotein (Flu-NP) from 4 healthy donors. IFN-γ-secretion-based enrichment of antigen-specific cells, optionally combined with tetramer staining, was the most efficient way for generating T-cell clones. In contrast, the commonly used limiting dilution approach was least efficient. TCR genes were isolated from T-cell clones and cloned into both a previously used gammaretroviral LTR-vector, MP91 and the novel lentiviral self-inactivating vector LeGO-MP that contains MP91-derived promotor and regulatory elements. To directly compare their functional efficiencies, we in parallel transduced T-cell lines and primary T cells with the two vectors encoding identical TCRs. Transduction efficiencies were approximately twice higher with the gammaretroviral vector. Secretion of high amounts of IFN-γ, IL-2 and TNF-α by transduced cells after exposure to the respective influenza target epitope proved efficient specificity transfer of the isolated TCRs to primary T-cells for both vectors, at the same time indicating superior functionality of MP91-transduced cells. In conclusion, we have developed optimized strategies to obtain and transfer antigen-specific TCRs as well as designed a novel lentiviral vector for TCR-gene transfer. Our data may help to improve adoptive T-cell therapies. PMID:23428899

Effects of the deletion of early region 4 (E4) open reading frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on virus-host cell interaction, transgene expression, and immunogenicity of replicating adenovirus HIV vaccine vectors.

PubMed

Thomas, Michael A; Song, Rui; Demberg, Thorsten; Vargas-Inchaustegui, Diego A; Venzon, David; Robert-Guroff, Marjorie

2013-01-01

The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1) through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and immunogenicity in a replicating Ad vector.

Effects of the Deletion of Early Region 4 (E4) Open Reading Frame 1 (orf1), orf1-2, orf1-3 and orf1-4 on Virus-Host Cell Interaction, Transgene Expression, and Immunogenicity of Replicating Adenovirus HIV Vaccine Vectors

PubMed Central

Thomas, Michael A.; Song, Rui; Demberg, Thorsten; Vargas-Inchaustegui, Diego A.; Venzon, David; Robert-Guroff, Marjorie

2013-01-01

The global health burden engendered by human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS) is a sobering reminder of the pressing need for a preventative vaccine. In non-human primate models replicating adenovirus (Ad)-HIV/SIV recombinant vaccine vectors have been shown to stimulate potent immune responses culminating in protection against challenge exposures. Nonetheless, an increase in the transgene carrying capacity of these Ad vectors, currently limited to approximately 3000 base pairs, would greatly enhance their utility. Using a replicating, E3-deleted Ad type 5 host range mutant (Ad5 hr) encoding full-length single-chain HIVBaLgp120 linked to the D1 and D2 domains of rhesus macaque CD4 (rhFLSC) we systematically deleted the genes encoding early region 4 open reading frame 1 (E4orf1) through E4orf4. All the Ad-rhFLSC vectors produced similar levels of viral progeny. Cell cycle analysis of infected human and monkey cells revealed no differences in virus-host interaction. The parental and E4-deleted viruses expressed comparable levels of the transgene with kinetics similar to Ad late proteins. Similar levels of cellular immune responses and transgene-specific antibodies were elicited in vaccinated mice. However, differences in recognition of Ad proteins and induced antibody subtypes were observed, suggesting that the E4 gene products might modulate antibody responses by as yet unknown mechanisms. In short, we have improved the transgene carrying capacity by one thousand base pairs while preserving the replicability, levels of transgene expression, and immunogenicity critical to these vaccine vectors. This additional space allows for flexibility in vaccine design that could not be obtained with the current vector and as such should facilitate the goal of improving vaccine efficacy. To the best of our knowledge, this is the first report describing the effects of these E4 deletions on transgene expression and immunogenicity in a replicating Ad vector. PMID:24143187

Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice.

PubMed

Boylan, Brendan T; Moreira, Fernando R; Carlson, Tim W; Bernard, Kristen A

2017-02-01

Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses.

Infectious mutants of cassava latent virus generated in vivo from intact recombinant DNA clones containing single copies of the genome.

PubMed Central

Stanley, J; Townsend, R

1986-01-01

Intact recombinant DNAs containing single copies of either component of the cassava latent virus genome can elicit infection when mechanically inoculated to host plants in the presence of the appropriate second component. Characterisation of infectious mutant progeny viruses, by analysis of virus-specific supercoiled DNA intermediates, indicates that most if not all of the cloning vector has been deleted, achieved at least in some cases by intermolecular recombination in vivo between DNAs 1 and 2. Significant rearrangements within the intergenic region of DNA 2, predominantly external to the common region, can be tolerated without loss of infectivity suggesting a somewhat passive role in virus multiplication for the sequences in question. Although packaging constraints might impose limits on the amount of DNA within geminate particles, isolation of an infectious coat protein mutant defective in virion production suggests that packaging is not essential for systemic spread of the viral DNA. Images PMID:2875435

Generation of Human Induced Pluripotent Stem Cells Using RNA-Based Sendai Virus System and Pluripotency Validation of the Resulting Cell Population.

PubMed

Chichagova, Valeria; Sanchez-Vera, Irene; Armstrong, Lyle; Steel, David; Lako, Majlinda

2016-01-01

Human induced pluripotent stem cells (hiPSCs) provide a platform for studying human disease in vitro, increase our understanding of human embryonic development, and provide clinically relevant cell types for transplantation, drug testing, and toxicology studies. Since their discovery, numerous advances have been made in order to eliminate issues such as vector integration into the host genome, low reprogramming efficiency, incomplete reprogramming and acquisition of genomic instabilities. One of the ways to achieve integration-free reprogramming is by using RNA-based Sendai virus. Here we describe a method to generate hiPSCs with Sendai virus in both feeder-free and feeder-dependent culture systems. Additionally, we illustrate methods by which to validate pluripotency of the resulting stem cell population.

Protein and modified vaccinia virus Ankara-based influenza virus nucleoprotein vaccines are differentially immunogenic in BALB/c mice.

PubMed

Altenburg, A F; Magnusson, S E; Bosman, F; Stertman, L; de Vries, R D; Rimmelzwaan, G F

2017-10-01

Because of the high variability of seasonal influenza viruses and the eminent threat of influenza viruses with pandemic potential, there is great interest in the development of vaccines that induce broadly protective immunity. Most probably, broadly protective influenza vaccines are based on conserved proteins, such as nucleoprotein (NP). NP is a vaccine target of interest as it has been shown to induce cross-reactive antibody and T cell responses. Here we tested and compared various NP-based vaccine preparations for their capacity to induce humoral and cellular immune responses to influenza virus NP. The immunogenicity of protein-based vaccine preparations with Matrix-M™ adjuvant as well as recombinant viral vaccine vector modified Vaccinia virus Ankara (MVA) expressing the influenza virus NP gene, with or without modifications that aim at optimization of CD8 + T cell responses, was addressed in BALB/c mice. Addition of Matrix-M™ adjuvant to NP wild-type protein-based vaccines significantly improved T cell responses. Furthermore, recombinant MVA expressing the influenza virus NP induced strong antibody and CD8 + T cell responses, which could not be improved further by modifications of NP to increase antigen processing and presentation. © 2017 British Society for Immunology.

Viral Vector-Based Targeting of miR-21 in Cardiac Nonmyocyte Cells Reduces Pathologic Remodeling of the Heart

PubMed Central

Ramanujam, Deepak; Sassi, Yassine; Laggerbauer, Bernhard; Engelhardt, Stefan

2016-01-01

Systemic inhibition of miR-21 has proven effective against myocardial fibrosis and dysfunction, while studies in cardiac myocytes suggested a protective role in this cell type. Considering potential implications for therapy, we aimed to determine the cell fraction where miR-21 exerts its pathological activity. We developed a viral vector-based strategy for gene targeting of nonmyocyte cardiac cells in vivo and compared global to cardiac myocyte-specific and nonmyocyte-specific deletion of miR-21 in chronic left ventricular pressure overload. Murine moloney virus and serotype 9 of adeno-associated virus were engineered to encode improved Cre recombinase for genetic deletion in miR-21fl/fl mice. Pericardial injection of murine moloney virus-improved Cre recombinase to neonates achieved highly selective genetic ablation of miR-21 in nonmyocyte cardiac cells, identified as cardiac fibroblasts and endothelial cells. Upon left ventricular pressure overload, cardiac function was only preserved in mice with miR-21 deficiency in nonmyocyte cardiac cells, but not in mice with global or cardiac myocyte-specific ablation. Our data demonstrate that miR-21 exerts its pathologic activity directly in cardiac nonmyocytes and encourage further development of antimiR-21 therapy toward cellular tropism. PMID:27545313

Creation of Rift Valley Fever Viruses with Four-Segmented Genomes Reveals Flexibility in Bunyavirus Genome Packaging

PubMed Central

Oreshkova, Nadia; Moormann, Rob J. M.; Kortekaas, Jeroen

2014-01-01

ABSTRACT Bunyavirus genomes comprise a small (S), a medium (M), and a large (L) RNA segment of negative polarity. Although the untranslated regions have been shown to comprise signals required for transcription, replication, and encapsidation, the mechanisms that drive the packaging of at least one S, M, and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). We studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising two S-type segments in the absence of an M-type segment to a virus consisting of four segments (RVFV-4s), of which three are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines. IMPORTANCE Rift Valley fever virus (RVFV) causes devastating disease in ruminants and occasionally humans. Virions capable of productive infection comprise at least one copy of the small (S), medium (M), and large (L) RNA genome segments. The M segment encodes a glycoprotein precursor (GPC) protein that is cotranslationally cleaved into Gn and Gc, which are required for virus entry and fusion. We studied the flexibility of RVFV genome packaging and developed experimental live-attenuated vaccines by applying a unique strategy based on the splitting of the GnGc open reading frame. Several RVFV variants, varying from viruses comprising two S-type segments to viruses consisting of four segments (RVFV-4s), of which three are M-type, could be rescued and were shown to induce a rapid protective immune response. Altogether, the segmentation of bunyavirus GPCs provides a new method for studying bunyavirus genome packaging and facilitates the development of novel live-attenuated bunyavirus vaccines. PMID:25008937

Creation of Rift Valley fever viruses with four-segmented genomes reveals flexibility in bunyavirus genome packaging.

PubMed

Wichgers Schreur, Paul J; Oreshkova, Nadia; Moormann, Rob J M; Kortekaas, Jeroen

2014-09-01

Bunyavirus genomes comprise a small (S), a medium (M), and a large (L) RNA segment of negative polarity. Although the untranslated regions have been shown to comprise signals required for transcription, replication, and encapsidation, the mechanisms that drive the packaging of at least one S, M, and L segment into a single virion to generate infectious virus are largely unknown. One of the most important members of the Bunyaviridae family that causes devastating disease in ruminants and occasionally humans is the Rift Valley fever virus (RVFV). We studied the flexibility of RVFV genome packaging by splitting the glycoprotein precursor gene, encoding the (NSm)GnGc polyprotein, into two individual genes encoding either (NSm)Gn or Gc. Using reverse genetics, six viruses with a segmented glycoprotein precursor gene were rescued, varying from a virus comprising two S-type segments in the absence of an M-type segment to a virus consisting of four segments (RVFV-4s), of which three are M-type. Despite that all virus variants were able to grow in mammalian cell lines, they were unable to spread efficiently in cells of mosquito origin. Moreover, in vivo studies demonstrated that RVFV-4s is unable to cause disseminated infection and disease in mice, even in the presence of the main virulence factor NSs, but induced a protective immune response against a lethal challenge with wild-type virus. In summary, splitting bunyavirus glycoprotein precursor genes provides new opportunities to study bunyavirus genome packaging and offers new methods to develop next-generation live-attenuated bunyavirus vaccines. Rift Valley fever virus (RVFV) causes devastating disease in ruminants and occasionally humans. Virions capable of productive infection comprise at least one copy of the small (S), medium (M), and large (L) RNA genome segments. The M segment encodes a glycoprotein precursor (GPC) protein that is cotranslationally cleaved into Gn and Gc, which are required for virus entry and fusion. We studied the flexibility of RVFV genome packaging and developed experimental live-attenuated vaccines by applying a unique strategy based on the splitting of the GnGc open reading frame. Several RVFV variants, varying from viruses comprising two S-type segments to viruses consisting of four segments (RVFV-4s), of which three are M-type, could be rescued and were shown to induce a rapid protective immune response. Altogether, the segmentation of bunyavirus GPCs provides a new method for studying bunyavirus genome packaging and facilitates the development of novel live-attenuated bunyavirus vaccines. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Plant Virus-Insect Vector Interactions: Current and Potential Future Research Directions.

PubMed

Dietzgen, Ralf G; Mann, Krin S; Johnson, Karyn N

2016-11-09

Acquisition and transmission by an insect vector is central to the infection cycle of the majority of plant pathogenic viruses. Plant viruses can interact with their insect host in a variety of ways including both non-persistent and circulative transmission; in some cases, the latter involves virus replication in cells of the insect host. Replicating viruses can also elicit both innate and specific defense responses in the insect host. A consistent feature is that the interaction of the virus with its insect host/vector requires specific molecular interactions between virus and host, commonly via proteins. Understanding the interactions between plant viruses and their insect host can underpin approaches to protect plants from infection by interfering with virus uptake and transmission. Here, we provide a perspective focused on identifying novel approaches and research directions to facilitate control of plant viruses by better understanding and targeting virus-insect molecular interactions. We also draw parallels with molecular interactions in insect vectors of animal viruses, and consider technical advances for their control that may be more broadly applicable to plant virus vectors.

Transient expression of the influenza A virus PB1-F2 protein using a plum pox virus-based vector in Nicotiana benthamiana.

PubMed

Kamencayová, M; Košík, I; Hunková, J; Subr, Z W

2014-01-01

PB1-F2 protein of influenza A virus (IAV) was cloned in a plum pox virus (PPV) genome-based vector and attempts to express it in biolistically transfected Nicotiana benthamiana plants were performed. The vector-insert construct replicated in infected plants properly and was stable during repeated passage by mechanical inoculation, as demonstrated by disease symptoms and immunoblot detection of PPV capsid protein, while PB1-F2-specific band was more faint. We showed that it was due its low solubility. Modification of sample preparation (denaturation/solubilization preceding the centrifugation of cell debris) led to substantial signal enhancement. Maximal level of PB1-F2 expression in plants was observed 12 days post inoculation (dpi). Only 1% SDS properly solubilized the protein, other detergents were much less efficient. Solubilization with 8M urea released approximately 50% of PB1-F2 from the plant tissues, thus the treatment with this removable chaotropic agent may be a good starting point for the purification of the protein for eventual functional studies in the future.

Development of a GFP expression vector for Cucurbit chlorotic yellows virus.

PubMed

Wei, Ying; Han, Xiaoyu; Wang, Zhenyue; Gu, Qinsheng; Li, Honglian; Chen, Linlin; Sun, Bingjian; Shi, Yan

2018-05-24

Cucurbit chlorotic yellows virus (CCYV), a bipartite crinivirus, causes chlorotic leaf spots and yellowing symptoms on cucurbit leaves. We previously developed an infectious clone of CCYV. Limited work has been conducted on the construction of a crinivirus green fluorescence protein (GFP) expression vector to date. We constructed a CCYV GFP expression vector using the "add a gene" strategy based on CCYV RNA2 cDNA constrcut. Three resultant clones, pCCYVGFP SGC , pCCYVGFP CGC , and pCCYVGFP CGS, were constructed with different promoters used to initiate GFP and CP expression. At 25 dpi GFP fluorescence was detectable not only in leaf veins but also in the surrounding cells. pCCYVGFP CGC -infected cucumber leaves exhibited cell spread at 25 dpi, whereas pCCYVGFP SGC and pCCYVGFP CGS were mainly found in single cells. Further observation of pCCYVGFP CGC GFP expression at 30 dpi, 40 dpi, and 50 dpi showed phloem-limited localization in the systemic leaves. We developed of a CCYV GFP expression vector that will be useful for further study of CCYV movement in cucurbits.

Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles

PubMed Central

Johnson, Reed F.; McCarthy, Sarah E.; Godlewski, Peter J.; Harty, Ronald N.

2006-01-01

The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minignome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions. PMID:16698994

Geminivirus vectors for high-level expression of foreign proteins in plant cells.

PubMed

Mor, Tsafrir S; Moon, Yong-Sun; Palmer, Kenneth E; Mason, Hugh S

2003-02-20

Bean yellow dwarf virus (BeYDV) is a monopartite geminivirus that can infect dicotyledonous plants. We have developed a high-level expression system that utilizes elements of the replication machinery of this single-stranded DNA virus. The replication initiator protein (Rep) mediates release and replication of a replicon from a DNA construct ("LSL vector") that contains an expression cassette for a gene of interest flanked by cis-acting elements of the virus. We used tobacco NT1 cells and biolistic delivery of plasmid DNA for evaluation of replication and expression of reporter genes contained within an LSL vector. By codelivery of a GUS reporter-LSL vector and a Rep-supplying vector, we obtained up to 40-fold increase in expression levels compared to delivery of the reporter-LSL vectors alone. High-copy replication of the LSL vector was correlated with enhanced expression of GUS. Rep expression using a whole BeYDV clone, a cauliflower mosaic virus 35S promoter driving either genomic rep or an intron-deleted rep gene, or 35S-rep contained in the LSL vector all achieved efficient replication and enhancement of GUS expression. We anticipate that this system can be adapted for use in transgenic plants or plant cell cultures with appropriately regulated expression of Rep, with the potential to greatly increase yield of recombinant proteins. Copyright 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 430-437, 2003.

Selection of transduced CD34+ progenitors and enzymatic correction of cells from Gaucher patients, with bicistronic vectors.

PubMed Central

Migita, M; Medin, J A; Pawliuk, R; Jacobson, S; Nagle, J W; Anderson, S; Amiri, M; Humphries, R K; Karlsson, S

1995-01-01

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected. Images Fig. 2 Fig. 3 PMID:8618847

Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector.

PubMed

Liang, Bo; Ngwuta, Joan O; Herbert, Richard; Swerczek, Joanna; Dorward, David W; Amaro-Carambot, Emerito; Mackow, Natalie; Kabatova, Barbora; Lingemann, Matthias; Surman, Sonja; Yang, Lijuan; Chen, Man; Moin, Syed M; Kumar, Azad; McLellan, Jason S; Kwong, Peter D; Graham, Barney S; Schaap-Nutt, Anne; Collins, Peter L; Munir, Shirin

2016-11-01

Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major pediatric respiratory pathogens that lack vaccines. A chimeric bovine/human PIV3 (rB/HPIV3) virus expressing the unmodified, wild-type (wt) RSV fusion (F) protein from an added gene was previously evaluated in seronegative children as a bivalent intranasal RSV/HPIV3 vaccine, and it was well tolerated but insufficiently immunogenic for RSV F. We recently showed that rB/HPIV3 expressing a partially stabilized prefusion form (pre-F) of RSV F efficiently induced "high-quality" RSV-neutralizing antibodies, defined as antibodies that neutralize RSV in vitro without added complement (B. Liang et al., J Virol 89:9499-9510, 2015, doi:10.1128/JVI.01373-15). In the present study, we modified RSV F by replacing its cytoplasmic tail (CT) domain or its CT and transmembrane (TM) domains (TMCT) with counterparts from BPIV3 F, with or without pre-F stabilization. This resulted in RSV F being packaged in the rB/HPIV3 particle with an efficiency similar to that of RSV particles. Enhanced packaging was substantially attenuating in hamsters (10- to 100-fold) and rhesus monkeys (100- to 1,000-fold). Nonetheless, TMCT-directed packaging substantially increased the titers of high-quality RSV-neutralizing serum antibodies in hamsters. In rhesus monkeys, a strongly additive immunogenic effect of packaging and pre-F stabilization was observed, as demonstrated by 8- and 30-fold increases of RSV-neutralizing serum antibody titers in the presence and absence of added complement, respectively, compared to pre-F stabilization alone. Analysis of vaccine-induced F-specific antibodies by binding assays indicated that packaging conferred substantial stabilization of RSV F in the pre-F conformation. This provides an improved version of this well-tolerated RSV/HPIV3 vaccine candidate, with potently improved immunogenicity, which can be returned to clinical trials. Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major viral agents of acute pediatric bronchiolitis and pneumonia worldwide that lack vaccines. A bivalent intranasal RSV/HPIV3 vaccine candidate consisting of a chimeric bovine/human PIV3 (rB/HPIV3) strain expressing the RSV fusion (F) protein was previously shown to be well tolerated by seronegative children but was insufficiently immunogenic for RSV F. In the present study, the RSV F protein was engineered to be packaged efficiently into vaccine virus particles. This resulted in a significantly enhanced quantity and quality of RSV-neutralizing antibodies in hamsters and nonhuman primates. In nonhuman primates, this effect was strongly additive to the previously described stabilization of the prefusion conformation of the F protein. The improved immunogenicity of RSV F by packaging appeared to involve prefusion stabilization. These findings provide a potently more immunogenic version of this well-tolerated vaccine candidate and should be applicable to other vectored vaccines. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Packaging and Prefusion Stabilization Separately and Additively Increase the Quantity and Quality of Respiratory Syncytial Virus (RSV)-Neutralizing Antibodies Induced by an RSV Fusion Protein Expressed by a Parainfluenza Virus Vector

PubMed Central

Liang, Bo; Ngwuta, Joan O.; Herbert, Richard; Swerczek, Joanna; Dorward, David W.; Amaro-Carambot, Emerito; Mackow, Natalie; Kabatova, Barbora; Lingemann, Matthias; Surman, Sonja; Yang, Lijuan; Chen, Man; Moin, Syed M.; Kumar, Azad; McLellan, Jason S.; Kwong, Peter D.; Graham, Barney S.; Schaap-Nutt, Anne; Collins, Peter L.

2016-01-01

ABSTRACT Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major pediatric respiratory pathogens that lack vaccines. A chimeric bovine/human PIV3 (rB/HPIV3) virus expressing the unmodified, wild-type (wt) RSV fusion (F) protein from an added gene was previously evaluated in seronegative children as a bivalent intranasal RSV/HPIV3 vaccine, and it was well tolerated but insufficiently immunogenic for RSV F. We recently showed that rB/HPIV3 expressing a partially stabilized prefusion form (pre-F) of RSV F efficiently induced “high-quality” RSV-neutralizing antibodies, defined as antibodies that neutralize RSV in vitro without added complement (B. Liang et al., J Virol 89:9499–9510, 2015, doi:10.1128/JVI.01373-15). In the present study, we modified RSV F by replacing its cytoplasmic tail (CT) domain or its CT and transmembrane (TM) domains (TMCT) with counterparts from BPIV3 F, with or without pre-F stabilization. This resulted in RSV F being packaged in the rB/HPIV3 particle with an efficiency similar to that of RSV particles. Enhanced packaging was substantially attenuating in hamsters (10- to 100-fold) and rhesus monkeys (100- to 1,000-fold). Nonetheless, TMCT-directed packaging substantially increased the titers of high-quality RSV-neutralizing serum antibodies in hamsters. In rhesus monkeys, a strongly additive immunogenic effect of packaging and pre-F stabilization was observed, as demonstrated by 8- and 30-fold increases of RSV-neutralizing serum antibody titers in the presence and absence of added complement, respectively, compared to pre-F stabilization alone. Analysis of vaccine-induced F-specific antibodies by binding assays indicated that packaging conferred substantial stabilization of RSV F in the pre-F conformation. This provides an improved version of this well-tolerated RSV/HPIV3 vaccine candidate, with potently improved immunogenicity, which can be returned to clinical trials. IMPORTANCE Human respiratory syncytial virus (RSV) and human parainfluenza virus type 3 (HPIV3) are major viral agents of acute pediatric bronchiolitis and pneumonia worldwide that lack vaccines. A bivalent intranasal RSV/HPIV3 vaccine candidate consisting of a chimeric bovine/human PIV3 (rB/HPIV3) strain expressing the RSV fusion (F) protein was previously shown to be well tolerated by seronegative children but was insufficiently immunogenic for RSV F. In the present study, the RSV F protein was engineered to be packaged efficiently into vaccine virus particles. This resulted in a significantly enhanced quantity and quality of RSV-neutralizing antibodies in hamsters and nonhuman primates. In nonhuman primates, this effect was strongly additive to the previously described stabilization of the prefusion conformation of the F protein. The improved immunogenicity of RSV F by packaging appeared to involve prefusion stabilization. These findings provide a potently more immunogenic version of this well-tolerated vaccine candidate and should be applicable to other vectored vaccines. PMID:27581977

Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice

PubMed Central

Ren, Shoufeng; Wei, Qimei; Cai, Liya; Yang, Xuejing; Xing, Cuicui; Tan, Feng; Leavenworth, Jianmei W.; Liang, Shaohui; Liu, Wenquan

2018-01-01

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP) is the major protective antigen of EBOV, and can generate virus-like particles (VLPs) by co-expression with matrix protein (VP40). In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV) replicon vector DREP to express EBOV GP and matrix viral protein (VP40). EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40). Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8+ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention. PMID:29375526

Development of a Reverse Genetic System for Infectious Salmon Anemia Virus: Rescue of Recombinant Fluorescent Virus by Using Salmon Internal Transcribed Spacer Region 1 as a Novel Promoter

PubMed Central

Toro-Ascuy, Daniela; Tambley, Carolina; Beltran, Carolina; Mascayano, Carolina; Sandoval, Nicolas; Olivares, Eduardo; Medina, Rafael A.; Spencer, Eugenio

2014-01-01

Infectious salmon anemia (ISA) is a serious disease of marine-farmed Atlantic salmon (Salmo salar) caused by ISA virus (ISAV), belonging to the genus Isavirus, family Orthomyxoviridae. There is an urgent need to understand the virulence factors and pathogenic mechanisms of ISAV and to develop new vaccine approaches. Using a recombinant molecular biology approach, we report the development of a plasmid-based reverse genetic system for ISAV, which includes the use of a novel fish promoter, the Atlantic salmon internal transcribed spacer region 1 (ITS-1). Salmon cells cotransfected with pSS-URG-based vectors expressing the eight viral RNA segments and four cytomegalovirus (CMV)-based vectors that express the four proteins of the ISAV ribonucleoprotein complex allowed the generation of infectious recombinant ISAV (rISAV). We generated three recombinant viruses, wild-type rISAV901_09 and rISAVrS6-NotI-HPR containing a NotI restriction site and rISAVS6/EGFP-HPR harboring the open reading frame of enhanced green fluorescent protein (EGFP), both within the highly polymorphic region (HPR) of segment 6. All rescued viruses showed replication activity and cytopathic effect in Atlantic salmon kidney-infected cells. The fluorescent recombinant viruses also showed a characteristic cytopathic effect in salmon cells, and the viruses replicated to a titer of 6.5 × 105 PFU/ml, similar to that of the wild-type virus. This novel reverse genetics system offers a powerful tool to study the molecular biology of ISAV and to develop a new generation of ISAV vaccines to prevent and mitigate ISAV infection, which has had a profound effect on the salmon industry. PMID:25480750

Peptide-Based Technologies to Alter Adenoviral Vector Tropism: Ways and Means for Systemic Treatment of Cancer

PubMed Central

Reetz, Julia; Herchenröder, Ottmar; Pützer, Brigitte M.

2014-01-01

Due to the fundamental progress in elucidating the molecular mechanisms of human diseases and the arrival of the post-genomic era, increasing numbers of therapeutic genes and cellular targets are available for gene therapy. Meanwhile, the most important challenge is to develop gene delivery vectors with high efficiency through target cell selectivity, in particular under in situ conditions. The most widely used vector system to transduce cells is based on adenovirus (Ad). Recent endeavors in the development of selective Ad vectors that target cells or tissues of interest and spare the alteration of all others have focused on the modification of the virus broad natural tropism. A popular way of Ad targeting is achieved by directing the vector towards distinct cellular receptors. Redirecting can be accomplished by linking custom-made peptides with specific affinity to cellular surface proteins via genetic integration, chemical coupling or bridging with dual-specific adapter molecules. Ideally, targeted vectors are incapable of entering cells via their native receptors. Such altered vectors offer new opportunities to delineate functional genomics in a natural environment and may enable efficient systemic therapeutic approaches. This review provides a summary of current state-of-the-art techniques to specifically target adenovirus-based gene delivery vectors. PMID:24699364

New frontiers in oncolytic viruses: optimizing and selecting for virus strains with improved efficacy

PubMed Central

2018-01-01

Oncolytic viruses have demonstrated selective replication and killing of tumor cells. Different types of oncolytic viruses – adenoviruses, alphaviruses, herpes simplex viruses, Newcastle disease viruses, rhabdoviruses, Coxsackie viruses, and vaccinia viruses – have been applied as either naturally occurring or engineered vectors. Numerous studies in animal-tumor models have demonstrated substantial tumor regression and prolonged survival rates. Moreover, clinical trials have confirmed good safety profiles and therapeutic efficacy for oncolytic viruses. Most encouragingly, the first cancer gene-therapy drug – Gendicine, based on oncolytic adenovirus type 5 – was approved in China. Likewise, a second-generation oncolytic herpes simplex virus-based drug for the treatment of melanoma has been registered in the US and Europe as talimogene laherparepvec. PMID:29445265

Adeno-associated virus-mediated gene transfer.

PubMed

Srivastava, Arun

2008-09-01

Although the remarkable versatility and efficacy of recombinant adeno-associated virus 2 (AAV2) vectors in transducing a wide variety of cells and tissues in vitro, and in numerous pre-clinical animal models of human diseases in vivo, have been well established, the published literature is replete with controversies with regard to the efficacy of AAV2 vectors in hematopoietic stem cell (HSC) transduction. A number of factors have contributed to these controversies, the molecular bases of which have begun to come to light in recent years. With the availability of several novel serotypes (AAV1 through AAV12), rational design of AAV capsid mutants, and strategies (self-complementary vector genomes, hematopoietic cell-specific promoters), it is indeed becoming feasible to achieve efficient transduction of HSC by AAV vectors. Using a murine serial bone marrow transplantation model in vivo, we have recently documented stable integration of the proviral AAV genome into mouse chromosomes, which does not lead to any overt hematological abnormalities. Thus, a better understanding of the AAV-HSC interactions, and the availability of a vast repertoire of novel serotype and capsid mutant vectors, are likely to have significant implications in the use of AAV vectors in high-efficiency transduction of HSCs as well as in gene therapy applications involving the hematopoietic system. (c) 2008 Wiley-Liss, Inc.

CRISPR/Cas9—Advancing Orthopoxvirus Genome Editing for Vaccine and Vector Development

PubMed Central

Okoli, Arinze; Okeke, Malachy I.; Tryland, Morten; Moens, Ugo

2018-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them. PMID:29361752

Unique Safety Issues Associated with Virus Vectored Vaccines: Potential for and Theoretical Consequences of Recombination with Wild Type Virus Strains

PubMed Central

Condit, Richard C.; Williamson, Anna-Lise; Sheets, Rebecca; Seligman, Stephen J.; Monath, Thomas P.; Excler, Jean-Louis; Gurwith, Marc; Bok, Karin; Robertson, James S.; Kim, Denny; Hendry, Michael; Singh, Vidisha; Mac, Lisa M.; Chen, Robert T.

2016-01-01

In 2003 and 2013, the World Health Organization convened informal consultations on characterization and quality aspects of vaccines based on live virus vectors. In the resulting reports, one of several issues raised for future study was the potential for recombination of virus-vectored vaccines with wild type pathogenic virus strains. This paper presents an assessment of this issue formulated by the Brighton Collaboration. To provide an appropriate context for understanding the potential for recombination of virus-vectored vaccines, we review briefly the current status of virus vectored vaccines, mechanisms of recombination between viruses, experience with recombination involving live attenuated vaccines in the field, and concerns raised previously in the literature regarding recombination of virus-vectored vaccines with wild type virus strains. We then present a discussion of the major variables that could influence recombination between a virus-vectored vaccine and circulating wild type virus and the consequences of such recombination, including intrinsic recombination properties of the parent virus used as a vector; sequence relatedness of vector and wild virus; virus host range, pathogenesis and transmission; replication competency of vector in target host; mechanism of vector attenuation; additional factors potentially affecting virulence; and circulation of multiple recombinant vectors in the same target population. Finally, we present some guiding principles for vector design and testing intended to anticipate and mitigate the potential for and consequences of recombination of virus-vectored vaccines with wild type pathogenic virus strains. PMID:27346303

Plant Virus–Insect Vector Interactions: Current and Potential Future Research Directions

PubMed Central

Dietzgen, Ralf G.; Mann, Krin S.; Johnson, Karyn N.

2016-01-01

Acquisition and transmission by an insect vector is central to the infection cycle of the majority of plant pathogenic viruses. Plant viruses can interact with their insect host in a variety of ways including both non-persistent and circulative transmission; in some cases, the latter involves virus replication in cells of the insect host. Replicating viruses can also elicit both innate and specific defense responses in the insect host. A consistent feature is that the interaction of the virus with its insect host/vector requires specific molecular interactions between virus and host, commonly via proteins. Understanding the interactions between plant viruses and their insect host can underpin approaches to protect plants from infection by interfering with virus uptake and transmission. Here, we provide a perspective focused on identifying novel approaches and research directions to facilitate control of plant viruses by better understanding and targeting virus–insect molecular interactions. We also draw parallels with molecular interactions in insect vectors of animal viruses, and consider technical advances for their control that may be more broadly applicable to plant virus vectors. PMID:27834855

9 CFR 112.5 - Review and approval of labeling.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Review and approval of labeling. 112.5 Section 112.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING...

9 CFR 112.5 - Review and approval of labeling.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Review and approval of labeling. 112.5 Section 112.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING...

9 CFR 112.5 - Review and approval of labeling.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Review and approval of labeling. 112.5 Section 112.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING...

9 CFR 112.5 - Review and approval of labeling.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Review and approval of labeling. 112.5 Section 112.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING...

9 CFR 112.5 - Review and approval of labeling.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Review and approval of labeling. 112.5 Section 112.5 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS PACKAGING AND LABELING...

Genome-Wide RNAi Screen Identifies Broadly-Acting Host Factors That Inhibit Arbovirus Infection

PubMed Central

Yasunaga, Ari; Hanna, Sheri L.; Li, Jianqing; Cho, Hyelim; Rose, Patrick P.; Spiridigliozzi, Anna; Gold, Beth; Diamond, Michael S.; Cherry, Sara

2014-01-01

Vector-borne viruses are an important class of emerging and re-emerging pathogens; thus, an improved understanding of the cellular factors that modulate infection in their respective vertebrate and insect hosts may aid control efforts. In particular, cell-intrinsic antiviral pathways restrict vector-borne viruses including the type I interferon response in vertebrates and the RNA interference (RNAi) pathway in insects. However, it is likely that additional cell-intrinsic mechanisms exist to limit these viruses. Since insects rely on innate immune mechanisms to inhibit virus infections, we used Drosophila as a model insect to identify cellular factors that restrict West Nile virus (WNV), a flavivirus with a broad and expanding geographical host range. Our genome-wide RNAi screen identified 50 genes that inhibited WNV infection. Further screening revealed that 17 of these genes were antiviral against additional flaviviruses, and seven of these were antiviral against other vector-borne viruses, expanding our knowledge of invertebrate cell-intrinsic immunity. Investigation of two newly identified factors that restrict diverse viruses, dXPO1 and dRUVBL1, in the Tip60 complex, demonstrated they contributed to antiviral defense at the organismal level in adult flies, in mosquito cells, and in mammalian cells. These data suggest the existence of broadly acting and functionally conserved antiviral genes and pathways that restrict virus infections in evolutionarily divergent hosts. PMID:24550726

Oncolytic Herpes Simplex Virus Vectors Fully Retargeted to Tumor- Associated Antigens.

PubMed

Uchida, Hiroaki; Hamada, Hirofumi; Nakano, Kenji; Kwon, Heechung; Tahara, Hideaki; Cohen, Justus B; Glorioso, Joseph C

2018-01-01

Oncolytic virotherapy is a novel therapeutic modality for malignant diseases that exploits selective viral replication in cancer cells. Herpes simplex virus (HSV) is a promising agent for oncolytic virotherapy due to its broad cell tropism and the identification of mutations that favor its replication in tumor over normal cells. However, these attenuating mutations also tend to limit the potency of current oncolytic HSV vectors that have entered clinical studies. As an alternative, vector retargeting to novel entry receptors has the potential to achieve tumor specificity at the stage of virus entry, eliminating the need for replication-attenuating mutations. Here, we summarize the molecular mechanism of HSV entry and recent advances in the development of fully retargeted HSV vectors for oncolytic virotherapy. Retargeted HSV vectors offer an attractive platform for the creation of a new generation of oncolytic HSV with improved efficacy and specificity. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Characterization of complete particles (VSV-G/SIN-GFP) and empty particles (VSV-G/EMPTY) in human immunodeficiency virus type 1-based lentiviral products for gene therapy: potential applications for improvement of product quality and safety.

PubMed

Zhao, Yuan; Keating, Kenneth; Dolman, Carl; Thorpe, Robin

2008-05-01

Lentiviral vectors persist in the host and are therefore ideally suited for long-term gene therapy. To advance the use of lentiviral vectors in humans, improvement of their production, purification, and characterization has become increasingly important and challenging. In addition to cellular contaminants derived from packaging cells, empty particles without therapeutic function are the major impurities that compromise product safety and efficacy. Removal of empty particles is difficult because of their innate similarity in particle size and protein composition to the complete particles. We propose that comparison of the properties of lentiviral products with those of purposely expressed empty particles may reveal potential differences between empty and complete particles. For this, three forms of recombinant lentiviral samples, that is, recombinant vesicular stomatitis virus glycoprotein (VSV-G) proteins, empty particles (VSV-G/Empty), and complete particles (VSV-G/SIN-GFP) carrying viral RNA, were purified by size-exclusion chromatography (SEC). The SEC-purified samples were further analyzed by immunoblotting with six antibodies to examine viral and cellular proteins associated with the particles. This study has demonstrated, for the first time, important differences between VSV-G/Empty particles and complete VSV-G/SIN-GFP particles. Differences include the processing of Gag protein and the inclusion of cellular proteins in the particles. Our findings support the development of improved production, purification, and characterization methods for lentiviral products.

Priming-boosting vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin and a nonreplicating vaccinia virus recombinant leads to long-lasting and effective immunity.

PubMed

Ami, Yasushi; Izumi, Yasuyuki; Matsuo, Kazuhiro; Someya, Kenji; Kanekiyo, Masaru; Horibata, Shigeo; Yoshino, Naoto; Sakai, Koji; Shinohara, Katsuaki; Matsumoto, Sohkichi; Yamada, Takeshi; Yamazaki, Shudo; Yamamoto, Naoki; Honda, Mitsuo

2005-10-01

Virus-specific T-cell responses can limit immunodeficiency virus type 1 (HIV-1) transmission and prevent disease progression and so could serve as the basis for an affordable, safe, and effective vaccine in humans. To assess their potential for a vaccine, we used Mycobacterium bovis bacillus Calmette-Guérin (BCG)-Tokyo and a replication-deficient vaccinia virus strain (DIs) as vectors to express full-length gag from simian immunodeficiency viruses (SIVs) (rBCG-SIVgag and rDIsSIVgag). Cynomolgus macaques were vaccinated with either rBCG-SIVgag dermally as a single modality or in combination with rDIsSIVgag intravenously. When cynomologus macaques were primed with rBCG-SIVgag and then boosted with rDIsSIVgag, high levels of gamma interferon (IFN-gamma) spot-forming cells specific for SIV Gag were induced. This combination regimen elicited effective protective immunity against mucosal challenge with pathogenic simian-human immunodeficiency virus for the 1 year the macaques were under observation. Antigen-specific intracellular IFN-gamma activity was similarly induced in each of the macaques with the priming-boosting regimen. Other groups receiving the opposite combination or the single-modality vaccines were not effectively protected. These results suggest that a recombinant M. bovis BCG-based vector may have potential as an HIV/AIDS vaccine when administered in combination with a replication-deficient vaccinia virus DIs vector in a priming-boosting strategy.

Progress and prospects: foamy virus vectors enter a new age.

PubMed

Erlwein, O; McClure, M O

2010-12-01

Foamy viruses, distantly related to the major subfamily of Retroviruses, Orthoretroviruses that include oncoviruses (for example, murine leukemia virus (MLV)) and lentiviruses (human immunodeficiency virus (HIV)), are endemic in mammalian species, but not in human populations. Humans infected by accidental or occupational exposure remain well. The virus is not transmitted to others, nor is it associated with any disease. These features added to its broad host range, efficient transduction of progenitor cells and an integration profile less likely to induce insertional mutagenesis, make these viruses attractive as vectors. Long-term reversal of disease phenotype in dogs with the genetic defect, leukocyte adhesion deficiency, by foamy virus vector therapy strengthens the case for their clinical exploitation.

Generation of a Lineage II Powassan Virus (Deer Tick Virus) cDNA Clone: Assessment of Flaviviral Genetic Determinants of Tick and Mosquito Vector Competence.

PubMed

Kenney, Joan L; Anishchenko, Michael; Hermance, Meghan; Romo, Hannah; Chen, Ching-I; Thangamani, Saravanan; Brault, Aaron C

2018-05-21

The Flavivirus genus comprises a diverse group of viruses that utilize a wide range of vertebrate hosts and arthropod vectors. The genus includes viruses that are transmitted solely by mosquitoes or vertebrate hosts as well as viruses that alternate transmission between mosquitoes or ticks and vertebrates. Nevertheless, the viral genetic determinants that dictate these unique flaviviral host and vector specificities have been poorly characterized. In this report, a cDNA clone of a flavivirus that is transmitted between ticks and vertebrates (Powassan lineage II, deer tick virus [DTV]) was generated and chimeric viruses between the mosquito/vertebrate flavivirus, West Nile virus (WNV), were constructed. These chimeric viruses expressed the prM and E genes of either WNV or DTV in the heterologous nonstructural (NS) backbone. Recombinant chimeric viruses rescued from cDNAs were characterized for their capacity to grow in vertebrate and arthropod (mosquito and tick) cells as well as for in vivo vector competence in mosquitoes and ticks. Results demonstrated that the NS elements were insufficient to impart the complete mosquito or tick growth phenotypes of parental viruses; however, these NS genetic elements did contribute to a 100- and 100,000-fold increase in viral growth in vitro in tick and mosquito cells, respectively. Mosquito competence was observed only with parental WNV, while infection and transmission potential by ticks were observed with both DTV and WNV-prME/DTV chimeric viruses. These data indicate that NS genetic elements play a significant, but not exclusive, role for vector usage of mosquito- and tick-borne flaviviruses.

Aedes-Borne Virus-Mosquito Interactions: Mass Spectrometry Strategies and Findings.

PubMed

Pando-Robles, Victoria; Batista, Cesar V

2017-06-01

Aedes-borne viruses are responsible for high-impact neglected tropical diseases and unpredictable outbreaks such as the ongoing Zika epidemics. Aedes mosquitoes spread different arboviruses such as Dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus, among others, and are responsible for the continuous emergence and reemergence of these pathogens. These viruses have complex transmission cycles that include two hosts, namely the Aedes mosquito as a vector and susceptible vertebrate hosts. Human infection with arboviruses causes diseases that range from subclinical or mild to febrile diseases, encephalitis, and hemorrhagic fever. Infected mosquitoes do not show detectable signs of disease, even though the virus maintains a lifelong persistent infection. The infection of the Aedes mosquito by viruses involves a molecular crosstalk between cell and viral proteins. An understanding of how mosquito vectors and viruses interact is of fundamental interest, and it also offers novel perspectives for disease control. In recent years, mass spectrometry (MS)-based strategies in combination with bioinformatics have been successfully applied to identify and quantify global changes in cellular proteins, lipids, peptides, and metabolites in response to viral infection. Although the information about proteomics in the Aedes mosquito is limited, the information that has been reported can set up the basis for future studies. This review reflects how MS-based approaches have extended our understanding of Aedes mosquito biology and the development of DENV and CHIKV infection in the vector. Finally, this review discusses future challenges in the field.

Studies towards the potential of poliovirus as a vector for the expression of HPV 16 virus-like-particles.

PubMed

van Kuppeveld, Frank J M; de Jong, Arjan; Dijkman, Henri B P M; Andino, Raul; Melchers, Willem J G

2002-11-15

Development of human cervical carcinomas is associated with infection by certain human papillomavirus (HPV) types. Thus, protection against HPV infection through vaccination may prevent development of cervical cancer. The purpose of this study was to investigate the possibility of using a poliovirus recombinant vector to induce immunity against HPV. A poliovirus recombinant was constructed which contained the complete coding sequence of the HPV 16 major capsid protein L1, between the P1 and P2 region of the poliovirus polyprotein. A replication-competent virus was obtained after transfection of the recombinant RNA into tissue culture cells. Electron microscopically examination of cells infected with the poliovirus-HPV L1 recombinant indicated that HPV 16 L1 self-assembles into virus-like particles. To investigate the immunological response in vivo, susceptible transgenic mice carrying the poliovirus receptor were infected with the recombinant poliovirus. In all mice a modest but consistent immune response against HPV 16 was observed. Based on these results, the potential for picornavirus-derived vectors in vaccine development against HPV infection is discussed.

[Efficient packaging retrovirus and construction of transgenic chicken technical platform].

PubMed

Man, Chaolai; Zhang, Qing; Chen, Yan; Zhu, Dahai

2007-10-01

Transgenic chicken and oviduct bioreactor are growing to be one of the hotspot of scientific study in the field of biology. The most successful method of producing transgenic chicken is pseudotyped retrovirus vector system, but no one has reported the production of transgenic chicken by retrovirus system recently in our country. In order to accelerate our study in this field, we introduced the relevant technical methods such as packaging retrovirus and vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped retrovirus, optimizing the conditions of packaging retrovirus, concentrating VSV-G pseudotyped retrovirus, helper virus assays, and microinjection of retrovirus. Furthermore, we successfully conducted in vivo study for detecting the marker gene EGFP of chicken embryo as well as in vitro study for detecting that gene of chicken embryo myoblast (CFM), thus we have provided an applied technical platform for studies of transgenic chicken in the future.

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells.

PubMed

Grosse, Stefanie; Penaud-Budloo, Magalie; Herrmann, Anne-Kathrin; Börner, Kathleen; Fakhiri, Julia; Laketa, Vibor; Krämer, Chiara; Wiedtke, Ellen; Gunkel, Manuel; Ménard, Lucie; Ayuso, Eduard; Grimm, Dirk

2017-10-15

The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans -complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production. Copyright © 2017 American Society for Microbiology.

Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

PubMed Central

Grosse, Stefanie; Penaud-Budloo, Magalie; Herrmann, Anne-Kathrin; Börner, Kathleen; Fakhiri, Julia; Laketa, Vibor; Krämer, Chiara; Wiedtke, Ellen; Gunkel, Manuel; Ménard, Lucie; Ayuso, Eduard

2017-01-01

ABSTRACT The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids. IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding of AAV/AAP biology, and it concomitantly provides insights into the importance of AAP for AAV vector production. PMID:28768875

Predicting the host of influenza viruses based on the word vector.

PubMed

Xu, Beibei; Tan, Zhiying; Li, Kenli; Jiang, Taijiao; Peng, Yousong

2017-01-01

Newly emerging influenza viruses continue to threaten public health. A rapid determination of the host range of newly discovered influenza viruses would assist in early assessment of their risk. Here, we attempted to predict the host of influenza viruses using the Support Vector Machine (SVM) classifier based on the word vector, a new representation and feature extraction method for biological sequences. The results show that the length of the word within the word vector, the sequence type (DNA or protein) and the species from which the sequences were derived for generating the word vector all influence the performance of models in predicting the host of influenza viruses. In nearly all cases, the models built on the surface proteins hemagglutinin (HA) and neuraminidase (NA) (or their genes) produced better results than internal influenza proteins (or their genes). The best performance was achieved when the model was built on the HA gene based on word vectors (words of three-letters long) generated from DNA sequences of the influenza virus. This results in accuracies of 99.7% for avian, 96.9% for human and 90.6% for swine influenza viruses. Compared to the method of sequence homology best-hit searches using the Basic Local Alignment Search Tool (BLAST), the word vector-based models still need further improvements in predicting the host of influenza A viruses.

Synthetic Virology: Engineering Viruses for Gene Delivery

PubMed Central

Guenther, Caitlin M.; Kuypers, Brianna E.; Lam, Michael T.; Robinson, Tawana M.; Zhao, Julia; Suh, Junghae

2014-01-01

The success of gene therapy relies heavily on the performance of vectors that can effectively deliver transgenes to desired cell populations. As viruses have evolved to deliver genetic material into cells, a prolific area of research has emerged over the last several decades to leverage the innate properties of viruses as well as to engineer new features into them. Specifically, the field of synthetic virology aims to capitalize on knowledge accrued from fundamental virology research in order to design functionally enhanced gene delivery vectors. The enhanced viral vectors, or “bionic” viruses, feature engineered components, or “parts”, that are natural (intrinsic to viruses or from other organisms) and synthetic (such as man-made polymers or inorganic nanoparticles). Various design strategies – rational, combinatorial, and pseudo-rational – have been pursued to create the hybrid viruses. The gene delivery vectors of the future will likely criss-cross the boundaries between natural and synthetic domains to harness the unique strengths afforded by the various functional parts that can be grafted onto virus capsids. Such research endeavours will further expand and enable enhanced control over the functional capacity of these nanoscale devices for biomedicine. PMID:25195922

Synthetic virology: engineering viruses for gene delivery.

PubMed

Guenther, Caitlin M; Kuypers, Brianna E; Lam, Michael T; Robinson, Tawana M; Zhao, Julia; Suh, Junghae

2014-01-01

The success of gene therapy relies heavily on the performance of vectors that can effectively deliver transgenes to desired cell populations. As viruses have evolved to deliver genetic material into cells, a prolific area of research has emerged over the last several decades to leverage the innate properties of viruses as well as to engineer new features into them. Specifically, the field of synthetic virology aims to capitalize on knowledge accrued from fundamental virology research in order to design functionally enhanced gene delivery vectors. The enhanced viral vectors, or 'bionic' viruses, feature engineered components, or 'parts', that are natural (intrinsic to viruses or from other organisms) and synthetic (such as man-made polymers or inorganic nanoparticles). Various design strategies--rational, combinatorial, and pseudo-rational--have been pursued to create the hybrid viruses. The gene delivery vectors of the future will likely criss-cross the boundaries between natural and synthetic domains to harness the unique strengths afforded by the various functional parts that can be grafted onto virus capsids. Such research endeavors will further expand and enable enhanced control over the functional capacity of these nanoscale devices for biomedicine. © 2014 Wiley Periodicals, Inc.

Cryo-electron Microscopy Reconstruction and Stability Studies of the Wild Type and the R432A Variant of Adeno-associated Virus Type 2 Reveal that Capsid Structural Stability Is a Major Factor in Genome Packaging.

PubMed

Drouin, Lauren M; Lins, Bridget; Janssen, Maria; Bennett, Antonette; Chipman, Paul; McKenna, Robert; Chen, Weijun; Muzyczka, Nicholas; Cardone, Giovanni; Baker, Timothy S; Agbandje-McKenna, Mavis

2016-10-01

The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ∼5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ∼10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the βA strand region under the icosahedral 2-fold axis rather than antiparallel to the βB strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency. The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ∼1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Transduction of Human Primitive Repopulating Hematopoietic Cells With Lentiviral Vectors Pseudotyped With Various Envelope Proteins

PubMed Central

Kim, Yoon-Sang; Wielgosz, Matthew M; Hargrove, Phillip; Kepes, Steven; Gray, John; Persons, Derek A; Nienhuis, Arthur W

2010-01-01

Lentiviral vectors are useful for transducing primitive hematopoietic cells. We examined four envelope proteins for their ability to mediate lentiviral transduction of mobilized human CD34+ peripheral blood cells. Lentiviral particles encoding green fluorescent protein (GFP) were pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G), the amphotropic (AMPHO) murine leukemia virus envelope protein, the endogenous feline leukemia viral envelope protein or the feline leukemia virus type C envelope protein. Because the relative amount of genome RNA per ml was similar for each pseudotype, we transduced CD34+ cells with a fixed volume of each vector preparation. Following an overnight transduction, CD34+ cells were transplanted into immunodeficient mice which were sacrificed 12 weeks later. The average percentages of engrafted human CD45+ cells in total bone marrow were comparable to that of the control, mock-transduced group (37–45%). Lenti-particles pseudotyped with the VSV-G envelope protein transduced engrafting cells two- to tenfold better than particles pseudotyped with any of the γ-retroviral envelope proteins. There was no correlation between receptor mRNA levels for the γ-retroviral vectors and transduction efficiency of primitive hematopoietic cells. These results support the use of the VSV-G envelope protein for the development of lentiviral producer cell lines for manufacture of clinical-grade vector. PMID:20372106

Zika Virus in Salivary Glands of Five Different Species of Wild-Caught Mosquitoes from Mexico.

PubMed

Elizondo-Quiroga, Darwin; Medina-Sánchez, Aarón; Sánchez-González, Jorge M; Eckert, Kristen Allison; Villalobos-Sánchez, Erendira; Navarro-Zúñiga, Antonio Rigoberto; Sánchez-Tejeda, Gustavo; Correa-Morales, Fabián; González-Acosta, Cassandra; Arias, Carlos F; López, Susana; Del Ángel, Rosa María; Pando-Robles, Victoria; Elizondo-Quiroga, Armando E

2018-01-16

Zika virus (ZIKV) is a mosquito-borne pathogen, and Aedes aegypti has been identified as the main vector of the disease. Other mosquito species in the Aedes and Culex genera have been suggested to have the potential for being competent vectors based on experimental exposition of mosquitoes to an infectious blood meal containing ZIKV. Here, we report the isolation in cell culture of ZIKV obtained from different body parts of wild-caught female mosquitoes (Ae. aegypti, Ae. vexans, Cx. quinquefasciatus, Cx. coronator, and Cx. tarsalis) and whole male mosquitoes (Ae. aegypti and Cx. quinquefasciatus) in Mexico. Importantly, this is the first report that shows the presence of the virus in the salivary glands of the wild-caught female mosquitoes species, Cx. coronator, Cx. tarsalis, and Ae. vexans. Our findings strongly suggest that all the species reported herein are potential vectors for ZIKV.

An efficient Foxtail mosaic virus vector system with reduced environmental risk

PubMed Central

2010-01-01

Background Plant viral vectors offer high-yield expression of pharmaceutical and commercially important proteins with a minimum of cost and preparation time. The use of Agrobacterium tumefaciens has been introduced to deliver the viral vector as a transgene to each plant cell via a simple, nonsterile infiltration technique called "agroinoculation". With agroinoculation, a full length, systemically moving virus is no longer necessary for excellent protein yield, since the viral transgene is transcribed and replicates in every infiltrated cell. Viral genes may therefore be deleted to decrease the potential for accidental spread and persistence of the viral vector in the environment. Results In this study, both the coat protein (CP) and triple gene block (TGB) genetic segments were eliminated from Foxtail mosaic virus to create the "FECT" vector series, comprising a deletion of 29% of the genome. This viral vector is highly crippled and expresses little or no marker gene within the inoculated leaf. However, when co-agroinoculated with a silencing suppressor (p19 or HcPro), FECT expressed GFP at 40% total soluble protein in the tobacco host, Nicotiana benthamiana. The modified FoMV vector retained the full-length replicase ORF, the TGB1 subgenomic RNA leader sequence and either 0, 22 or 40 bases of TGB1 ORF (in vectors FECT0, FECT22 and FECT40, respectively). As well as N. benthamiana, infection of legumes was demonstrated. Despite many attempts, expression of GFP via syringe agroinoculation of various grass species was very low, reflecting the low Agrobacterium-mediated transformation rate of monocots. Conclusions The FECT/40 vector expresses foreign genes at a very high level, and yet has a greatly reduced biohazard potential. It can form no virions and can effectively replicate only in a plant with suppressed silencing. PMID:21162736

Estimation of the size of genetic bottlenecks in cell-to-cell movement of soil-borne wheat mosaic virus and the possible role of the bottlenecks in speeding up selection of variations in trans-acting genes or elements.

PubMed

Miyashita, Shuhei; Kishino, Hirohisa

2010-02-01

Genetic bottlenecks facilitate the fixation and extinction of variants in populations, and viral populations are no exception to this theory. To examine the existence of genetic bottlenecks in cell-to-cell movement of plant RNA viruses, we prepared constructs for Soil-borne wheat mosaic virus RNA2 vectors carrying two different fluorescent proteins, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP). Coinoculation of host plant leaves with the two RNA2 vectors and the wild-type RNA1 showed separation of the two vector RNA2s, mostly within seven to nine cell-to-cell movements from individual initially coinfected cells. Our statistical analysis showed that the number of viral RNA genomes establishing infection in adjacent cells after the first cell-to-cell movement from an initially infected cell was 5.97 +/- 0.22 on average and 5.02 +/- 0.29 after the second cell-to-cell movement. These results indicate that plant RNA viruses may generally face narrow genetic bottlenecks in every cell-to-cell movement. Furthermore, our model suggests that, rather than suffering from fitness losses caused by the bottlenecks, the plant RNA viruses are utilizing the repeated genetic bottlenecks as an essential element of rapid selection of their adaptive variants in trans-acting genes or elements to respond to host shifting and changes in the growth conditions of the hosts.

Reversal of blindness in animal models of leber congenital amaurosis using optimized AAV2-mediated gene transfer.

PubMed

Bennicelli, Jeannette; Wright, John Fraser; Komaromy, Andras; Jacobs, Jonathan B; Hauck, Bernd; Zelenaia, Olga; Mingozzi, Federico; Hui, Daniel; Chung, Daniel; Rex, Tonia S; Wei, Zhangyong; Qu, Guang; Zhou, Shangzhen; Zeiss, Caroline; Arruda, Valder R; Acland, Gregory M; Dell'Osso, Lou F; High, Katherine A; Maguire, Albert M; Bennett, Jean

2008-03-01

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 x 10(10) vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations.

Reversal of Blindness in Animal Models of Leber Congenital Amaurosis Using Optimized AAV2-mediated Gene Transfer

PubMed Central

Bennicelli, Jeannette; Wright, John Fraser; Komaromy, Andras; Jacobs, Jonathan B; Hauck, Bernd; Zelenaia, Olga; Mingozzi, Federico; Hui, Daniel; Chung, Daniel; Rex, Tonia S; Wei, Zhangyong; Qu, Guang; Zhou, Shangzhen; Zeiss, Caroline; Arruda, Valder R; Acland, Gregory M; Dell’Osso, Lou F; High, Katherine A; Maguire, Albert M; Bennett, Jean

2010-01-01

We evaluated the safety and efficacy of an optimized adeno-associated virus (AAV; AAV2.RPE65) in animal models of the RPE65 form of Leber congenital amaurosis (LCA). Protein expression was optimized by addition of a modified Kozak sequence at the translational start site of hRPE65. Modifications in AAV production and delivery included use of a long stuffer sequence to prevent reverse packaging from the AAV inverted-terminal repeats, and co-injection with a surfactant. The latter allows consistent and predictable delivery of a given dose of vector. We observed improved electroretinograms (ERGs) and visual acuity in Rpe65 mutant mice. This has not been reported previously using AAV2 vectors. Subretinal delivery of 8.25 × 1010 vector genomes in affected dogs was well tolerated both locally and systemically, and treated animals showed improved visual behavior and pupillary responses, and reduced nystagmus within 2 weeks of injection. ERG responses confirmed the reversal of visual deficit. Immunohistochemistry confirmed transduction of retinal pigment epithelium cells and there was minimal toxicity to the retina as judged by histopathologic analysis. The data demonstrate that AAV2.RPE65 delivers the RPE65 transgene efficiently and quickly to the appropriate target cells in vivo in animal models. This vector holds great promise for treatment of LCA due to RPE65 mutations. PMID:18209734

Generation of an Adenovirus-Parvovirus Chimera with Enhanced Oncolytic Potential

PubMed Central

El-Andaloussi, Nazim; Bonifati, Serena; Kaufmann, Johanna K.; Mailly, Laurent; Daeffler, Laurent; Deryckère, François; Nettelbeck, Dirk M.; Rommelaere, Jean

2012-01-01

In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells. PMID:22787235

[Construction and expression of a recombinant adenovirus with LZP3].

PubMed

Chen, Bang-dang; Zhang, Fu-chun; Sun, Mei-yu; Li, Yi-jie; Ma, Zheng-hai

2007-08-01

To explore a new immunocontraceptive vaccine and construct an attenuated recombinant adenoviral vaccine against Lagurus lagurus zona pellucida 3(LZP3). LZP3 gene was subcloned into the shuttle vector pShuttle-CMV, and then a two-step transformation procedure was employed to construct a recombinant adenoviral plasmid with LZP3, which was digested with Pac I and transfected into HEK293 cells to package recombinant adenovirus particles. Finally, HeLa cells were infected by the recombinant adenovirus. LZP3 gene was detected from the recombinant virus by PCR, and its transcription and expression were analyzed by RT-PCR and Western blot. Recombinant adenovirus vector pAd-LZP3 with LZP3 gene was constructed by homologous recombination in E.coli, and a recombinant adenovirus was obtained by transfecting HEK293 cells with pAd-LZP3. PCR test indicated that LZP3 gene was successfully integrated into the adenoviral genome, and the titer of the recombinant adenovirus reached 1.2x10(10) pfu/L. The transcription and expression of LZP3 gene in the infected HeLa cells were confirmed by RT-PCR and Western blot. The recombinant adenovirus RAd-LZP3 can be successfully expressed in the infected HeLa cells, which lays the foundation for further researches into immunizing animals with RAd-LZP3.

Generation of an adenovirus-parvovirus chimera with enhanced oncolytic potential.

PubMed

El-Andaloussi, Nazim; Bonifati, Serena; Kaufmann, Johanna K; Mailly, Laurent; Daeffler, Laurent; Deryckère, François; Nettelbeck, Dirk M; Rommelaere, Jean; Marchini, Antonio

2012-10-01

In this study, our goal was to generate a chimeric adenovirus-parvovirus (Ad-PV) vector that combines the high-titer and efficient gene transfer of adenovirus with the anticancer potential of rodent parvovirus. To this end, the entire oncolytic PV genome was inserted into a replication-defective E1- and E3-deleted Ad5 vector genome. As we found that parvoviral NS expression inhibited Ad-PV chimera production, we engineered the parvoviral P4 early promoter, which governs NS expression, by inserting into its sequence tetracycline operator elements. As a result of these modifications, P4-driven expression was blocked in the packaging T-REx-293 cells, which constitutively express the tetracycline repressor, allowing high-yield chimera production. The chimera effectively delivered the PV genome into cancer cells, from which fully infectious replication-competent parvovirus particles were generated. Remarkably, the Ad-PV chimera exerted stronger cytotoxic activities against various cancer cell lines, compared with the PV and Ad parental viruses, while being still innocuous to a panel of tested healthy primary human cells. This Ad-PV chimera represents a novel versatile anticancer agent which can be subjected to further genetic manipulations in order to reinforce its enhanced oncolytic capacity through arming with transgenes or retargeting into tumor cells.

Enhancement of antitumor activity of gammaretrovirus carrying IL-12 gene through genetic modification of envelope targeting HER2 receptor: a promising strategy for bladder cancer therapy.

PubMed

Tsai, Y-S; Shiau, A-L; Chen, Y-F; Tsai, H-T; Tzai, T-S; Wu, C-L

2010-01-01

The objective of this study was to develop an HER2-targeted, envelope-modified Moloney murine leukemia virus (MoMLV)-based gammaretroviral vector carrying interleukin (IL)-12 gene for bladder cancer therapy. It displayed a chimeric envelope protein containing a single-chain variable fragment (scFv) antibody to the HER2 receptor and carried the mouse IL-12 gene. The fragment of anti-erbB2scFv was constructed into the proline-rich region of the viral envelope of the packaging vector lacking a transmembrane subunit of the carboxyl terminal region of surface subunit. As compared with envelope-unmodified gammaretroviruses, envelope-modified ones had extended viral tropism to human HER2-expressing bladder cancer cell lines, induced apoptosis, and affected cell cycle progression despite lower viral titers. Moreover, animal studies showed that envelope-modified gammaretroviruses carrying IL-12 gene exerted higher antitumor activity in terms of retarding tumor growth and prolonging the survival of tumor-bearing mice than unmodified ones, which were associated with enhanced tumor cell apoptosis as well as increased intratumoral levels of IL-12, interferon-gamma, IL-1beta, and tumor necrosis factor-alpha proteins. Therefore, the antitumor activity of gammaretroviruses carrying the IL-12 gene was enhanced through genetic modification of the envelope targeting HER2 receptor, which may be a promising strategy for bladder cancer therapy.

Membrane-bound SIV envelope trimers are immunogenic in ferrets after intranasal vaccination with a replication-competent canine distemper virus vector.

PubMed

Zhang, Xinsheng; Wallace, Olivia; Wright, Kevin J; Backer, Martin; Coleman, John W; Koehnke, Rebecca; Frenk, Esther; Domi, Arban; Chiuchiolo, Maria J; DeStefano, Joanne; Narpala, Sandeep; Powell, Rebecca; Morrow, Gavin; Boggiano, Cesar; Zamb, Timothy J; Richter King, C; Parks, Christopher L

2013-11-01

We are investigating canine distemper virus (CDV) as a vaccine vector for the delivery of HIV envelope (Env) that closely resembles the native trimeric spike. We selected CDV because it will promote vaccine delivery to lymphoid tissues, and because human exposure is infrequent, reducing potential effects of pre-existing immunity. Using SIV Env as a model, we tested a number of vector and gene insert designs. Vectors containing a gene inserted between the CDV H and L genes, which encoded Env lacking most of its cytoplasmic tail, propagated efficiently in Vero cells, expressed the immunogen on the cell surface, and incorporated the SIV glycoprotein into progeny virus particles. When ferrets were vaccinated intranasally, there were no signs of distress, vector replication was observed in the gut-associated lymphoid tissues, and the animals produced anti-SIV Env antibodies. These data show that live CDV-SIV Env vectors can safely induce anti-Env immune responses following intranasal vaccination. © 2013 Elsevier Inc. All rights reserved.

Interleukin-Encoding Adenoviral Vectors as Genetic Adjuvant for Vaccination against Retroviral Infection

PubMed Central

Ohs, Inga; Windmann, Sonja; Wildner, Oliver; Dittmer, Ulf; Bayer, Wibke

2013-01-01

Interleukins (IL) are cytokines with stimulatory and modulatory functions in the immune system. In this study, we have chosen interleukins which are involved in the enhancement of TH2 responses and B cell functions to analyze their potential to improve a prophylactic adenovirus-based anti-retroviral vaccine with regard to antibody and virus-specific CD4+ T cell responses. Mice were vaccinated with an adenoviral vector which encodes and displays the Friend Virus (FV) surface envelope protein gp70 (Ad.pIXgp70) in combination with adenoviral vectors encoding the interleukins IL4, IL5, IL6, IL7 or IL23. Co-application of Ad.pIXgp70 with Ad.IL5, Ad.IL6 or Ad.IL23 resulted in improved protection with high control over FV-induced splenomegaly and reduced viral loads. Mice co-immunized with adenoviral vectors encoding IL5 or IL23 showed increased neutralizing antibody responses while mice co-immunized with Ad.IL6 or Ad.IL23 showed improved FV-specific CD4+ T cell responses compared to mice immunized with Ad.pIXgp70 alone. We show that the co-application of adenoviral vectors encoding specific interleukins is suitable to improve the vaccination efficacy of an anti-retroviral vaccine. Improved protection correlated with improved CD4+ T cell responses and especially with higher neutralizing antibody titers. The co-application of selected interleukin-encoding adenoviral vectors is a valuable tool for vaccination with regard to enhancement of antibody mediated immunity. PMID:24349306

Characterization of retroviral infectivity and superinfection resistance during retrovirus-mediated transduction of mammalian cells.

PubMed

Liao, J; Wei, Q; Fan, J; Zou, Y; Song, D; Liu, J; Liu, F; Ma, C; Hu, X; Li, L; Yu, Y; Qu, X; Chen, L; Yu, X; Zhang, Z; Zhao, C; Zeng, Z; Zhang, R; Yan, S; Wu, T; Wu, X; Shu, Y; Lei, J; Li, Y; Zhang, W; Wang, J; Reid, R R; Lee, M J; Huang, W; Wolf, J M; He, T-C; Wang, J

2017-06-01

Retroviral vectors including lentiviral vectors are commonly used tools to stably express transgenes or RNA molecules in mammalian cells. Their utilities are roughly divided into two categories, stable overexpression of transgenes and RNA molecules, which requires maximal transduction efficiency, or functional selection with retrovirus (RV)-based libraries, which takes advantage of retroviral superinfection resistance. However, the dynamic features of RV-mediated transduction are not well characterized. Here, we engineered two murine stem cell virus-based retroviral vectors expressing dual fluorescence proteins and antibiotic markers, and analyzed virion production efficiency and virion stability, dynamic infectivity and superinfection resistance in different cell types, and strategies to improve transduction efficiency. We found that the highest virion production occurred between 60 and 72 h after transfection. The stability of the collected virion supernatant decreased by >60% after 3 days in storage. We found that RV infectivity varied drastically in the tested human cancer lines, while low transduction efficiency was partially overcome with increased virus titer, prolonged infection duration and/or repeated infections. Furthermore, we demonstrated that RV receptors PIT1 and PIT2 were lowly expressed in the analyzed cells, and that PIT1 and/or PIT2 overexpression significantly improved transduction efficiency in certain cell lines. Thus, our findings provide resourceful information for the optimal conditions of retroviral-mediated gene delivery.

Influenza A and B Virus Intertypic Reassortment through Compatible Viral Packaging Signals

PubMed Central

Baker, Steven F.; Nogales, Aitor; Finch, Courtney; Tuffy, Kevin M.; Domm, William; Perez, Daniel R.; Topham, David J.

2014-01-01

ABSTRACT Influenza A and B viruses cocirculate in humans and together cause disease and seasonal epidemics. These two types of influenza viruses are evolutionarily divergent, and exchange of genetic segments inside coinfected cells occurs frequently within types but never between influenza A and B viruses. Possible mechanisms inhibiting the intertypic reassortment of genetic segments could be due to incompatible protein functions of segment homologs, a lack of processing of heterotypic segments by influenza virus RNA-dependent RNA polymerase, an inhibitory effect of viral proteins on heterotypic virus function, or an inability to specifically incorporate heterotypic segments into budding virions. Here, we demonstrate that the full-length hemagglutinin (HA) of prototype influenza B viruses can complement the function of multiple influenza A viruses. We show that viral noncoding regions were sufficient to drive gene expression for either type A or B influenza virus with its cognate or heterotypic polymerase. The native influenza B virus HA segment could not be incorporated into influenza A virus virions. However, by adding the influenza A virus packaging signals to full-length influenza B virus glycoproteins, we rescued influenza A viruses that possessed HA, NA, or both HA and NA of influenza B virus. Furthermore, we show that, similar to single-cycle infectious influenza A virus, influenza B virus cannot incorporate heterotypic transgenes due to packaging signal incompatibilities. Altogether, these results demonstrate that the lack of influenza A and B virus reassortants can be attributed at least in part to incompatibilities in the virus-specific packaging signals required for effective segment incorporation into nascent virions. IMPORTANCE Reassortment of influenza A or B viruses provides an evolutionary strategy leading to unique genotypes, which can spawn influenza A viruses with pandemic potential. However, the mechanism preventing intertypic reassortment or gene exchange between influenza A and B viruses is not well understood. Nucleotides comprising the coding termini of each influenza A virus gene segment are required for specific segment incorporation during budding. Whether influenza B virus shares a similar selective packaging strategy or if packaging signals prevent intertypic reassortment remains unknown. Here, we provide evidence suggesting a similar mechanism of influenza B virus genome packaging. Furthermore, by appending influenza A virus packaging signals onto influenza B virus segments, we rescued recombinant influenza A/B viruses that could reassort in vitro with another influenza A virus. These findings suggest that the divergent evolution of packaging signals aids with the speciation of influenza A and B viruses and is in part responsible for the lack of intertypic viral reassortment. PMID:25008914

A stable RNA virus-based vector for citrus trees

DOE Office of Scientific and Technical Information (OSTI.GOV)

Folimonov, Alexey S.; Folimonova, Svetlana Y.; Bar-Joseph, Moshe

Virus-based vectors are important tools in plant molecular biology and plant genomics. A number of vectors based on viruses that infect herbaceous plants are in use for expression or silencing of genes in plants as well as screening unknown sequences for function. Yet there is a need for useful virus-based vectors for woody plants, which demand much greater stability because of the longer time required for systemic infection and analysis. We examined several strategies to develop a Citrus tristeza virus (CTV)-based vector for transient expression of foreign genes in citrus trees using a green fluorescent protein (GFP) as a reporter.more » These strategies included substitution of the p13 open reading frame (ORF) by the ORF of GFP, construction of a self-processing fusion of GFP in-frame with the major coat protein (CP), or expression of the GFP ORF as an extra gene from a subgenomic (sg) mRNA controlled either by a duplicated CTV CP sgRNA controller element (CE) or an introduced heterologous CE of Beet yellows virus. Engineered vector constructs were examined for replication, encapsidation, GFP expression during multiple passages in protoplasts, and for their ability to infect, move, express GFP, and be maintained in citrus plants. The most successful vectors based on the 'add-a-gene' strategy have been unusually stable, continuing to produce GFP fluorescence after more than 4 years in citrus trees.« less

Insertion of targeting domains into the envelope glycoprotein of Moloney murine leukemia virus (MoMLV)-based vectors modulates the route of mCAT-1-mediated viral entry.

PubMed

Viejo-Borbolla, A; Pizzato, M; Blair, E D; Schulz, T F

2005-03-01

Several groups have inserted targeting domains into the envelope glycoprotein (Env) of Moloney murine leukemia virus (MoMLV) in an attempt to produce targeted retroviral vectors for human gene therapy. While binding of these modified Envs to the target molecule expressed on the surface of human cells was observed, specific high-titer infection of human cells expressing the target molecule was not achieved. Here we investigate the initial steps in the entry process of targeted MoMLV vectors both in murine and human cells expressing the MoMLV receptor, the mouse cationic amino acid transporter-1 (mCAT-1). We show that insertion of a small ligand targeted to E-selectin and of a single chain antibody (scFv) targeted to folate-binding protein (FBP) into the N-terminus of MoMLV Env results in the reduction of the infectivity and the kinetics of entry of the MoMLV vectors. The use of soluble receptor-binding domain (sRBD), bafilomycin A1 (BafA1) and methyl-beta-cyclodextrin (MbetaC) increase the infectivity of the MoMLV vectors targeted to FBP (MoMLV-FBP) suggesting that the scFv targeted to FBP increases the threshold for fusion and might re-route entry of the targeted MoMLV-FBP vector towards an endocytic, non-productive pathway.

Rabies Virus Envelope Glycoprotein Targets Lentiviral Vectors to the Axonal Retrograde Pathway in Motor Neurons*

PubMed Central

Hislop, James N.; Islam, Tarin A.; Eleftheriadou, Ioanna; Carpentier, David C. J.; Trabalza, Antonio; Parkinson, Michael; Schiavo, Giampietro; Mazarakis, Nicholas D.

2014-01-01

Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors. PMID:24753246

Modified Vaccinia Virus Ankara: History, Value in Basic Research, and Current Perspectives for Vaccine Development.

PubMed

Volz, A; Sutter, G

2017-01-01

Safety tested Modified Vaccinia virus Ankara (MVA) is licensed as third-generation vaccine against smallpox and serves as a potent vector system for development of new candidate vaccines against infectious diseases and cancer. Historically, MVA was developed by serial tissue culture passage in primary chicken cells of vaccinia virus strain Ankara, and clinically used to avoid the undesirable side effects of conventional smallpox vaccination. Adapted to growth in avian cells MVA lost the ability to replicate in mammalian hosts and lacks many of the genes orthopoxviruses use to conquer their host (cell) environment. As a biologically well-characterized mutant virus, MVA facilitates fundamental research to elucidate the functions of poxvirus host-interaction factors. As extremely safe viral vectors MVA vaccines have been found immunogenic and protective in various preclinical infection models. Multiple recombinant MVA currently undergo clinical testing for vaccination against human immunodeficiency viruses, Mycobacterium tuberculosis or Plasmodium falciparum. The versatility of the MVA vector vaccine platform is readily demonstrated by the swift development of experimental vaccines for immunization against emerging infections such as the Middle East Respiratory Syndrome. Recent advances include promising results from the clinical testing of recombinant MVA-producing antigens of highly pathogenic avian influenza virus H5N1 or Ebola virus. This review summarizes our current knowledge about MVA as a unique strain of vaccinia virus, and discusses the prospects of exploiting this virus as research tool in poxvirus biology or as safe viral vector vaccine to challenge existing and future bottlenecks in vaccinology. © 2017 Elsevier Inc. All rights reserved.

Pseudotyped Lentiviral Vectors for Retrograde Gene Delivery into Target Brain Regions

PubMed Central

Kobayashi, Kenta; Inoue, Ken-ichi; Tanabe, Soshi; Kato, Shigeki; Takada, Masahiko; Kobayashi, Kazuto

2017-01-01

Gene transfer through retrograde axonal transport of viral vectors offers a substantial advantage for analyzing roles of specific neuronal pathways or cell types forming complex neural networks. This genetic approach may also be useful in gene therapy trials by enabling delivery of transgenes into a target brain region distant from the injection site of the vectors. Pseudotyping of a lentiviral vector based on human immunodeficiency virus type 1 (HIV-1) with various fusion envelope glycoproteins composed of different combinations of rabies virus glycoprotein (RV-G) and vesicular stomatitis virus glycoprotein (VSV-G) enhances the efficiency of retrograde gene transfer in both rodent and nonhuman primate brains. The most recently developed lentiviral vector is a pseudotype with fusion glycoprotein type E (FuG-E), which demonstrates highly efficient retrograde gene transfer in the brain. The FuG-E–pseudotyped vector permits powerful experimental strategies for more precisely investigating the mechanisms underlying various brain functions. It also contributes to the development of new gene therapy approaches for neurodegenerative disorders, such as Parkinson’s disease, by delivering genes required for survival and protection into specific neuronal populations. In this review article, we report the properties of the FuG-E–pseudotyped vector, and we describe the application of the vector to neural circuit analysis and the potential use of the FuG-E vector in gene therapy for Parkinson’s disease. PMID:28824385

Optimal packaging of FIV genomic RNA depends upon a conserved long-range interaction and a palindromic sequence within gag.

PubMed

Rizvi, Tahir A; Kenyon, Julia C; Ali, Jahabar; Aktar, Suriya J; Phillip, Pretty S; Ghazawi, Akela; Mustafa, Farah; Lever, Andrew M L

2010-10-15

The feline immunodeficiency virus (FIV) is a lentivirus that is related to human immunodeficiency virus (HIV), causing a similar pathology in cats. It is a potential small animal model for AIDS and the FIV-based vectors are also being pursued for human gene therapy. Previous studies have mapped the FIV packaging signal (ψ) to two or more discontinuous regions within the 5' 511 nt of the genomic RNA and structural analyses have determined its secondary structure. The 5' and 3' sequences within ψ region interact through extensive long-range interactions (LRIs), including a conserved heptanucleotide interaction between R/U5 and gag. Other secondary structural elements identified include a conserved 150 nt stem-loop (SL2) and a small palindromic stem-loop within gag open reading frame that might act as a viral dimerization initiation site. We have performed extensive mutational analysis of these sequences and structures and ascertained their importance in FIV packaging using a trans-complementation assay. Disrupting the conserved heptanucleotide LRI to prevent base pairing between R/U5 and gag reduced packaging by 2.8-5.5 fold. Restoration of pairing using an alternative, non-wild type (wt) LRI sequence restored RNA packaging and propagation to wt levels, suggesting that it is the structure of the LRI, rather than its sequence, that is important for FIV packaging. Disrupting the palindrome within gag reduced packaging by 1.5-3-fold, but substitution with a different palindromic sequence did not restore packaging completely, suggesting that the sequence of this region as well as its palindromic nature is important. Mutation of individual regions of SL2 did not have a pronounced effect on FIV packaging, suggesting that either it is the structure of SL2 as a whole that is necessary for optimal packaging, or that there is redundancy within this structure. The mutational analysis presented here has further validated the previously predicted RNA secondary structure of FIV ψ. Copyright © 2010 Elsevier Ltd. All rights reserved.

Alternative cell lines to improve the rescue of infectious human astrovirus from a cDNA clone.

PubMed

Velázquez-Moctezuma, Rodrigo; Baños-Lara, Ma del Rocío; Acevedo, Yunuén; Méndez, Ernesto

2012-02-01

A reverse genetics system for human astrovirus (HAstV) was established previously; however, it has not been exploited mainly because cells used for virus packaging are not permissive, requiring several rounds of replication to obtain acceptable infectious virus. In this work, in the search for alternative permissive cell lines to be used as packaging cells, Hek-293 and Huh7.5.1 were tested. Given that HAstV infection in Hek-293 showed differences with that in Caco-2, the gold standard for HAstV growth but scarcely transfectable, and it was more similar to that observed in the hepatoma Huh7.5.1 cell line, these last cells were further used to transfect viral RNA. Virus titers near to 10(8) infectious particles per ml (ffu/ml) were obtained at 16-20 h after transfection with RNA from infected cells. However, virus titers close to 10(6) ffu/ml were obtained by using in vitro transcribed RNA from a cDNA HAstV-1 clone. In contrast, virus recovery in BHK-21, reported previously as the packaging cells, from this RNA was of about 10(4) ffu/ml, two logarithms less than in Huh7.5.1. Apparently, the 5'-end modification of the viral RNA determined its specific infectivity, since virus recovery was abolished when the total RNA was treated with proteinase-K, probably by removing a protein-linked genome protein, but it increased when capping of the in vitro transcribed RNA was more efficient. Thus, an alternative and more efficient reverse genetics system for HAstV was established by using Huh7.5.1 cells. Copyright © 2011. Published by Elsevier B.V.

Production of recombinant adenovirus containing human interlukin-4 gene.

PubMed

Mojarrad, Majid; Abdolazimi, Yassan; Hajati, Jamshid; Modarressi, Mohammad Hossein

2011-11-01

Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics that made it a good choice for using in cancer gene therapy, controlling inflammatory diseases, and studies on autoimmune diseases. In brief, IL-4 coding sequence was amplified by and cloned in pAd-Track-CMV. Then, by means of homologous recombination between recombinant pAd-Track-CMV and Adeasy-1 plasmid in bacteria, recombinant adenovirus complete genome was made and IL-4 containing shuttle vector was incorporated into the viral backbone. After linearization, for virus packaging, viral genome was transfected into HEK-293 cell line. Viral production was conveniently followed with the aid of green fluorescent protein. Recombinant adenovirus produced here, was capable to infecting cell lines and express interlukin-4 in cell. This system can be used as a powerful, easy, and cost benefit tool in various studies on cancer gene therapy and also studies on immunogenetics.

Retargeting Vesicular Stomatitis Virus Glycoprotein Pseudotyped Lentiviral Vectors with Enhanced Stability by In Situ Synthesized Polymer Shell

PubMed Central

Liang, Min; Yan, Ming; Lu, Yunfeng

2013-01-01

Abstract The ability to introduce transgenes with precise specificity to the desired target cells or tissues is key to a more facile application of genetic therapy. Here, we describe a novel method using nanotechnology to generate lentiviral vectors with altered recognition of host cell receptor specificity. Briefly, the infectivity of the vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vectors was shielded by a thin polymer shell synthesized in situ onto the viral envelope, and new binding ability was conferred to the shielded virus by introducing acrylamide-tailored cyclic arginine-glycine-aspartic acid (cRGD) peptide to the polymer shell. We termed the resulting virus “targeting nanovirus.” The targeting nanovirus had similar titer with VSV-G pseudotypes and specifically transduced Hela cells with high transduction efficiency. In addition, the encapsulation of the VSV-G pseudotyped lentivirus by the polymer shell did not change the pathway that VSV-G pseudotypes enter and fuse with cells, as well as later events such as reverse transcription and gene expression. Furthermore, the targeting nanovirus possessed enhanced stability in the presence of human serum, indicating protection of the virus by the polymer shell from human serum complement inactivation. This novel use of nanotechnology demonstrates proof of concept for an approach that could be more generally applied for redirecting viral vectors for laboratory and clinical purposes. PMID:23327104

Genome-wide RNAi screening identifies host restriction factors critical for in vivo AAV transduction

PubMed Central

Mano, Miguel; Ippodrino, Rudy; Zentilin, Lorena; Zacchigna, Serena; Giacca, Mauro

2015-01-01

Viral vectors based on the adeno-associated virus (AAV) hold great promise for in vivo gene transfer; several unknowns, however, still limit the vectors’ broader and more efficient application. Here, we report the results of a high-throughput, whole-genome siRNA screening aimed at identifying cellular factors regulating AAV transduction. We identified 1,483 genes affecting vector efficiency more than 4-fold and up to 50-fold, either negatively or positively. Most of these factors have not previously been associated to AAV infection. The most effective siRNAs were independent from the virus serotype or analyzed cell type and were equally evident for single-stranded and self-complementary AAV vectors. A common characteristic of the most effective siRNAs was the induction of cellular DNA damage and activation of a cell cycle checkpoint. This information can be exploited for the development of more efficient AAV-based gene delivery procedures. Administration of the most effective siRNAs identified by the screening to the liver significantly improved in vivo AAV transduction efficiency. PMID:26305933

Elimination of endogenous aberrant kappa chain transcripts from sp2/0-derived hybridoma cells by specific ribozyme cleavage: utility in genetic therapy of HIV-1 infections.

PubMed Central

Duan, L; Pomerantz, R J

1994-01-01

The pooled degenerate-primer polymerase chain reaction (PCR) technology is now widely used in the amplification and cloning of murine hybridoma-specific immunoglobulin gene cDNAs. The design of primers is mainly based on the highly conserved 5' terminus of immunoglobulin gene variable regions and the constant region in the 3' terminus. Of note, most murine hybridoma cell lines are derived from the Sp2/0 cell line, which is demonstrated to express endogenous aberrant kappa chains (abV kappa). This high-level endogenous abV kappa mixes with specific kappa chains in the hybridomas and interferes with the efficiency of the reverse transcriptase (RT)-PCR cloning strategy. In this report, during the cloning of murine anti-human immunodeficiency virus type I (HIV-1) hybridoma immunoglobulin cDNAs, a specific primer-PCR screening system was developed, based on the abV kappa complementarity-defining region (CDR), to eliminate abV kappa-carrying plasmids. Furthermore, an abV kappa sequence-specific derived ribozyme was developed and packaged in a retroviral expression vector system. This abV kappa ribozyme can be transduced into different murine hybridomas, and expressed intracellularly to potently eliminate endogenous abV kappa RNA. Images PMID:7816635

Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palella, T.D.; Silverman, L.J.; Schroll, C.T.

1988-01-01

The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolationmore » of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.« less

Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human

DOE Office of Scientific and Technical Information (OSTI.GOV)

Palmer, T.D.; Hock, R.A.; Osborne, W.R.A.

1987-02-01

Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. The authors studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vectormore » containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA/sup -/ fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.« less

A virus vector based on Canine Herpesvirus for vaccine applications in canids.

PubMed

Strive, T; Hardy, C M; Wright, J; Reubel, G H

2007-01-31

Canine Herpesvirus (CHV) is being developed as a virus vector for the vaccination of European red foxes. However, initial studies using recombinant CHV vaccines in foxes revealed viral attenuation and lack of antibody response to inserted foreign antigens. These findings were attributed both to inactivation of the thymidine kinase (TK) gene and excess foreign genetic material in the recombinant viral genome. In this study, we report an improved CHV-bacterial artificial chromosome (BAC) vector system designed to overcome attenuation in foxes. A non-essential region was identified in the CHV genome as an alternative insertion site for foreign genes. Replacement of a guanine/cytosine (GC)-rich intergenic region between UL21 and UL22 of CHV with a marker gene did not change growth behaviour in vitro, showing that this region is not essential for virus growth in cell culture. We subsequently produced a CHV-BAC vector with an intact TK gene in which the bacterial genes and the antigen expression cassette were inserted into this GC-rich locus. Unlike earlier constructs, the new CHV-BAC allowed self-excision of the bacterial genes via homologous recombination after transfection of BACs into cell culture. The BAC-CHV system was used to produce a recombinant virus that constitutively expressed porcine zona pellucida subunit C protein between the UL21 and UL22 genes of CHV. Complete self-excision of the bacterial genes from CHV was achieved within one round of replication whilst retaining antigen gene expression.

Efficient and stable expression of GFP through Wheat streak mosaic virus-based vectors in cereal hosts using a range of cleavage sites: Formation of dense fluorescent aggregates for sensitive virus tracking

USDA-ARS?s Scientific Manuscript database

A series of Wheat streak mosaic virus (WSMV)-based expression vectors were developed by engineering cycle 3 GFP (GFP) cistron between P1 and HC-Pro cistrons with several catalytic/cleavage peptides at the C-terminus of GFP. WSMV-GFP vectors with the Foot-and-mouth disease virus 1D/2A or 2A catalytic...

A scalable method for the production of high-titer and high-quality adeno-associated type 9 vectors using the HSV platform

PubMed Central

Adamson-Small, Laura; Potter, Mark; Falk, Darin J; Cleaver, Brian; Byrne, Barry J; Clément, Nathalie

2016-01-01

Recombinant adeno-associated vectors based on serotype 9 (rAAV9) have demonstrated highly effective gene transfer in multiple animal models of muscular dystrophies and other neurological indications. Current limitations in vector production and purification have hampered widespread implementation of clinical candidate vectors, particularly when systemic administration is considered. In this study, we describe a complete herpes simplex virus (HSV)-based production and purification process capable of generating greater than 1 × 1014 rAAV9 vector genomes per 10-layer CellSTACK of HEK 293 producer cells, or greater than 1 × 105 vector genome per cell, in a final, fully purified product. This represents a 5- to 10-fold increase over transfection-based methods. In addition, rAAV vectors produced by this method demonstrated improved biological characteristics when compared to transfection-based production, including increased infectivity as shown by higher transducing unit-to-vector genome ratios and decreased total capsid protein amounts, shown by lower empty-to-full ratios. Together, this data establishes a significant improvement in both rAAV9 yields and vector quality. Further, the method can be readily adapted to large-scale good laboratory practice (GLP) and good manufacturing practice (GMP) production of rAAV9 vectors to enable preclinical and clinical studies and provide a platform to build on toward late-phases and commercial production. PMID:27222839

Newcastle disease virus-vectored rabies vaccine is safe, highly immunogenic, and provides long-lasting protection in dogs and cats.

PubMed

Ge, Jinying; Wang, Xijun; Tao, Lihong; Wen, Zhiyuan; Feng, Na; Yang, Songtao; Xia, Xianzhu; Yang, Chinglai; Chen, Hualan; Bu, Zhigao

2011-08-01

Effective, safe, and affordable rabies vaccines are still being sought. Newcastle disease virus (NDV), an avian paramyxovirus, has shown promise as a vaccine vector for mammals. Here, we generated a recombinant avirulent NDV La Sota strain expressing the rabies virus glycoprotein (RVG) and evaluated its potential to serve as a vaccine against rabies. The recombinant virus, rL-RVG, retained its high-growth property in chicken eggs, with titers of up to 10⁹·⁸ 50% egg infective doses (EID₅₀)/ml of allantoic fluid. RVG expression enabled rL-RVG to spread from cell to cell in a rabies virus-like manner, and RVG was incorporated on the surface of the rL-RVG viral particle. RVG incorporation did not alter the trypsin-dependent infectivity of the NDV vector in mammalian cells. rL-RVG and La Sota NDV showed similar levels of sensitivity to a neutralization antibody against NDV and similar levels of resistance to a neutralization antibody against rabies virus. Animal studies demonstrated that rL-RVG is safe in several species, including cats and dogs, when administered as multiple high doses of recombinant vaccine. Intramuscular vaccination with rL-RVG induced a substantial rabies virus neutralization antibody response and provided complete protection from challenge with circulating rabies virus strains. Most importantly, rL-RVG induced strong and long-lasting protective neutralization antibody responses to rabies virus in dogs and cats. A low vaccine dose of 10⁸·³ EID₅₀ completely protected dogs from challenge with a circulating strain of rabies virus for more than a year. This is the first study to demonstrate that immunization with an NDV-vectored vaccine can induce long-lasting, systemic protective immunity against rabies.

Progress on adenovirus-vectored universal influenza vaccines.

PubMed

Xiang, Kui; Ying, Guan; Yan, Zhou; Shanshan, Yan; Lei, Zhang; Hongjun, Li; Maosheng, Sun

2015-01-01

Influenza virus (IFV) infection causes serious health problems and heavy financial burdens each year worldwide. The classical inactivated influenza virus vaccine (IIVV) and live attenuated influenza vaccine (LAIV) must be updated regularly to match the new strains that evolve due to antigenic drift and antigenic shift. However, with the discovery of broadly neutralizing antibodies that recognize conserved antigens, and the CD8(+) T cell responses targeting viral internal proteins nucleoprotein (NP), matrix protein 1 (M1) and polymerase basic 1 (PB1), it is possible to develop a universal influenza vaccine based on the conserved hemagglutinin (HA) stem, NP, and matrix proteins. Recombinant adenovirus (rAd) is an ideal influenza vaccine vector because it has an ideal stability and safety profile, induces balanced humoral and cell-mediated immune responses due to activation of innate immunity, provides 'self-adjuvanting' activity, can mimic natural IFV infection, and confers seamless protection against mucosal pathogens. Moreover, this vector can be developed as a low-cost, rapid-response vaccine that can be quickly manufactured. Therefore, an adenovirus vector encoding conserved influenza antigens holds promise in the development of a universal influenza vaccine. This review will summarize the progress in adenovirus-vectored universal flu vaccines and discuss future novel approaches.

“Stealth” Adenoviruses Blunt Cell-Mediated and Humoral Immune Responses against the Virus and Allow for Significant Gene Expression upon Readministration in the Lung

PubMed Central

Croyle, Maria A.; Chirmule, Narendra; Zhang, Yi; Wilson, James M.

2001-01-01

Most of the early gene therapy trials for cystic fibrosis have been with adenovirus vectors. First-generation viruses with E1a and E1b deleted are limited by transient expression of the transgene and substantial inflammatory responses. Gene transfer is also significantly curtailed following a second dose of virus. In an effort to reduce adenovirus-associated inflammation, capsids of first-generation vectors were modified with various activated monomethoxypolyethylene glycols. Cytotoxic T-lymphocyte production was significantly reduced in C57BL/6 mice after a single intratracheal administration of modified vectors, and length of gene expression was extended from 4 to 42 days. T-cell subsets from mice exposed to the conjugated vectors demonstrated a marked decrease in Th1 responses and slight enhancement of Th2 responses compared to animals dosed with native virus. Neutralizing antibodies (NAB) against adenovirus capsid proteins were reduced in serum and bronchoalveolar lavage fluid of animals after a single dose of modified virus, allowing significant levels of gene expression upon rechallenge with native adenovirus. Modification with polyethylene glycol (PEG) also allowed substantial gene expression from the new vectors in animals previously immunized with unmodified virus. However, gene expression was significantly reduced after two doses of the same PEG-conjugated vector. Alternating the activation group of PEG between doses did produce significant gene expression upon readministration. This technology in combination with second-generation or helper-dependent adenovirus could produce dosing strategies which promote successful readministration of vector in clinical trials and marked expression in patients with significant anti-adenovirus NAB levels and reduce the possibility of immune reactions against viral vectors for gene therapy. PMID:11312351

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin.

PubMed

Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G; Corydon, Thomas J; Mikkelsen, Jacob Giehm; Aagaard, Lars

2015-08-01

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo.

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin

PubMed Central

Jakobsen, Maria; Askou, Anne Louise; Stenderup, Karin; Rosada, Cecilia; Dagnæs-Hansen, Frederik; Jensen, Thomas G.; Corydon, Thomas J.; Mikkelsen, Jacob Giehm; Aagaard, Lars

2015-01-01

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo. PMID:26204415

Reporter gene expression in fish following cutaneous infection with pantropic retroviral vectors.

PubMed

Paul, T A; Burns, J C; Shike, H; Getchell, R; Bowser, P R; Whitlock, K E; Casey, J W

2001-06-01

A central issue in gene delivery systems is choosing promoters that will direct defined and sustainable levels of gene expression. Pantropic retroviral vectors provide a means to insert genes into either somatic or germline cells. In this study, we focused on somatic cell infection by evaluating the activity of 3 promoters inserted by vectors into fish cell lines and fish skin using pantropic retroviruses. In bluegill and zebrafish cell lines, the highest levels of luciferase expression were observed from the 5' murine leukemia virus long terminal repeat of the retroviral vector. The Rous sarcoma virus long terminal repeat and cytomegalovirus early promoter, as internal promoters, generated lower levels of luciferase. Luciferase reporter vectors infected zebrafish skin, as measured by the presence of viral DNA, and expressed luciferase. We infected developing walleye dermal sarcomas with retroviral vectors to provide an environment with enhanced cell proliferation, a condition necessary for integration of the provirus into the host genome. We demonstrated a 4-fold to 7-fold increase in luciferase gene expression in tumor tissue over infections in normal walleye skin.

RNA Interference Restricts Rift Valley Fever Virus in Multiple Insect Systems.

PubMed

Dietrich, Isabelle; Jansen, Stephanie; Fall, Gamou; Lorenzen, Stephan; Rudolf, Martin; Huber, Katrin; Heitmann, Anna; Schicht, Sabine; Ndiaye, El Hadji; Watson, Mick; Castelli, Ilaria; Brennan, Benjamin; Elliott, Richard M; Diallo, Mawlouth; Sall, Amadou A; Failloux, Anna-Bella; Schnettler, Esther; Kohl, Alain; Becker, Stefanie C

2017-01-01

The emerging bunyavirus Rift Valley fever virus (RVFV) is transmitted to humans and livestock by a large number of mosquito species. RNA interference (RNAi) has been characterized as an important innate immune defense mechanism used by mosquitoes to limit replication of positive-sense RNA flaviviruses and togaviruses; however, little is known about its role against negative-strand RNA viruses such as RVFV. We show that virus-specific small RNAs are produced in infected mosquito cells, in Drosophila melanogaster cells, and, most importantly, also in RVFV vector mosquitoes. By addressing the production of small RNAs in adult Aedes sp. and Culex quinquefasciatus mosquitoes, we showed the presence of virus-derived Piwi-interacting RNAs (piRNAs) not only in Aedes sp. but also in C. quinquefasciatus mosquitoes, indicating that antiviral RNA interference in C. quinquefasciatus mosquitoes is similar to the described activities of RNAi in Aedes sp. mosquitoes. We also show that these have antiviral activity, since silencing of RNAi pathway effectors enhances viral replication. Moreover, our data suggest that RVFV does not encode a suppressor of RNAi. These findings point toward a significant role of RNAi in the control of RVFV in mosquitoes. IMPORTANCE Rift Valley fever virus (RVFV; Phlebovirus , Bunyaviridae ) is an emerging zoonotic mosquito-borne pathogen of high relevance for human and animal health. Successful strategies of intervention in RVFV transmission by its mosquito vectors and the prevention of human and veterinary disease rely on a better understanding of the mechanisms that govern RVFV-vector interactions. Despite its medical importance, little is known about the factors that govern RVFV replication, dissemination, and transmission in the invertebrate host. Here we studied the role of the antiviral RNA interference immune pathways in the defense against RVFV in natural vector mosquitoes and mosquito cells and draw comparisons to the model insect Drosophila melanogaster . We found that RVFV infection induces both the exogenous small interfering RNA (siRNA) and piRNA pathways, which contribute to the control of viral replication in insects. Furthermore, we demonstrate the production of virus-derived piRNAs in Culex quinquefasciatus mosquitoes. Understanding these pathways and the targets within them offers the potential of the development of novel RVFV control measures in vector-based strategies.

RNA Interference Restricts Rift Valley Fever Virus in Multiple Insect Systems

PubMed Central

Jansen, Stephanie; Fall, Gamou; Lorenzen, Stephan; Rudolf, Martin; Huber, Katrin; Heitmann, Anna; Schicht, Sabine; Ndiaye, El Hadji; Watson, Mick; Castelli, Ilaria; Elliott, Richard M.; Diallo, Mawlouth; Sall, Amadou A.; Failloux, Anna-Bella; Schnettler, Esther

2017-01-01

ABSTRACT The emerging bunyavirus Rift Valley fever virus (RVFV) is transmitted to humans and livestock by a large number of mosquito species. RNA interference (RNAi) has been characterized as an important innate immune defense mechanism used by mosquitoes to limit replication of positive-sense RNA flaviviruses and togaviruses; however, little is known about its role against negative-strand RNA viruses such as RVFV. We show that virus-specific small RNAs are produced in infected mosquito cells, in Drosophila melanogaster cells, and, most importantly, also in RVFV vector mosquitoes. By addressing the production of small RNAs in adult Aedes sp. and Culex quinquefasciatus mosquitoes, we showed the presence of virus-derived Piwi-interacting RNAs (piRNAs) not only in Aedes sp. but also in C. quinquefasciatus mosquitoes, indicating that antiviral RNA interference in C. quinquefasciatus mosquitoes is similar to the described activities of RNAi in Aedes sp. mosquitoes. We also show that these have antiviral activity, since silencing of RNAi pathway effectors enhances viral replication. Moreover, our data suggest that RVFV does not encode a suppressor of RNAi. These findings point toward a significant role of RNAi in the control of RVFV in mosquitoes. IMPORTANCE Rift Valley fever virus (RVFV; Phlebovirus, Bunyaviridae) is an emerging zoonotic mosquito-borne pathogen of high relevance for human and animal health. Successful strategies of intervention in RVFV transmission by its mosquito vectors and the prevention of human and veterinary disease rely on a better understanding of the mechanisms that govern RVFV-vector interactions. Despite its medical importance, little is known about the factors that govern RVFV replication, dissemination, and transmission in the invertebrate host. Here we studied the role of the antiviral RNA interference immune pathways in the defense against RVFV in natural vector mosquitoes and mosquito cells and draw comparisons to the model insect Drosophila melanogaster. We found that RVFV infection induces both the exogenous small interfering RNA (siRNA) and piRNA pathways, which contribute to the control of viral replication in insects. Furthermore, we demonstrate the production of virus-derived piRNAs in Culex quinquefasciatus mosquitoes. Understanding these pathways and the targets within them offers the potential of the development of novel RVFV control measures in vector-based strategies. PMID:28497117

Alternative reverse genetics system for influenza viruses based on a synthesized swine 45S rRNA promoter.

PubMed

Wang, Kai; Huang, Qi; Yang, Zhiwei; Qi, Kezong; Liu, Hongmei; Chen, Hongjun

2017-08-01

We generated an alternative reverse genetics (RG) system based on a synthesized swine 45S rRNA promoter to rescue the H3N2 subtype swine influenza virus. All eight flanking segment cassettes of A/swine/Henan/7/2010 (H3N2) were amplified with ambisense expression elements from RG plasmids. All segments were then recombined with the pHC2014 vector, which contained the synthesized swine 45S rRNA promoter (spol1) and its terminal sequence (t1) in a pcDNA3 backbone. As a result, we obtained a set of RG plasmids carrying the corresponding eight-segment cassettes. We efficiently generated the H3N2 virus after transfection into 293T/PK15, PK15, and 293T cells. The efficiency of spol1-driven influenza virus rescue in PK15 cells was similar to that in 293T cells by titration using the human pol1 RG system. Our approach suggests that an alternative spol1-based RG system can produce influenza viruses.

Genome segregation and packaging machinery in Acanthamoeba polyphaga mimivirus is reminiscent of bacterial apparatus.

PubMed

Chelikani, Venkata; Ranjan, Tushar; Zade, Amrutraj; Shukla, Avi; Kondabagil, Kiran

2014-06-01

Genome packaging is a critical step in the virion assembly process. The putative ATP-driven genome packaging motor of Acanthamoeba polyphaga mimivirus (APMV) and other nucleocytoplasmic large DNA viruses (NCLDVs) is a distant ortholog of prokaryotic chromosome segregation motors, such as FtsK and HerA, rather than other viral packaging motors, such as large terminase. Intriguingly, APMV also encodes other components, i.e., three putative serine recombinases and a putative type II topoisomerase, all of which are essential for chromosome segregation in prokaryotes. Based on our analyses of these components and taking the limited available literature into account, here we propose for the first time a model for genome segregation and packaging in APMV that can possibly be extended to NCLDV subfamilies, except perhaps Poxviridae and Ascoviridae. This model might represent a unique variation of the prokaryotic system acquired and contrived by the large DNA viruses of eukaryotes. It is also consistent with previous observations that unicellular eukaryotes, such as amoebae, are melting pots for the advent of chimeric organisms with novel mechanisms. Extremely large viruses with DNA genomes infect a wide range of eukaryotes, from human beings to amoebae and from crocodiles to algae. These large DNA viruses, unlike their much smaller cousins, have the capability of making most of the protein components required for their multiplication. Once they infect the cell, these viruses set up viral replication centers, known as viral factories, to carry out their multiplication with very little help from the host. Our sequence analyses show that there is remarkable similarity between prokaryotes (bacteria and archaea) and large DNA viruses, such as mimivirus, vaccinia virus, and pandoravirus, in the way that they process their newly synthesized genetic material to make sure that only one copy of the complete genome is generated and is meticulously placed inside the newly synthesized viral particle. These findings have important evolutionary implications about the origin and evolution of large viruses.

Genome Segregation and Packaging Machinery in Acanthamoeba polyphaga Mimivirus Is Reminiscent of Bacterial Apparatus

PubMed Central

Chelikani, Venkata; Ranjan, Tushar; Zade, Amrutraj; Shukla, Avi

2014-01-01

ABSTRACT Genome packaging is a critical step in the virion assembly process. The putative ATP-driven genome packaging motor of Acanthamoeba polyphaga mimivirus (APMV) and other nucleocytoplasmic large DNA viruses (NCLDVs) is a distant ortholog of prokaryotic chromosome segregation motors, such as FtsK and HerA, rather than other viral packaging motors, such as large terminase. Intriguingly, APMV also encodes other components, i.e., three putative serine recombinases and a putative type II topoisomerase, all of which are essential for chromosome segregation in prokaryotes. Based on our analyses of these components and taking the limited available literature into account, here we propose for the first time a model for genome segregation and packaging in APMV that can possibly be extended to NCLDV subfamilies, except perhaps Poxviridae and Ascoviridae. This model might represent a unique variation of the prokaryotic system acquired and contrived by the large DNA viruses of eukaryotes. It is also consistent with previous observations that unicellular eukaryotes, such as amoebae, are melting pots for the advent of chimeric organisms with novel mechanisms. IMPORTANCE Extremely large viruses with DNA genomes infect a wide range of eukaryotes, from human beings to amoebae and from crocodiles to algae. These large DNA viruses, unlike their much smaller cousins, have the capability of making most of the protein components required for their multiplication. Once they infect the cell, these viruses set up viral replication centers, known as viral factories, to carry out their multiplication with very little help from the host. Our sequence analyses show that there is remarkable similarity between prokaryotes (bacteria and archaea) and large DNA viruses, such as mimivirus, vaccinia virus, and pandoravirus, in the way that they process their newly synthesized genetic material to make sure that only one copy of the complete genome is generated and is meticulously placed inside the newly synthesized viral particle. These findings have important evolutionary implications about the origin and evolution of large viruses. PMID:24623441

Human Bocavirus Type-1 Capsid Facilitates the Transduction of Ferret Airways by Adeno-Associated Virus Genomes.

PubMed

Yan, Ziying; Feng, Zehua; Sun, Xingshen; Zhang, Yulong; Zou, Wei; Wang, Zekun; Jensen-Cody, Chandler; Liang, Bo; Park, Soo-Yeun; Qiu, Jianming; Engelhardt, John F

2017-08-01

Human bocavirus type-1 (HBoV1) has a high tropism for the apical membrane of human airway epithelia. The packaging of a recombinant adeno-associated virus 2 (rAAV2) genome into HBoV1 capsid produces a chimeric vector (rAAV2/HBoV1) that also efficiently transduces human airway epithelia. As such, this vector is attractive for use in gene therapies to treat lung diseases such as cystic fibrosis. However, preclinical development of rAAV2/HBoV1 vectors has been hindered by the fact that humans are the only known host for HBoV1 infection. This study reports that rAAV2/HBoV1 vector is capable of efficiently transducing the lungs of both newborn (3- to 7-day-old) and juvenile (29-day-old) ferrets, predominantly in the distal airways. Analyses of in vivo, ex vivo, and in vitro models of the ferret proximal airway demonstrate that infection of this particular region is less effective than it is in humans. Studies of vector binding and endocytosis in polarized ferret proximal airway epithelial cultures revealed that a lack of effective vector endocytosis is the main cause of inefficient transduction in vitro. While transgene expression declined proportionally with growth of the ferrets following infection at 7 days of age, reinfection of ferrets with rAAV2/HBoV1 at 29 days gave rise to approximately 5-fold higher levels of transduction than observed in naive infected 29-day-old animals. The findings presented here lay the foundation for clinical development of HBoV1 capsid-based vectors for lung gene therapy in cystic fibrosis using ferret models.

Efficient transduction of equine adipose-derived mesenchymal stem cells by VSV-G pseudotyped lentiviral vectors.

PubMed

Petersen, Gayle F; Hilbert, Bryan; Trope, Gareth; Kalle, Wouter; Strappe, Padraig

2014-12-01

Equine adipose-derived mesenchymal stem cells (EADMSC) provide a unique cell-based approach for treatment of a variety of equine musculoskeletal injuries, via regeneration of diseased or damaged tissue, or the secretion of immunomodulatory molecules. These capabilities can be further enhanced by genetic modification using lentiviral vectors, which provide a safe and efficient method of gene delivery. We investigated the suitability of lentiviral vector technology for gene delivery into EADMSC, using GFP expressing lentiviral vectors pseudotyped with the G glycoprotein from the vesicular stomatitis virus (V-GFP) or, for the first time, the baculovirus gp64 envelope protein (G-GFP). In this study, we produced similarly high titre V-GFP and G-GFP lentiviral vectors. Flow cytometric analysis showed efficient transduction using V-GFP; however G-GFP exhibited a poor ability to transduce EADMSC. Transduction resulted in sustained GFP expression over four passages, with minimal effects on cell viability and doubling time, and an unaltered chondrogenic differentiation potential. Copyright © 2014 Elsevier Ltd. All rights reserved.

Inhibition of histone deacetylation and DNA methylation improves gene expression mediated by the adeno-associated virus/phage in cancer cells.

PubMed

Kia, Azadeh; Yata, Teerapong; Hajji, Nabil; Hajitou, Amin

2013-10-22

Bacteriophage (phage), viruses that infect bacteria only, have become promising vectors for targeted systemic delivery of genes to cancer, although, with poor efficiency. We previously designed an improved phage vector by incorporating cis genetic elements of adeno-associated virus (AAV). This novel AAV/phage hybrid (AAVP) specifically targeted systemic delivery of therapeutic genes into tumors. To advance the AAVP vector, we recently introduced the stress-inducible Grp78 tumor specific promoter and found that this dual tumor-targeted AAVP provides persistent gene expression, over time, in cancer cells compared to silenced gene expression from the CMV promoter in the parental AAVP. Herein, we investigated the effect of histone deacetylation and DNA methylation on AAVP-mediated gene expression in cancer cells and explored the effect of cell confluence state on AAVP gene expression efficacy. Using a combination of AAVP expressing the GFP reporter gene, flow cytometry, inhibitors of histone deacetylation, and DNA methylation, we have demonstrated that histone deacetylation and DNA methylation are associated with silencing of gene expression from the CMV promoter in the parental AAVP. Importantly, inhibitors of histone deacetylases boost gene expression in cancer cells from the Grp78 promoter in the dual tumor-targeted AAVP. However, cell confluence had no effect on AAVP-guided gene expression. Our findings prove that combination of histone deacetylase inhibitor drugs with the Grp78 promoter is an effective approach to improve AAVP-mediated gene expression in cancer cells and should be considered for AAVP-based clinical cancer gene therapy.

Viral Vectors for Gene Delivery to the Central Nervous System

PubMed Central

Lentz, Thomas B.; Gray, Steven J.; Samulski, R. Jude

2011-01-01

The potential benefits of gene therapy for neurological diseases such as Parkinson’s, Amyotrophic Lateral Sclerosis (ALS), Epilepsy, and Alzheimer’s are enormous. Even a delay in the onset of severe symptoms would be invaluable to patients suffering from these and other diseases. Significant effort has been placed in developing vectors capable of delivering therapeutic genes to the CNS in order to treat neurological disorders. At the forefront of potential vectors, viral systems have evolved to efficiently deliver their genetic material to a cell. The biology of different viruses offers unique solutions to the challenges of gene therapy, such as cell targeting, transgene expression and vector production. It is important to consider the natural biology of a vector when deciding whether it will be the most effective for a specific therapeutic function. In this review, we outline desired features of the ideal vector for gene delivery to the CNS and discuss how well available viral vectors compare to this model. Adeno-associated virus, retrovirus, adenovirus and herpesvirus vectors are covered. Focus is placed on features of the natural biology that have made these viruses effective tools for gene delivery with emphasis on their application in the CNS. Our goal is to provide insight into features of the optimal vector and which viral vectors can provide these features. PMID:22001604

Encapsidated Host Factors in Alphavirus Particles Influence Midgut Infection of Aedes aegypti.

PubMed

Mackenzie-Liu, David; Sokoloski, Kevin J; Purdy, Sarah; Hardy, Richard W

2018-05-16

Transmission of mosquito-borne viruses requires the efficient infection of both a permissive vertebrate host and a competent mosquito vector. The infectivity of Sindbis virus (SINV), the type species of the Alphavirus genus, is influenced by both the original and new host cell. We have shown that infection of vertebrate cells by SINV, chikungunya virus (CHIKV), and Ross River virus (RRV) produces two subpopulations of virus particles separable based on density. In contrast, a single population of viral particles is produced by mosquito cells. Previous studies demonstrated that the denser vertebrate-derived particles and the mosquito-derived particles contain components of the small subunit of the host cell ribosome, whereas the less dense vertebrate-derived particles do not. Infection of mice with RRV showed that both particle subpopulations are produced in an infected vertebrate, but in a tissue specific manner with serum containing only the less dense version of the virus particles. Previous infectivity studies using SINV particles have shown that the denser particles (SINV Heavy ) and mosquito derived particles SINV C6/36 are significantly more infectious in vertebrate cells than the less dense vertebrate derived particles (SINV Light ). The current study shows that SINV Light particles, initiate the infection of the mosquito midgut more efficiently than SINV Heavy particles and that this enhanced infectivity is associated with an exacerbated immune response to SINV Light infection in midgut tissues. The enhanced infection of SINV Light is specific to the midgut as intrathoracically injected virus do not exhibit the same fitness advantage. Together, our data indicate a biologically significant role for the SINV Light subpopulation in the efficient transmission from infected vertebrates to the mosquito vector.

Rabies virus envelope glycoprotein targets lentiviral vectors to the axonal retrograde pathway in motor neurons.

PubMed

Hislop, James N; Islam, Tarin A; Eleftheriadou, Ioanna; Carpentier, David C J; Trabalza, Antonio; Parkinson, Michael; Schiavo, Giampietro; Mazarakis, Nicholas D

2014-06-06

Rabies pseudotyped lentiviral vectors have great potential in gene therapy, not least because of their ability to transduce neurons following their distal axonal application. However, very little is known about the molecular processes that underlie their retrograde transport and cell transduction. Using multiple labeling techniques and confocal microscopy, we demonstrated that pseudotyping with rabies virus envelope glycoprotein (RV-G) enabled the axonal retrograde transport of two distinct subtypes of lentiviral vector in motor neuron cultures. Analysis of this process revealed that these vectors trafficked through Rab5-positive endosomes and accumulated within a non-acidic Rab7 compartment. RV-G pseudotyped vectors were co-transported with both the tetanus neurotoxin-binding fragment and the membrane proteins thought to mediate rabies virus endocytosis (neural cell adhesion molecule, nicotinic acetylcholine receptor, and p75 neurotrophin receptor), thus demonstrating that pseudotyping with RV-G targets lentiviral vectors for transport along the same pathway exploited by several toxins and viruses. Using motor neurons cultured in compartmentalized chambers, we demonstrated that axonal retrograde transport of these vectors was rapid and efficient; however, it was not able to transduce the targeted neurons efficiently, suggesting that impairment in processes occurring after arrival of the viral vector in the soma is responsible for the low transduction efficiency seen in vivo, which suggests a novel area for improvement of gene therapy vectors. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Elucidating the Potential of Plant Rhabdoviruses as Vector Expressions Systems

USDA-ARS?s Scientific Manuscript database

Maize fine streak virus (MFSV) is a member of the genus Nucleorhabdovirus that is transmitted by the leafhopper Graminella nigrifons. The virus replicates in both its maize host and its insect vector. To determine whether Drosophila S2 cells support the production of full-length MFSV proteins, we ...

Correction of mutant Fanconi anemia gene by homologous recombination in human hematopoietic cells using adeno-associated virus vector.

PubMed

Paiboonsukwong, Kittiphong; Ohbayashi, Fumi; Shiiba, Haruka; Aizawa, Emi; Yamashita, Takayuki; Mitani, Kohnosuke

2009-11-01

Adeno-associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR. We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells. We subsequently constructed an AAV vector encoding the normal sequences from the Fanconi anemia group A (FANCA) locus to correct a mutation in the gene in BCL derived from a FANCA patient. Under optimal conditions, approximately 50% of BCL cells were transduced with an AAV serotype 2 (AAV-2) vector. In FANCA BCL cells, up to 0.016% of infected cells were gene-corrected by HR. AAV-mediated restoration of normal genotypic and phenotypic characteristics in FANCA-mutant cells was confirmed at the DNA, protein and functional levels. The results obtained in the present study indicate that AAV vectors may be applicable for gene correction therapy of inherited hematopoietic disorders.

A heterotypic bystander effect for tumor cell killing after adeno-associated virus/phage-mediated, vascular-targeted suicide gene transfer.

PubMed

Trepel, Martin; Stoneham, Charlotte A; Eleftherohorinou, Hariklia; Mazarakis, Nicholas D; Pasqualini, Renata; Arap, Wadih; Hajitou, Amin

2009-08-01

Suicide gene transfer is the most commonly used cytotoxic approach in cancer gene therapy; however, a successful suicide gene therapy depends on the generation of efficient targeted systemic gene delivery vectors. We recently reported that selective systemic delivery of suicide genes such as herpes simplex virus thymidine kinase (HSVtk) to tumor endothelial cells through a novel targeted adeno-associated virus/phage vector leads to suppression of tumor growth. This marked effect has been postulated to result primarily from the death of cancer cells by hypoxia following the targeted disruption of tumor blood vessels. Here, we investigated whether an additional mechanism of action is involved. We show that there is a heterotypic "bystander" effect between endothelial cells expressing the HSVtk suicide gene and tumor cells. Treatment of cocultures of HSVtk-transduced endothelial cells and non-HSVtk-transduced tumor cells with ganciclovir results in the death of both endothelial and tumor cells. Blocking of this effect by 18alpha-glycyrrhetinic acid indicates that gap junctions between endothelial and tumor cells are largely responsible for this phenomenon. Moreover, the observed bystander killing is mediated by connexins 43 and 26, which are expressed in endothelial and tumor cell types. Finally, this heterotypic bystander effect is accompanied by a suppression of tumor growth in vivo that is independent of primary gene transfer into host-derived tumor vascular endothelium. These findings add an alternative nonmutually exclusive and potentially synergistic cytotoxic mechanism to cancer gene therapy based on targeted adeno-associated virus/phage and further support the promising role of nonmalignant tumor stromal cells as therapeutic targets.

Transient dominant host-range selection using Chinese hamster ovary cells to generate marker-free recombinant viral vectors from vaccinia virus.

PubMed

Liu, Liang; Cooper, Tamara; Eldi, Preethi; Garcia-Valtanen, Pablo; Diener, Kerrilyn R; Howley, Paul M; Hayball, John D

2017-04-01

Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Replicative ability is restored by co-insertion of the host-range genes, providing for dominant selection of the recombinant viruses. Here, we describe a new method for the construction of rVACVs using the cowpox CP77 protein and unmodified VACV as the starting material. Our selection system will expand the range of tools available for positive selection of rVACV during vector construction, and it is substantially more high-fidelity than approaches based on selection for drug resistance.

Biosafety considerations for attenuated measles virus vectors used in virotherapy and vaccination.

PubMed

Baldo, Aline; Galanis, Evanthia; Tangy, Frédéric; Herman, Philippe

2016-05-03

Attenuated measles virus (MV) is one of the most effective and safe vaccines available, making it attractive candidate vector to prevent infectious diseases. Attenuated MV have acquired the ability to use the complement regulator CD46 as a major receptor to mediate virus entry and intercellular fusion. Therefore, attenuated MV strains preferentially infect and destroy a wide variety of cancer cells making them also attractive oncolytic vectors. The use of recombinant MV vector has to comply with various regulatory requirements, particularly relating to the assessment of potential risks for human health and the environment. The present article highlights the main characteristics of MV and recombinant MV vectors used for vaccination and virotherapy and discusses these features from a biosafety point of view.

Evaluation of two strains of Marek's disease virus serotype 1 for the development of recombinant vaccines against very virulent infectious bursal disease virus.

PubMed

Li, Kai; Liu, Yongzhen; Liu, Changjun; Gao, Li; Gao, Yulong; Zhang, Yanping; Cui, Hongyu; Qi, Xiaole; Zhong, Li; Wang, Xiaomei

2017-03-01

Attenuated strains of Marek's disease virus serotype 1 (MDV1), and the closely related herpesvirus of turkeys, are among the most potent vectors for development of recombinant vaccines for poultry. To investigate the effects of MDV1 strain characteristics on the protective efficacy of the recombinant vaccines, we developed two recombinant MDV1 vaccines for expressing the VP2 gene of infectious bursal disease virus (IBDV) based on two different MDV1 strains, the attenuated strain 814 and the Meq gene-deleted recombinant MDV1 strain rLMS△Meq, as the viral vectors. The r814-VP2 virus based on the 814 strain exhibited higher replication efficiency in cell culture while lower viral titers in chickens, compared to rLMS△Meq-VP2 derived from the rLMS△Meq strain. Further studies indicated that r814-VP2 produced higher levels of VP2 protein in cells and elicited stronger immune responses against IBDV in chickens than rLMS△Meq-VP2. After IBDV challenge, rLMS△Meq-VP2 provided 50% protection against mortality, and the birds that survived developed bursal atrophy and gross lesions. In contrast, r814-VP2 conferred complete protection not only against development of clinical signs and mortality, but also against the formation of bursal lesions. The results indicate that different MDV1 vector influences the protective efficacy of recombinant MDV1 vaccines. The r814-VP2 has the potential to serve as a bivalent vaccine against two important lethal pathogens of chickens. Copyright © 2016 Elsevier B.V. All rights reserved.

Immunogenic HSV-mediated oncolysis shapes the antitumor immune response and contributes to therapeutic efficacy.

PubMed

Workenhe, Samuel T; Simmons, Graydon; Pol, Jonathan G; Lichty, Brian D; Halford, William P; Mossman, Karen L

2014-01-01

Within the oncolytic virus field, the extent of virus replication that is essential for immune stimulation to control tumor growth remains unresolved. Using infected cell protein 0 (ICP0)-defective oncolytic Herpes simplex virus type 1 (HSV-1) and HSV-2 viruses (dICP0 and dNLS) that show differences in their in vitro replication and cytotoxicity, we investigated the inherent features of oncolytic HSV viruses that are required for potent antitumor activity. In vitro, the HSV-2 vectors showed rapid cytotoxicity despite lower viral burst sizes compared to HSV-1 vectors. In vivo, although both of the dICP0 vectors initially replicated to a similar level, HSV-1 dICP0 was rapidly cleared from the tumors. In spite of this rapid clearance, HSV-1 dICP0 treatment conferred significant survival benefit. HSV-1 dICP0-treated tumors showed significantly higher levels of danger-associated molecular patterns that correlated with higher numbers of antigen-presenting cells within the tumor and increased antigen-specific CD8+ T-cell levels in the peripheral blood. This study suggests that, at least in the context of oncolytic HSV, the initial stages of immunogenic virus replication leading to activation of antitumor immunity are more important than persistence of a replicating virus within the tumor. This knowledge provides important insight for the design of therapeutically successful oncolytic viruses.

Effects of pre-existing orthopoxvirus-specific immunity on the performance of Modified Vaccinia virus Ankara-based influenza vaccines.

PubMed

Altenburg, Arwen F; van Trierum, Stella E; de Bruin, Erwin; de Meulder, Dennis; van de Sandt, Carolien E; van der Klis, Fiona R M; Fouchier, Ron A M; Koopmans, Marion P G; Rimmelzwaan, Guus F; de Vries, Rory D

2018-04-24

The replication-deficient orthopoxvirus modified vaccinia virus Ankara (MVA) is a promising vaccine vector against various pathogens and has an excellent safety record. However, pre-existing vector-specific immunity is frequently suggested to be a drawback of MVA-based vaccines. To address this issue, mice were vaccinated with MVA-based influenza vaccines in the presence or absence of orthopoxvirus-specific immunity. Importantly, protective efficacy of an MVA-based influenza vaccine against a homologous challenge was not impaired in the presence of orthopoxvirus-specific pre-existing immunity. Nonetheless, orthopoxvirus-specific pre-existing immunity reduced the induction of antigen-specific antibodies under specific conditions and completely prevented induction of antigen-specific T cell responses by rMVA-based vaccination. Notably, antibodies induced by vaccinia virus vaccination, both in mice and humans, were not capable of neutralizing MVA. Thus, when using rMVA-based vaccines it is important to consider the main correlate of protection induced by the vaccine, the vaccine dose and the orthopoxvirus immune status of vaccine recipients.

In vivo production of recombinant proteins using occluded recombinant AcMNPV-derived baculovirus vectors.

PubMed

Guijarro-Pardo, Eva; Gómez-Sebastián, Silvia; Escribano, José M

2017-12-01

Trichoplusia ni insect larvae infected with vectors derived from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV), are an excellent alternative to insect cells cultured in conventional bioreactors to produce recombinant proteins because productivity and cost-efficiency reasons. However, there is still a lot of work to do to reduce the manual procedures commonly required in this production platform that limit its scalability. To increase the scalability of this platform technology, a current bottleneck to be circumvented in the future is the need of injection for the inoculation of larvae with polyhedrin negative baculovirus vectors (Polh-) because of the lack of oral infectivity of these viruses, which are commonly used for production in insect cell cultures. In this work we have developed a straightforward alternative to obtain orally infective vectors derived from AcMNPV and expressing recombinant proteins that can be administered to the insect larvae (Trichoplusia ni) by feeding, formulated in the insect diet. The approach developed was based on the use of a recombinant polyhedrin protein expressed by a recombinant vector (Polh+), able to co-occlude any recombinant Polh- baculovirus vector expressing a recombinant protein. A second alternative was developed by the generation of a dual vector co-expressing the recombinant polyhedrin protein and the foreign gene of interest to obtain the occluded viruses. Additionally, by the incorporation of a reporter gene into the helper Polh+ vector, it was possible the follow-up visualization of the co-occluded viruses infection in insect larvae and will help to homogenize infection conditions. By using these methodologies, the production of recombinant proteins in per os infected larvae, without manual infection procedures, was very similar in yield to that obtained by manual injection of recombinant Polh- AcMNPV-based vectors expressing the same proteins. However, further analyses will be required for a detailed comparison of production yields reached by injection vs oral infections for different recombinant proteins. In conclusion, these results open the possibility of future industrial scaling-up production of recombinant proteins in insect larvae by reducing manual operations. Copyright © 2017 Elsevier B.V. All rights reserved.

Improved methods of AAV-mediated gene targeting for human cell lines using ribosome-skipping 2A peptide

PubMed Central

Karnan, Sivasundaram; Ota, Akinobu; Konishi, Yuko; Wahiduzzaman, Md; Hosokawa, Yoshitaka; Konishi, Hiroyuki

2016-01-01

The adeno-associated virus (AAV)-based targeting vector has been one of the tools commonly used for genome modification in human cell lines. It allows for relatively efficient gene targeting associated with 1–4-log higher ratios of homologous-to-random integration of targeting vectors (H/R ratios) than plasmid-based targeting vectors, without actively introducing DNA double-strand breaks. In this study, we sought to improve the efficiency of AAV-mediated gene targeting by introducing a 2A-based promoter-trap system into targeting constructs. We generated three distinct AAV-based targeting vectors carrying 2A for promoter trapping, each targeting a GFP-based reporter module incorporated into the genome, PIGA exon 6 or PIGA intron 5. The absolute gene targeting efficiencies and H/R ratios attained using these vectors were assessed in multiple human cell lines and compared with those attained using targeting vectors carrying internal ribosome entry site (IRES) for promoter trapping. We found that the use of 2A for promoter trapping increased absolute gene targeting efficiencies by 3.4–28-fold and H/R ratios by 2–5-fold compared to values obtained with IRES. In CRISPR-Cas9-assisted gene targeting using plasmid-based targeting vectors, the use of 2A did not enhance the H/R ratios but did upregulate the absolute gene targeting efficiencies compared to the use of IRES. PMID:26657635

Newcastle Disease Virus as a Vaccine Vector for Development of Human and Veterinary Vaccines

PubMed Central

Kim, Shin-Hee; Samal, Siba K.

2016-01-01

Viral vaccine vectors have shown to be effective in inducing a robust immune response against the vaccine antigen. Newcastle disease virus (NDV), an avian paramyxovirus, is a promising vaccine vector against human and veterinary pathogens. Avirulent NDV strains LaSota and B1 have long track records of safety and efficacy. Therefore, use of these strains as vaccine vectors is highly safe in avian and non-avian species. NDV replicates efficiently in the respiratory track of the host and induces strong local and systemic immune responses against the foreign antigen. As a vaccine vector, NDV can accommodate foreign sequences with a good degree of stability and as a RNA virus, there is limited possibility for recombination with host cell DNA. Using NDV as a vaccine vector in humans offers several advantages over other viral vaccine vectors. NDV is safe in humans due to host range restriction and there is no pre-existing antibody to NDV in the human population. NDV is antigenically distinct from common human pathogens. NDV replicates to high titer in a cell line acceptable for human vaccine development. Therefore, NDV is an attractive vaccine vector for human pathogens for which vaccines are currently not available. NDV is also an attractive vaccine vector for animal pathogens. PMID:27384578

Newcastle Disease Virus as a Vaccine Vector for Development of Human and Veterinary Vaccines.

PubMed

Kim, Shin-Hee; Samal, Siba K

2016-07-04

Viral vaccine vectors have shown to be effective in inducing a robust immune response against the vaccine antigen. Newcastle disease virus (NDV), an avian paramyxovirus, is a promising vaccine vector against human and veterinary pathogens. Avirulent NDV strains LaSota and B1 have long track records of safety and efficacy. Therefore, use of these strains as vaccine vectors is highly safe in avian and non-avian species. NDV replicates efficiently in the respiratory track of the host and induces strong local and systemic immune responses against the foreign antigen. As a vaccine vector, NDV can accommodate foreign sequences with a good degree of stability and as a RNA virus, there is limited possibility for recombination with host cell DNA. Using NDV as a vaccine vector in humans offers several advantages over other viral vaccine vectors. NDV is safe in humans due to host range restriction and there is no pre-existing antibody to NDV in the human population. NDV is antigenically distinct from common human pathogens. NDV replicates to high titer in a cell line acceptable for human vaccine development. Therefore, NDV is an attractive vaccine vector for human pathogens for which vaccines are currently not available. NDV is also an attractive vaccine vector for animal pathogens.

Transmissible Gastroenteritis Coronavirus Genome Packaging Signal Is Located at the 5′ End of the Genome and Promotes Viral RNA Incorporation into Virions in a Replication-Independent Process

PubMed Central

Morales, Lucia; Mateos-Gomez, Pedro A.; Capiscol, Carmen; del Palacio, Lorena; Sola, Isabel

2013-01-01

Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5′ end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3′ end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies. PMID:23966403

Development of siRNA expression vector utilizing rock bream beta-actin promoter: a potential therapeutic tool against viral infection in fish.

PubMed

Zenke, Kosuke; Nam, Yoon Kwon; Kim, Ki Hong

2010-01-01

In the present study, we have developed short interfering RNA (siRNA) expression vector utilizing rock bream beta-actin promoter and examined the possible use for the inhibition of highly pathogenic fish virus, rock bream iridovirus (RBIV), replication in vitro. Initially, in order to express siRNA effectively, we added several modifications to wild-type rock bream beta-actin promoter. Next, we succeeded in knocking down the expression of enhanced green fluorescent protein reporter gene expression in fish cells using newly developed vector more effectively than the fugu U6 promoter-driven vector we described previously. Finally, we could observe that cells transfected with modified rock bream beta-actin promoter-driven siRNA expression vector targeting major capsid protein (MCP) gene of RBIV exhibited more resistance to RBIV challenge than other control cells. Our results indicate that this novel siRNA expression vector can be used as a new tool for therapeutics in virus infection in fish species.

Tissue-specific expression of silkmoth chorion genes in vivo using Bombyx mori nuclear polyhedrosis virus as a transducing vector.

PubMed Central

Iatrou, K; Meidinger, R G

1990-01-01

A pair of silkmoth chorion chromosomal genes, HcA.12-HcB.12, was inserted into a baculovirus transfer vector, pBmp2, derived from the nuclear polyhedrosis virus of Bombyx mori. This vector, which permits the insertion of foreign genetic material in the vicinity of a mutationally inactivated polyhedrin gene, was used to acquire the corresponding recombinant virus. Injection of mutant silkmoth pupae that lack all Hc chorion genes with the recombinant virus resulted in the infection of all internal organs including follicular tissue. Analysis of RNA from infected tissues has demonstrated that the two chorion genes present in the viral genome are correctly transcribed under the control of their own promoter in follicular cells, the tissue in which chorion genes are normally expressed. The chorion primary transcripts are also correctly processed in the infected follicular cells and yield mature mRNAs indistinguishable from authentic chorion mRNAs present in wild-type follicles. These results demonstrate that recombinant nuclear polyhedrosis viruses can be used as transducing vectors for introducing genetic material of host origin into the cells of the organism and that the transduced genes are transiently expressed in a tissue-specific manner under the control of their resident regulatory sequences. Thus we show the in vivo expression of cloned genes under cellular promoter control in an insect other than Drosophila melanogaster. The approach should be applicable to all insect systems that are subject to nuclear polyhedrosis virus infection. Images PMID:2187186

Development of renal-targeted vectors through combined in vivo phage display and capsid engineering of adenoviral fibers from serotype 19p.

PubMed

Denby, Laura; Work, Lorraine M; Seggern, Dan J Von; Wu, Eugene; McVey, John H; Nicklin, Stuart A; Baker, Andrew H

2007-09-01

The potential efficacy of gene delivery is dictated by the infectivity profile of existing vectors, which is often restrictive. In order to target cells and organs for which no efficient vector is currently available, a promising approach would be to engineer vectors with novel transduction profiles. Applications that involve injecting adenovirus (Ad) vectors into the bloodstream require that native tropism for the liver be removed, and that targeting moieties be engineered into the capsid. We previously reported that pseudotyping the Ad serotype 5 fiber for that of Ad19p results in reduced hepatic transduction. In this study we show that this may be caused, at least in part, by a reduction in the capacity of the Ad19p-based virus to bind blood coagulation factors. It is therefore a potential candidate for vector retargeting, focusing on the kidney as a therapeutic target. We used in vivo phage display in rats, and identified peptides HTTHREP and HITSLLS that homed to the kidneys following intravenous injection. We engineered the HI loop of Ad19p to accommodate peptide insertions and clones. Intravenous delivery of each peptide-modified virus resulted in selective renal targeting, with HTTHREP and HITSLLS-targeted viruses selectively transducing tubular epithelium and glomeruli, respectively. Our study has important implications for the use of genetic engineering of Ad fibers to produce targeted gene delivery vectors.

Heterologous Packaging Signals on Segment 4, but Not Segment 6 or Segment 8, Limit Influenza A Virus Reassortment.

PubMed

White, Maria C; Steel, John; Lowen, Anice C

2017-06-01

Influenza A virus (IAV) RNA packaging signals serve to direct the incorporation of IAV gene segments into virus particles, and this process is thought to be mediated by segment-segment interactions. These packaging signals are segment and strain specific, and as such, they have the potential to impact reassortment outcomes between different IAV strains. Our study aimed to quantify the impact of packaging signal mismatch on IAV reassortment using the human seasonal influenza A/Panama/2007/99 (H3N2) and pandemic influenza A/Netherlands/602/2009 (H1N1) viruses. Focusing on the three most divergent segments, we constructed pairs of viruses that encoded identical proteins but differed in the packaging signal regions on a single segment. We then evaluated the frequency with which segments carrying homologous versus heterologous packaging signals were incorporated into reassortant progeny viruses. We found that, when segment 4 (HA) of coinfecting parental viruses was modified, there was a significant preference for the segment containing matched packaging signals relative to the background of the virus. This preference was apparent even when the homologous HA constituted a minority of the HA segment population available in the cell for packaging. Conversely, when segment 6 (NA) or segment 8 (NS) carried modified packaging signals, there was no significant preference for homologous packaging signals. These data suggest that movement of NA and NS segments between the human H3N2 and H1N1 lineages is unlikely to be restricted by packaging signal mismatch, while movement of the HA segment would be more constrained. Our results indicate that the importance of packaging signals in IAV reassortment is segment dependent. IMPORTANCE Influenza A viruses (IAVs) can exchange genes through reassortment. This process contributes to both the highly diverse population of IAVs found in nature and the formation of novel epidemic and pandemic IAV strains. Our study sought to determine the extent to which IAV packaging signal divergence impacts reassortment between seasonal IAVs. Our knowledge in this area is lacking, and insight into the factors that influence IAV reassortment will inform and strengthen ongoing public health efforts to anticipate the emergence of new viruses. We found that the packaging signals on the HA segment, but not the NA or NS segments, restricted IAV reassortment. Thus, the packaging signals of the HA segment could be an important factor in determining the likelihood that two IAV strains of public health interest will undergo reassortment. Copyright © 2017 American Society for Microbiology.

Heterologous Packaging Signals on Segment 4, but Not Segment 6 or Segment 8, Limit Influenza A Virus Reassortment

PubMed Central

White, Maria C.; Steel, John

2017-01-01

ABSTRACT Influenza A virus (IAV) RNA packaging signals serve to direct the incorporation of IAV gene segments into virus particles, and this process is thought to be mediated by segment-segment interactions. These packaging signals are segment and strain specific, and as such, they have the potential to impact reassortment outcomes between different IAV strains. Our study aimed to quantify the impact of packaging signal mismatch on IAV reassortment using the human seasonal influenza A/Panama/2007/99 (H3N2) and pandemic influenza A/Netherlands/602/2009 (H1N1) viruses. Focusing on the three most divergent segments, we constructed pairs of viruses that encoded identical proteins but differed in the packaging signal regions on a single segment. We then evaluated the frequency with which segments carrying homologous versus heterologous packaging signals were incorporated into reassortant progeny viruses. We found that, when segment 4 (HA) of coinfecting parental viruses was modified, there was a significant preference for the segment containing matched packaging signals relative to the background of the virus. This preference was apparent even when the homologous HA constituted a minority of the HA segment population available in the cell for packaging. Conversely, when segment 6 (NA) or segment 8 (NS) carried modified packaging signals, there was no significant preference for homologous packaging signals. These data suggest that movement of NA and NS segments between the human H3N2 and H1N1 lineages is unlikely to be restricted by packaging signal mismatch, while movement of the HA segment would be more constrained. Our results indicate that the importance of packaging signals in IAV reassortment is segment dependent. IMPORTANCE Influenza A viruses (IAVs) can exchange genes through reassortment. This process contributes to both the highly diverse population of IAVs found in nature and the formation of novel epidemic and pandemic IAV strains. Our study sought to determine the extent to which IAV packaging signal divergence impacts reassortment between seasonal IAVs. Our knowledge in this area is lacking, and insight into the factors that influence IAV reassortment will inform and strengthen ongoing public health efforts to anticipate the emergence of new viruses. We found that the packaging signals on the HA segment, but not the NA or NS segments, restricted IAV reassortment. Thus, the packaging signals of the HA segment could be an important factor in determining the likelihood that two IAV strains of public health interest will undergo reassortment. PMID:28331085

SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption.

PubMed

Ho, Michelle L; Adler, Benjamin A; Torre, Michael L; Silberg, Jonathan J; Suh, Junghae

2013-12-20

Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions.

SCHEMA computational design of virus capsid chimeras: calibrating how genome packaging, protection, and transduction correlate with calculated structural disruption

PubMed Central

Ho, Michelle L.; Adler, Benjamin A.; Torre, Michael L.; Silberg, Jonathan J.; Suh, Junghae

2013-01-01

Adeno-associated virus (AAV) recombination can result in chimeric capsid protein subunits whose ability to assemble into an oligomeric capsid, package a genome, and transduce cells depends on the inheritance of sequence from different AAV parents. To develop quantitative design principles for guiding site-directed recombination of AAV capsids, we have examined how capsid structural perturbations predicted by the SCHEMA algorithm correlate with experimental measurements of disruption in seventeen chimeric capsid proteins. In our small chimera population, created by recombining AAV serotypes 2 and 4, we found that protection of viral genomes and cellular transduction were inversely related to calculated disruption of the capsid structure. Interestingly, however, we did not observe a correlation between genome packaging and calculated structural disruption; a majority of the chimeric capsid proteins formed at least partially assembled capsids and more than half packaged genomes, including those with the highest SCHEMA disruption. These results suggest that the sequence space accessed by recombination of divergent AAV serotypes is rich in capsid chimeras that assemble into 60-mer capsids and package viral genomes. Overall, the SCHEMA algorithm may be useful for delineating quantitative design principles to guide the creation of libraries enriched in genome-protecting virus nanoparticles that can effectively transduce cells. Such improvements to the virus design process may help advance not only gene therapy applications, but also other bionanotechnologies dependent upon the development of viruses with new sequences and functions. PMID:23899192

Comparative Study of the Pathological Effects of Western Equine Encephalomyelitis Virus in Four Strains of Culex tarsalis Coquillett (Diptera: Culicidae)

PubMed Central

Neira, Marco V.; Mahmood, Farida; Reisen, William K.; James, Calvin B. L.; Romoser, William S.

2014-01-01

Early reports suggested that mosquito cells infected with arboviruses remain viable and undamaged. However, more recent experimental evidence suggests that arboviral infection of mosquito tissues might indeed result in pathological changes, with potential implications for vector survival and virus transmission. Here, we compare the pathological effects of western equine encephalomyelitis virus (WEEV) infection in four strains of Culex tarsalis previously reported to differ in their competence as WEEV vectors. Pathological effects were observed in cells of the midgut epithelium, salivary glands, and eggs. Cell rounding and sloughing of midgut epithelial cells was associated with those strains reported to be the least susceptible to WEEV infection, whereas midgut necrosis and vacuolation upon infection were associated with strains showing higher susceptibility. Although pathological effects were sporadically observed in infected salivary glands, further studies are required to evaluate their impact on vector competence. Additionally, the potential implications of observed C. tarsalis egg infection with WEEV are discussed. PMID:25346928

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yokoo, Masako; Fujita, Ryosuke; Innate Immunity Laboratory, Graduate School of Life Science and Creative Research Institution, Hokkaido University, Sapporo 001-0021

Highlights: •The baculovirus vector infiltrates the cells of economic important fishes. •Drosophila Mos1 transposase expressed in fish cells maintains its ability to localize to the nucleus. •The baculoviral vector carrying Mos1 is a useful tool to stably transform fish cells. -- Abstract: Drosophila Mos1 belongs to the mariner family of transposons, which are one of the most ubiquitous transposons among eukaryotes. We first determined nuclear transportation of the Drosophila Mos1-EGFP fusion protein in fish cell lines because it is required for a function of transposons. We next constructed recombinant baculoviral vectors harboring the Drosophila Mos1 transposon or marker genes locatedmore » between Mos1 inverted repeats. The infectivity of the recombinant virus to fish cells was assessed by monitoring the expression of a fluorescent protein encoded in the viral genome. We detected transgene expression in CHSE-214, HINAE, and EPC cells, but not in GF or RTG-2 cells. In the co-infection assay of the Mos1-expressing virus and reporter gene-expressing virus, we successfully transformed CHSE-214 and HINAE cells. These results suggest that the combination of a baculovirus and Mos1 transposable element may be a tool for transgenesis in fish cells.« less

Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector

PubMed Central

Grieger, Joshua C; Soltys, Stephen M; Samulski, Richard Jude

2016-01-01

Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of preclinical and clinical applications. Many developments including new serotypes as well as self-complementary vectors are now entering the clinic. With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high-titer, highly pure, and potent quantities of rAAV vectors. Utilizing the relatively simple and efficient transfection system of HEK293 cells as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in animal component-free suspension conditions in shaker flasks and WAVE bioreactors that allows for rapid and scalable rAAV production. Using the triple transfection method, the suspension HEK293 cell line generates greater than 1 × 105 vector genome containing particles (vg)/cell or greater than 1 × 1014 vg/l of cell culture when harvested 48 hours post-transfection. To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent, transfection conditions and cell density. A universal purification strategy, based on ion exchange chromatography methods, was also developed that results in high-purity vector preps of AAV serotypes 1–6, 8, 9 and various chimeric capsids tested. This user-friendly process can be completed within 1 week, results in high full to empty particle ratios (>90% full particles), provides postpurification yields (>1 × 1013 vg/l) and purity suitable for clinical applications and is universal with respect to all serotypes and chimeric particles. To date, this scalable manufacturing technology has been utilized to manufacture GMP phase 1 clinical AAV vectors for retinal neovascularization (AAV2), Hemophilia B (scAAV8), giant axonal neuropathy (scAAV9), and retinitis pigmentosa (AAV2), which have been administered into patients. In addition, we report a minimum of a fivefold increase in overall vector production by implementing a perfusion method that entails harvesting rAAV from the culture media at numerous time-points post-transfection. PMID:26437810

Vaccination with Replication Deficient Adenovectors Encoding YF-17D Antigens Induces Long-Lasting Protection from Severe Yellow Fever Virus Infection in Mice.

PubMed

Bassi, Maria R; Larsen, Mads A B; Kongsgaard, Michael; Rasmussen, Michael; Buus, Søren; Stryhn, Anette; Thomsen, Allan R; Christensen, Jan P

2016-02-01

The live attenuated yellow fever vaccine (YF-17D) has been successfully used for more than 70 years. It is generally considered a safe vaccine, however, recent reports of serious adverse events following vaccination have raised concerns and led to suggestions that even safer YF vaccines should be developed. Replication deficient adenoviruses (Ad) have been widely evaluated as recombinant vectors, particularly in the context of prophylactic vaccination against viral infections in which induction of CD8+ T-cell mediated immunity is crucial, but potent antibody responses may also be elicited using these vectors. In this study, we present two adenobased vectors targeting non-structural and structural YF antigens and characterize their immunological properties. We report that a single immunization with an Ad-vector encoding the non-structural protein 3 from YF-17D could elicit a strong CD8+ T-cell response, which afforded a high degree of protection from subsequent intracranial challenge of vaccinated mice. However, full protection was only observed using a vector encoding the structural proteins from YF-17D. This vector elicited virus-specific CD8+ T cells as well as neutralizing antibodies, and both components were shown to be important for protection thus mimicking the situation recently uncovered in YF-17D vaccinated mice. Considering that Ad-vectors are very safe, easy to produce and highly immunogenic in humans, our data indicate that a replication deficient adenovector-based YF vaccine may represent a safe and efficient alternative to the classical live attenuated YF vaccine and should be further tested.

Copy number determination of genetically-modified hematopoietic stem cells.

PubMed

Schuesler, Todd; Reeves, Lilith; Kalle, Christof von; Grassman, Elke

2009-01-01

Human gene transfer with gammaretroviral, murine leukemia virus (MLV) based vectors has been shown to effectively insert and express transgene sequences at a level of therapeutic benefit. However, there are numerous reports of disruption of the normal cellular processes caused by the viral insertion, even of replication deficient gammaretroviral vectors. Current gammaretroviral and lentiviral vectors do not control the site of insertion into the genome, hence, the possibility of disruption of the target cell genome. Risk related to viral insertions is linked to the number of insertions of the transgene into the cellular DNA, as has been demonstrated for replication competent and replication deficient retroviruses in experiments. At high number of insertions per cell, cell transformation due to vector induced activation of proto-oncogenes is more likely to occur, in particular since more than one transforming event is needed for oncogenesis. Thus, determination of the vector copy number in bulk transduced populations, individual colony forming units, and tissue from the recipient of the transduced cells is an increasingly important safety assay and has become a standard, though not straightforward assay, since the inception of quantitative PCR.

Single-Vector, Single-Injection Recombinant Vesicular Stomatitis Virus Vaccines Against High-Containment Viruses.

PubMed

Whitt, Michael A; Geisbert, Thomas W; Mire, Chad E

2016-01-01

There are many avenues for making an effective vaccine against viruses. Depending on the virus these can include one of the following: inactivation of whole virions; attenuation of viruses; recombinant viral proteins; non-replication-competent virus particles; or surrogate virus vector systems such as vesicular stomatitis virus (VSV). VSV is a prototypic enveloped animal virus that has been used for over four decades to study virus replication, entry, and assembly due to its ability to replicate to high titers in a wide variety of mammalian and insect cells. The use of reverse genetics to recover infectious and single-cycle replicating VSV from plasmid DNA transfected in cell culture began a revolution in the study of recombinant VSV (rVSV). This platform can be manipulated to study the viral genetic sequences and proteins important in the virus life cycle. Additionally, foreign genes can be inserted between naturally occurring or generated start/stop signals and polyadenylation sites within the VSV genome. VSV has a tolerance for foreign gene expression which has led to numerous rVSVs reported in the literature. Of particular interest are the very effective single-dose rVSV vaccine vectors against high-containment viruses such as filoviruses, henipaviruses, and arenaviruses. Herein we describe the methods for selecting foreign antigenic genes, selecting the location within the VSV genome for insertion, generation of rVSV using reverse genetics, and proper vaccine study designs.

Development and characterization of a packaging cell line for pseudo-infectious yellow fever virus particle generation.

PubMed

Queiroz, Sabrina Ribeiro de Almeida; Silva Júnior, José Valter Joaquim; Silva, Andréa Nazaré Monteiro Rangel da; Carvalho, Amanda Gomes de Oliveira; Santos, Jefferson José da Silva; Gil, Laura Helena Vega Gonzales

2018-01-01

Pseudo-infectious yellow fever viral particles (YFV-PIVs) have been used to study vaccines and viral packaging. Here, we report the development of a packaging cell line, which expresses the YFV prM/E proteins. HEK293 cells were transfected with YFV prM/E and C (84 nt) genes to generate HEK293-YFV-PrM/E-opt. The cells were evaluated for their ability to express the heterologous proteins and to package the replicon repYFV-17D-LucIRES, generating YFV-PIVs. The expression of prM/E proteins was confirmed, and the cell line trans-packaged the replicon for recovery of a reporter for the YFV-PIVs. HEK293-YFV-prM/E-opt trans-packaging capacity demonstrates its possible biotechnology application.

A lentiviral vector that activates latent human immunodeficiency virus-1 proviruses by the overexpression of tat and that kills the infected cells.

PubMed

Macías, David; Oya, Ricardo; Saniger, Luisa; Martín, Francisco; Luque, Francisco

2009-11-01

Despite the efficient HIV-1 replication blockage achieved with current highly active antiretroviral therapy (HAART) therapies, HIV-1 persists in the body and survives in a latent state that can last for the entire life of the patient. A long-lived reservoir of latently infected CD4(+) memory T cells represents the most important sanctuary for the virus and the greatest obstacle for viral eradication. In this work, we present an initial step toward a gene therapy approach aimed at the activation of latent provirus to induce the death of latently infected T cells. Latent HIV-1 infection is characterized by the failure of viral gene expression as a consequence of uninitiated or aborted transcription. We have constructed an HIV-1-based lentiviral vector (p5p53RTAT3) that expresses the viral trans-activating protein Tat in a drug-regulated manner and p53 in a Rev-dependent manner. We have demonstrated that the Tat-expressed protein from p5p53RTAT3 vector reactivates latent HIV-1 proviruses in J1.1 and ACH-2 cell lines and promotes p53-induced apoptosis in the presence of Rev. Our system was able to trigger the trans-activation of the provirus 5' long terminal repeat (LTR), stimulate the expression of the Rev protein from a tat-defective provirus, and provoke apoptosis selectively in the cells transfected with a tat-defective HIV-1 provirus in contrast to those with no HIV-1 provirus. However, the Rev-dependent p53 killing of latently infected cells was not effective enough for complete elimination of the awakened HIV-1 viruses. In summary, we have developed a vector system that is efficient in activating latent HIV-1 proviruses but that needs further improvement to kill infected cells.

Avipoxviruses: infection biology and their use as vaccine vectors.

PubMed

Weli, Simon C; Tryland, Morten

2011-02-03

Avipoxviruses (APVs) belong to the Chordopoxvirinae subfamily of the Poxviridae family. APVs are distributed worldwide and cause disease in domestic, pet and wild birds of many species. APVs are transmitted by aerosols and biting insects, particularly mosquitoes and arthropods and are usually named after the bird species from which they were originally isolated. The virus species Fowlpox virus (FWPV) causes disease in poultry and associated mortality is usually low, but in flocks under stress (other diseases, high production) mortality can reach up to 50%. APVs are also major players in viral vaccine vector development for diseases in human and veterinary medicine. Abortive infection in mammalian cells (no production of progeny viruses) and their ability to accommodate multiple gene inserts are some of the characteristics that make APVs promising vaccine vectors. Although abortive infection in mammalian cells conceivably represents a major vaccine bio-safety advantage, molecular mechanisms restricting APVs to certain hosts are not yet fully understood. This review summarizes the current knowledge relating to APVs, including classification, morphogenesis, host-virus interactions, diagnostics and disease, and also highlights the use of APVs as recombinant vaccine vectors.

Design, Construction and Evaluation of 1a/JFH1 HCV Chimera by Replacing the Intergenotypic Variable Region

PubMed Central

Ghasemi, Faezeh; Ghayour-Mobarhan, Majid; Pasdar, Alireza; Pourianfar, Hamid; Reza Aghasadeghi, Mohammad; Gouklani, Hamed; Meshkat, Zahra

2016-01-01

Background The E2 glycoprotein is an important encoded hepatitis C virus (HCV) protein that contains three different variable regions. Objectives The aim of the present study was to construct an HCV 1a/JFH1 chimeric virus by replacing the intergenotypic variable region (igVR) fragment of the highly variable region of the E2 gene of the Japanese Fulminant hepatitis genotype 2a JFH1 virus with a similar region of HCV genotype 1a. This chimera was produced as a model virus with the ability to be cultured. We analyzed the adapted virus and the variations of nucleic acids within it. Methods Specific primers were designed for the igVR of HCV genotype 1a followed by the overlap-PCR method for the synthesis of the desired DNA fragment. The amplified igVR-1a chimera gene and pFL-J6/JFH were digested by KpnI and BsiWI restriction enzymes, and the fragment was ligated into pFL-J6/JFH. The recombinant vector was transformed into Escherichia coli JM109 strain competent cells. All clones were confirmed by colony PCR using specific primers, and the confirmed recombinant vector was sequenced. The recombinant vector was targeted for RNA synthesis by T7 RNA polymerase enzyme. RNA transfection was performed in the Huh7.5 cell line. Virus production in several passages and the evaluated viral load were studied using quantitative real-time PCR and ELISA methods. After 30 passages, the RNA virus was extracted and cloned in PCDNA3.1 vector, and was then sequenced Results Quantitative real-time PCR results showed 11,292,514 copies/mL of chimeric virus production in cell culture. The virus production was confirmed using ELISA, which showed a virus core production of 808.2 pg/mL. The results of cloning and sequencing showed that some of the nucleic acids in the chimera virus were changed, affecting the viral behavior in the cell culture. Conclusions Real-time PCR and ELISA showed high levels of production of 1a/JFH1 chimeric HCV in the Huh7.5 cell culture. The constructed virus can be used for future studies, including the development of new HCV drugs and vaccines. PMID:27882063

The antiretrovirus drug 3'-azido-3'-deoxythymidine increases the retrovirus mutation rate.

PubMed Central

Julias, J G; Kim, T; Arnold, G; Pathak, V K

1997-01-01

It was previously observed that the nucleoside analog 5-azacytidine increased the spleen necrosis virus (SNV) mutation rate 13-fold in one cycle of retrovirus replication (V. K. Pathak and H. M. Temin, J. Virol. 66:3093-3100, 1992). Based on this observation, we hypothesized that nucleoside analogs used as antiviral drugs may also increase retrovirus mutation rates. We sought to determine if 3'-azido-3'-deoxythymidine (AZT), the primary treatment for human immunodeficiency virus type 1 (HIV-1) infection, increases the retrovirus mutation rate. Two assays were used to determine the effects of AZT on retrovirus mutation rates. The strategy of the first assay involved measuring the in vivo rate of inactivation of the lacZ gene in one replication cycle of SNV- and murine leukemia virus-based retroviral vectors. We observed 7- and 10-fold increases in the SNV mutant frequency following treatment of target cells with 0.1 and 0.5 microM AZT, respectively. The murine leukemia virus mutant frequency increased two- and threefold following treatment of target cells with 0.5 and 1.0 microM AZT, respectively. The second assay used an SNV-based shuttle vector containing the lacZ alpha gene. Proviruses were recovered as plasmids in Escherichia coli, and the rate of inactivation of lacZ alpha was measured. The results indicated that treatment of target cells increased the overall mutation rate two- to threefold. DNA sequence analysis of mutant proviruses indicated that AZT increased both the deletion and substitution rates. These results suggest that AZT treatment of HIV-1 infection may increase the degree of viral variation and alter virus evolution or pathogenesis. PMID:9151812

Dual AAV Vectors for Stargardt Disease.

PubMed

Trapani, Ivana

2018-01-01

Stargardt disease (STGD1), due to mutations in the large ABCA4 gene, is the most common inherited macular degeneration in humans. Attempts at developing gene therapy approaches for treatment of STGD1 are currently ongoing. Among all the vectors available for gene therapy of inherited retinal diseases, those based on adeno-associated viruses (AAV) are the most promising given the efficacy shown in various animal models and their excellent safety profile in humans, as confirmed in many ongoing clinical trials. However, one of the main obstacles for the use of AAV is their limited effective packaging capacity of about 5 kb. Taking advantage of the AAV genome's ability to concatemerize , others and we have recently developed dual AAV vectors to overcome this limit. We tested dual AAV vectors for ABCA4 delivery, and found that they transduce efficiently both mouse and pig photoreceptors , and rescue the Abca4-/- mouse retinal phenotype, indicating their potential for gene therapy of STGD1. This chapter details how we designed dual AAV vectors for the delivery of the ABCA4 gene and describes the techniques that can be explored to evaluate dual AAV transduction efficiency in vitro and in the retina, and their efficacy in the mouse model of STGD1.

Transcriptomic profiles of human foreskin fibroblast cells in response to orf virus.

PubMed

Chen, Daxiang; Long, Mingjian; Xiao, Bin; Xiong, Yufeng; Chen, Huiqin; Chen, Yu; Kuang, Zhenzhan; Li, Ming; Wu, Yingsong; Rock, Daniel L; Gong, Daoyuan; Wang, Yong; He, Haijian; Liu, Fang; Luo, Shuhong; Hao, Wenbo

2017-08-29

Orf virus has been utilized as a safe and efficient viral vector against not only diverse infectious diseases, but also against tumors. However, the nature of the genes triggered by the vector in human cells is poorly characterized. Using RNA sequencing technology, we compared specific changes in the transcriptomic profiles in human foreskin fibroblast cells following infection by the orf virus. The results indicated that orf virus upregulates or downregulates expression of a variety of genes, including genes involved in antiviral immune response, apoptosis, cell cycle and a series of signaling pathways, such as the IFN and p53-signaling pathways. The orf virus stimulates or inhibits immune gene expression such as chemokines, chemokine receptors, cytokines, cytokine receptors, and molecules involved in antigen uptake and processing after infection. Expression of pro-apoptotic genes increased at 8 hours post-infection. The p53 signaling pathway was activated to induce apoptosis at the same time. However, the cell cycle program was promoted after infection, which may be due to the immunomodulatory genes of the orf virus. This presents the first description of transcription profile changes in human foreskin fibroblast cells after orf virus infection and provides an in-depth analysis of the interaction between the host and orf virus. These data offer new insights into the understanding of the mechanisms of infection by orf virus and identify potential targets for future studies.

Assessment of bovine herpesvirus 4 based vector in chicken.

PubMed

Donofrio, Gaetano; Manarolla, Giovanni; Ravanetti, Lara; Sironi, Giuseppe; Cavirani, Sandro; Cabassi, Clotilde Silvia; Flammini, Cesidio Filippo; Rampin, Tiziana

2008-03-01

The biological characteristics of BoHV-4 make it a good candidate as a gene delivery vector for vaccination purposes. These characteristics include little or no pathogenicity, unlikely oncogenicity, the ability to accommodate large amounts of foreign genetic material, the ability to infect several cell types from different animal species, such as sheep, goats, swine, cats, dogs, rabbits, mink, horses, turkeys, ferrets, monkeys, hamsters, rats, mice, and chickens. In this report, the feasibility to use BoHV-4 based vector in chicken was investigated. Although BoHV-4 was able to replicate, leading to a cytopathic effect in a chicken cell line and infect the chorion allantoic membrane of embryonated eggs, however it was not pathogenic even when a large dose of virus was injected into the chicken. An immune response could be produced against heterologous antigen delivered by a recombinant BoHV-4. These data suggest the feasibility of using BoHV-4 based vector for vaccination purposes in chickens.

Wolbachia significantly impacts the vector competence of Aedes aegypti for Mayaro virus.

PubMed

Pereira, Thiago Nunes; Rocha, Marcele Neves; Sucupira, Pedro Henrique Ferreira; Carvalho, Fabiano Duarte; Moreira, Luciano Andrade

2018-05-02

Wolbachia, an intracellular endosymbiont present in up to 70% of all insect species, has been suggested as a sustainable strategy for the control of arboviruses such as Dengue, Zika and Chikungunya. As Mayaro virus outbreaks have also been reported in Latin American countries, the objective of this study was to evaluate the vector competence of Brazilian field-collected Ae. aegypti and the impact of Wolbachia (wMel strain) upon this virus. Our in vitro studies with Aag2 cells showed that Mayaro virus can rapidly multiply, whereas in wMel-infected Aag2 cells, viral growth was significantly impaired. In addition, C6/36 cells seem to have alterations when infected by Mayaro virus. In vivo experiments showed that field-collected Ae. aegypti mosquitoes are highly permissive to Mayaro virus infection, and high viral prevalence was observed in the saliva. On the other hand, Wolbachia-harboring mosquitoes showed significantly impaired capability to transmit Mayaro virus. Our results suggest that the use of Wolbachia-harboring mosquitoes may represent an effective mechanism for the reduction of Mayaro virus transmission throughout Latin America.

Quantum dot coating of baculoviral vectors enables visualization of transduced cells and tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Ying; Lo, Seong Loong; Zheng, Yuangang

2013-04-26

Highlights: •The use of quantum dot (QD)-labeled viral vectors for in vivo imaging is not well investigated. •A new method to label enveloped baculovirus with glutathione-capped CdTe QDs is developed. •The labeling enables the identification of transduced, cultured cells based on fluorescence. •The labeling also allows evaluation of viral transduction in a real-time manner in living mice. •The method has the potential to assess viral vector-based gene therapy protocols in future. -- Abstract: Imaging of transduced cells and tissues is valuable in developing gene transfer vectors and evaluating gene therapy efficacy. We report here a simple method to use brightmore » and photostable quantum dots to label baculovirus, an emerging gene therapy vector. The labeling was achieved through the non-covalent interaction of glutathione-capped CdTe quantum dots with the virus envelope, without the use of chemical conjugation. The quantum dot labeling was nondestructive to viral transduction function and enabled the identification of baculoviral vector-transduced, living cells based on red fluorescence. When the labeled baculoviral vectors were injected intravenously or intraventricularly for in vivo delivery of a transgene into mice, quantum dot fluorescence signals allow us monitor whether or not the injected tissues were transduced. More importantly, using a dual-color whole-body imaging technology, we demonstrated that in vivo viral transduction could be evaluated in a real-time manner in living mice. Thus, our method of labeling a read-to-use gene delivery vector with quantum dots could be useful towards the improvement of vector design and will have the potential to assess baculovirus-based gene therapy protocols in future.« less

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor

PubMed Central

Zhao, Wei; Jardine, Paul J.

2015-01-01

ABSTRACT During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. IMPORTANCE During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs. PMID:26423956

An RNA Domain Imparts Specificity and Selectivity to a Viral DNA Packaging Motor.

PubMed

Zhao, Wei; Jardine, Paul J; Grimes, Shelley

2015-12-01

During assembly, double-stranded DNA viruses, including bacteriophages and herpesviruses, utilize a powerful molecular motor to package their genomic DNA into a preformed viral capsid. An integral component of the packaging motor in the Bacillus subtilis bacteriophage ϕ29 is a viral genome-encoded pentameric ring of RNA (prohead RNA [pRNA]). pRNA is a 174-base transcript comprised of two domains, domains I and II. Early studies initially isolated a 120-base form (domain I only) that retains high biological activity in vitro; hence, no function could be assigned to domain II. Here we define a role for this domain in the packaging process. DNA packaging using restriction digests of ϕ29 DNA showed that motors with the 174-base pRNA supported the correct polarity of DNA packaging, selectively packaging the DNA left end. In contrast, motors containing the 120-base pRNA had compromised specificity, packaging both left- and right-end fragments. The presence of domain II also provides selectivity in competition assays with genomes from related phages. Furthermore, motors with the 174-base pRNA were restrictive, in that they packaged only one DNA fragment into the head, whereas motors with the 120-base pRNA packaged several fragments into the head, indicating multiple initiation events. These results show that domain II imparts specificity and stringency to the motor during the packaging initiation events that precede DNA translocation. Heteromeric rings of pRNA demonstrated that one or two copies of domain II were sufficient to impart this selectivity/stringency. Although ϕ29 differs from other double-stranded DNA phages in having an RNA motor component, the function provided by pRNA is carried on the motor protein components in other phages. During virus assembly, genome packaging involves the delivery of newly synthesized viral nucleic acid into a protein shell. In the double-stranded DNA phages and herpesviruses, this is accomplished by a powerful molecular motor that translocates the viral DNA into a preformed viral shell. A key event in DNA packaging is recognition of the viral DNA among other nucleic acids in the host cell. Commonly, a DNA-binding protein mediates the interaction of viral DNA with the motor/head shell. Here we show that for the bacteriophage ϕ29, this essential step of genome recognition is mediated by a viral genome-encoded RNA rather than a protein. A domain of the prohead RNA (pRNA) imparts specificity and stringency to the motor by ensuring the correct orientation of DNA packaging and restricting initiation to a single event. Since this assembly step is unique to the virus, DNA packaging is a novel target for the development of antiviral drugs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Baculovirus GP64-mediated entry into mammalian cells.

PubMed

Kataoka, Chikako; Kaname, Yuuki; Taguwa, Shuhei; Abe, Takayuki; Fukuhara, Takasuke; Tani, Hideki; Moriishi, Kohji; Matsuura, Yoshiharu

2012-03-01

The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.

A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meador, Lydia R.

Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viralmore » vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1. - Highlights: • We devised a prime/boost anti HIV-1 vaccination strategy modeled after RV144. • We used plant-derived virus-like particles (VLPs) consisting of Gag and dgp41. • We used attenuated, replicating vaccinia virus vectors expressing the same antigens. • The immunogens elicited strong cellular and humoral immune responses.« less

Intracellular Crosslinking of Filoviral Nucleoproteins with Xintrabodies Restricts Viral Packaging

PubMed Central

Darling, Tamarand Lee; Sherwood, Laura Jo; Hayhurst, Andrew

2017-01-01

Viruses assemble large macromolecular repeat structures that become part of the infectious particles or virions. Ribonucleocapsids (RNCs) of negative strand RNA viruses are a prime example where repetition of nucleoprotein (NP) along the genome creates a core polymeric helical scaffold that accommodates other nucleocapsid proteins including viral polymerase. The RNCs are transported through the cytosol for packaging into virions through association with viral matrix proteins at cell membranes. We hypothesized that RNC would be ideal targets for crosslinkers engineered to promote aberrant protein–protein interactions, thereby blocking their orderly transport and packaging. Previously, we had generated single-domain antibodies (sdAbs) against Filoviruses that have all targeted highly conserved C-terminal regions of NP known to be repetitively exposed along the length of the RNCs of Marburgvirus (MARV) and Ebolavirus (EBOV). Our crosslinker design consisted of dimeric sdAb expressed intracellularly, which we call Xintrabodies (X- for crosslinking). Electron microscopy of purified NP polymers incubated with purified sdAb constructs showed NP aggregation occurred in a genus-specific manner with dimeric and not monomeric sdAb. A virus-like particle (VLP) assay was used for initial evaluation where we found that dimeric sdAb inhibited NP incorporation into VP40-based VLPs whereas monomeric sdAb did not. Inhibition of NP packaging was genus specific. Confocal microscopy revealed dimeric sdAb was diffuse when expressed alone but focused on pools of NP when the two were coexpressed, while monomeric sdAb showed ambivalent partition. Infection of stable Vero cell lines expressing dimeric sdAb specific for either MARV or EBOV NP resulted in smaller plaques and reduced progeny of cognate virus relative to wild-type Vero cells. Though the impact was marginal at later time-points, the collective data suggest that viral replication can be reduced by crosslinking intracellular NP using relatively small amounts of dimeric sdAb to restrict NP packaging. The stoichiometry and ease of application of the approach would likely benefit from transitioning away from intracellular expression of crosslinking sdAb to exogenous delivery of antibody. By retuning sdAb specificity, the approach of crosslinking highly conserved regions of assembly critical proteins may well be applicable to inhibiting replication processes of a broad spectrum of viruses. PMID:29021793

A new rabies vaccine based on a recombinant ORF virus (parapoxvirus) expressing the rabies virus glycoprotein.

PubMed

Amann, Ralf; Rohde, Jörg; Wulle, Ulrich; Conlee, Douglas; Raue, Rudiger; Martinon, Olivier; Rziha, Hanns-Joachim

2013-02-01

The present study describes the generation of a new Orf virus (ORFV) recombinant, D1701-V-RabG, expressing the rabies virus (RABV) glycoprotein that is correctly presented on the surface of infected cells without the need of replication or production of infectious recombinant virus. One single immunization with recombinant ORFV can stimulate high RABV-specific virus-neutralizing antibody (VNA) titers in mice, cats, and dogs, representing all nonpermissive hosts for the ORFV vector. The protective immune response against severe lethal challenge infection was analyzed in detail in mice using different dosages, numbers, and routes for immunization with the ORFV recombinant. Long-term levels of VNA could be elicited that remained greater than 0.5 IU per ml serum, indicative for the protective status. Single applications of higher doses (10(7) PFU) can be sufficient to confer complete protection against intracranial (i.c.) challenge, whereas booster immunization was needed for protection by the application of lower dosages. Anamnestic immune responses were achieved by each of the seven tested routes of inoculation, including oral application. Finally, in vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T cell subpopulations during immunization and/or challenge infection attested the importance of CD4 T cells for the induction of protective immunity by D1701-V-RabG. This report demonstrates another example of the potential of the ORFV vector and also indicates the capability of the new recombinant for vaccination of animals.

A New Rabies Vaccine Based on a Recombinant Orf Virus (Parapoxvirus) Expressing the Rabies Virus Glycoprotein

PubMed Central

Amann, Ralf; Rohde, Jörg; Wulle, Ulrich; Conlee, Douglas; Raue, Rudiger; Martinon, Olivier

2013-01-01

The present study describes the generation of a new Orf virus (ORFV) recombinant, D1701-V-RabG, expressing the rabies virus (RABV) glycoprotein that is correctly presented on the surface of infected cells without the need of replication or production of infectious recombinant virus. One single immunization with recombinant ORFV can stimulate high RABV-specific virus-neutralizing antibody (VNA) titers in mice, cats, and dogs, representing all nonpermissive hosts for the ORFV vector. The protective immune response against severe lethal challenge infection was analyzed in detail in mice using different dosages, numbers, and routes for immunization with the ORFV recombinant. Long-term levels of VNA could be elicited that remained greater than 0.5 IU per ml serum, indicative for the protective status. Single applications of higher doses (107 PFU) can be sufficient to confer complete protection against intracranial (i.c.) challenge, whereas booster immunization was needed for protection by the application of lower dosages. Anamnestic immune responses were achieved by each of the seven tested routes of inoculation, including oral application. Finally, in vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T cell subpopulations during immunization and/or challenge infection attested the importance of CD4 T cells for the induction of protective immunity by D1701-V-RabG. This report demonstrates another example of the potential of the ORFV vector and also indicates the capability of the new recombinant for vaccination of animals. PMID:23175365

Adenovirus vector infection of non-small-cell lung cancer cells is a trigger for multi-drug resistance mediated by P-glycoprotein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tomono, Takumi; Kajita, Masahiro; Yano, Kentaro

P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNAmore » expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. -- Highlights: •Adenovirus vector infection induced P-gp mRNA expression in three NSCLC cell lines. •Adenovirus vector infection enhanced P-gp-mediated doxorubicin efflux from the cells. •The increase of P-gp was not mediated by nuclear receptors (PXR, CAR) or COX-2.« less

Distinct Immunogenicity and Efficacy of Poxvirus-Based Vaccine Candidates against Ebola Virus Expressing GP and VP40 Proteins.

PubMed

Lázaro-Frías, Adrián; Gómez-Medina, Sergio; Sánchez-Sampedro, Lucas; Ljungberg, Karl; Ustav, Mart; Liljeström, Peter; Muñoz-Fontela, César; Esteban, Mariano; García-Arriaza, Juan

2018-06-01

Zaire and Sudan ebolavirus species cause a severe disease in humans and nonhuman primates (NHPs) characterized by a high mortality rate. There are no licensed therapies or vaccines against Ebola virus disease (EVD), and the recent 2013 to 2016 outbreak in West Africa highlighted the need for EVD-specific medical countermeasures. Here, we generated and characterized head-to-head the immunogenicity and efficacy of five vaccine candidates against Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV) based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing either the virus glycoprotein (GP) or GP together with the virus protein 40 (VP40) forming virus-like particles (VLPs). In a human monocytic cell line, the different MVA vectors (termed MVA-EBOVs and MVA-SUDVs) triggered robust innate immune responses, with production of beta interferon (IFN-β), proinflammatory cytokines, and chemokines. Additionally, several innate immune cells, such as dendritic cells, neutrophils, and natural killer cells, were differentially recruited in the peritoneal cavity of mice inoculated with MVA-EBOVs. After immunization of mice with a homologous prime/boost protocol (MVA/MVA), total IgG antibodies against GP or VP40 from Zaire and Sudan ebolavirus were differentially induced by these vectors, which were mainly of the IgG1 and IgG3 isotypes. Remarkably, an MVA-EBOV construct coexpressing GP and VP40 protected chimeric mice challenged with EBOV to a greater extent than a vector expressing GP alone. These results support the consideration of MVA-EBOVs and MVA-SUDVs expressing GP and VP40 and producing VLPs as best-in-class potential vaccine candidates against EBOV and SUDV. IMPORTANCE EBOV and SUDV cause a severe hemorrhagic fever affecting humans and NHPs. Since their discovery in 1976, they have caused several sporadic epidemics, with the recent outbreak in West Africa from 2013 to 2016 being the largest and most severe, with more than 11,000 deaths being reported. Although some vaccines are in advanced clinical phases, less expensive, safer, and more effective licensed vaccines are desirable. We generated and characterized head-to-head the immunogenicity and efficacy of five novel vaccines against EBOV and SUDV based on the poxvirus MVA expressing GP or GP and VP40. The expression of GP and VP40 leads to the formation of VLPs. These MVA-EBOV and MVA-SUDV recombinants triggered robust innate and humoral immune responses in mice. Furthermore, MVA-EBOV recombinants expressing GP and VP40 induced high protection against EBOV in a mouse challenge model. Thus, MVA expressing GP and VP40 and producing VLPs is a promising vaccine candidate against EBOV and SUDV. Copyright © 2018 American Society for Microbiology.

A baculovirus dual expression vector derived from the Autographa californica nuclear polyhedrosis virus polyhedrin and p10 promoters: co-expression of two influenza virus genes in insect cells.

PubMed

Weyer, U; Possee, R D

1991-12-01

A baculovirus transfer vector, pAcUW3, was developed to facilitate the insertion of two influenza virus genes, those encoding the haemagglutinin (HA) and neuraminidase (NA) membrane glycoproteins, into the Autographa californica nuclear polyhedrosis virus genome in a single cotransfection experiment. The NA gene was inserted in place of the polyhedrin coding sequences under the control of the polyhedrin promoter, whereas the HA gene was placed under the control of a copy of the p10 promoter at a site upstream of and in opposite orientation to the polyhedrin promoter. After infection of Spodoptera frugiperda cells with the recombinant virus, AcUW3HANA, both HA and NA were expressed in the very late phase of infection and were shown to be functional in appropriate assays. Immunofluorescence assays demonstrated their localization at the surface of infected insect cells. The expression of both foreign genes in the recombinant virus was found to be stable for at least 12 passages in cell culture.

Biosafety considerations for attenuated measles virus vectors used in virotherapy and vaccination

PubMed Central

Baldo, Aline; Galanis, Evanthia; Tangy, Frédéric; Herman, Philippe

2016-01-01

ABSTRACT Attenuated measles virus (MV) is one of the most effective and safe vaccines available, making it attractive candidate vector to prevent infectious diseases. Attenuated MV have acquired the ability to use the complement regulator CD46 as a major receptor to mediate virus entry and intercellular fusion. Therefore, attenuated MV strains preferentially infect and destroy a wide variety of cancer cells making them also attractive oncolytic vectors. The use of recombinant MV vector has to comply with various regulatory requirements, particularly relating to the assessment of potential risks for human health and the environment. The present article highlights the main characteristics of MV and recombinant MV vectors used for vaccination and virotherapy and discusses these features from a biosafety point of view. PMID:26631840

The Host Range of Gammaretroviruses and Gammaretroviral Vectors Includes Post-Mitotic Neural Cells

PubMed Central

Liu, Xiu-Huai; Xu, Wenqin; Russ, Jill; Eiden, Lee E.; Eiden, Maribeth V.

2011-01-01

Background Gammaretroviruses and gammaretroviral vectors, in contrast to lentiviruses and lentiviral vectors, are reported to be restricted in their ability to infect growth-arrested cells. The block to this restriction has never been clearly defined. The original assessment of the inability of gammaretroviruses and gammaretroviral vectors to infect growth-arrested cells was carried out using established cell lines that had been growth-arrested by chemical means, and has been generalized to neurons, which are post-mitotic. We re-examined the capability of gammaretroviruses and their derived vectors to efficiently infect terminally differentiated neuroendocrine cells and primary cortical neurons, a target of both experimental and therapeutic interest. Methodology/Principal Findings Using GFP expression as a marker for infection, we determined that both growth-arrested (NGF-differentiated) rat pheochromocytoma cells (PC12 cells) and primary rat cortical neurons could be efficiently transduced, and maintained long-term protein expression, after exposure to murine leukemia virus (MLV) and MLV-based retroviral vectors. Terminally differentiated PC12 cells transduced with a gammaretroviral vector encoding the anti-apoptotic protein Bcl-xL were protected from cell death induced by withdrawal of nerve growth factor (NGF), demonstrating gammaretroviral vector-mediated delivery and expression of genes at levels sufficient for therapeutic effect in non-dividing cells. Post-mitotic rat cortical neurons were also shown to be susceptible to transduction by murine replication-competent gammaretroviruses and gammaretroviral vectors. Conclusions/Significance These findings suggest that the host range of gammaretroviruses includes post-mitotic and other growth-arrested cells in mammals, and have implications for re-direction of gammaretroviral gene therapy to neurological disease. PMID:21464894

Cotransduction with MGMT and Ubiquitous or Erythroid-Specific GFP Lentiviruses Allows Enrichment of Dual-Positive Hematopoietic Progenitor Cells In Vivo

PubMed Central

Roth, Justin C.; Ismail, Mourad; Reese, Jane S.; Lingas, Karen T.; Ferrari, Giuliana; Gerson, Stanton L.

2012-01-01

The P140K point mutant of MGMT allows robust hematopoietic stem cell (HSC) enrichment in vivo. Thus, dual-gene vectors that couple MGMT and therapeutic gene expression have allowed enrichment of gene-corrected HSCs in animal models. However, expression levels from dual-gene vectors are often reduced for one or both genes. Further, it may be desirable to express selection and therapeutic genes at distinct stages of cell differentiation. In this regard, we evaluated whether hematopoietic cells could be efficiently cotransduced using low MOIs of two separate single-gene lentiviruses, including MGMT for dual-positive cell enrichment. Cotransduction efficiencies were evaluated using a range of MGMT : GFP virus ratios, MOIs, and selection stringencies in vitro. Cotransduction was optimal when equal proportions of each virus were used, but low MGMT : GFP virus ratios resulted in the highest proportion of dual-positive cells after selection. This strategy was then evaluated in murine models for in vivo selection of HSCs cotransduced with a ubiquitous MGMT expression vector and an erythroid-specific GFP vector. Although the MGMT and GFP expression percentages were variable among engrafted recipients, drug selection enriched MGMT-positive leukocyte and GFP-positive erythroid cell populations. These data demonstrate cotransduction as a mean to rapidly enrich and evaluate therapeutic lentivectors in vivo. PMID:22888445

Simple and effective generation of transgene-free induced pluripotent stem cells using an auto-erasable Sendai virus vector responding to microRNA-302.

PubMed

Nishimura, Ken; Ohtaka, Manami; Takada, Hitomi; Kurisaki, Akira; Tran, Nhi Vo Kieu; Tran, Yen Thi Hai; Hisatake, Koji; Sano, Masayuki; Nakanishi, Mahito

2017-08-01

Transgene-free induced pluripotent stem cells (iPSCs) are valuable for both basic research and potential clinical applications. We previously reported that a replication-defective and persistent Sendai virus (SeVdp) vector harboring four reprogramming factors (SeVdp-iPS) can efficiently induce generation of transgene-free iPSCs. This vector can express all four factors stably and simultaneously without chromosomal integration and can be eliminated completely from reprogrammed cells by suppressing vector-derived RNA-dependent RNA polymerase. Here, we describe an improved SeVdp-iPS vector (SeVdp(KOSM)302L) that is automatically erased in response to microRNA-302 (miR-302), uniquely expressed in pluripotent stem cells (PSCs). Gene expression and genome replication of the SeVdp-302L vector, which contains miRNA-302a target sequences at the 3' untranslated region of L mRNA, are strongly suppressed in PSCs. Consequently, SeVdp(KOSM)302L induces expression of reprogramming factors in somatic cells, while it is automatically erased from cells successfully reprogrammed to express miR-302. As this vector can reprogram somatic cells into transgene-free iPSCs without the aid of exogenous short interfering RNA (siRNA), the results we present here demonstrate that this vector may become an invaluable tool for the generation of human iPSCs for future clinical applications. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Adenovirus vector infection of non-small-cell lung cancer cells is a trigger for multi-drug resistance mediated by P-glycoprotein.

PubMed

Tomono, Takumi; Kajita, Masahiro; Yano, Kentaro; Ogihara, Takuo

2016-08-05

P-glycoprotein (P-gp) is an ATP-binding cassette protein involved in cancer multi-drug resistance (MDR). It has been reported that infection with some bacteria and viruses induces changes in the activities of various drug-metabolizing enzymes and transporters, including P-gp. Although human adenoviruses (Ad) cause the common cold, the effect of Ad infection on MDR in cancer has not been established. In this study, we investigated whether Ad infection is a cause of MDR in A549, H441 and HCC827 non-small-cell lung cancer (NSCLC) cell lines, using an Ad vector system. We found that Ad vector infection of NSCLC cell lines induced P-gp mRNA expression, and the extent of induction was dependent on the number of Ad vector virus particles and the infection time. Heat-treated Ad vector, which is not infectious, did not alter P-gp mRNA expression. Uptake experiments with doxorubicin (DOX), a P-gp substrate, revealed that DOX accumulation was significantly decreased in Ad vector-infected A549 cells. The decrease of DOX uptake was blocked by verapamil, a P-gp inhibitor. Our results indicated that Ad vector infection of NSCLC cells caused MDR mediated by P-gp overexpression. The Ad vector genome sequence is similar to that of human Ad, and therefore human Ad infection of lung cancer patients may lead to chemoresistance in the clinical environment. Copyright © 2016 Elsevier Inc. All rights reserved.

Foamy Virus Vector Carries a Strong Insulator in Its Long Terminal Repeat Which Reduces Its Genotoxic Potential

PubMed Central

2017-01-01

ABSTRACT Strong viral enhancers in gammaretrovirus vectors have caused cellular proto-oncogene activation and leukemia, necessitating the use of cellular promoters in “enhancerless” self-inactivating integrating vectors. However, cellular promoters result in relatively low transgene expression, often leading to inadequate disease phenotype correction. Vectors derived from foamy virus, a nonpathogenic retrovirus, show higher preference for nongenic integrations than gammaretroviruses/lentiviruses and preferential integration near transcriptional start sites, like gammaretroviruses. We found that strong viral enhancers/promoters placed in foamy viral vectors caused extremely low immortalization of primary mouse hematopoietic stem/progenitor cells compared to analogous gammaretrovirus/lentivirus vectors carrying the same enhancers/promoters, an effect not explained solely by foamy virus' modest insertional site preference for nongenic regions compared to gammaretrovirus/lentivirus vectors. Using CRISPR/Cas9-mediated targeted insertion of analogous proviral sequences into the LMO2 gene and then measuring LMO2 expression, we demonstrate a sequence-specific effect of foamy virus, independent of insertional bias, contributing to reduced genotoxicity. We show that this effect is mediated by a 36-bp insulator located in the foamy virus long terminal repeat (LTR) that has high-affinity binding to the CCCTC-binding factor. Using our LMO2 activation assay, LMO2 expression was significantly increased when this insulator was removed from foamy virus and significantly reduced when the insulator was inserted into the lentiviral LTR. Our results elucidate a mechanism underlying the low genotoxicity of foamy virus, identify a novel insulator, and support the use of foamy virus as a vector for gene therapy, especially when strong enhancers/promoters are required. IMPORTANCE Understanding the genotoxic potential of viral vectors is important in designing safe and efficacious vectors for gene therapy. Self-inactivating vectors devoid of viral long-terminal-repeat enhancers have proven safe; however, transgene expression from cellular promoters is often insufficient for full phenotypic correction. Foamy virus is an attractive vector for gene therapy. We found foamy virus vectors to be remarkably less genotoxic, well below what was expected from their integration site preferences. We demonstrate that the foamy virus long terminal repeats contain an insulator element that binds CCCTC-binding factor and reduces its insertional genotoxicity. Our study elucidates a mechanism behind the low genotoxic potential of foamy virus, identifies a unique insulator, and supports the use of foamy virus as a vector for gene therapy. PMID:29046446

Production of Recombinant Adenovirus Containing Human Interlukin-4 Gene

PubMed Central

Mojarrad, Majid; Abdolazimi, Yassan; Hajati, Jamshid; Modarressi, Mohammad Hossein

2011-01-01

Objective(s) Recombinant adenoviruses are currently used for a variety of purposes, including in vitro gene transfer, in vivo vaccination, and gene therapy. Ability to infect many cell types, high efficiency in gene transfer, entering both dividing and non dividing cells, and growing to high titers make this virus a good choice for using in various experiments. In the present experiment, a recombinant adenovirus containing human IL-4 coding sequence was made. IL-4 has several characteristics that made it a good choice for using in cancer gene therapy, controlling inflammatory diseases, and studies on autoimmune diseases. Materials and Methods In brief, IL-4 coding sequence was amplified by and cloned in pAd-Track-CMV. Then, by means of homologous recombination between recombinant pAd-Track-CMV and Adeasy-1 plasmid in bacteria, recombinant adenovirus complete genome was made and IL-4 containing shuttle vector was incorporated into the viral backbone. After linearization, for virus packaging, viral genome was transfected into HEK-293 cell line. Viral production was conveniently followed with the aid of green fluorescent protein. Results Recombinant adenovirus produced here, was capable to infecting cell lines and express interlukin-4 in cell. Conclusion This system can be used as a powerful, easy, and cost benefit tool in various studies on cancer gene therapy and also studies on immunogenetics. PMID:23493491

Vaccine strategies against Babesia bovis based on prime-boost immunizations in mice with modified vaccinia Ankara vector and recombinant proteins.

PubMed

Jaramillo Ortiz, José Manuel; Del Médico Zajac, María Paula; Zanetti, Flavia Adriana; Molinari, María Paula; Gravisaco, María José; Calamante, Gabriela; Wilkowsky, Silvina Elizabeth

2014-08-06

In this study, a recombinant modified vaccinia virus Ankara vector expressing a chimeric multi-antigen was obtained and evaluated as a candidate vaccine in homologous and heterologous prime-boost immunizations with a recombinant protein cocktail. The chimeric multi-antigen comprises immunodominant B and T cell regions of three Babesia bovis proteins. Humoral and cellular immune responses were evaluated in mice to compare the immunogenicity induced by different immunization schemes. The best vaccination scheme was achieved with a prime of protein cocktail and a boost with the recombinant virus. This scheme induced high level of specific IgG antibodies and secreted IFN and a high degree of activation of IFNγ(+) CD4(+) and CD8(+) specific T cells. This is the first report in which a novel vaccine candidate was constructed based on a rationally designed multi-antigen and evaluated in a prime-boost regime, optimizing the immune response necessary for protection against bovine babesiosis. Copyright © 2014 Elsevier Ltd. All rights reserved.

Bovine Herpesvirus-4-Based Vector Delivering Peste des Petits Ruminants Virus Hemagglutinin ORF Induces both Neutralizing Antibodies and Cytotoxic T Cell Responses

PubMed Central

Macchi, Francesca; Rojas, José Manuel; Verna, Andrea Elizabeth; Sevilla, Noemí; Franceschi, Valentina; Tebaldi, Giulia; Cavirani, Sandro; Martín, Verónica; Donofrio, Gaetano

2018-01-01

Peste des Petits Ruminants Virus (PPRV) is an extremely infective morbillivirus that primarily affects goats and sheep. In underdeveloped countries where livestock are the main economical resource, PPRV causes considerable economic losses. Protective live attenuated vaccines are currently available but they induce antibody responses similar to those produced in PPRV naturally infected animals. Effective vaccines able to distinguish between vaccinated and naturally infected animals are required to PPRV control and eradication programs. Hemagglutinin (H) is a highly immunogenic PPRV envelope glycoprotein displaying both hemagglutinin and neuraminidase activities, playing a crucial role in virus attachment and penetration. In this study, a recombinant Bovine Herpesvirus-4 (BoHV-4)-based vector delivering an optimized PPRV-Hemagglutinin expression cassette, BoHV-4-A-PPRV-H-ΔTK, was assessed in immunocompetent C57BL/6 mice. BoHV-4-A-PPRV-H-ΔTK-immunization elicited both cellular and humoral immune responses with specific T cell, cytotoxic T lymphocyte, and sero-neutralizing antibody against PPRV. These data suggest recombinant BoHV-4-A-PPRV-H-ΔTK as an effective vaccine candidate to protect against PPRV herd infection and potentially applicable for eradication programs. PMID:29556236

The natural dietary genistein boosts bacteriophage-mediated cancer cell killing by improving phage-targeted tumor cell transduction.

PubMed

Tsafa, Effrosyni; Al-Bahrani, Mariam; Bentayebi, Kaoutar; Przystal, Justyna; Suwan, Keittisak; Hajitou, Amin

2016-08-09

Gene therapy has long been regarded as a promising treatment for cancer. However, cancer gene therapy is still facing the challenge of targeting gene delivery vectors specifically to tumors when administered via clinically acceptable non-invasive systemic routes (i.e. intravenous). The bacteria virus, bacteriophage (phage), represents a new generation of promising vectors in systemic gene delivery since their targeting can be achieved through phage capsid display ligands, which enable them to home to specific tumor receptors without the need to ablate any native eukaryotic tropism. We have previously reported a tumor specific bacteriophage vector named adeno-associated virus/phage, or AAVP, in which gene expression is under a recombinant human rAAV2 virus genome targeted to tumors via a ligand-directed phage capsid. However, cancer gene therapy with this tumor-targeted vector achieved variable outcomes ranging from tumor regression to no effect in both experimental and natural preclinical models. Herein, we hypothesized that combining the natural dietary genistein, with proven anticancer activity, would improve bacteriophage anticancer safe therapy. We show that combination treatment with genistein and AAVP increased targeted cancer cell killing by AAVP carrying the gene for Herpes simplex virus thymidine kinase (HSVtk) in 2D tissue cultures and 3D tumor spheroids. We found this increased tumor cell killing was associated with enhanced AAVP-mediated gene expression. Next, we established that genistein protects AAVP against proteasome degradation and enhances vector genome accumulation in the nucleus. Combination of genistein and phage-guided virotherapy is a safe and promising strategy that should be considered in anticancer therapy with AAVP.

Development of canine herpesvirus based antifertility vaccines for foxes using bacterial artificial chromosomes.

PubMed

Strive, Tanja; Hardy, Christopher M; French, Nigel; Wright, John D; Nagaraja, Nitin; Reubel, Gerhard H

2006-02-13

Using bacterial artificial chromosome (BAC) technology, a canine herpesvirus (CHV)-based recombinant vaccine vector was produced for the development of an antifertility vaccine for foxes. Infectious viruses were recovered following transfection of canid cells with a BAC plasmid carrying the complete CHV genome. In vitro growth characteristics of BAC-derived viruses were similar to that of wildtype (wt)-CHV. Two recombinant antigens, fox zona pellucida protein subunit 3 (fZPC) and enhanced green fluorescent protein (EGFP) as control antigen, were inserted into thymidine kinase (TK) locus of the CHV genome and shown to be efficiently expressed in vitro. Inoculation of foxes with transgenic CHVs induced CHV specific antibodies, but was innocuous and failed to elicit transgene-specific antibody responses. Infectious virus or viral DNA was not detected in mucosal secretions or tissues of vaccinated foxes. The CHV-BAC system proved to be a quick and reliable method to manipulate the CHV genome. It will help to readily apply changes in the vector design in order to improve virus replication in vivo.

Development of a Newcastle disease virus vector expressing a foreign gene through an internal ribosomal entry site provides direct proof for a sequential transcription mechanism.

PubMed

Zhang, Zhenyu; Zhao, Wei; Li, Deshan; Yang, Jinlong; Zsak, Laszlo; Yu, Qingzhong

2015-08-01

In the present study, we developed a novel approach for foreign gene expression by Newcastle disease virus (NDV) from a second ORF through an internal ribosomal entry site (IRES). Six NDV LaSota strain-based recombinant viruses vectoring the IRES and a red fluorescence protein (RFP) gene behind the nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin-neuraminidase (HN) or large polymerase (L) gene ORF were generated using reverse genetics technology. The insertion of the second ORF slightly attenuated virus pathogenicity, but did not affect ability of the virus to grow. Quantitative measurements of RFP expression in virus-infected DF-1 cells revealed that the abundance of viral mRNAs and red fluorescence intensity were positively correlated with the gene order of NDV, 3'-NP-P-M-F-HN-L-5', proving the sequential transcription mechanism for NDV. The results herein suggest that the level of foreign gene expression could be regulated by selecting the second ORF insertion site to maximize the efficacy of vaccine and gene therapy.

[Adeno-associated viral vectors: methods for production and purification for gene therapy applications].

PubMed

Mena-Enriquez, Mayra; Flores-Contreras, Lucia; Armendáriz-Borunda, Juan

2012-01-01

Viral vectors based on adeno-associated virus (AAV) are widely used in gene therapy protocols, because they have characteristics that make them valuable for the treatment of genetic and chronic degenerative diseases. AAV2 serotype had been the best characterized to date. However, the AAV vectors developed from other serotypes is of special interest, since they have organ-specific tropism which increases their potential for transgene delivery to target cells for performing their therapeutic effects. This article summarizes AAV generalities, methods for their production and purification. It also discusses the use of these vectors in vitro, in vivo and their application in gene therapy clinical trials.

The Adenovirus Genome Contributes to the Structural Stability of the Virion

PubMed Central

Saha, Bratati; Wong, Carmen M.; Parks, Robin J.

2014-01-01

Adenovirus (Ad) vectors are currently the most commonly used platform for therapeutic gene delivery in human gene therapy clinical trials. Although these vectors are effective, many researchers seek to further improve the safety and efficacy of Ad-based vectors through detailed characterization of basic Ad biology relevant to its function as a vector system. Most Ad vectors are deleted of key, or all, viral protein coding sequences, which functions to not only prevent virus replication but also increase the cloning capacity of the vector for foreign DNA. However, radical modifications to the genome size significantly decreases virion stability, suggesting that the virus genome plays a role in maintaining the physical stability of the Ad virion. Indeed, a similar relationship between genome size and virion stability has been noted for many viruses. This review discusses the impact of the genome size on Ad virion stability and emphasizes the need to consider this aspect of virus biology in Ad-based vector design. PMID:25254384

The Human Immunodeficiency Virus Type 1 Vif Protein Reduces Intracellular Expression and Inhibits Packaging of APOBEC3G (CEM15), a Cellular Inhibitor of Virus Infectivity

PubMed Central

Kao, Sandra; Khan, Mohammad A.; Miyagi, Eri; Plishka, Ron; Buckler-White, Alicia; Strebel, Klaus

2003-01-01

Replication of human immunodeficiency virus type 1 (HIV-1) in most primary cells and some immortalized T-cell lines depends on the activity of the viral infectivity factor (Vif). Vif has the ability to counteract a cellular inhibitor, recently identified as CEM15, that blocks infectivity of Vif-defective HIV-1 variants. CEM15 is identical to APOBEC3G and belongs to a family of proteins involved in RNA and DNA deamination. We cloned APOBEC3G from a human kidney cDNA library and confirmed that the protein acts as a potent inhibitor of HIV replication and is sensitive to the activity of Vif. We found that wild-type Vif inhibits packaging of APOBEC3G into virus particles in a dose-dependent manner. In contrast, biologically inactive variants carrying in-frame deletions in various regions of Vif or mutation of two highly conserved cysteine residues did not inhibit packaging of APOBEC3G. Interestingly, expression of APOBEC3G in the presence of wild-type Vif not only affected viral packaging but also reduced its intracellular expression level. This effect was not seen in the presence of biologically inactive Vif variants. Pulse-chase analyses did not reveal a significant difference in the stability of APOBEC3G in the presence or absence of Vif. However, in the presence of Vif, the rate of synthesis of APOBEC3G was slightly reduced. The reduction of intracellular APOBEC3G in the presence of Vif does not fully account for the Vif-induced reduction of virus-associated APOBEC3G, suggesting that Vif may function at several levels to prevent packaging of APOBEC3G into virus particles. PMID:14557625

Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System

PubMed Central

Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M.

2015-01-01

Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health. PMID:26458221

Improved Production Efficiency of Virus-Like Particles by the Baculovirus Expression Vector System.

PubMed

López-Vidal, Javier; Gómez-Sebastián, Silvia; Bárcena, Juan; Nuñez, Maria del Carmen; Martínez-Alonso, Diego; Dudognon, Benoit; Guijarro, Eva; Escribano, José M

2015-01-01

Vaccines based on virus-like particles (VLPs) have proven effective in humans and animals. In this regard, the baculovirus expression vector system (BEVS) is one of the technologies of choice to generate such highly immunogenic vaccines. The extended use of these vaccines for human and animal populations is constrained because of high production costs, therefore a significant improvement in productivity is crucial to ensure their commercial viability. Here we describe the use of the previously described baculovirus expression cassette, called TB, to model the production of two VLP-forming vaccine antigens in insect cells. Capsid proteins from porcine circovirus type 2 (PCV2 Cap) and from the calicivirus that causes rabbit hemorrhagic disease (RHDV VP60) were expressed in insect cells using baculoviruses genetically engineered with the TB expression cassette. Productivity was compared to that obtained using standard counterpart vectors expressing the same proteins under the control of the polyhedrin promoter. Our results demonstrate that the use of the TB expression cassette increased the production yields of these vaccine antigens by around 300% with respect to the standard vectors. The recombinant proteins produced by TB-modified vectors were fully functional, forming VLPs identical in size and shape to those generated by the standard baculoviruses, as determined by electron microscopy analysis. The use of the TB expression cassette implies a simple modification of the baculovirus vectors that significantly improves the cost efficiency of VLP-based vaccine production, thereby facilitating the commercial viability and broad application of these vaccines for human and animal health.

Spatial and Temporal Analysis of Alphavirus Replication and Assembly in Mammalian and Mosquito Cells.

PubMed

Jose, Joyce; Taylor, Aaron B; Kuhn, Richard J

2017-02-14

Sindbis virus (SINV [genus Alphavirus , family Togaviridae ]) is an enveloped, mosquito-borne virus. Alphaviruses cause cytolytic infections in mammalian cells while establishing noncytopathic, persistent infections in mosquito cells. Mosquito vector adaptation of alphaviruses is a major factor in the transmission of epidemic strains of alphaviruses. Though extensive studies have been performed on infected mammalian cells, the morphological and structural elements of alphavirus replication and assembly remain poorly understood in mosquito cells. Here we used high-resolution live-cell imaging coupled with single-particle tracking and electron microscopy analyses to delineate steps in the alphavirus life cycle in both the mammalian host cell and insect vector cells. Use of dually labeled SINV in conjunction with cellular stains enabled us to simultaneously determine the spatial and temporal differences of alphavirus replication complexes (RCs) in mammalian and insect cells. We found that the nonstructural viral proteins and viral RNA in RCs exhibit distinct spatial organization in mosquito cytopathic vacuoles compared to replication organelles from mammalian cells. We show that SINV exploits filopodial extensions for virus dissemination in both cell types. Additionally, we propose a novel mechanism for replication complex formation around glycoprotein-containing vesicles in mosquito cells that produced internally released particles that were seen budding from the vesicles by live imaging. Finally, by characterizing mosquito cell lines that were persistently infected with fluorescent virus, we show that the replication and assembly machinery are highly modified, and this allows continuous production of alphaviruses at reduced levels. IMPORTANCE Reemerging mosquito-borne alphaviruses cause serious human epidemics worldwide. Several structural and imaging studies have helped to define the life cycle of alphaviruses in mammalian cells, but the mode of virus replication and assembly in the invertebrate vector and mechanisms producing two disease outcomes in two types of cells are yet to be identified. Using transmission electron microscopy and live-cell imaging with dual fluorescent protein-tagged SINV, we show that while insect and mammalian cells display similarities in entry and exit, they present distinct spatial and temporal organizations in virus replication and assembly. By characterizing acutely and persistently infected cells, we provide new insights into alphavirus replication and assembly in two distinct hosts, resulting in high-titer virus production in mammalian cells and continuous virus production at reduced levels in mosquito cells-presumably a prerequisite for alphavirus maintenance in nature. Copyright © 2017 Jose et al.

Foamy Virus Vector-mediated Gene Correction of a Mouse Model of Wiskott–Aldrich Syndrome

PubMed Central

Uchiyama, Toru; Adriani, Marsilio; Jagadeesh, G Jayashree; Paine, Adam; Candotti, Fabio

2012-01-01

The Wiskott–Aldrich syndrome (WAS) is an X-linked disorder characterized by eczema, thrombocytopenia and immunodeficiency. Hematopoietic cell transplantation can cure the disease and gene therapy is being tested as an alternative treatment option. In this study, we assessed the use of foamy virus (FV) vectors as a gene transfer system for WAS, using a Was knockout (KO) mouse model. Preliminary experiments using FV vectors expressing the green fluorescent protein under the transcriptional control of the endogenous WAS promoter or a ubiquitously acting chromatin opening element allowed us to define transduction conditions resulting in high (>40%) and long-term in-vivo marking of blood cells after transplantation. In following experiments, Was KO mice were treated with FV vectors containing the human WAS complementary DNA (cDNA). Transplanted animals expressed the WAS protein (WASp) in T and B lymphocytes, as well as platelets and showed restoration of both T-cell receptor-mediated responses and B-cell migration. We also observed recovery of platelet adhesion and podosome formation in dendritic cells (DCs) of treated mice. These data demonstrate that FV vectors can be effective for hematopoietic stem cell (HSC)-directed gene correction of WAS. PMID:22215016

Transduction of rat pancreatic islets with pseudotyped adeno-associated virus vectors

PubMed Central

Craig, Anthony T; Gavrilova, Oksana; Dwyer, Nancy K; Jou, William; Pack, Stephanie; Liu, Eric; Pechhold, Klaus; Schmidt, Michael; McAlister, Victor J; Chiorini, John A; Blanchette-Mackie, E Joan; Harlan, David M; Owens, Roland A

2009-01-01

Background Pancreatic islet transplantation is a promising treatment for type I diabetes mellitus, but current immunosuppressive strategies do not consistently provide long-term survival of transplanted islets. We are therefore investigating the use of adeno-associated viruses (AAVs) as gene therapy vectors to transduce rat islets with immunosuppressive genes prior to transplantation into diabetic mice. Results We compared the transduction efficiency of AAV2 vectors with an AAV2 capsid (AAV2/2) to AAV2 vectors pseudotyped with AAV5 (AAV2/5), AAV8 (AAV2/8) or bovine adeno-associated virus (BAAV) capsids, or an AAV2 capsid with an insertion of the low density lipoprotein receptor ligand from apolipoprotein E (AAV2apoE), on cultured islets, in the presence of helper adenovirus infection to speed expression of a GFP transgene. Confocal microscopy and flow cytometry were used. The AAV2/5 vector was superior to AAV2/2 and AAV2/8 in rat islets. Flow cytometry indicated AAV2/5-mediated gene expression in approximately 9% of rat islet cells and almost 12% of insulin-positive cells. The AAV2/8 vector had a higher dependence on the helper virus multiplicity of infection than the AAV 2/5 vector. In addition, the BAAV and AAV2apoE vectors were superior to AAV2/2 for transducing rat islets. Rat islets (300 per mouse) transduced with an AAV2/5 vector harboring the immunosuppressive transgene, tgfβ1, retain the ability to correct hyperglycemia when transplanted into immune-deficient diabetic mice. Conclusion AAV2/5 vectors may therefore be useful for pre-treating donor islets prior to transplantation. PMID:19450275

Engineering HSV-1 vectors for gene therapy.

PubMed

Goins, William F; Huang, Shaohua; Cohen, Justus B; Glorioso, Joseph C

2014-01-01

Virus vectors have been employed as gene transfer vehicles for various preclinical and clinical gene therapy applications, and with the approval of Glybera (alipogene tiparvovec) as the first gene therapy product as a standard medical treatment (Yla-Herttuala, Mol Ther 20: 1831-1832, 2013), gene therapy has reached the status of being a part of standard patient care. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing tumor cells have been used in Phase I-III human trials in patients with glioblastoma multiforme, a fatal form of brain cancer, and in malignant melanoma. In fact, T-VEC (talimogene laherparepvec, formerly known as OncoVex GM-CSF) displayed efficacy in a recent Phase III trial when compared to standard GM-CSF treatment alone (Andtbacka et al. J Clin Oncol 31: sLBA9008, 2013) and may soon become the second FDA-approved gene therapy product used in standard patient care. In addition to the replication-competent oncolytic HSV vectors like T-VEC, replication-defective HSV vectors have been employed in Phase I-II human trials and have been explored as delivery vehicles for disorders such as pain, neuropathy, and other neurodegenerative conditions. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are totally replication defective, nontoxic, and capable of long-term transgene expression in neurons. This chapter describes methods for the construction of recombinant genomic HSV vectors based on the HSV-1 replication-defective vector backbones, steps in their purification, and their small-scale production for use in cell culture experiments as well as preclinical animal studies.

Inhibition of West Nile Virus replication by retrovirus-delivered small interfering RNA in human neuroblastoma cells.

PubMed

Yang, Yongbo; Wu, Chengxiang; Wu, Jianguo; Nerurkar, Vivek R; Yanagihara, Richard; Lu, Yuanan

2008-05-01

West Nile virus (WNV) has been responsible for the largest outbreaks of arboviral encephalitis in U.S. history. No specific drug is currently available for the effective treatment of WNV infection. To exploit RNA interference as a potential therapeutic approach, a Moloney murine leukemia virus-based retrovirus vector was used to effectively deliver WNV-specific small interfering RNA (siRNA) into human neuroblastoma HTB-11 cells. Viral plaque assays demonstrated that transduced cells were significantly refractory to WNV replication, as compared to untransduced control cells (P < 0.05), which correlated with the reduced expression of target viral genes and respective viral proteins. Therefore, retrovirus-mediated delivery of siRNA for gene silencing can be used to study the specific functions of viral genes associated with replication and may have potential therapeutic applications.

Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications.

PubMed

Strods, Arnis; Ose, Velta; Bogans, Janis; Cielens, Indulis; Kalnins, Gints; Radovica, Ilze; Kazaks, Andris; Pumpens, Paul; Renhofa, Regina

2015-06-26

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.

Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications

PubMed Central

Strods, Arnis; Ose, Velta; Bogans, Janis; Cielens, Indulis; Kalnins, Gints; Radovica, Ilze; Kazaks, Andris; Pumpens, Paul; Renhofa, Regina

2015-01-01

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications. PMID:26113394

Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications

NASA Astrophysics Data System (ADS)

Strods, Arnis; Ose, Velta; Bogans, Janis; Cielens, Indulis; Kalnins, Gints; Radovica, Ilze; Kazaks, Andris; Pumpens, Paul; Renhofa, Regina

2015-06-01

Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.

Double-stranded RNA transcribed from vector-based oligodeoxynucleotide acts as transcription factor decoy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xiao, Xiao; Gang, Yi; Department of Infectious Diseases, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, Shaanxi Province

2015-02-06

Highlights: • A shRNA vector based transcription factor decoy, VB-ODN, was designed. • VB-ODN for NF-κB inhibited cell viability in HEK293 cells. • VB-ODN inhibited expression of downstream genes of target transcription factors. • VB-ODN may enhance nuclear entry ratio for its feasibility of virus production. - Abstract: In this study, we designed a short hairpin RNA vector-based oligodeoxynucleotide (VB-ODN) carrying transcription factor (TF) consensus sequence which could function as a decoy to block TF activity. Specifically, VB-ODN for Nuclear factor-κB (NF-κB) could inhibit cell viability and decrease downstream gene expression in HEK293 cells without affecting expression of NF-κB itself.more » The specific binding between VB-ODN produced double-stranded RNA and NF-κB was evidenced by electrophoretic mobility shift assay. Moreover, similar VB-ODNs designed for three other TFs also inhibit their downstream gene expression but not that of themselves. Our study provides a new design of decoy for blocking TF activity.« less

Design and construction of targeted AAVP vectors for mammalian cell transduction.

PubMed

Hajitou, Amin; Rangel, Roberto; Trepel, Martin; Soghomonyan, Suren; Gelovani, Juri G; Alauddin, Mian M; Pasqualini, Renata; Arap, Wadih

2007-01-01

Bacteriophage (phage) evolved as bacterial viruses, but can be adapted to transduce mammalian cells through ligand-directed targeting to a specific receptor. We have recently reported a new generation of hybrid prokaryotic-eukaryotic vectors, which are chimeras of genetic cis-elements of recombinant adeno-associated virus and phage (termed AAVP). This protocol describes the design and construction of ligand-directed AAVP vectors, production of AAVP particles and the methodology to transduce mammalian cells in vitro and to target tissues in vivo after systemic administration. Targeted AAVP particles are made in a two-step process. First, a ligand peptide of choice is displayed on the coat protein to generate a targeted backbone phage vector. Then, a recombinant AAV carrying a mammalian transgene cassette is inserted into an intergenomic region. High-titer suspensions (approximately 10(10)-10(11) transducing units per microl) can be produced within 3 days after vector construction. Transgene expression by targeted AAVP usually reaches maximum levels within 1 week.

Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs

PubMed Central

Jiang, Xiaohong; Dalebout, Tim J.; Bredenbeek, Peter J.; Carrion, Ricardo; Brasky, Kathleen; Patterson, Jean; Goicochea, Marco; Bryant, Joseph; Salvato, Maria S.; Lukashevich, Igor S.

2010-01-01

Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV GP1 and GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and –GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF and LASV GP proteins in infected cells. YF17D/LASV-GP1&GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1&GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa. PMID:21145373

Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection

PubMed Central

Millet, Jean Kaoru; Whittaker, Gary R.

2016-01-01

Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors. The basis for their generation lies in the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins of other viruses into a pseudotyped virus particle. These can be engineered to contain reporter genes such as luciferase, enabling quantification of virus entry events upon pseudotyped particle infection with susceptible cells. Here, we detail a protocol enabling generation of MLV-based pseudotyped particles, using the Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) as an example of a heterologous envelope glycoprotein to be incorporated. We also describe how these particles are used to infect susceptible cells and to perform a quantitative infectivity readout by a luciferase assay. PMID:28018942

Adeno-associated virus vectors can be efficiently produced without helper virus.

PubMed

Matsushita, T; Elliger, S; Elliger, C; Podsakoff, G; Villarreal, L; Kurtzman, G J; Iwaki, Y; Colosi, P

1998-07-01

The purpose of this work was to develop an efficient method for the production of adeno-associated virus (AAV) vectors in the absence of helper virus. The adenovirus regions that mediate AAV vector replication were identified and assembled into a helper plasmid. These included the VA, E2A and E4 regions. When this helper plasmid was cotransfected into 293 cells, along with plasmids encoding the AAV vector, and rep and cap genes, AAV vector was produced as efficiently as when using adenovirus infection as a source of help. CMV-driven constructs expressing the E4orf6 and the 72-M(r), E2A proteins were able to functionally replace the E4 and E2A regions, respectively. Therefore the minimum set of genes required to produce AAV helper activity equivalent to that provided by adenovirus infection consists of, or is a subset of, the following genes: the E4orf6 gene, the 72-M(r), E2A protein gene, the VA RNA genes and the E1 region. AAV vector preparations made with adenovirus and by the helper virus-free method were essentially indistinguishable with respect to particle density, particle to infectivity ratio, capsimer ratio and efficiency of muscle transduction in vivo. Only AAV vector preparations made by the helper virus-free method were not reactive with anti-adenovirus sera.

Immunogenicity and efficacy of a chimpanzee adenovirus-vectored Rift Valley fever vaccine in mice.

PubMed

Warimwe, George M; Lorenzo, Gema; Lopez-Gil, Elena; Reyes-Sandoval, Arturo; Cottingham, Matthew G; Spencer, Alexandra J; Collins, Katharine A; Dicks, Matthew D J; Milicic, Anita; Lall, Amar; Furze, Julie; Turner, Alison V; Hill, Adrian V S; Brun, Alejandro; Gilbert, Sarah C

2013-12-05

Rift Valley Fever (RVF) is a viral zoonosis that historically affects livestock production and human health in sub-Saharan Africa, though epizootics have also occurred in the Arabian Peninsula. Whilst an effective live-attenuated vaccine is available for livestock, there is currently no licensed human RVF vaccine. Replication-deficient chimpanzee adenovirus (ChAd) vectors are an ideal platform for development of a human RVF vaccine, given the low prevalence of neutralizing antibodies against them in the human population, and their excellent safety and immunogenicity profile in human clinical trials of vaccines against a wide range of pathogens. Here, in BALB/c mice, we evaluated the immunogenicity and efficacy of a replication-deficient chimpanzee adenovirus vector, ChAdOx1, encoding the RVF virus envelope glycoproteins, Gn and Gc, which are targets of virus neutralizing antibodies. The ChAdOx1-GnGc vaccine was assessed in comparison to a replication-deficient human adenovirus type 5 vector encoding Gn and Gc (HAdV5-GnGc), a strategy previously shown to confer protective immunity against RVF in mice. A single immunization with either of the vaccines conferred protection against RVF virus challenge eight weeks post-immunization. Both vaccines elicited RVF virus neutralizing antibody and a robust CD8+ T cell response. Together the results support further development of RVF vaccines based on replication-deficient adenovirus vectors, with ChAdOx1-GnGc being a potential candidate for use in future human clinical trials.

HIV-1-based defective lentiviral vectors efficiently transduce human monocytes-derived macrophages and suppress replication of wild-type HIV-1

PubMed Central

Zeng, Lingbing; Planelles, Vicente; Sui, Ziye; Gartner, Suzanne; Maggirwar, Sanjay B.; Dewhurst, Stephen; Ye, Linbai; Nerurkar, Vivek R.; Yanagihara, Richard; Lu, Yuanan

2010-01-01

Background Human monocytes play an important role in mediating human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS), and monocytes-derived macrophages (MDM) represent a major viral reservoir within the brain and other target organs. Current gene transduction of MDM is hindered by a limited efficiency. In this study we established a lentiviral vector-based technique for improved gene transfer into human MDM cultures in vitro and demonstrated significant protection of transduced MDM from super-infection with wild-type HIV-1. Methods HIV-1-based lentiviral vector stocks were prepared in 293T cells by the established calcium phosphate transfection method. Human monocytes were isolated from donors' blood by Ficoll-Paque separation and cultured in vitro. To establish an effective technique for vector-mediated gene transfer, primary cultures of human MDM were transduced at varying multiplicities of infection (MOI) and at a range of time points following initial isolation of cells (time-in-culture). Transduced cells were then examined for transgene (green fluorescent protein (GFP)) expression by fluorescent microscopy and reverse transcription polymerase chain reaction (RT-PCR). These cultures were then exposed to wild-type HIV-1, and viral replication was quantitated by p24 assay; production of neurotoxic effector molecules by the transduced MDM was also examined, using indicator neurons. Results We have demonstrated that primary human MDM could be efficiently transduced (>50%) with concentrated HIV-1-based defective lentiviral vectors (DLV). Furthermore, DLV-mediated gene transduction was stable, and the transduced cells exhibited no apparent difference from normal MDM in terms of their morphology, viability and neurotoxin secretion. Challenge of DLV-transduced MDM cultures with HIV-1Ba-L revealed a 4- to 5-fold reduction in viral replication, as measured by p24 antigen production. This effect was associated with the mobilization of the GFP-expressing DLV construct by the wild-type virus. Conclusions These data demonstrate the inhibition of HIV-1 replication in primary MDM, by a DLV vector that lacks any anti-HIV-1 transgene. These findings lay the initial groundwork for future studies on the ability of DLV-modified monocytes to introduce anti-HIV-1 genes into the CNS. Lentiviral vector-mediated gene delivery to the CNS by monocytes/macrophages is a promising, emerging strategy for treating neuro-AIDS. PMID:16142830

Amplified and persistent immune responses generated by single-cycle replicating adenovirus vaccines.

PubMed

Crosby, Catherine M; Nehete, Pramod; Sastry, K Jagannadha; Barry, Michael A

2015-01-01

Replication-competent adenoviral (RC-Ad) vectors generate exceptionally strong gene-based vaccine responses by amplifying the antigen transgenes they carry. While they are potent, they also risk causing adenovirus infections. More common replication-defective Ad (RD-Ad) vectors with deletions of E1 avoid this risk but do not replicate their transgene and generate markedly weaker vaccine responses. To amplify vaccine transgenes while avoiding production of infectious progeny viruses, we engineered "single-cycle" adenovirus (SC-Ad) vectors by deleting the gene for IIIa capsid cement protein of lower-seroprevalence adenovirus serotype 6. In mouse, human, hamster, and macaque cells, SC-Ad6 still replicated its genome but prevented genome packaging and virion maturation. When used for mucosal intranasal immunization of Syrian hamsters, both SC-Ad and RC-Ad expressed transgenes at levels hundreds of times higher than that of RD-Ad. Surprisingly, SC-Ad, but not RC-Ad, generated higher levels of transgene-specific antibody than RD-Ad, which notably climbed in serum and vaginal wash samples over 12 weeks after single mucosal immunization. When RD-Ad and SC-Ad were tested by single sublingual immunization in rhesus macaques, SC-Ad generated higher gamma interferon (IFN-γ) responses and higher transgene-specific serum antibody levels. These data suggest that SC-Ad vectors may have utility as mucosal vaccines. This work illustrates the utility of our recently developed single-cycle adenovirus (SC-Ad6) vector as a new vaccine platform. Replication-defective (RD-Ad6) vectors produce low levels of transgene protein, which leads to minimal antibody responses in vivo. This study shows that replicating SC-Ad6 produces higher levels of luciferase and induces higher levels of green fluorescent protein (GFP)-specific antibodies than RD in a permissive Syrian hamster model. Surprisingly, although a replication-competent (RC-Ad6) vector produces more luciferase than SC-Ad6, it does not elicit comparable levels of anti-GFP antibodies in permissive hamsters. When tested in the larger rhesus macaque model, SC-Ad6 induces higher transgene-specific antibody and T cell responses. Together, these data suggest that SC-Ad6 could be a more effective platform for developing vaccines against more relevant antigens. This could be especially beneficial for developing vaccines for pathogens for which traditional replication-defective adenovirus vectors have not been effective. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Sequence of retrovirus provirus resembles that of bacterial transposable elements

NASA Astrophysics Data System (ADS)

Shimotohno, Kunitada; Mizutani, Satoshi; Temin, Howard M.

1980-06-01

The nucleotide sequences of the terminal regions of an infectious integrated retrovirus cloned in the modified λ phage cloning vector Charon 4A have been elucidated. There is a 569-base pair direct repeat at both ends of the viral DNA. The cell-virus junctions at each end consist of a 5-base pair direct repeat of cell DNA next to a 3-base pair inverted repeat of viral DNA. This structure resembles that of a transposable element and is consistent with the protovirus hypothesis that retroviruses evolved from the cell genome.

Adenovirus-Vectored Broadly Neutralizing Antibodies Directed Against gp120 Prevent Human Immunodeficiency Virus Type 1 Acquisition in Humanized Mice.

PubMed

Liu, Shan; Jackson, Andrew; Beloor, Jagadish; Kumar, Priti; Sutton, Richard E

2015-09-01

Despite nearly three decades of research, a safe and effective vaccine against human immunodeficiency virus type 1 (HIV-1) has yet to be achieved. More recently, the discovery of highly potent anti-gp160 broadly neutralizing antibodies (bNAbs) has garnered renewed interest in using antibody-based prophylactic and therapeutic approaches. Here, we encoded bNAbs in first-generation adenoviral (ADV) vectors, which have the distinctive features of a large coding capacity and ease of propagation. A single intramuscular injection of ADV-vectorized bNAbs in humanized mice generated high serum levels of bNAbs that provided protection against multiple repeated challenges with a high dose of HIV-1, prevented depletion of peripheral CD4(+) T cells, and reduced plasma viral loads to below detection limits. Our results suggest that ADV vectors may be a viable option for the prophylactic and perhaps therapeutic use of bNAbs in humans.

Engineering adeno-associated virus 2 vectors for targeted gene delivery to atherosclerotic lesions.

PubMed

White, K; Büning, H; Kritz, A; Janicki, H; McVey, J; Perabo, L; Murphy, G; Odenthal, M; Work, L M; Hallek, M; Nicklin, S A; Baker, A H

2008-03-01

Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.

Toward exascale production of recombinant adeno-associated virus for gene transfer applications.

PubMed

Cecchini, S; Negrete, A; Kotin, R M

2008-06-01

To gain acceptance as a medical treatment, adeno-associated virus (AAV) vectors require a scalable and economical production method. Recent developments indicate that recombinant AAV (rAAV) production in insect cells is compatible with current good manufacturing practice production on an industrial scale. This platform can fully support development of rAAV therapeutics from tissue culture to small animal models, to large animal models, to toxicology studies, to Phase I clinical trials and beyond. Efforts to characterize, optimize and develop insect cell-based rAAV production have culminated in successful bioreactor-scale production of rAAV, with total yields potentially capable of approaching the exa-(10(18)) scale. These advances in large-scale AAV production will allow us to address specific catastrophic, intractable human diseases such as Duchenne muscular dystrophy, for which large amounts of recombinant vector are essential for successful outcome.

Virus Database and Online Inquiry System Based on Natural Vectors.

PubMed

Dong, Rui; Zheng, Hui; Tian, Kun; Yau, Shek-Chung; Mao, Weiguang; Yu, Wenping; Yin, Changchuan; Yu, Chenglong; He, Rong Lucy; Yang, Jie; Yau, Stephen St

2017-01-01

We construct a virus database called VirusDB (http://yaulab.math.tsinghua.edu.cn/VirusDB/) and an online inquiry system to serve people who are interested in viral classification and prediction. The database stores all viral genomes, their corresponding natural vectors, and the classification information of the single/multiple-segmented viral reference sequences downloaded from National Center for Biotechnology Information. The online inquiry system serves the purpose of computing natural vectors and their distances based on submitted genomes, providing an online interface for accessing and using the database for viral classification and prediction, and back-end processes for automatic and manual updating of database content to synchronize with GenBank. Submitted genomes data in FASTA format will be carried out and the prediction results with 5 closest neighbors and their classifications will be returned by email. Considering the one-to-one correspondence between sequence and natural vector, time efficiency, and high accuracy, natural vector is a significant advance compared with alignment methods, which makes VirusDB a useful database in further research.

Coated microneedle arrays for transcutaneous delivery of live virus vaccines

PubMed Central

Vrdoljak, Anto; McGrath, Marie G.; Carey, John B.; Draper, Simon J.; Hill, Adrian V.S.; O’Mahony, Conor; Crean, Abina M.; Moore, Anne C.

2016-01-01

Vaccines are sensitive biologics that require continuous refrigerated storage to maintain their viability. The vast majority of vaccines are also administered using needles and syringes. The need for cold chain storage and the significant logistics surrounding needle-and-syringe vaccination is constraining the success of immunization programs. Recombinant live viral vectors are a promising platform for the development of vaccines against a number of infectious diseases, however these viruses must retain infectivity to be effective. Microneedles offer an effective and painless method for delivery of vaccines directly into skin that in the future could provide solutions to current vaccination issues. Here we investigated methods of coating live recombinant adenovirus and modified vaccinia virus Ankara (MVA) vectors onto solid microneedle arrays. An effective spray-coating method, using conventional pharmaceutical processes, was developed, in tandem with suitable sugar-based formulations, which produces arrays with a unique coating of viable virus in a dry form around the shaft of each microneedle on the array. Administration of live virus-coated microneedle arrays successfully resulted in virus delivery, transcutaneous infection and induced an antibody or CD8+ T cell response in mice that was comparable to that obtained by needle-and-syringe intradermal immunization. To our knowledge, this is the first report of successful vaccination with recombinant live viral vectored vaccines coated on microneedle delivery devices. PMID:22245683

Coated microneedle arrays for transcutaneous delivery of live virus vaccines.

PubMed

Vrdoljak, Anto; McGrath, Marie G; Carey, John B; Draper, Simon J; Hill, Adrian V S; O'Mahony, Conor; Crean, Abina M; Moore, Anne C

2012-04-10

Vaccines are sensitive biologics that require continuous refrigerated storage to maintain their viability. The vast majority of vaccines are also administered using needles and syringes. The need for cold chain storage and the significant logistics surrounding needle-and-syringe vaccination is constraining the success of immunization programs. Recombinant live viral vectors are a promising platform for the development of vaccines against a number of infectious diseases, however these viruses must retain infectivity to be effective. Microneedles offer an effective and painless method for delivery of vaccines directly into skin that in the future could provide solutions to current vaccination issues. Here we investigated methods of coating live recombinant adenovirus and modified vaccinia virus Ankara (MVA) vectors onto solid microneedle arrays. An effective spray-coating method, using conventional pharmaceutical processes, was developed, in tandem with suitable sugar-based formulations, which produces arrays with a unique coating of viable virus in a dry form around the shaft of each microneedle on the array. Administration of live virus-coated microneedle arrays successfully resulted in virus delivery, transcutaneous infection and induced an antibody or CD8(+) T cell response in mice that was comparable to that obtained by needle-and-syringe intradermal immunization. To our knowledge, this is the first report of successful vaccination with recombinant live viral vectored vaccines coated on microneedle delivery devices. Copyright © 2011 Elsevier B.V. All rights reserved.

Herpes Simplex Virus DNA Packaging without Measurable DNA Synthesis

PubMed Central

Church, Geoffrey A.; Dasgupta, Anindya; Wilson, Duncan W.

1998-01-01

Herpes simplex virus (HSV) type 1 DNA synthesis and packaging occur within the nuclei of infected cells; however, the extent to which the two processes are coupled remains unclear. Correct packaging is thought to be dependent upon DNA debranching or other repair processes, and such events commonly involve new DNA synthesis. Furthermore, the HSV UL15 gene product, essential for packaging, nevertheless localizes to sites of active DNA replication and may link the two events. It has previously been difficult to determine whether packaging requires concomitant DNA synthesis due to the complexity of these processes and of the viral life cycle; however, we have recently described a model system which simplifies the study of HSV assembly. Cells infected with HSV strain tsProt.A accumulate unpackaged capsids at the nonpermissive temperature of 39°C. Following release of the temperature block, these capsids proceed to package viral DNA in a single, synchronous wave. Here we report that, when DNA replication was inhibited prior to release of the temperature block, DNA packaging and later events in viral assembly nevertheless occurred at near-normal levels. We conclude that, under our conditions, HSV DNA packaging does not require detectable levels of DNA synthesis. PMID:9525593

Recombinant Measles AIK-C Vaccine Strain Expressing the prM-E Antigen of Japanese Encephalitis Virus.

PubMed

Higuchi, Akira; Toriniwa, Hiroko; Komiya, Tomoyoshi; Nakayama, Tetsuo

2016-01-01

An inactivated Japanese encephalitis virus (JEV) vaccine, which induces neutralizing antibodies, has been used for many years in Japan. In the present study, the JEV prM-E protein gene was cloned, inserted at the P/M junction of measles AIK-C cDNA, and an infectious virus was recovered. The JEV E protein was expressed in B95a cells infected with the recombinant virus. Cotton rats were inoculated with recombinant virus. Measles PA antibodies were detected three weeks after immunization. Neutralizing antibodies against JEV developed one week after inoculation, and EIA antibodies were detected three weeks after immunization. The measles AIK-C-based recombinant virus simultaneously induced measles and JEV immune responses, and may be a candidate for infant vaccines. Therefore, the present strategy of recombinant viruses based on a measles vaccine vector would be applicable to the platform for vaccine development.

The next step in gene delivery: molecular engineering of adeno-associated virus serotypes.

PubMed

Wang, Jinhui; Faust, Susan M; Rabinowitz, Joseph E

2011-05-01

Delivery is at the heart of gene therapy. Viral DNA delivery systems are asked to avoid the immune system, transduce specific target cell types while avoiding other cell types, infect dividing and non-dividing cells, insert their cargo within the host genome without mutagenesis or to remain episomal, and efficiently express transgenes for a substantial portion of a lifespan. These sought-after features cannot be associated with a single delivery system, or can they? The Adeno-associated virus family of gene delivery vehicles has proven to be highly malleable. Pseudotyping, using AAV serotype 2 terminal repeats to generate designer shells capable of transducing selected cell types, enables the packaging of common genomes into multiple serotypes virions to directly compare gene expression and tropism. In this review the ability to manipulate this virus will be examined from the inside out. The influence of host cell factors and organism biology including the immune response on the molecular fate of the viral genome will be discussed as well as differences in cellular trafficking patterns and uncoating properties that influence serotype transduction. Re-engineering the prototype vector AAV2 using epitope insertion, chemical modification, and molecular evolution not only demonstrated the flexibility of the best-studied serotype, but now also expanded the tool kit for molecular modification of all AAV serotypes. Current AAV research has changed its focus from examination of wild-type AAV biology to the feedback of host cell/organism on the design and development of a new generation of recombinant AAV delivery vehicles. This article is part of a Special Section entitled "Special Section: Cardiovascular Gene Therapy". Copyright © 2010 Elsevier Ltd. All rights reserved.

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences

PubMed Central

Kalloush, Rawan M.; Vivet-Boudou, Valérie; Ali, Lizna M.; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A.

2016-01-01

MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2′hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5′ region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. PMID:27095024

Disabled infectious single cycle-herpes simplex virus (DISC-HSV) as a vector for immunogene therapy of cancer.

PubMed

Rees, Robert C; McArdle, Stephanie; Mian, Shahid; Li, Geng; Ahmad, Murrium; Parkinson, Richard; Ali, Selman A

2002-02-01

Disabled infectious single cycle-herpes simplex viruses (DISC-HSV) have been shown to be safe for use in humans and may be considered efficacious as vectors for immunogene therapy in cancer. Preclinical studies show that DISC-HSV is an efficient delivery system for cytokine genes and antigens. DISC-HSV infects a high proportion of cells, resulting in rapid gene expression for at least 72 h. The DISC-HSV-mGM-CSF vector, when inoculated into tumors, induces tumor regression in a high percentage of animals, concomitant with establishing a cytotoxic T-cell response, which is MHC class I restricted and directed against peptides of known tumor antigens. The inherent properties of DISC-HSV makes it a suitable vector for consideration in human immunogene therapy trials.

Self-assembled pentablock copolymers for selective and sustained gene delivery

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Bingqi

2011-05-15

The poly(diethylaminoethyl methacrylate) (PDEAEM) - Pluronic F127 - PDEAEM pentablock copolymer (PB) gene delivery vector system has been found to possess an inherent selectivity in transfecting cancer cells over non-cancer cells in vitro, without attaching any targeting ligands. In order to understand the mechanism of this selective transfection, three possible intracellular barriers to transfection were investigated in both cancer and non-cancer cells. We concluded that escape from the endocytic pathway served as the primary intracellular barrier for PB-mediated transfection. Most likely, PB vectors were entrapped and rendered non-functional in acidic lysosomes of non-cancer cells, but survived in less acidic lysosomesmore » of cancer cells. The work highlights the importance of identifying intracellular barriers for different gene delivery systems and provides a new paradigm for designing targeting vectors based on intracellular differences between cell types, rather than through the use of targeting ligands. The PB vector was further developed to simultaneously deliver anticancer drugs and genes, which showed a synergistic effect demonstrated by significantly enhanced gene expression in vitro. Due to the thermosensitive gelation behavior, the PB vector packaging both drug and gene was also investigated for its in vitro sustained release properties by using polyethylene glycol diacrylate as a barrier gel to mimic the tumor matrix in vivo. Overall, this work resulted in the development of a gene delivery vector for sustained and selective gene delivery to tumor cells for cancer therapy.« less

Artificial Virus as Trump-card to Resolve Exigencies in Targeted Gene Delivery.

PubMed

Ajithkumar, K C; Pramod, Kannissery

2018-01-01

Viruses are potent pathogens that can effectively deliver the genetic material to susceptible host cells. This capability is beneficially utilized to successfully deliver the genetic material. However, the use of virus mediated gene delivery is considered divisive, because the potentially replicable genomes recombine or integrate with the cell DNA resulting in immunogenicity, ranging from inflammation to death. Thus, the need for potentially effective non-viral gene delivery vehicles arises. Non-viral vectors, protein only particles and virus like particles (VLP) can be constructed which contain all the necessary functional moieties. These resemble viruses and are called artificial or synthetic virus. The artificial virus eliminates the disadvantages of viral vectors but retain the beneficial effects of the viruses. Need for further functionalization can be avoided by this approach because incorporation of requisite agents such as cell ligands, membrane active peptides, etc. into proteins is possible. The protein- DNA complexes resemble bacterial inclusion bodies. Nucleic acids influence conformation of protein units which subsequently result in cell uptake and finally to the cell nucleus. Such tunable systems mimic the activities of infected viruses and are used for the safe and effective delivery of drugs and genetic material in gene therapy. The versatility, stability and biocompatible nature of artificial virus along with high transfection efficacy have made it favorite for gene delivery purposes, in addition to being useful for various biomedical and drug delivery applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Production of transgenic Korean native cattle expressing enhanced green fluorescent protein using a FIV-based lentiviral vector injected into MII oocytes.

PubMed

Xu, Yong-Nan; Uhm, Sang-Jun; Koo, Bon-Chul; Kwon, Mo-Sun; Roh, Ji-Yeol; Yang, Jung-Seok; Choi, Hyun-Yong; Heo, Young-Tae; Cui, Xiang-Shun; Yoon, Joon-Ho; Ko, Dae-Hwan; Kim, Teoan; Kim, Nam-Hyung

2013-01-20

The potential benefits of generating and using transgenic cattle range from improvements in agriculture to the production of large quantities of pharmaceutically relevant proteins. Previous studies have attempted to produce transgenic cattle and other livestock by pronuclear injection and somatic cell nuclear transfer, but these approaches have been largely ineffective; however, a third approach, lentivirus-mediated transgenesis, has successfully produced transgenic livestock. In this study, we generated transgenic (TG) Korean native cattle using perivitelline space injection of viral vectors, which expressed enhanced green fluorescent protein (EGFP) systemically. Two different types of lentiviral vectors derived from feline immunodeficiency virus (FIV) and human immunodeficiency virus (HIV) carrying EGFP were injected into the perivitelline space of MII oocytes. EGFP expression at 8-cell stage was significantly higher in the FIV group compared to the HIV group (47.5%±2.2% v.s. 22.9%±2.9%). Eight-cell embryos that expressed EGFP were cultured into blastocysts and then transferred into 40 heifers. Ten heifers were successfully impregnated and delivered 10 healthy calves. All of these calves expressed EGFP as detected by in vivo imaging, PCR and Southern blotting. In addition, we established an EGFP-expressing cell line from TG calves, which was followed by nuclear transfer (NT). Recloned 8-cell embryos also expressed EGFP, and there were no differences in the rates of fusion, cleavage and development between cells derived from TG and non-TG calves, which were subsequently used for NT. These results illustrate that FIV-based lentiviruses are useful for the production of TG cattle. Moreover, our established EGFP cell line can be used for additional studies that involve induced pluripotent stem cells. Copyright © 2013. Published by Elsevier Ltd.

A plasmid-based reverse genetics system for influenza A virus.

PubMed Central

Pleschka, S; Jaskunas, R; Engelhardt, O G; Zürcher, T; Palese, P; García-Sastre, A

1996-01-01

A reverse genetics system for negative-strand RNA viruses was first successfully developed for influenza viruses. This technology involved the transfection of in vitro-reconstituted ribonucleoprotein (RNP) complexes into influenza virus-infected cells. We have now developed a method that allows intracellular reconstitution of RNP complexes from plasmid-based expression vectors. Expression of a viral RNA-like transcript is achieved from a plasmid containing a truncated human polymerase I (polI) promoter and a ribozyme sequence that generates the desired 3' end by autocatalytic cleavage. The polI-driven plasmid is cotransfected into human 293 cells with polII-responsive plasmids that express the viral PB1, PB2, PA, and NP proteins. This exclusively plasmid-driven system results in the efficient transcription and replication of the viral RNA-like reporter and allows the study of cis- and trans-acting signals involved in the transcription and replication of influenza virus RNAs. Using this system, we have also been able to rescue a synthetic neuraminidase gene into a recombinant influenza virus. This method represents a convenient alternative to the previously established RNP transfection system. PMID:8648766

Initial viral load determines the magnitude of the human CD8 T cell response to yellow fever vaccination.

PubMed

Akondy, Rama S; Johnson, Philip L F; Nakaya, Helder I; Edupuganti, Srilatha; Mulligan, Mark J; Lawson, Benton; Miller, Joseph D; Pulendran, Bali; Antia, Rustom; Ahmed, Rafi

2015-03-10

CD8 T cells are a potent tool for eliminating intracellular pathogens and tumor cells. Thus, eliciting robust CD8 T-cell immunity is the basis for many vaccines under development. However, the relationship between antigen load and the magnitude of the CD8 T-cell response is not well-described in a human immune response. Here we address this issue by quantifying viral load and the CD8 T-cell response in a cohort of 80 individuals immunized with the live attenuated yellow fever vaccine (YFV-17D) by sampling peripheral blood at days 0, 1, 2, 3, 5, 7, 9, 11, 14, 30, and 90. When the virus load was below a threshold (peak virus load < 225 genomes per mL, or integrated virus load < 400 genome days per mL), the magnitude of the CD8 T-cell response correlated strongly with the virus load (R(2) ∼ 0.63). As the virus load increased above this threshold, the magnitude of the CD8 T-cell responses saturated. Recent advances in CD8 T-cell-based vaccines have focused on replication-incompetent or single-cycle vectors. However, these approaches deliver relatively limited amounts of antigen after immunization. Our results highlight the requirement that T-cell-based vaccines should deliver sufficient antigen during the initial period of the immune response to elicit a large number of CD8 T cells that may be needed for protection.

Formation of newly synthesized adeno-associated virus capsids in the cell nucleus.

PubMed

Bell, Peter; Vandenberghe, Luk H; Wilson, James M

2014-06-01

Adeno-associated virus (AAV) particles inside the nucleus of a HEK 293 cell are shown by electron microscopy. Cells have been triple-transfected for vector production and were analyzed for capsid formation three days later. Newly assembled particle are visible as seemingly unstructured conglomerates or crystal-like arrays.

A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles.

PubMed

Thuenemann, Eva C; Meyers, Ann E; Verwey, Jeanette; Rybicki, Edward P; Lomonossoff, George P

2013-09-01

Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Leucine-rich repeat-containing G protein-coupled receptor 4 facilitates vesicular stomatitis virus infection by binding vesicular stomatitis virus glycoprotein.

PubMed

Zhang, Na; Huang, Hongjun; Tan, Binghe; Wei, Yinglei; Xiong, Qingqing; Yan, Yan; Hou, Lili; Wu, Nannan; Siwko, Stefan; Cimarelli, Andrea; Xu, Jianrong; Han, Honghui; Qian, Min; Liu, Mingyao; Du, Bing

2017-10-06

Vesicular stomatitis virus (VSV) and rabies and Chandipura viruses belong to the Rhabdovirus family. VSV is a common laboratory virus to study viral evolution and host immune responses to viral infection, and recombinant VSV-based vectors have been widely used for viral oncolysis, vaccination, and gene therapy. Although the tropism of VSV is broad, and its envelope glycoprotein G is often used for pseudotyping other viruses, the host cellular components involved in VSV infection remain unclear. Here, we demonstrate that the host protein leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is essential for VSV and VSV-G pseudotyped lentivirus (VSVG-LV) to infect susceptible cells. Accordingly, Lgr4-deficient mice had dramatically decreased VSV levels in the olfactory bulb. Furthermore, Lgr4 knockdown in RAW 264.7 cells also significantly suppressed VSV infection, and Lgr4 overexpression in RAW 264.7 cells enhanced VSV infection. Interestingly, only VSV infection relied on Lgr4, whereas infections with Newcastle disease virus, influenza A virus (A/WSN/33), and herpes simplex virus were unaffected by Lgr4 status. Of note, assays of virus entry, cell ELISA, immunoprecipitation, and surface plasmon resonance indicated that VSV bound susceptible cells via the Lgr4 extracellular domain. Pretreating cells with an Lgr4 antibody, soluble LGR4 extracellular domain, or R-spondin 1 blocked VSV infection by competitively inhibiting VSV binding to Lgr4. Taken together, the identification of Lgr4 as a VSV-specific host factor provides important insights into understanding VSV entry and its pathogenesis and lays the foundation for VSV-based gene therapy and viral oncolytic therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Replication-Competent Controlled Herpes Simplex Virus

PubMed Central

Bloom, David C.; Feller, Joyce; McAnany, Peterjon; Vilaboa, Nuria

2015-01-01

ABSTRACT We present the development and characterization of a replication-competent controlled herpes simplex virus 1 (HSV-1). Replication-essential ICP4 and ICP8 genes of HSV-1 wild-type strain 17syn+ were brought under the control of a dually responsive gene switch. The gene switch comprises (i) a transactivator that is activated by a narrow class of antiprogestins, including mifepristone and ulipristal, and whose expression is mediated by a promoter cassette that comprises an HSP70B promoter and a transactivator-responsive promoter and (ii) transactivator-responsive promoters that drive the ICP4 and ICP8 genes. Single-step growth experiments in different cell lines demonstrated that replication of the recombinant virus, HSV-GS3, is strictly dependent on an activating treatment consisting of administration of a supraphysiological heat dose in the presence of an antiprogestin. The replication-competent controlled virus replicates with an efficiency approaching that of the wild-type virus from which it was derived. Essentially no replication occurs in the absence of activating treatment or if HSV-GS3-infected cells are exposed only to heat or antiprogestin. These findings were corroborated by measurements of amounts of viral DNA and transcripts of the regulated ICP4 gene and the glycoprotein C (gC) late gene, which was not regulated. Similar findings were made in experiments with a mouse footpad infection model. IMPORTANCE The alphaherpesviruses have long been considered vectors for recombinant vaccines and oncolytic therapies. The traditional approach uses vector backbones containing attenuating mutations that restrict replication to ensure safety. The shortcoming of this approach is that the attenuating mutations tend to limit both the immune presentation and oncolytic properties of these vectors. HSV-GS3 represents a novel type of vector that, when activated, replicates with the efficiency of a nonattenuated virus and whose safety is derived from deliberate, stringent regulation of multiple replication-essential genes. By directing activating heat to the region of virus administration, replication is strictly confined to infected cells within this region. The requirement for antiprogestin provides an additional level of safety, ensuring that virus replication cannot be triggered inadvertently. Replication-competent controlled vectors such as HSV-GS3 may have the potential to be superior to conventional attenuated HSV vaccine and oncolytic vectors without sacrificing safety. PMID:26269179

Nuclease-free Adeno-Associated Virus-Mediated Il2rg Gene Editing in X-SCID Mice.

PubMed

Hiramoto, Takafumi; Li, Li B; Funk, Sarah E; Hirata, Roli K; Russell, David W

2018-05-02

X-linked severe combined immunodeficiency (X-SCID) has been successfully treated by hematopoietic stem cell (HSC) transduction with retroviral vectors expressing the interleukin-2 receptor subunit gamma gene (IL2RG), but several patients developed malignancies due to vector integration near cellular oncogenes. This adverse side effect could in principle be avoided by accurate IL2RG gene editing with a vector that does not contain a functional promoter or IL2RG gene. Here, we show that adeno-associated virus (AAV) gene editing vectors can insert a partial Il2rg cDNA at the endogenous Il2rg locus in X-SCID murine bone marrow cells and that these ex vivo-edited cells repopulate transplant recipients and produce CD4 + and CD8 + T cells. Circulating, edited lymphocytes increased over time and appeared in secondary transplant recipients, demonstrating successful editing in long-term repopulating cells. Random vector integration events were nearly undetectable, and malignant transformation of the transplanted cells was not observed. Similar editing frequencies were observed in human hematopoietic cells. Our results demonstrate that therapeutically relevant HSC gene editing can be achieved by AAV vectors in the absence of site-specific nucleases and suggest that this may be a safe and effective therapy for hematopoietic diseases where in vivo selection can increase edited cell numbers. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

The natural dietary genistein boosts bacteriophage-mediated cancer cell killing by improving phage-targeted tumor cell transduction

PubMed Central

Tsafa, Effrosyni; Al-Bahrani, Mariam; Bentayebi, Kaoutar; Przystal, Justyna; Suwan, Keittisak; Hajitou, Amin

2016-01-01

Gene therapy has long been regarded as a promising treatment for cancer. However, cancer gene therapy is still facing the challenge of targeting gene delivery vectors specifically to tumors when administered via clinically acceptable non-invasive systemic routes (i.e. intravenous). The bacteria virus, bacteriophage (phage), represents a new generation of promising vectors in systemic gene delivery since their targeting can be achieved through phage capsid display ligands, which enable them to home to specific tumor receptors without the need to ablate any native eukaryotic tropism. We have previously reported a tumor specific bacteriophage vector named adeno-associated virus/phage, or AAVP, in which gene expression is under a recombinant human rAAV2 virus genome targeted to tumors via a ligand-directed phage capsid. However, cancer gene therapy with this tumor-targeted vector achieved variable outcomes ranging from tumor regression to no effect in both experimental and natural preclinical models. Herein, we hypothesized that combining the natural dietary genistein, with proven anticancer activity, would improve bacteriophage anticancer safe therapy. We show that combination treatment with genistein and AAVP increased targeted cancer cell killing by AAVP carrying the gene for Herpes simplex virus thymidine kinase (HSVtk) in 2D tissue cultures and 3D tumor spheroids. We found this increased tumor cell killing was associated with enhanced AAVP-mediated gene expression. Next, we established that genistein protects AAVP against proteasome degradation and enhances vector genome accumulation in the nucleus. Combination of genistein and phage-guided virotherapy is a safe and promising strategy that should be considered in anticancer therapy with AAVP. PMID:27437775

Trans-packaging of human immunodeficiency virus type 1 genome into Gag virus-like particles in Saccharomyces cerevisiae.

PubMed

Tomo, Naoki; Goto, Toshiyuki; Morikawa, Yuko

2013-03-26

Yeast is recognized as a generally safe microorganism and is utilized for the production of pharmaceutical products, including vaccines. We previously showed that expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts released Gag virus-like particles (VLPs) extracellularly, suggesting that the production system could be used in vaccine development. In this study, we further establish HIV-1 genome packaging into Gag VLPs in a yeast cell system. The nearly full-length HIV-1 genome containing the entire 5' long terminal repeat, U3-R-U5, did not transcribe gag mRNA in yeast. Co-expression of HIV-1 Tat, a transcription activator, did not support the transcription. When the HIV-1 promoter U3 was replaced with the promoter for the yeast glyceraldehyde-3-phosphate dehydrogenase gene, gag mRNA transcription was restored, but no Gag protein expression was observed. Co-expression of HIV-1 Rev, a factor that facilitates nuclear export of gag mRNA, did not support the protein synthesis. Progressive deletions of R-U5 and its downstream stem-loop-rich region (SL) to the gag start ATG codon restored Gag protein expression, suggesting that a highly structured noncoding RNA generated from the R-U5-SL region had an inhibitory effect on gag mRNA translation. When a plasmid containing the HIV-1 genome with the R-U5-SL region was coexpressed with an expression plasmid for Gag protein, the HIV-1 genomic RNA was transcribed and incorporated into Gag VLPs formed by Gag protein assembly, indicative of the trans-packaging of HIV-1 genomic RNA into Gag VLPs in a yeast cell system. The concentration of HIV-1 genomic RNA in Gag VLPs released from yeast was approximately 500-fold higher than that in yeast cytoplasm. The deletion of R-U5 to the gag gene resulted in the failure of HIV-1 RNA packaging into Gag VLPs, indicating that the packaging signal of HIV-1 genomic RNA present in the R-U5 to gag region functions similarly in yeast cells. Our data indicate that selective trans-packaging of HIV-1 genomic RNA into Gag VLPs occurs in a yeast cell system, analogous to a mammalian cell system, suggesting that yeast may provide an alternative packaging system for lentiviral RNA.

An Exosome-Based Vaccine Platform Imparts Cytotoxic T Lymphocyte Immunity Against Viral Antigens.

PubMed

Anticoli, Simona; Manfredi, Francesco; Chiozzini, Chiara; Arenaccio, Claudia; Olivetta, Eleonora; Ferrantelli, Flavia; Capocefalo, Antonio; Falcone, Emiliana; Ruggieri, Anna; Federico, Maurizio

2018-04-01

Exosomes are 50-150 nm sized nanovesicles released by all eukaryotic cells. The authors very recently described a method to engineer exosomes in vivo with the E7 protein of Human Papilloma Virus (HPV). This technique consists in the intramuscular injection of a DNA vector expressing HPV-E7 fused at the C-terminus of an exosome-anchoring protein, that is, Nef mut , the authors previously characterized for its high levels of incorporation in exosomes. In this configuration, the ≈11 kDa E7 protein elicited a both strong and effective antigen-specific cytotoxic T lymphocyte (CTL) immunity. Attempting to establish whether this method could have general applicability, the authors expanded the immunogenicity studies toward an array of viral products of various origin and size including Ebola Virus VP24, VP40 and NP, Influenza Virus NP, Crimean-Congo Hemorrhagic Fever NP, West Nile Virus NS3, and Hepatitis C Virus NS3. All antigens appeared stable upon fusion with Nef mut , and are uploaded in exosomes at levels comparable to Nef mut . When injected in mice, DNA vectors expressing the diverse fusion products elicited a well detectable antigen-specific CD8 + T cell response associating with a cytotoxic activity potent enough to kill peptide-loaded and/or antigen-expressing syngeneic cells. These data definitely proven both effectiveness and flexibility of this innovative CTL vaccine platform. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

BacMam immunization partially protects pigs against sublethal challenge with African swine fever virus.

PubMed

Argilaguet, Jordi M; Pérez-Martín, Eva; López, Sergio; Goethe, Martin; Escribano, J M; Giesow, Katrin; Keil, Günther M; Rodríguez, Fernando

2013-04-01

Lack of vaccines and efficient control measures complicate the control and eradication of African swine fever (ASF). Limitations of conventional inactivated and attenuated virus-based vaccines against African swine fever virus (ASFV) highlight the need to use new technologies to develop efficient and safe vaccines against this virus. With this aim in mind, in this study we have constructed BacMam-sHAPQ, a baculovirus based vector for gene transfer into mammalian cells, expressing a fusion protein comprising three in tandem ASFV antigens: p54, p30 and the extracellular domain of the viral hemagglutinin (secretory hemagglutinin, sHA), under the control of the human cytomegalovirus immediate early promoter (CMVie). Confirming its correct in vitro expression, BacMam-sHAPQ induced specific T-cell responses directly after in vivo immunization. Conversely, no specific antibody responses were detectable prior to ASFV challenge. The protective potential of this recombinant vaccine candidate was tested by a homologous sublethal challenge with ASFV following immunization. Four out of six immunized pigs remained viremia-free after ASFV infection, while the other two pigs showed similar viremic titres to control animals. The protection afforded correlated with the presence of a large number of virus-specific IFNγ-secreting T-cells in blood at 17 days post-infection. In contrast, the specific antibody levels observed after ASFV challenge in sera from BacMam-sHAPQ immunized pigs were indistinguishable from those found in control pigs. These results highlight the importance of the cellular responses in protection against ASFV and point towards BacMam vectors as potential tools for future vaccine development. Copyright © 2013 Elsevier B.V. All rights reserved.

Development of recombinant adeno-associated virus vectors carrying small interfering RNA (shHec1)-mediated depletion of kinetochore Hec1 protein in tumor cells.

PubMed

Li, L; Yang, L; Scudiero, D A; Miller, S A; Yu, Z-X; Stukenberg, P T; Shoemaker, R H; Kotin, R M

2007-05-01

Transcript depletion using small interfering RNA (siRNA) technology represents a potentially valuable technique for the treatment of cancer. However, delivering therapeutic quantities of siRNA into solid tumors by chemical transfection is not feasible, whereas viral vectors efficiently transduce many human tumor cell lines. Yet producing sufficient quantities of viral vectors that elicit acute and selective cytotoxicity remains a major obstacle for preclinical and clinical trials. Using the invertebrate Spodoptera frugiperda (Sf9) cell line, we were able to produce high titer stocks of cytotoxic recombinant adeno-associated virus (rAAV) that express short hairpin RNA (shRNA) and that efficiently deplete Hec1 (highly expressed in cancer 1), or Kntc2 (kinetochore-associated protein 2), a kinetochore protein directly involved in kinetochore microtubule interactions, chromosome congression and spindle checkpoint signaling. Depletion of Hec1 protein results in persistent spindle checkpoint activation followed by cell death. Because Hec1 expression and activity are only present in mitotic cells, non-dividing cells were not affected by rAAV treatment. On the basis of the results of screening 56 human tumor cell lines with three different serotype vectors, we used a tumor xenograft model to test the effects in vivo. The effects of the shHec1 vector were evident in sectioned and stained tumors. The experiments with rAAV-shRNA vectors demonstrate the utility of producing vectors in invertebrate cells to obtain sufficient concentrations and quantities for solid tumor therapy. This addresses an important requirement for cancer gene therapy, to produce cytotoxic vectors in sufficient quantities and concentrations to enable quantitative transduction and selective killing of solid tumor cells.

Vector platforms for gene therapy of inherited retinopathies

PubMed Central

Trapani, Ivana; Puppo, Agostina; Auricchio, Alberto

2014-01-01

Inherited retinopathies (IR) are common untreatable blinding conditions. Most of them are inherited as monogenic disorders, due to mutations in genes expressed in retinal photoreceptors (PR) and in retinal pigment epithelium (RPE). The retina’s compatibility with gene transfer has made transduction of different retinal cell layers in small and large animal models via viral and non-viral vectors possible. The ongoing identification of novel viruses as well as modifications of existing ones based either on rational design or directed evolution have generated vector variants with improved transduction properties. Dozens of promising proofs of concept have been obtained in IR animal models with both viral and non-viral vectors, and some of them have been relayed to clinical trials. To date, recombinant vectors based on the adeno-associated virus (AAV) represent the most promising tool for retinal gene therapy, given their ability to efficiently deliver therapeutic genes to both PR and RPE and their excellent safety and efficacy profiles in humans. However, AAVs’ limited cargo capacity has prevented application of the viral vector to treatments requiring transfer of genes with a coding sequence larger than 5 kb. Vectors with larger capacity, i.e. nanoparticles, adenoviral and lentiviral vectors are being exploited for gene transfer to the retina in animal models and, more recently, in humans. This review focuses on the available platforms for retinal gene therapy to fight inherited blindness, highlights their main strengths and examines the efforts to overcome some of their limitations. PMID:25124745

Mosaic H5 Hemagglutinin Provides Broad Humoral and Cellular Immune Responses against Influenza Viruses

PubMed Central

Kamlangdee, Attapon; Kingstad-Bakke, Brock

2016-01-01

ABSTRACT The most effective way to prevent influenza virus infection is via vaccination. However, the constant mutation of influenza viruses due to antigenic drift and shift compromises vaccine efficacy. This represents a major challenge to the development of a cross-protective vaccine that can protect against circulating viral antigenic diversity. Using the modified vaccinia Ankara (MVA) virus, we had previously generated a recombinant vaccine against highly pathogenic avian influenza virus (H5N1) based on an in silico mosaic approach. This MVA-H5M construct protected mice against multiple clades of H5N1 and H1N1 viruses. We have now further characterized the immune responses using immunodepletion of T cells and passive serum transfer, and these studies indicate that antibodies are the main contributors in homosubtypic protection (H5N1 clades). Compared to a MVA construct expressing hemagglutinin (HA) from influenza virus A/VN/1203/04 (MVA-HA), the MVA-H5M vaccine markedly increased and broadened B cell and T cell responses against H5N1 virus. The MVA-H5M also provided effective protection with no morbidity against H5N1 challenge, whereas MVA-HA-vaccinated mice showed clinical signs and experienced significant weight loss. In addition, MVA-H5M induced CD8+ T cell responses that play a major role in heterosubtypic protection (H1N1). Finally, expression of the H5M gene as either a DNA vaccine or a subunit protein protected mice against H5N1 challenge, indicating the effectiveness of the mosaic sequence without viral vectors for the development of a universal influenza vaccine. IMPORTANCE Influenza viruses infect up to one billion people around the globe each year and are responsible for 300,000 to 500,000 deaths annually. Vaccines are still the main intervention to prevent infection, but they fail to provide effective protection against heterologous strains of viruses. We developed broadly reactive H5N1 vaccine based on an in silico mosaic approach and previously demonstrated that modified vaccinia Ankara expressing an H5 mosaic hemagglutinin prevented infection with multiple clades of H5N1 and limited severe disease after H1N1 infection. Further characterization revealed that antibody responses and T cells are main contributors to protection against H5N1 and H1N1 viruses, respectively. The vaccine also broadens both T cell and B cell responses compared to native H5 vaccine from influenza virus A/Vietnam/1203/04. Finally, delivering the H5 mosaic as a DNA vaccine or as a purified protein demonstrated effective protection similar to the viral vector approach. PMID:27194759

Alphavirus vector-based replicon particles expressing multivalent cross-protective Lassa virus glycoproteins

PubMed Central

Wang, Min; Jokinen, Jenny; Tretyakova, Irina; Pushko, Peter; Lukashevich, Igor S.

2018-01-01

Lassa virus (LASV) is the most prevalent rodent-borne arenavirus circulated in West Africa. With population at risk from Senegal to Nigeria, LASV causes Lassa fever and is responsible for thousands of deaths annually. High genetic diversity of LASV is one of the challenges for vaccine R&D. We developed multivalent virus-like particle vectors (VLPVs) derived from the human Venezuelan equine encephalitis TC-83 IND vaccine (VEEV) as the next generation of alphavirus-based bicistronic RNA replicon particles. The genes encoding VEEV structural proteins were replaced with LASV glycoproteins (GPC) from distantly related clades I and IV with individual 26S promoters. Bicistronic RNA replicons encoding wild-type LASV GPC (GPCwt) and C-terminally deleted, non-cleavable modified glycoprotein (ΔGPfib), were encapsidated into VLPV particles using VEEV capsid and glycoproteins provided in trans. In transduced cells, VLPVs induced simultaneous expression of LASV GPCwt and ΔGPfib from 26S alphavirus promoters. LASV ΔGPfib was predominantly expressed as trimers, accumulated in the endoplasmic reticulum, induced ER stress and apoptosis promoting antigen cross-priming. VLPV vaccines were immunogenic and protective in mice and upregulated CD11c+/CD8+ dendritic cells playing the major role in cross-presentation. Notably, VLPV vaccination resulted in induction of cross-reactive multifunctional T cell responses after stimulation of immune splenocytes with peptide cocktails derived from LASV from clades I-IV. Multivalent RNA replicon-based LASV vaccines can be applicable for first responders, international travelers visiting endemic areas, military and lab personnel. PMID:29287681

The Use of Adenovirus Dodecahedron in the Delivery of an Enzymatic Activity in the Cell

PubMed Central

Sumarheni; Gallet, Benoit; Fender, Pascal

2016-01-01

Penton-dodecahedron (Pt-Dd) derived from adenovirus type 3 is a symmetric complex of pentameric penton base plus fiber which can be produced in the baculovirus system at a high concentration. The size of Pt-Dd is smaller than the virus, but this virus-like particle (VLP) has the major proteins recognized by specific receptors on the surface of almost all types of cell. In this study, by direct observation with fluorescence microscopy on a fixed and living cell, the intracellular trafficking and localization of Pt-Dd labeled with fluorescence dyes in the cytoplasm of HeLa Tub-GFP showed a rapid internalization characteristic. Subsequently, the linkage of horseradish peroxidase (HRP) with Pt-Dd as the vector demonstrated an efficient system to deliver this enzyme into the cell without interfering its enzymatic activity as shown by biochemical and cellular experiments. These results were supported by additional studies using Bs-Dd or free form of the HRP used as the control. Overall, this study strengthens the potential role of Pt-Dd as an alternative vector for delivering therapeutic agents. PMID:27242929

Genetically modified rabies virus-vectored Ebola virus disease vaccines are safe and induce efficacious immune responses in mice and dogs.

PubMed

Shuai, Lei; Wang, Xijun; Wen, Zhiyuan; Ge, Jinying; Wang, Jinliang; Zhao, Dandan; Bu, Zhigao

2017-10-01

Ebola viruses (EBOVs) are zoonotic pathogens that cause EBOV disease (EVD) with high case fatality in humans. Currently, EVD vaccines are still under development in several countries. Here, we generated two recombinant rabies viruses (RABVs), rERAG 333E /ZGP and rERAG 333E /SGP, expressing the Zaire EBOV glycoprotein (ZGP) or Sudan EBOV glycoprotein (SGP) gene based on a modified ERA vaccine strain (rERAG 333E ) vector platform. The recombinant RABVs retained growth properties similar to those of the vector virus in BSR cell culture and efficiently expressed ZGP or SGP. After intracerebral (i.c.) inoculation with rERAG 333E /ZGP or rERAG 333E /SGP, all adult mice showed no signs of disease or weight loss and suckling mice maintained similar survivorship curve as those mice inoculated with control vector rERAG 333E , demonstrating that ZGP or SGP expression did not increase the virulence of the vector. Mouse immunization studies showed that vaccination with rERAG 333E /ZGP and rERAG 333E /SGP induced Zaire or Sudan EBOV neutralizing antibody (VNA) responses and IgG, IgG2a responses to ZGP or SGP, suggesting their potential as oral or inactivated bivalent vaccines against rabies and EVD. Most importantly, all dogs immunized orally with rERAG 333E /ZGP developed long-lasting ZEBOV and RABV VNA responses with or without previous rabies vaccine immunization history. Live rERAG 333E with EBOV GP thus appear to have the potential to be oral vaccines for free-roaming animals in endemic areas of EVD and rabies, and may serve as inactivated vaccines for use in humans. Copyright © 2017. Published by Elsevier B.V.

Vector design for liver specific expression of multiple interfering RNAs that target hepatitis B virus transcripts

PubMed Central

Snyder, Lindsey L.; Esser, Jonathan M.; Pachuk, Catherine J.; Steel, Laura F.

2008-01-01

RNA interference (RNAi) is a process that can target intracellular RNAs for degradation in a highly sequence specific manner, making it a powerful tool that is being pursued in both research and therapeutic applications. Hepatitis B virus (HBV) is a serious public health problem in need of better treatment options, and aspects of its life cycle make it an excellent target for RNAi-based therapeutics. We have designed a vector that expresses interfering RNAs that target HBV transcripts, including both viral RNA replicative intermediates and mRNAs encoding viral proteins. Our vector design incorporates many features of endogenous microRNA (miRNA) gene organization that are proving useful for the development of reagents for RNAi. In particular, our vector contains an RNA pol II driven gene cassette that leads to tissue specific expression and efficient processing of multiple interfering RNAs from a single transcript, without the co-expression of any protein product. This vector shows potent silencing of HBV targets in cell culture models of HBV infection. The vector design will be applicable to silencing of additional cellular or disease-related genes. PMID:18499277

Novel Recombinant Mycobacterium bovis BCG, Ovine Atadenovirus, and Modified Vaccinia Virus Ankara Vaccines Combine To Induce Robust Human Immunodeficiency Virus-Specific CD4 and CD8 T-Cell Responses in Rhesus Macaques▿

PubMed Central

Rosario, Maximillian; Hopkins, Richard; Fulkerson, John; Borthwick, Nicola; Quigley, Máire F.; Joseph, Joan; Douek, Daniel C.; Greenaway, Hui Yee; Venturi, Vanessa; Gostick, Emma; Price, David A.; Both, Gerald W.; Sadoff, Jerald C.; Hanke, Tomáš

2010-01-01

Mycobacterium bovis bacillus Calmette-Guérin (BCG), which elicits a degree of protective immunity against tuberculosis, is the most widely used vaccine in the world. Due to its persistence and immunogenicity, BCG has been proposed as a vector for vaccines against other infections, including HIV-1. BCG has a very good safety record, although it can cause disseminated disease in immunocompromised individuals. Here, we constructed a recombinant BCG vector expressing HIV-1 clade A-derived immunogen HIVA using the recently described safer and more immunogenic BCG strain AERAS-401 as the parental mycobacterium. Using routine ex vivo T-cell assays, BCG.HIVA401 as a stand-alone vaccine induced undetectable and weak CD8 T-cell responses in BALB/c mice and rhesus macaques, respectively. However, when BCG.HIVA401 was used as a priming component in heterologous vaccination regimens together with recombinant modified vaccinia virus Ankara-vectored MVA.HIVA and ovine atadenovirus-vectored OAdV.HIVA vaccines, robust HIV-1-specific T-cell responses were elicited. These high-frequency T-cell responses were broadly directed and capable of proliferation in response to recall antigen. Furthermore, multiple antigen-specific T-cell clonotypes were efficiently recruited into the memory pool. These desirable features are thought to be associated with good control of HIV-1 infection. In addition, strong and persistent T-cell responses specific for the BCG-derived purified protein derivative (PPD) antigen were induced. This work is the first demonstration of immunogenicity for two novel vaccine vectors and the corresponding candidate HIV-1 vaccines BCG.HIVA401 and OAdV.HIVA in nonhuman primates. These results strongly support their further exploration. PMID:20375158

Virus Capsids as Targeted Nanoscale Delivery Vessels of Photoactive Compounds for Site-Specific Photodynamic Therapy

NASA Astrophysics Data System (ADS)

Cohen, Brian A.

The research presented in this work details the use of a viral capsid as an addressable delivery vessel of photoactive compounds for use in photodynamic therapy. Photodynamic therapy is a treatment that involves the interaction of light with a photosensitizing molecule to create singlet oxygen, a reactive oxygen species. Overproduction of singlet oxygen in cells can cause oxidative damage leading to cytotoxicity and eventually cell death. Challenges with the current generation of FDA-approved photosensitizers for photodynamic therapy primarily stem from their lack of tissue specificity. This work describes the packaging of photoactive cationic porphyrins inside the MS2 bacteriophage capsid, followed by external modification of the capsid with cancer cell-targeting G-quadruplex DNA aptamers to generate a tumor-specific photosensitizing agent. First, a cationic porphyrin is loaded into the capsids via nucleotide-driven packaging, a process that involves charge interaction between the porphyrin and the RNA inside the capsid. Results show that over 250 porphyrin molecules associate with the RNA within each MS2 capsid. Removal of RNA from the capsid severely inhibits the packaging of the cationic porphyrins. Porphyrin-virus constructs were then shown to photogenerate singlet oxygen, and cytotoxicity in non-targeted photodynamic treatment experiments. Next, each porphyrin-loaded capsid is externally modified with approximately 60 targeting DNA aptamers by employing a heterobifunctional crosslinking agent. The targeting aptamer is known to bind the protein nucleolin, a ubiquitous protein that is overexpressed on the cell surface by many cancer cell types. MCF-7 human breast carcinoma cells and MCF-10A human mammary epithelial cells were selected as an in vitro model for breast cancer and normal tissue, respectively. Fluorescently tagged virus-aptamer constructs are shown to selectively target MCF-7 cells versus MCF-10A cells. Finally, results are shown in which porphyrin-virus-aptamer constructs selectively target and kill cancer cells versus non-cancer cells. Specifically, the results show that MS2 is a viable candidate as an addressable nanodelivery vessel of photoactive compounds, and the implications are that the nucleotide-driven packaging approach for modifying MS2 can be used to impart new functionalities for a host of diagnostic or therapeutic applications.

An adenovirus prime/plasmid boost strategy for induction of equipotent immune responses to two dengue virus serotypes.

PubMed

Khanam, Saima; Rajendra, Pilankatta; Khanna, Navin; Swaminathan, Sathyamangalam

2007-02-15

Dengue is a public health problem of global significance for which there is neither an effective antiviral therapy nor a preventive vaccine. It is a mosquito-borne viral disease, caused by dengue (DEN) viruses, which are members of the Flaviviridae family. There are four closely related serotypes, DEN-1, DEN-2, DEN-3 and DEN-4, each of which is capable of causing disease. As immunity to any one serotype can potentially sensitize an individual to severe disease during exposure to a heterologous serotype, the general consensus is that an effective vaccine should be tetravalent, that is, it must be capable of affording protection against all four serotypes. The current strategy of creating tetravalent vaccine formulations by mixing together four monovalent live attenuated vaccine viruses has revealed the phenomenon of viral interference leading to the manifestation of immune responses biased towards a single serotype. This work stems from the emergence of (i) the DEN virus envelope (E) domain III (EDIII) as the most important region of the molecule from a vaccine perspective and (ii) the adenovirus (Ad) as a promising vaccine vector platform. We describe the construction of a recombinant, replication-defective Ad (rAd) vector encoding a chimeric antigen made of in-frame linked EDIIIs of DEN virus serotypes 2 and 4. Using this rAd vector, in conjunction with a plasmid vector encoding the same chimeric bivalent antigen, in a prime-boost strategy, we show that it is possible to elicit equipotent neutralizing and T cell responses specific to both DEN serotypes 2 and 4. Our data support the hypothesis that a DEN vaccine targeting more than one serotype may be based on a single DNA-based vector to circumvent viral interference. This work lays the foundation for developing a single Ad vector encoding EDIIIs of all four DEN serotypes to evoke a balanced immune response against each one of them. Thus, this work has implications for the development of safe and effective tetravalent dengue vaccines.

Development of Sendai Virus Vectors and their Potential Applications in Gene Therapy and Regenerative Medicine

PubMed Central

Nakanishi, Mahito; Otsu, Makoto

2012-01-01

Gene delivery/expression vectors have been used as fundamental technologies in gene therapy since the 1980s. These technologies are also being applied in regenerative medicine as tools to reprogram cell genomes to a pluripotent state and to other cell lineages. Rapid progress in these new research areas and expectations for their translation into clinical applications have facilitated the development of more sophisticated gene delivery/expression technologies. Since its isolation in 1953 in Japan, Sendai virus (SeV) has been widely used as a research tool in cell biology and in industry, but the application of SeV as a recombinant viral vector has been investigated only recently. Recombinant SeV vectors have various unique characteristics, such as low pathogenicity, powerful capacity for gene expression and a wide host range. In addition, the cytoplasmic gene expression mediated by this vector is advantageous for applications, in that chromosomal integration of exogenous genes can be undesirable. In this review, we introduce a brief historical background on the development of recombinant SeV vectors and describe their current applications in gene therapy. We also describe the application of SeV vectors in advanced nuclear reprogramming and introduce a defective and persistent SeV vector (SeVdp) optimized for such reprogramming. PMID:22920683

Enhancers Are Major Targets for Murine Leukemia Virus Vector Integration

PubMed Central

De Ravin, Suk See; Su, Ling; Theobald, Narda; Choi, Uimook; Macpherson, Janet L.; Poidinger, Michael; Symonds, Geoff; Pond, Susan M.; Ferris, Andrea L.; Hughes, Stephen H.

2014-01-01

ABSTRACT Retroviral vectors have been used in successful gene therapies. However, in some patients, insertional mutagenesis led to leukemia or myelodysplasia. Both the strong promoter/enhancer elements in the long terminal repeats (LTRs) of murine leukemia virus (MLV)-based vectors and the vector-specific integration site preferences played an important role in these adverse clinical events. MLV integration is known to prefer regions in or near transcription start sites (TSS). Recently, BET family proteins were shown to be the major cellular proteins responsible for targeting MLV integration. Although MLV integration sites are significantly enriched at TSS, only a small fraction of the MLV integration sites (<15%) occur in this region. To resolve this apparent discrepancy, we created a high-resolution genome-wide integration map of more than one million integration sites from CD34+ hematopoietic stem cells transduced with a clinically relevant MLV-based vector. The integration sites form ∼60,000 tight clusters. These clusters comprise ∼1.9% of the genome. The vast majority (87%) of the integration sites are located within histone H3K4me1 islands, a hallmark of enhancers. The majority of these clusters also have H3K27ac histone modifications, which mark active enhancers. The enhancers of some oncogenes, including LMO2, are highly preferred targets for integration without in vivo selection. IMPORTANCE We show that active enhancer regions are the major targets for MLV integration; this means that MLV preferentially integrates in regions that are favorable for viral gene expression in a variety of cell types. The results provide insights for MLV integration target site selection and also explain the high risk of insertional mutagenesis that is associated with gene therapy trials using MLV vectors. PMID:24501411

Heat Shock Protein 70 Enhances Mucosal Immunity against Human Norovirus When Coexpressed from a Vesicular Stomatitis Virus Vector

PubMed Central

Ma, Yuanmei; Duan, Yue; Wei, Yongwei; Liang, Xueya; Niewiesk, Stefan; Oglesbee, Michael

2014-01-01

ABSTRACT Human norovirus (NoV) accounts for 95% of nonbacterial gastroenteritis worldwide. Currently, there is no vaccine available to combat human NoV as it is not cultivable and lacks a small-animal model. Recently, we demonstrated that recombinant vesicular stomatitis virus (rVSV) expressing human NoV capsid protein (rVSV-VP1) induced strong immunities in mice (Y. Ma and J. Li, J. Virol. 85:2942–2952, 2011). To further improve the safety and efficacy of the vaccine candidate, heat shock protein 70 (HSP70) was inserted into the rVSV-VP1 backbone vector. A second construct was generated in which the firefly luciferase (Luc) gene was inserted in place of HSP70 as a control for the double insertion. The resultant recombinant viruses (rVSV-HSP70-VP1 and rVSV-Luc-VP1) were significantly more attenuated in cell culture and viral spread in mice than rVSV-VP1. At the inoculation dose of 1.0 × 106 PFU, rVSV-HSP70-VP1 triggered significantly higher vaginal IgA than rVSV-VP1 and significantly higher fecal and vaginal IgA responses than rVSV-Luc-VP1, although serum IgG and T cell responses were similar. At the inoculation dose of 5.0 × 106 PFU, rVSV-HSP70-VP1 stimulated significantly higher T cell, fecal, and vaginal IgA responses than rVSV-VP1. Fecal and vaginal IgA responses were also significantly increased when combined vaccination of rVSV-VP1 and rVSV-HSP70 was used. Collectively, these data indicate that (i) insertion of an additional gene (HSP70 or Luc) into the rVSV-VP1 backbone further attenuates the VSV-based vaccine in vitro and in vivo, thus improving the safety of the vaccine candidate, and (ii) HSP70 enhances the human NoV-specific mucosal and T cell immunities triggered by a VSV-based human NoV vaccine. IMPORTANCE Human norovirus (NoV) is responsible for more than 95% of acute nonbacterial gastroenteritis worldwide. Currently, there is no vaccine for this virus. Development of a live attenuated vaccine for human NoV has not been possible because it is uncultivable. Thus, a live vector-based vaccine may provide an alternative vaccine strategy. In this study, we developed a vesicular stomatitis virus (VSV)-based human NoV vaccine candidate. We constructed rVSV-HSP70-VP1, coexpressing heat shock protein (HSP70) and capsid (VP1) genes of human NoV, and rVSV-Luc-VP1, coexpressing firefly luciferase (Luc) and VP1 genes. We found that VSVs with a double gene insertion were significantly more attenuated than VSV with a single VP1 insertion (rVSV-VP1). Furthermore, we found that coexpression or coadministration of HSP70 from VSV vector significantly enhanced human NoV-specific mucosal immunity. Collectively, we developed an improved live vectored vaccine candidate for human NoV which will be useful for future clinical studies. PMID:24574391

Elements in the Development of a Production Process for Modified Vaccinia Virus Ankara

PubMed Central

Jordan, Ingo; Lohr, Verena; Genzel, Yvonne; Reichl, Udo; Sandig, Volker

2013-01-01

The production of several viral vaccines depends on chicken embryo fibroblasts or embryonated chicken eggs. To replace this logistically demanding substrate, we created continuous anatine suspension cell lines (CR and CR.pIX), developed chemically-defined media, and established production processes for different vaccine viruses. One of the processes investigated in greater detail was developed for modified vaccinia virus Ankara (MVA). MVA is highly attenuated for human recipients and an efficient vector for reactogenic expression of foreign genes. Because direct cell-to-cell spread is one important mechanism for vaccinia virus replication, cultivation of MVA in bioreactors is facilitated if cell aggregates are induced after infection. This dependency may be the mechanism behind our observation that a novel viral genotype (MVA-CR) accumulates with serial passage in suspension cultures. Sequencing of a major part of the genomic DNA of the new strain revealed point mutations in three genes. We hypothesize that these changes confer an advantage because they may allow a greater fraction of MVA-CR viruses to escape the host cells for infection of distant targets. Production and purification of MVA-based vaccines may be simplified by this combination of designed avian cell line, chemically defined media and the novel virus strain. PMID:27694766

Elements in the Development of a Production Process for Modified Vaccinia Virus Ankara.

PubMed

Jordan, Ingo; Lohr, Verena; Genzel, Yvonne; Reichl, Udo; Sandig, Volker

2013-11-01

The production of several viral vaccines depends on chicken embryo fibroblasts or embryonated chicken eggs. To replace this logistically demanding substrate, we created continuous anatine suspension cell lines (CR and CR.pIX), developed chemically-defined media, and established production processes for different vaccine viruses. One of the processes investigated in greater detail was developed for modified vaccinia virus Ankara (MVA). MVA is highly attenuated for human recipients and an efficient vector for reactogenic expression of foreign genes. Because direct cell-to-cell spread is one important mechanism for vaccinia virus replication, cultivation of MVA in bioreactors is facilitated if cell aggregates are induced after infection. This dependency may be the mechanism behind our observation that a novel viral genotype (MVA-CR) accumulates with serial passage in suspension cultures. Sequencing of a major part of the genomic DNA of the new strain revealed point mutations in three genes. We hypothesize that these changes confer an advantage because they may allow a greater fraction of MVA-CR viruses to escape the host cells for infection of distant targets. Production and purification of MVA-based vaccines may be simplified by this combination of designed avian cell line, chemically defined media and the novel virus strain.

Effective photodynamic therapy in drug-resistant prostate cancer cells utilizing a non-viral antitumor vector (a secondary publication).

PubMed

Yamauchi, Masaya; Honda, Norihiro; Hazama, Hisanao; Tachikawa, Shoji; Nakamura, Hiroyuki; Kaneda, Yasufumi; Awazu, Kunio

2016-03-31

There is an urgent need to develop an efficient strategy for the treatment of drug-resistant prostate cancer. Photodynamic therapy (PDT), in which low incident levels of laser energy are used to activate a photosensitizer taken up by tumor cells, is expected as a novel therapy for the treatment of prostate cancer because of the minimal invasive nature of PDT. The present study was designed to assess the efficacy of a novel vector approach combined with a conventional porphyrin-based photosensitizer. Our group focused on a non-viral vector (hemagglutinating virus of Japan envelope; HVJ-E) combined with protoporphyrin IX (PpIX) lipid, termed the porphyrus envelope (PE). It has been previously confirmed that HVJ-E has drug-delivering properties and can induce cancer-specific cell death. The PE (HVJ-E contained in PpIX lipid) was developed as a novel photosensitizer. In this study, the antitumor and PDT efficacy of the PE against hormone-antagonistic human prostate cancer cells (PC-3) were evaluated. Our results demonstrated that, under specific circumstances, PDT using the PE was very effective against PC-3 cells. A novel therapy for drug-resistant prostate cancer based on this vector approach is eagerly anticipated.

Identification of the subgenomic promoter of the coat protein gene of cucumber fruit mottle mosaic virus and development of a heterologous expression vector.

PubMed

Rhee, Sun-Ju; Jang, Yoon Jeong; Lee, Gung Pyo

2016-06-01

Heterologous gene expression using plant virus vectors enables research on host-virus interactions and the production of useful proteins, but the host range of plant viruses limits the practical applications of such vectors. Here, we aimed to develop a viral vector based on cucumber fruit mottle mosaic virus (CFMMV), a member of the genus Tobamovirus, whose members infect cucurbits. The subgenomic promoter (SGP) in the coat protein (CP) gene, which was used to drive heterologous expression, was mapped by analyzing deletion mutants from a CaMV 35S promoter-driven infectious CFMMV clone. The region from nucleotides (nt) -55 to +160 relative to the start codon of the open reading frame (ORF) of CP was found to be a fully active promoter, and the region from nt -55 to +100 was identified as the active core promoter. Based on these SGPs, we constructed a cloning site in the CFMMV vector and successfully expressed enhanced green fluorescent protein (EGFP) in Nicotiana benthamiana and watermelon (Citrullus lanatus). Co-inoculation with the P19 suppressor increased EGFP expression and viral replication by blocking degradation of the viral genome. Our CFMMV vector will be useful as an expression vector in cucurbits.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression.

PubMed

Trinh, Alice T; Ball, Bret G; Weber, Erin; Gallaher, Timothy K; Gluzman-Poltorak, Zoya; Anderson, French; Basile, Lena A

2009-12-30

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

Efficacy of Antiretroviral Agents against Murine Replication-Competent Retrovirus Infection in Human Cells

PubMed Central

Powell, Sharon K.; Artlip, Moria; Kaloss, Michele; Brazinski, Scott; Lyons, Russette; McGarrity, Gerard J.; Otto, Edward

1999-01-01

Retroviral vectors for gene therapy are designed to minimize the occurrence of replication-competent retrovirus (RCR); nonetheless, it is possible that a vector-derived RCR could establish an infection in a patient. Since the efficacy of antiretroviral agents can be impacted by interactions between virus, host cell, and drug, five commonly used antiretroviral drugs were evaluated for their abilities to inhibit the replication of a murine leukemia virus (MLV)-derived RCR in human cells. The results obtained indicate that the combination of nucleoside analogs zidovudine and dideoxyinosine with the protease inhibitor indinavir effectively inhibits MLV-derived RCR replication in three human cell lines. In addition, MLV-derived RCR was found to be inherently resistant to the nucleoside analogs lamivudine and stavudine, suggesting that mutations conferring resistance to nucleoside analogs in human immunodeficiency virus type 1 have the same effect even in an alternative viral backbone. PMID:10482636

Current Good Manufacturing Practice Production of an Oncolytic Recombinant Vesicular Stomatitis Viral Vector for Cancer Treatment

PubMed Central

Meseck, M.; Derecho, I.; Lopez, P.; Knoblauch, C.; McMahon, R.; Anderson, J.; Dunphy, N.; Quezada, V.; Khan, R.; Huang, P.; Dang, W.; Luo, M.; Hsu, D.; Woo, S.L.C.; Couture, L.

2011-01-01

Abstract Vesicular stomatitis virus (VSV) is an oncolytic virus currently being investigated as a promising tool to treat cancer because of its ability to selectively replicate in cancer cells. To enhance the oncolytic property of the nonpathologic laboratory strain of VSV, we generated a recombinant vector [rVSV(MΔ51)-M3] expressing murine gammaherpesvirus M3, a secreted viral chemokine-binding protein that binds to a broad range of mammalian chemokines with high affinity. As previously reported, when rVSV(MΔ51)-M3 was used in an orthotopic model of hepatocellular carcinoma (HCC) in rats, it suppressed inflammatory cell migration to the virus-infected tumor site, which allowed for enhanced intratumoral virus replication leading to increased tumor necrosis and substantially prolonged survival. These encouraging results led to the development of this vector for clinical translation in patients with HCC. However, a scalable current Good Manufacturing Practice (cGMP)-compliant manufacturing process has not been described for this vector. To produce the quantities of high-titer virus required for clinical trials, a process that is amenable to GMP manufacturing and scale-up was developed. We describe here a large-scale (50-liter) vector production process capable of achieving crude titers on the order of 109 plaque-forming units (PFU)/ml under cGMP. This process was used to generate a master virus seed stock and a clinical lot of the clinical trial agent under cGMP with an infectious viral titer of approximately 2 × 1010 PFU/ml (total yield, 1 × 1013 PFU). The lot has passed all U.S. Food and Drug Administration-mandated release testing and will be used in a phase 1 clinical translational trial in patients with advanced HCC. PMID:21083425

Current good manufacturing practice production of an oncolytic recombinant vesicular stomatitis viral vector for cancer treatment.

PubMed

Ausubel, L J; Meseck, M; Derecho, I; Lopez, P; Knoblauch, C; McMahon, R; Anderson, J; Dunphy, N; Quezada, V; Khan, R; Huang, P; Dang, W; Luo, M; Hsu, D; Woo, S L C; Couture, L

2011-04-01

Vesicular stomatitis virus (VSV) is an oncolytic virus currently being investigated as a promising tool to treat cancer because of its ability to selectively replicate in cancer cells. To enhance the oncolytic property of the nonpathologic laboratory strain of VSV, we generated a recombinant vector [rVSV(MΔ51)-M3] expressing murine gammaherpesvirus M3, a secreted viral chemokine-binding protein that binds to a broad range of mammalian chemokines with high affinity. As previously reported, when rVSV(MΔ51)-M3 was used in an orthotopic model of hepatocellular carcinoma (HCC) in rats, it suppressed inflammatory cell migration to the virus-infected tumor site, which allowed for enhanced intratumoral virus replication leading to increased tumor necrosis and substantially prolonged survival. These encouraging results led to the development of this vector for clinical translation in patients with HCC. However, a scalable current Good Manufacturing Practice (cGMP)-compliant manufacturing process has not been described for this vector. To produce the quantities of high-titer virus required for clinical trials, a process that is amenable to GMP manufacturing and scale-up was developed. We describe here a large-scale (50-liter) vector production process capable of achieving crude titers on the order of 10(9) plaque-forming units (PFU)/ml under cGMP. This process was used to generate a master virus seed stock and a clinical lot of the clinical trial agent under cGMP with an infectious viral titer of approximately 2 × 10(10) PFU/ml (total yield, 1 × 10(13) PFU). The lot has passed all U.S. Food and Drug Administration-mandated release testing and will be used in a phase 1 clinical translational trial in patients with advanced HCC.

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS1[OPEN

PubMed Central

2018-01-01

Virus-induced gene silencing (VIGS) is used extensively for gene function studies in plants. VIGS is inexpensive and rapid compared with silencing conducted through stable transformation, but many virus-silencing vectors, especially in grasses, induce only transient silencing phenotypes. A major reason for transient phenotypes is the instability of the foreign gene fragment (insert) in the vector during VIGS. Here, we report the development of a Brome mosaic virus (BMV)-based vector that better maintains inserts through modification of the original BMV vector RNA sequence. Modification of the BMV RNA3 sequence yielded a vector, BMVCP5, that better maintained phytoene desaturase and heat shock protein70-1 (HSP70-1) inserts in Nicotiana benthamiana and maize (Zea mays). Longer maintenance of inserts was correlated with greater target gene silencing and more extensive visible silencing phenotypes displaying greater tissue penetration and involving more leaves. The modified vector accumulated similarly to the original vector in N. benthamiana after agroinfiltration, thus maintaining a high titer of virus in this intermediate host used to produce virus inoculum for grass hosts. For HSP70, silencing one family member led to a large increase in the expression of another family member, an increase likely related to the target gene knockdown and not a general effect of virus infection. The cause of the increased insert stability in the modified vector is discussed in relationship to its recombination and accumulation potential. The modified vector will improve functional genomic studies in grasses, and the conceptual methods used to improve the vector may be applied to other VIGS vectors. PMID:29127260

Retargeting of adenovirus vectors through genetic fusion of a single-chain or single-domain antibody to capsid protein IX.

PubMed

Poulin, Kathy L; Lanthier, Robert M; Smith, Adam C; Christou, Carin; Risco Quiroz, Milagros; Powell, Karen L; O'Meara, Ryan W; Kothary, Rashmi; Lorimer, Ian A; Parks, Robin J

2010-10-01

Adenovirus (Ad) vectors are the most commonly used system for gene therapy applications, due in part to their ability to infect a wide array of cell types and tissues. However, many therapies would benefit from the ability to target the Ad vector only to specific cells, such as tumor cells for cancer gene therapy. In this study, we investigated the utility of capsid protein IX (pIX) as a platform for the presentation of single-chain variable-fragment antibodies (scFv) and single-domain antibodies (sdAb) for virus retargeting. We show that scFv can be displayed on the capsid through genetic fusion to native pIX but that these molecules fail to retarget the virus, due to improper folding of the scFv. Redirecting expression of the fusion protein to the endoplasmic reticulum (ER) results in correct folding of the scFv and allows it to recognize its epitope; however, ER-targeted pIX-scFv was incorporated into the Ad capsid at a very low level which was not sufficient to retarget virus infection. In contrast, a pIX-sdAb construct was efficiently incorporated into the Ad capsid and enhanced virus infection of cells expressing the targeted receptor. Taken together, our data indicate that pIX is an effective platform for presentation of large targeting polypeptides on the surface of the virus capsid, but the nature of the ligand can significantly affect its association with virions.

Adeno-Associated Virus Type 2 Wild-Type and Vector-Mediated Genomic Integration Profiles of Human Diploid Fibroblasts Analyzed by Third-Generation PacBio DNA Sequencing

PubMed Central

Hüser, Daniela; Gogol-Döring, Andreas; Chen, Wei

2014-01-01

ABSTRACT Genome-wide analysis of adeno-associated virus (AAV) type 2 integration in HeLa cells has shown that wild-type AAV integrates at numerous genomic sites, including AAVS1 on chromosome 19q13.42. Multiple GAGY/C repeats, resembling consensus AAV Rep-binding sites are preferred, whereas rep-deficient AAV vectors (rAAV) regularly show a random integration profile. This study is the first study to analyze wild-type AAV integration in diploid human fibroblasts. Applying high-throughput third-generation PacBio-based DNA sequencing, integration profiles of wild-type AAV and rAAV are compared side by side. Bioinformatic analysis reveals that both wild-type AAV and rAAV prefer open chromatin regions. Although genomic features of AAV integration largely reproduce previous findings, the pattern of integration hot spots differs from that described in HeLa cells before. DNase-Seq data for human fibroblasts and for HeLa cells reveal variant chromatin accessibility at preferred AAV integration hot spots that correlates with variant hot spot preferences. DNase-Seq patterns of these sites in human tissues, including liver, muscle, heart, brain, skin, and embryonic stem cells further underline variant chromatin accessibility. In summary, AAV integration is dependent on cell-type-specific, variant chromatin accessibility leading to random integration profiles for rAAV, whereas wild-type AAV integration sites cluster near GAGY/C repeats. IMPORTANCE Adeno-associated virus type 2 (AAV) is assumed to establish latency by chromosomal integration of its DNA. This is the first genome-wide analysis of wild-type AAV2 integration in diploid human cells and the first to compare wild-type to recombinant AAV vector integration side by side under identical experimental conditions. Major determinants of wild-type AAV integration represent open chromatin regions with accessible consensus AAV Rep-binding sites. The variant chromatin accessibility of different human tissues or cell types will have impact on vector targeting to be considered during gene therapy. PMID:25031342

A Herpes Simplex Virus-Derived Replicative Vector Expressing LIF Limits Experimental Demyelinating Disease and Modulates Autoimmunity

PubMed Central

Nygårdas, Michaela; Paavilainen, Henrik; Müther, Nadine; Nagel, Claus-Henning; Röyttä, Matias; Sodeik, Beate; Hukkanen, Veijo

2013-01-01

Herpes simplex virus type 1 (HSV-1) has properties that can be exploited for the development of gene therapy vectors. The neurotropism of HSV enables delivery of therapeutic genes to the nervous system. Using a bacterial artificial chromosome (BAC), we constructed an HSV-1(17+)-based replicative vector deleted of the neurovirulence gene γ134.5, and expressing leukemia inhibitory factor (LIF) as a transgene for treatment of experimental autoimmune encephalomyelitis (EAE). EAE is an inducible T-cell mediated autoimmune disease of the central nervous system (CNS) and is used as an animal model for multiple sclerosis. Demyelination and inflammation are hallmarks of both diseases. LIF is a cytokine that has the potential to limit demyelination and oligodendrocyte loss in CNS autoimmune diseases and to affect the T-cell mediated autoimmune response. In this study SJL/J mice, induced for EAE, were treated with a HSV-LIF vector intracranially and the subsequent changes in disease parameters and immune responses during the acute disease were investigated. Replicating HSV-LIF and its DNA were detected in the CNS during the acute infection, and the vector spread to the spinal cord but was non-virulent. The HSV-LIF significantly ameliorated the EAE and contributed to a higher number of oligodendrocytes in the brains when compared to untreated mice. The HSV-LIF therapy also induced favorable changes in the expression of immunoregulatory cytokines and T-cell population markers in the CNS during the acute disease. These data suggest that BAC-derived HSV vectors are suitable for gene therapy of CNS disease and can be used to test the therapeutic potential of immunomodulatory factors for treatment of EAE. PMID:23700462

Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus.

PubMed

Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian; Wei, Taiyun

2016-01-15

Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an incompetent vector, the small brown planthopper (SBPH). Here, we show that silencing of the core component Dicer-2 of the small interfering RNA (siRNA) pathway increases viral titers in the midgut epithelium past the threshold (1.99 × 10(9) copies of the SRBSDV P10 gene/μg of midgut RNA) for viral dissemination into the midgut muscles and then into the salivary glands, allowing the SBPH to become a competent vector of SRBSDV. This result is the first evidence that the siRNA antiviral pathway has a direct role in the control of viral dissemination from the midgut epithelium and that it affects the competence of the virus's vector. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Small Interfering RNA Pathway Modulates Initial Viral Infection in Midgut Epithelium of Insect after Ingestion of Virus

PubMed Central

Lan, Hanhong; Chen, Hongyan; Liu, Yuyan; Jiang, Chaoyang; Mao, Qianzhuo; Jia, Dongsheng; Chen, Qian

2015-01-01

ABSTRACT Numerous viruses are transmitted in a persistent manner by insect vectors. Persistent viruses establish their initial infection in the midgut epithelium, from where they disseminate to the midgut visceral muscles. Although propagation of viruses in insect vectors can be controlled by the small interfering RNA (siRNA) antiviral pathway, whether the siRNA pathway can control viral dissemination from the midgut epithelium is unknown. Infection by a rice virus (Southern rice black streaked dwarf virus [SRBSDV]) of its incompetent vector (the small brown planthopper [SBPH]) is restricted to the midgut epithelium. Here, we show that the siRNA pathway is triggered by SRBSDV infection in continuously cultured cells derived from the SBPH and in the midgut of the intact insect. Knockdown of the expression of the core component Dicer-2 of the siRNA pathway by RNA interference strongly increased the ability of SRBSDV to propagate in continuously cultured SBPH cells and in the midgut epithelium, allowing viral titers in the midgut epithelium to reach the threshold (1.99 × 109 copies of the SRBSDV P10 gene/μg of midgut RNA) needed for viral dissemination into the SBPH midgut muscles. Our results thus represent the first elucidation of the threshold for viral dissemination from the insect midgut epithelium. Silencing of Dicer-2 further facilitated the transmission of SRBSDV into rice plants by SBPHs. Taken together, our results reveal the new finding that the siRNA pathway can control the initial infection of the insect midgut epithelium by a virus, which finally affects the competence of the virus's vector. IMPORTANCE Many viral pathogens that cause significant global health and agricultural problems are transmitted via insect vectors. The first bottleneck in viral infection, the midgut epithelium, is a principal determinant of the ability of an insect species to transmit a virus. Southern rice black streaked dwarf virus (SRBSDV) is restricted exclusively to the midgut epithelium of an incompetent vector, the small brown planthopper (SBPH). Here, we show that silencing of the core component Dicer-2 of the small interfering RNA (siRNA) pathway increases viral titers in the midgut epithelium past the threshold (1.99 × 109 copies of the SRBSDV P10 gene/μg of midgut RNA) for viral dissemination into the midgut muscles and then into the salivary glands, allowing the SBPH to become a competent vector of SRBSDV. This result is the first evidence that the siRNA antiviral pathway has a direct role in the control of viral dissemination from the midgut epithelium and that it affects the competence of the virus's vector. PMID:26537672

Diagnosis and Prevention of Infection by Phlebotomus Fever Group Viruses

DTIC Science & Technology

1991-05-03

proficient, but non- optimal expression vectors [e.g., pAcRP6, see below]. The two types of recombinant viruses were used to infect Spodoptera ... frugiperda cells and the expressed PT viral proteins characterised (Overton et al., 1987). Recombinant AcNPV having the S DNA in one orientation expressed...proteins virus were raised in mice using the corresponding S. frugiperda infected cell extracts and were employed to identify N and NSs proteins in PT virus

Vaccine Efficacy against Malaria by the Combination of Porcine Parvovirus-Like Particles and Vaccinia Virus Vectors Expressing CS of Plasmodium

PubMed Central

Rodríguez, Dolores; González-Aseguinolaza, Gloria; Rodríguez, Juan R.; Vijayan, Aneesh; Gherardi, Magdalena; Rueda, Paloma; Casal, J. Ignacio; Esteban, Mariano

2012-01-01

With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs) carrying the CD8+ T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS), and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8+ T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV) vectors from the Western Reserve (WR) and modified virus Ankara (MVA) strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria. PMID:22529915

Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium.

PubMed

Rodríguez, Dolores; González-Aseguinolaza, Gloria; Rodríguez, Juan R; Vijayan, Aneesh; Gherardi, Magdalena; Rueda, Paloma; Casal, J Ignacio; Esteban, Mariano

2012-01-01

With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs) carrying the CD8(+) T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS), and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8(+) T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV) vectors from the Western Reserve (WR) and modified virus Ankara (MVA) strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria.

Resistance to human immunodeficiency virus type 1 (HIV-1) generated by lentivirus vector-mediated delivery of the CCR5Δ32 gene despite detectable expression of the HIV-1 co-receptors

PubMed Central

Jin, Qingwen; Marsh, Jon; Cornetta, Kenneth; Alkhatib, Ghalib

2009-01-01

It has previously been demonstrated that there are two distinct mechanisms for genetic resistance to human immunodeficiency virus type 1 (HIV-1) conferred by the CCR5Δ32 gene: the loss of wild-type CCR5 surface expression and the generation of CCR5Δ32 protein, which interacts with CXCR4. To analyse the protective effects of long-term expression of the CCR5Δ32 protein, recombinant lentiviral vectors were used to deliver the CCR5Δ32 gene into human cell lines and primary peripheral blood mononuclear cells that had been immortalized by human T-cell leukemia virus type 1. Blasticidin S-resistant cell lines expressing the lentivirus-encoded CCR5Δ32 showed a significant reduction in HIV-1 Env-mediated fusion assays. It was shown that CD4+ T lymphocytes expressing the lentivirus-encoded CCR5Δ32 gene were highly resistant to infection by a primary but not by a laboratory-adapted X4 strain, suggesting different infectivity requirements. In contrast to previous studies that analysed the CCR5Δ32 protective effects in a transient expression system, this study showed that long-term expression of CCR5Δ32 conferred resistance to HIV-1 despite cell-surface expression of the HIV co-receptors. The results suggest an additional unknown mechanism for generating the CCR5Δ32 resistance phenotype and support the hypothesis that the CCR5Δ32 protein acts as an HIV-suppressive factor by altering the stoichiometry of the molecules involved in HIV-1 entry. The lentiviral-CCR5Δ32 vectors offer a method of generating HIV-resistant cells by delivery of the CCR5Δ32 gene that may be useful for stem cell- or T-cell-based gene therapy for HIV-1 infection. PMID:18796731

Radiation increases the activity of oncolytic adenovirus cancer gene therapy vectors that overexpress the ADP (E3-11.6K) protein.

PubMed

Toth, Karoly; Tarakanova, Vera; Doronin, Konstantin; Ward, Peter; Kuppuswamy, Mohan; Locke, Jacob E; Dawson, Julie E; Kim, Han J; Wold, William S M

2003-03-01

We have described three potential adenovirus type 5 (Ad5)-based replication-competent cancer gene therapy vectors named KD1, KD3, and VRX-007. All three vectors overexpress an Ad5 protein named Adenovirus Death Protein (ADP, also named E3-11.6 K protein). ADP is required for efficient lysis of Ad5-infected cells and spread of virus from cell to cell, and thus its overexpression increases the oncolytic activity of the vectors. KD1 and KD3 contain mutations in the Ad5 E1A gene that knock out binding of the E1A proteins to cellular p300/CBP and pRB; these mutations allow KD1 and KD3 to grow well in cancer cells but not in normal cells. VRX-007 has wild-type E1A. Here we report that radiation increases the oncolytic activity of KD1, KD3, and VRX-007. This increased activity was observed in cultured cells, and it was not because of radiation-induced replication of the vectors. The combination of radiation plus KD3 suppressed the growth of A549 lung adenocarcinoma xenografts in nude mice more efficiently than radiation alone or KD3 alone. The combination of ADP-overexpressing vectors and radiation may have potential in treating cancer.

Complementation between avirulent Newcastle disease virus and a fusion protein gene expressed from a retrovirus vector: requirements for membrane fusion.

PubMed Central

Morrison, T; McQuain, C; McGinnes, L

1991-01-01

The cDNA derived from the fusion gene of the virulent AV strain of Newcastle disease virus (NDV) was expressed in chicken embryo cells by using a retrovirus vector. The fusion protein expressed in this system was transported to the cell surface and was efficiently cleaved into the disulfide-linked F1-F2 form found in infectious virions. The cells expressing the fusion gene grew normally and could be passaged many times. Monolayers of these cells would plaque, in the absence of trypsin, avirulent NDV strains (strains which encode a fusion protein which is not cleaved in tissue culture). Fusion protein-expressing cells would not fuse if mixed with uninfected cells or uninfected cells expressing the hemagglutinin-neuraminidase (HN) protein. However, the fusion protein-expressing cells, if infected with avirulent strains of NDV, would fuse with uninfected cells, suggesting that fusion requires both the fusion protein and another viral protein expressed in the same cell. Fusion was also seen after transfection of the HN protein gene into fusion protein-expressing cells. Thus, the expressed fusion protein gene is capable of complementing the virus infection, providing an active cleaved fusion protein required for the spread of infection. However, the fusion protein does not mediate cell fusion unless the cell also expresses the HN protein. Fusion protein-expressing cells would not plaque influenza virus in the absence of trypsin, nor would influenza virus-infected fusion protein-expressing cells fuse with uninfected cells. Thus, the influenza virus HA protein will not substitute for the NDV HN protein in cell-to-cell fusion. Images PMID:1987376

Modified Newcastle disease virus vectors expressing the H5 hemagglutinin induce enhanced protection against highly pathogenic H5N1 avian influenza virus in chickens

PubMed Central

Kim, Shin-Hee; Paldurai, Anandan; Xiao, Sa; Collins, Peter L.; Samal, Siba K.

2016-01-01

Naturally-occurring attenuated strains of Newcastle disease virus (NDV) are being developed as vaccine vectors for use in poultry and humans. However, some NDV strains, such as Beaudette C (BC), may retain too much virulence in poultry for safe use, and more highly attenuated strains may be suboptimally immunogenic. We therefore modified the BC strain by changing the multibasic cleavage site sequence of the F protein to the dibasic sequence of avirulent strain LaSota. Additionally, the BC, F, and HN proteins were modified in several ways to enhance virus replication. These modified BC-derived vectors and the LaSota strain were engineered to express the hemagglutin (HA) protein of H5N1 highly pathogenic influenza virus (HPAIV). In general, the modified BC-based vectors expressing HA replicated better than LaSota/HA, and expressed higher levels of HA protein. Pathogenicity tests indicated that all the modified viruses were highly attenuated in chickens. Based on in vitro characterization, two of the modified BC vectors were chosen for evaluation in chickens as vaccine vectors against H5N1 HPAIV A/Vietnam/1203/04. Immunization of chickens with rNDV vector vaccines followed by challenge with HPAIV demonstrated high levels of protection against clinical disease and mortality. However, only those chickens immunized with modified BC/HA in which residues 271–330 from the F protein had been replaced with the corresponding sequence from the NDV AKO strain conferred complete protection against challenge virus shedding. Our findings suggest that this modified rNDV can be used safely as a vaccine vector with enhanced replication, expression, and protective efficacy in avian species, and potentially in humans. PMID:24968158

Resting lymphocyte transduction with measles virus glycoprotein pseudotyped lentiviral vectors relies on CD46 and SLAM

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou Qi; Schneider, Irene C.; Gallet, Manuela

2011-05-10

The measles virus (MV) glycoproteins hemagglutinin (H) and fusion (F) were recently shown to mediate transduction of resting lymphocytes by lentiviral vectors. MV vaccine strains use CD46 or signaling lymphocyte activation molecule (SLAM) as receptor for cell entry. A panel of H protein mutants derived from vaccine strain or wild-type MVs that lost or gained CD46 or SLAM receptor usage were investigated for their ability to mediate gene transfer into unstimulated T lymphocytes. The results demonstrate that CD46 is sufficient for efficient vector particle association with unstimulated lymphocytes. For stable gene transfer into these cells, however, both MV receptors weremore » found to be essential.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gokumakulapalle, Madhuri; Mei, Ya-Fang, E-mail: ya-fang.mei@umu.se

The use of continuous cell lines derived from the African green monkey kidney (AGMK) has led to major advances in virus vaccine development. However, to date, these cells have not been used to facilitate the creation of human adenoviruses because most human adenoviruses undergo abortive infections in them. Here, we report the susceptibility of AGMK-derived cells to adenovirus 11p (Ad11p) infection. First, we showed that CD46 molecules, which act as receptors for Ad11p, are expressed in AGMK cells. We then monitored Ad11p replication by measuring GFP expression as an indicator of viral transcription. We found that AGMK-derived cells were asmore » capable as carcinoma cells at propagating full-length replication-competent Ad11p (RCAd11p) DNA. Of the AGMK cell lines tested, Vero cells had the greatest capacity for adenovirus production. Thus, AGMK cells can be used to evaluate RCAd11p-mediated gene delivery, and Vero cells can be used for the production of RCAd11pGFP vectors at relatively high yields. - Highlights: • Africa green monkey cell lines were monitored for human adenovirus 11p GFP vector infection. • Human CD46 molecules were detectable in these monkey cell lines. • Adenovirus 11p GFP vector can be propagated in Vero cells increases the safety of Ad11p-based vectors for clinical trials. • To use Vero cells for preparation of Ad11p vector avoids the potential inclusion of oncogenes from tumor cells.« less

Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs.

PubMed

Jiang, Xiaohong; Dalebout, Tim J; Bredenbeek, Peter J; Carrion, Ricardo; Brasky, Kathleen; Patterson, Jean; Goicochea, Marco; Bryant, Joseph; Salvato, Maria S; Lukashevich, Igor S

2011-02-01

Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV-GP1 and -GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and -GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF proteins and LASV GP antigens in infected cells. YF17D/LASV-GP1 and -GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1 and -GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa. Copyright © 2010 Elsevier Ltd. All rights reserved.

Peptide Processing Is Critical for T-Cell Memory Inflation and May Be Optimized to Improve Immune Protection by CMV-Based Vaccine Vectors.

PubMed

Dekhtiarenko, Iryna; Ratts, Robert B; Blatnik, Renata; Lee, Lian N; Fischer, Sonja; Borkner, Lisa; Oduro, Jennifer D; Marandu, Thomas F; Hoppe, Stephanie; Ruzsics, Zsolt; Sonnemann, Julia K; Mansouri, Mandana; Meyer, Christine; Lemmermann, Niels A W; Holtappels, Rafaela; Arens, Ramon; Klenerman, Paul; Früh, Klaus; Reddehase, Matthias J; Riemer, Angelika B; Cicin-Sain, Luka

2016-12-01

Cytomegalovirus (CMV) elicits long-term T-cell immunity of unparalleled strength, which has allowed the development of highly protective CMV-based vaccine vectors. Counterintuitively, experimental vaccines encoding a single MHC-I restricted epitope offered better immune protection than those expressing entire proteins, including the same epitope. To clarify this conundrum, we generated recombinant murine CMVs (MCMVs) encoding well-characterized MHC-I epitopes at different positions within viral genes and observed strong immune responses and protection against viruses and tumor growth when the epitopes were expressed at the protein C-terminus. We used the M45-encoded conventional epitope HGIRNASFI to dissect this phenomenon at the molecular level. A recombinant MCMV expressing HGIRNASFI on the C-terminus of M45, in contrast to wild-type MCMV, enabled peptide processing by the constitutive proteasome, direct antigen presentation, and an inflation of antigen-specific effector memory cells. Consequently, our results indicate that constitutive proteasome processing of antigenic epitopes in latently infected cells is required for robust inflationary responses. This insight allows utilizing the epitope positioning in the design of CMV-based vectors as a novel strategy for enhancing their efficacy.

Peptide Processing Is Critical for T-Cell Memory Inflation and May Be Optimized to Improve Immune Protection by CMV-Based Vaccine Vectors

PubMed Central

Blatnik, Renata; Lee, Lian N.; Fischer, Sonja; Borkner, Lisa; Oduro, Jennifer D.; Marandu, Thomas F.; Hoppe, Stephanie; Ruzsics, Zsolt; Sonnemann, Julia K.; Meyer, Christine; Holtappels, Rafaela; Arens, Ramon; Früh, Klaus; Reddehase, Matthias J.; Riemer, Angelika B.; Cicin-Sain, Luka

2016-01-01

Cytomegalovirus (CMV) elicits long-term T-cell immunity of unparalleled strength, which has allowed the development of highly protective CMV-based vaccine vectors. Counterintuitively, experimental vaccines encoding a single MHC-I restricted epitope offered better immune protection than those expressing entire proteins, including the same epitope. To clarify this conundrum, we generated recombinant murine CMVs (MCMVs) encoding well-characterized MHC-I epitopes at different positions within viral genes and observed strong immune responses and protection against viruses and tumor growth when the epitopes were expressed at the protein C-terminus. We used the M45-encoded conventional epitope HGIRNASFI to dissect this phenomenon at the molecular level. A recombinant MCMV expressing HGIRNASFI on the C-terminus of M45, in contrast to wild-type MCMV, enabled peptide processing by the constitutive proteasome, direct antigen presentation, and an inflation of antigen-specific effector memory cells. Consequently, our results indicate that constitutive proteasome processing of antigenic epitopes in latently infected cells is required for robust inflationary responses. This insight allows utilizing the epitope positioning in the design of CMV-based vectors as a novel strategy for enhancing their efficacy. PMID:27977791

A pepper mottle virus-based vector enables systemic expression of endoglucanase D in non-transgenic plants.

PubMed

Song, Eun Gyeong; Ryu, Ki Hyun

2017-12-01

Plant-virus-based expression vectors have been used as an alternative to the creation of transgenic plants. Using a virus-based vector, we investigated the feasibility of producing the endoglucanase D (EngD) from Clostridium cellulovorans in Nicotiana benthamiana. This protein has endoglucanase, xylanase, and exoglucanase activities and may be of value for cellulose digestion in the generation of biofuels from plant biomass. The EngD gene was cloned between the nuclear inclusion b (NIb)- and coat protein (CP)-encoding sequences of pSP6PepMoV-Vb1. In vitro transcripts derived from the clone (pSP6PepMoV-Vb1/EngD) were infectious in N. benthamiana but caused milder symptoms than wild-type PepMoV-Vb1. RT-PCR amplification of total RNA from non-inoculated upper leaves infected with PepMoV-Vb1/EngD produced the target band for the CP, partial NIb and EngD-CP regions of PepMoV-V1/EngD, in addition to nonspecific bands. Western blot analysis showed the CP target bands of PepMoV-Vb1/EngD as well as non-target bands. EngD enzymatic activity in infected plants was detected using a glucose assay. The plant leaves showed increased senescence compared with healthy and PepMoV-Vb1-infected plants. Our study suggests the feasibility of using a viral vector for systemic infection of plants for expression of heterologous engD for the purpose of digesting a cellulose substrate in plant cells for biomass production.

Biological and immunogenic properties of rabies virus glycoprotein expressed by canine herpesvirus vector.

PubMed

Xuan, X; Tuchiya, K; Sato, I; Nishikawa, Y; Onoderaz, Y; Takashima, Y; Yamamoto, A; Katsumata, A; Iwata, A; Ueda, S; Mikami, T; Otsuka, H

1998-01-01

In order to evaluate whether canine herpesvirus (CHV) could be used as a live vector for the expression of heterologous immunogenes, we constructed a recombinant canine herpesvirus (CHV) expressing glycoprotein (G protein) of rabies virus (RV). The gene of G protein was inserted within the thymidine kinase gene of CHV YP11mu strain under the control of the human cytomegalovirus immediate early promoter. The G protein expressed by the recombinant CHV was processed and transported to the cell surface as in RV infected cells, and showed the same biological activities such as low pH dependent cell fusion and hemadsorption. The antigenic authenticity of the recombinant G protein was confirmed by a panel of monoclonal antibodies specific for G protein. Dogs inoculated intransally with the recombinant CHV produced higher titres of virus neutralizing antibodies against RV than those inoculated with a commercial, inactivated rabies vaccine. These results suggest that the CHV recombinant expressing G protein can be used as a vaccine to control canine rabies and that CHV may be useful as a vector to develop live recombinant against other infectious diseases in dogs.

Hybrid biosynthetic gene therapy vector development and dual engineering capacity.

PubMed

Jones, Charles H; Ravikrishnan, Anitha; Chen, Mingfu; Reddinger, Ryan; Kamal Ahmadi, Mahmoud; Rane, Snehal; Hakansson, Anders P; Pfeifer, Blaine A

2014-08-26

Genetic vaccines offer a treatment opportunity based upon successful gene delivery to specific immune cell modulators. Driving the process is the vector chosen for gene cargo packaging and subsequent delivery to antigen-presenting cells (APCs) capable of triggering an immune cascade. As such, the delivery process must successfully navigate a series of requirements and obstacles associated with the chosen vector and target cell. In this work, we present the development and assessment of a hybrid gene delivery vector containing biological and biomaterial components. Each component was chosen to design and engineer gene delivery separately in a complimentary and fundamentally distinct fashion. A bacterial (Escherichia coli) inner core and a biomaterial [poly(beta-amino ester)]-coated outer surface allowed the simultaneous application of molecular biology and polymer chemistry to address barriers associated with APC gene delivery, which include cellular uptake and internalization, phagosomal escape, and intracellular cargo concentration. The approach combined and synergized normally disparate vector properties and tools, resulting in increased in vitro gene delivery beyond individual vector components or commercially available transfection agents. Furthermore, the hybrid device demonstrated a strong, efficient, and safe in vivo humoral immune response compared with traditional forms of antigen delivery. In summary, the flexibility, diversity, and potential of the hybrid design were developed and featured in this work as a platform for multivariate engineering at the vector and cellular scales for new applications in gene delivery immunotherapy.

Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Filone, Claire Marie; Heise, Mark; Doms, Robert W.

2006-12-20

Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expressionmore » of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by {beta}-galactosidase {alpha}-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion.« less

Packaging of Mason-Pfizer monkey virus (MPMV) genomic RNA depends upon conserved long-range interactions (LRIs) between U5 and gag sequences.

PubMed

Kalloush, Rawan M; Vivet-Boudou, Valérie; Ali, Lizna M; Mustafa, Farah; Marquet, Roland; Rizvi, Tahir A

2016-06-01

MPMV has great potential for development as a vector for gene therapy. In this respect, precisely defining the sequences and structural motifs that are important for dimerization and packaging of its genomic RNA (gRNA) are of utmost importance. A distinguishing feature of the MPMV gRNA packaging signal is two phylogenetically conserved long-range interactions (LRIs) between U5 and gag complementary sequences, LRI-I and LRI-II. To test their biological significance in the MPMV life cycle, we introduced mutations into these structural motifs and tested their effects on MPMV gRNA packaging and propagation. Furthermore, we probed the structure of key mutants using SHAPE (selective 2'hydroxyl acylation analyzed by primer extension). Disrupting base-pairing of the LRIs affected gRNA packaging and propagation, demonstrating their significance to the MPMV life cycle. A double mutant restoring a heterologous LRI-I was fully functional, whereas a similar LRI-II mutant failed to restore gRNA packaging and propagation. These results demonstrate that while LRI-I acts at the structural level, maintaining base-pairing is not sufficient for LRI-II function. In addition, in vitro RNA dimerization assays indicated that the loss of RNA packaging in LRI mutants could not be attributed to the defects in dimerization. Our findings suggest that U5-gag LRIs play an important architectural role in maintaining the structure of the 5' region of the MPMV gRNA, expanding the crucial role of LRIs to the nonlentiviral group of retroviruses. © 2016 Kalloush et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

Concurrent micro-RNA mediated silencing of tick-borne flavivirus replication in tick vector and in the brain of vertebrate host.

PubMed

Tsetsarkin, Konstantin A; Liu, Guangping; Kenney, Heather; Hermance, Meghan; Thangamani, Saravanan; Pletnev, Alexander G

2016-09-13

Tick-borne viruses include medically important zoonotic pathogens that can cause life-threatening diseases. Unlike mosquito-borne viruses, whose impact can be restrained via mosquito population control programs, for tick-borne viruses only vaccination remains the reliable means of disease prevention. For live vaccine viruses a concern exists, that spillovers from viremic vaccinees could result in introduction of genetically modified viruses into sustainable tick-vertebrate host transmission cycle in nature. To restrict tick-borne flavivirus (Langat virus, LGTV) vector tropism, we inserted target sequences for tick-specific microRNAs (mir-1, mir-275 and mir-279) individually or in combination into several distant regions of LGTV genome. This caused selective attenuation of viral replication in tick-derived cells. LGTV expressing combinations of target sequences for tick- and vertebrate CNS-specific miRNAs were developed. The resulting viruses replicated efficiently and remained stable in simian Vero cells, which do not express these miRNAs, however were severely restricted to replicate in tick-derived cells. In addition, simultaneous dual miRNA targeting led to silencing of virus replication in live Ixodes ricinus ticks and abolished virus neurotropism in highly permissive newborn mice. The concurrent restriction of adverse replication events in vertebrate and invertebrate hosts will, therefore, ensure the environmental safety of live tick-borne virus vaccine candidates.

Ins and Outs of Multipartite Positive-Strand RNA Plant Viruses: Packaging versus Systemic Spread

PubMed Central

Dall’Ara, Mattia; Ratti, Claudio; Bouzoubaa, Salah E.; Gilmer, David

2016-01-01

Viruses possessing a non-segmented genome require a specific recognition of their nucleic acid to ensure its protection in a capsid. A similar feature exists for viruses having a segmented genome, usually consisting of viral genomic segments joined together into one viral entity. While this appears as a rule for animal viruses, the majority of segmented plant viruses package their genomic segments individually. To ensure a productive infection, all viral particles and thereby all segments have to be present in the same cell. Progression of the virus within the plant requires as well a concerted genome preservation to avoid loss of function. In this review, we will discuss the “life aspects” of chosen phytoviruses and argue for the existence of RNA-RNA interactions that drive the preservation of viral genome integrity while the virus progresses in the plant. PMID:27548199

Lentiviral gene transduction of mouse and human hematopoietic stem cells.

PubMed

van Til, Niek P; Wagemaker, Gerard

2014-01-01

Lentiviral vectors can be used to genetically modify a broad range of cells. Hematopoietic stem cells (HSCs) are particularly suitable for lentiviral gene augmentation, because these cells can be enriched with relative ease from mouse bone marrow and human hematopoietic sources, and in principle require relatively limited cell numbers to completely reconstitute the hematopoietic system in vivo. Furthermore, lentiviral vectors are very efficient if pseudotyped with broad tropism envelope proteins. This chapter focuses on gene modification by the use of self-inactivating third-generation human immunodeficiency virus-derived lentiviral vectors for ex vivo HSC modification for both mouse and human application.

Anterograde or Retrograde Transsynaptic Circuit Tracing in Vertebrates with Vesicular Stomatitis Virus Vectors

PubMed Central

Beier, Kevin T.; Mundell, Nathan A.; Pan, Y. Albert; Cepko, Constance L.

2016-01-01

Viruses have been used as transsynaptic tracers, allowing one to map the inputs and outputs of neuronal populations, due to their ability to replicate in neurons and transmit in vivo only across synaptically connected cells. To date, their use has been largely restricted to mammals. In order to explore the use of such viruses in an expanded host range, we tested the transsynaptic tracing ability of recombinant vesicular stomatitis virus (rVSV) vectors in a variety of organisms. Successful infection and gene expression were achieved in a wide range of organisms, including vertebrate and invertebrate model organisms. Moreover, rVSV enabled transsynaptic tracing of neural circuitry in predictable directions dictated by the viral envelope glycoprotein (G), derived from either VSV or rabies virus (RABV). Anterograde and retrograde labeling, from initial infection and/or viral replication and transmission, was observed in Old and New World monkeys, seahorses, jellyfish, zebrafish, chickens, and mice. These vectors are widely applicable for gene delivery, afferent tract tracing, and/or directional connectivity mapping. Here, we detail the use of these vectors and provide protocols for propagating virus, changing the surface glycoprotein, and infecting multiple organisms using several injection strategies. PMID:26729030

Anterograde or Retrograde Transsynaptic Circuit Tracing in Vertebrates with Vesicular Stomatitis Virus Vectors.

PubMed

Beier, Kevin T; Mundell, Nathan A; Pan, Y Albert; Cepko, Constance L

2016-01-04

Viruses have been used as transsynaptic tracers, allowing one to map the inputs and outputs of neuronal populations, due to their ability to replicate in neurons and transmit in vivo only across synaptically connected cells. To date, their use has been largely restricted to mammals. In order to explore the use of such viruses in an expanded host range, we tested the transsynaptic tracing ability of recombinant vesicular stomatitis virus (rVSV) vectors in a variety of organisms. Successful infection and gene expression were achieved in a wide range of organisms, including vertebrate and invertebrate model organisms. Moreover, rVSV enabled transsynaptic tracing of neural circuitry in predictable directions dictated by the viral envelope glycoprotein (G), derived from either VSV or rabies virus (RABV). Anterograde and retrograde labeling, from initial infection and/or viral replication and transmission, was observed in Old and New World monkeys, seahorses, jellyfish, zebrafish, chickens, and mice. These vectors are widely applicable for gene delivery, afferent tract tracing, and/or directional connectivity mapping. Here, we detail the use of these vectors and provide protocols for propagating virus, changing the surface glycoprotein, and infecting multiple organisms using several injection strategies. Copyright © 2016 John Wiley & Sons, Inc.

Primate Neural Retina Upregulates IL-6 and IL-10 in Response to a Herpes Simplex Vector Suggesting the Presence of a Pro-/Anti-inflammatory Axis

PubMed Central

Sauter, Monica M.; Brandt, Curtis R.

2016-01-01

Injection of herpes simplex virus vectors into the vitreous of primate eyes induces an acute, transient uveitis. The purpose of this study was to characterize innate immune responses of macaque neural retina tissue to the herpes simplex virus type 1-based gene delivery vector hrR3. PCR array analysis demonstrated the induction of the pro-inflammatory cytokine IL-6, as well as the anti-inflammatory cytokine IL-10, following hrR3 exposure. Secretion of IL-6 was detected by ELISA and cone photoreceptors and Muller cells were the predominant IL-6 positive cell types. RNA in situ hybridization confirmed that IL-6 was expressed in photoreceptor and Muller cells. The IL-10 positive cells in the inner nuclear layer were identified as amacrine cells by immunofluorescence staining with calretinin antibody. hrR3 challenge resulted in activation of NFκB (p65) in Muller glial cells, but not in cone photoreceptors, suggesting a novel regulatory mechanism for IL-6 expression in cone cells. hrR3 replication was not required for IL-6 induction or NFκB (p65) activation. These data suggest a pro-inflammatory (IL-6)/anti-inflammatory (IL-10) axis exists in neural retina and the severity of acute posterior uveitis may be determined by this interaction. Further studies are needed to identify the trigger for IL-6 and IL-10 induction and the mechanism of IL-6 induction in cone cells. PMID:27170050

RNA interference-based functional knockdown of the voltage-gated potassium channel Kv7.2 in dorsal root ganglion neurons after in vitro and in vivo gene transfer by adeno-associated virus vectors.

PubMed

Valdor, Markus; Wagner, Anke; Röhrs, Viola; Berg, Johanna; Fechner, Henry; Schröder, Wolfgang; Tzschentke, Thomas M; Bahrenberg, Gregor; Christoph, Thomas; Kurreck, Jens

2018-01-01

Activation of the neuronal potassium channel Kv7.2 encoded by the KCNQ2 gene has recently been shown to be an attractive mechanism to inhibit nociceptive transmission. However, potent, selective, and clinically proven activators of Kv7.2/Kv7.3 currents with analgesic properties are still lacking. An important prerequisite for the development of new drugs is a model to test the selectivity of novel agonists by abrogating Kv7.2/Kv7.3 function. Since constitutive knockout mice are not viable, we developed a model based on RNA interference-mediated silencing of KCNQ2. By delivery of a KCNQ2-specific short hairpin RNA with adeno-associated virus vectors, we completely abolished the activity of the specific Kv7.2/Kv7.3-opener ICA-27243 in rat sensory neurons. Results obtained in the silencing experiments were consistent between freshly prepared and cryopreserved dorsal root ganglion neurons, as well as in dorsal root ganglion neurons dissociated and cultured after in vivo administration of the silencing vector by intrathecal injections into rats. Interestingly, the tested associated virus serotypes substantially differed with respect to their transduction capability in cultured neuronal cell lines and primary dorsal root ganglion neurons and the in vivo transfer of transgenes by intrathecal injection of associated virus vectors. However, our study provides the proof-of-concept that RNA interference-mediated silencing of KCNQ2 is a suitable approach to create an ex vivo model for testing the specificity of novel Kv7.2/Kv7.3 agonists.

Approaches to utilize mesenchymal progenitor cells as cellular vehicles.

PubMed

Pereboeva, L; Komarova, S; Mikheeva, G; Krasnykh, V; Curiel, D T

2003-01-01

Mammalian cells represent a novel vector approach for gene delivery that overcomes major drawbacks of viral and nonviral vectors and couples cell therapy with gene delivery. A variety of cell types have been tested in this regard, confirming that the ideal cellular vector system for ex vivo gene therapy has to comply with stringent criteria and is yet to be found. Several properties of mesenchymal progenitor cells (MPCs), such as easy access and simple isolation and propagation procedures, make these cells attractive candidates as cellular vehicles. In the current work, we evaluated the potential utility of MPCs as cellular vectors with the intent to use them in the cancer therapy context. When conventional adenoviral (Ad) vectors were used for MPC transduction, the highest transduction efficiency of MPCs was 40%. We demonstrated that Ad primary-binding receptors were poorly expressed on MPCs, while the secondary Ad receptors and integrins presented in sufficient amounts. By employing Ad vectors with incorporated integrin-binding motifs (Ad5lucRGD), MPC transduction was augmented tenfold, achieving efficient genetic loading of MPCs with reporter and anticancer genes. MPCs expressing thymidine kinase were able to exert a bystander killing effect on the cancer cell line SKOV3ip1 in vitro. In addition, we found that MPCs were able to support Ad replication, and thus can be used as cell vectors to deliver oncolytic viruses. Our results show that MPCs can foster expression of suicide genes or support replication of adenoviruses as potential anticancer therapeutic payloads. These findings are consistent with the concept that MPCs possess key properties that ensure their employment as cellular vehicles and can be used to deliver either therapeutic genes or viruses to tumor sites.

Safety mechanism assisted by the repressor of tetracycline (SMART) vaccinia virus vectors for vaccines and therapeutics.

PubMed

Grigg, Patricia; Titong, Allison; Jones, Leslie A; Yilma, Tilahun D; Verardi, Paulo H

2013-09-17

Replication-competent viruses, such as Vaccinia virus (VACV), are powerful tools for the development of oncolytic viral therapies and elicit superior immune responses when used as vaccine and immunotherapeutic vectors. However, severe complications from uncontrolled viral replication can occur, particularly in immunocompromised individuals or in those with other predisposing conditions. VACVs constitutively expressing interferon-γ (IFN-γ) replicate in cell culture indistinguishably from control viruses; however, they replicate in vivo to low or undetectable levels, and are rapidly cleared even in immunodeficient animals. In an effort to develop safe and highly effective replication-competent VACV vectors, we established a system to inducibly express IFN-γ. Our SMART (safety mechanism assisted by the repressor of tetracycline) vectors are designed to express the tetracycline repressor under a constitutive VACV promoter and IFN-γ under engineered tetracycline-inducible promoters. Immunodeficient SCID mice inoculated with VACVs not expressing IFN-γ demonstrated severe weight loss, whereas those given VACVs expressing IFN-γ under constitutive VACV promoters showed no signs of infection. Most importantly, mice inoculated with a VACV expressing the IFN-γ gene under an inducible promoter remained healthy in the presence of doxycycline, but exhibited severe weight loss in the absence of doxycycline. In this study, we developed a safety mechanism for VACV based on the conditional expression of IFN-γ under a tightly controlled tetracycline-inducible VACV promoter for use in vaccines and oncolytic cancer therapies.

Genetic engineering of stem cells for enhanced therapy.

PubMed

Nowakowski, Adam; Andrzejewska, Anna; Janowski, Miroslaw; Walczak, Piotr; Lukomska, Barbara

2013-01-01

Stem cell therapy is a promising strategy for overcoming the limitations of current treatment methods. The modification of stem cell properties may be necessary to fully exploit their potential. Genetic engineering, with an abundance of methodology to induce gene expression in a precise and well-controllable manner, is particularly attractive for this purpose. There are virus-based and non-viral methods of genetic manipulation. Genome-integrating viral vectors are usually characterized by highly efficient and long-term transgene expression, at a cost of safety. Non-integrating viruses are also highly efficient in transduction, and, while safer, offer only a limited duration of transgene expression. There is a great diversity of transfectable forms of nucleic acids; however, for efficient shuttling across cell membranes, additional manipulation is required. Both physical and chemical methods have been employed for this purpose. Stem cell engineering for clinical applications is still in its infancy and requires further research. There are two main strategies for inducing transgene expression in therapeutic cells: transient and permanent expression. In many cases, including stem cell trafficking and using cell therapy for the treatment of rapid-onset disease with a short healing process, transient transgene expression may be a sufficient and optimal approach. For that purpose, mRNA-based methods seem ideally suited, as they are characterized by a rapid, highly efficient transfection, with outstanding safety. Permanent transgene expression is primarily based on the application of viral vectors, and, due to safety concerns, these methods are more challenging. There is active, ongoing research toward the development of non-viral methods that would induce permanent expression, such as transposons and mammalian artificial chromosomes.

Development of replication-competent viral vectors for HIV vaccine delivery

PubMed Central

Parks, Christopher L.; Picker, Louis J.; King, C. Richter

2014-01-01

Purpose of review Briefly describe some of the replication-competent (RC) vectors being investigated for development of candidate HIV vaccines focusing primarily on technologies that have advanced to testing in macaques or have entered clinical trials. Recent findings RC viral vectors have advanced to the stage were decisions can be made regarding future development of HIV vaccines. The viruses being used as RC vector platforms vary considerably, and their unique attributes make it possible to test multiple vaccine design concepts and also mimic various aspects of an HIV infection. RC viral vectors encoding SIV or HIV proteins can be used to safely immunize macaques, and in some cases, there is evidence of significant vaccine efficacy in challenge protection studies. Several live HIV vaccine vectors are in clinical trials to evaluate immunogenicity, safety, the effect of mucosal delivery, and potential effects of pre-existing immunity. Summary A variety of DNA and RNA viruses are being used to develop RC viral vectors for HIV vaccine delivery. Multiple viral vector platforms have proven to be safe and immunogenic with evidence of efficacy in macaques. Some of the more advanced HIV vaccine prototypes based on vesicular stomatitis virus, vaccinia virus, measles virus, and Sendai virus are in clinical trials. PMID:23925000

Armored long non-coding RNA MEG3 targeting EGFR based on recombinant MS2 bacteriophage virus-like particles against hepatocellular carcinoma.

PubMed

Chang, Le; Wang, Guojing; Jia, Tingting; Zhang, Lei; Li, Yulong; Han, Yanxi; Zhang, Kuo; Lin, Guigao; Zhang, Rui; Li, Jinming; Wang, Lunan

2016-04-26

Hepatocellular carcinoma (HCC) is one of the most frequently diagnosed cancers worldwide. However, the treatment of patients with HCC is particularly challenging. Long non-coding RNA maternally expressed gene 3 (MEG3) has been identified as a potential suppressor of several types of tumors, but the delivery of long RNA remains problematic, limiting its applications. In the present study, we designed a novel delivery system based on MS2 virus-like particles (VLPs) crosslinked with GE11 polypeptide. This vector was found to be fast, effective and safe for the targeted delivery of lncRNA MEG3 RNA to the epidermal growth factor receptor (EGFR)-positive HCC cell lines without the activation of EGFR downstream pathways, and significantly attenuated both in vitro and in vivo tumor cell growth. Our study also revealed that the targeted delivery was mainly dependent on clathrin-mediated endocytosis and MEG3 RNA suppresses tumor growth mainly via increasing the expression of p53 and its downstream gene GDF15, but decreasing the expression of MDM2. Thus, this vector is promising as a novel delivery system and may facilitate a new approach to lncRNA based cancer therapy.

The role of gut bacteria in Schmallenberg virus transmission by Culicoides biting midges

USDA-ARS?s Scientific Manuscript database

When an arbo-virus enters a vector it will first enter the gut system of this insect before entering cells of the insect body. Once in the gut-system, arbo-viruses and gut microbiota can interact with each other. We wondered if different gut bacterial communities could influence virus infection of b...

Oncotargeting by Vesicular Stomatitis Virus (VSV): Advances in Cancer Therapy.

PubMed

Bishnoi, Suman; Tiwari, Ritudhwaj; Gupta, Sharad; Byrareddy, Siddappa N; Nayak, Debasis

2018-02-23

Modern oncotherapy approaches are based on inducing controlled apoptosis in tumor cells. Although a number of apoptosis-induction approaches are available, site-specific delivery of therapeutic agents still remain the biggest hurdle in achieving the desired cancer treatment benefit. Additionally, systemic treatment-induced toxicity remains a major limiting factor in chemotherapy. To specifically address drug-accessibility and chemotherapy side effects, oncolytic virotherapy (OV) has emerged as a novel cancer treatment alternative. In OV, recombinant viruses with higher replication capacity and stronger lytic properties are being considered for tumor cell-targeting and subsequent cell lysing. Successful application of OVs lies in achieving strict tumor-specific tropism called oncotropism, which is contingent upon the biophysical interactions of tumor cell surface receptors with viral receptors and subsequent replication of oncolytic viruses in cancer cells. In this direction, few viral vector platforms have been developed and some of these have entered pre-clinical/clinical trials. Among these, the Vesicular stomatitis virus (VSV)-based platform shows high promise, as it is not pathogenic to humans. Further, modern molecular biology techniques such as reverse genetics tools have favorably advanced this field by creating efficient recombinant VSVs for OV; some have entered into clinical trials. In this review, we discuss the current status of VSV based oncotherapy, challenges, and future perspectives regarding its therapeutic applications in the cancer treatment.

Oncotargeting by Vesicular Stomatitis Virus (VSV): Advances in Cancer Therapy

PubMed Central

Bishnoi, Suman; Tiwari, Ritudhwaj; Gupta, Sharad; Byrareddy, Siddappa N.; Nayak, Debasis

2018-01-01

Modern oncotherapy approaches are based on inducing controlled apoptosis in tumor cells. Although a number of apoptosis-induction approaches are available, site-specific delivery of therapeutic agents still remain the biggest hurdle in achieving the desired cancer treatment benefit. Additionally, systemic treatment-induced toxicity remains a major limiting factor in chemotherapy. To specifically address drug-accessibility and chemotherapy side effects, oncolytic virotherapy (OV) has emerged as a novel cancer treatment alternative. In OV, recombinant viruses with higher replication capacity and stronger lytic properties are being considered for tumor cell-targeting and subsequent cell lysing. Successful application of OVs lies in achieving strict tumor-specific tropism called oncotropism, which is contingent upon the biophysical interactions of tumor cell surface receptors with viral receptors and subsequent replication of oncolytic viruses in cancer cells. In this direction, few viral vector platforms have been developed and some of these have entered pre-clinical/clinical trials. Among these, the Vesicular stomatitis virus (VSV)-based platform shows high promise, as it is not pathogenic to humans. Further, modern molecular biology techniques such as reverse genetics tools have favorably advanced this field by creating efficient recombinant VSVs for OV; some have entered into clinical trials. In this review, we discuss the current status of VSV based oncotherapy, challenges, and future perspectives regarding its therapeutic applications in the cancer treatment. PMID:29473868

An Improved Brome mosaic virus Silencing Vector: Greater Insert Stability and More Extensive VIGS.

PubMed

Ding, Xin Shun; Mannas, Stephen W; Bishop, Bethany A; Rao, Xiaolan; Lecoultre, Mitchell; Kwon, Soonil; Nelson, Richard S

2018-01-01

Virus-induced gene silencing (VIGS) is used extensively for gene function studies in plants. VIGS is inexpensive and rapid compared with silencing conducted through stable transformation, but many virus-silencing vectors, especially in grasses, induce only transient silencing phenotypes. A major reason for transient phenotypes is the instability of the foreign gene fragment (insert) in the vector during VIGS. Here, we report the development of a Brome mosaic virus (BMV)-based vector that better maintains inserts through modification of the original BMV vector RNA sequence. Modification of the BMV RNA3 sequence yielded a vector, BMVCP5, that better maintained phytoene desaturase and heat shock protein70-1 ( HSP70-1 ) inserts in Nicotiana benthamiana and maize ( Zea mays ). Longer maintenance of inserts was correlated with greater target gene silencing and more extensive visible silencing phenotypes displaying greater tissue penetration and involving more leaves. The modified vector accumulated similarly to the original vector in N. benthamiana after agroinfiltration, thus maintaining a high titer of virus in this intermediate host used to produce virus inoculum for grass hosts. For HSP70 , silencing one family member led to a large increase in the expression of another family member, an increase likely related to the target gene knockdown and not a general effect of virus infection. The cause of the increased insert stability in the modified vector is discussed in relationship to its recombination and accumulation potential. The modified vector will improve functional genomic studies in grasses, and the conceptual methods used to improve the vector may be applied to other VIGS vectors. © 2018 American Society of Plant Biologists. All Rights Reserved.

Hypotrichosis and juvenile macular dystrophy caused by CDH3 mutation: A candidate disease for retinal gene therapy.

PubMed

Singh, Mandeep S; Broadgate, Suzanne; Mathur, Ranjana; Holt, Richard; Halford, Stephanie; MacLaren, Robert E

2016-05-09

Hypotrichosis with juvenile macular dystrophy (HJMD) is an autosomal recessive disorder that causes childhood visual impairment. HJMD is caused by mutations in CDH3 which encodes cadherin-3, a protein expressed in retinal pigment epithelium (RPE) cells that may have a key role in intercellular adhesion. We present a case of HJMD and analyse its phenotypic and molecular characteristics to assess the potential for retinal gene therapy as a means of preventing severe visual loss in this condition. Longitudinal in vivo imaging of the retina showed the relative anatomical preservation of the macula, which suggested the presence of a therapeutic window for gene augmentation therapy to preserve visual acuity. The coding sequence of CDH3 fits within the packaging limit of recombinant adeno-associated virus vectors that have been shown to be safe in clinical trials and can efficiently target RPE cells. This report expands the number of reported cases of HJMD and highlights the phenotypic characteristics to consider when selecting candidates for retinal gene therapy.

Do viruses use vectors to penetrate mucus barriers?

PubMed Central

Ribbeck, Katharina

2010-01-01

I propose a mechanism by which viruses successfully infect new individuals, despite being immotile particles with no ability for directed movement. Within cells, viral particle movements are directed by motors and elements of the cytoskeleton, but how viruses cross extracellular barriers, like mucus, remains a mystery. I propose that viruses cross these barriers by hitch-hiking on bacteria or sperm cells which can transport themselves across mucosal layers designed to protect the underlying cells from pathogen attack. An important implication of this hypothesis is that agents that block interactions between viruses and bacteria or sperm may be new tools for disease prevention. PMID:20190864

Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by Food and Drug Administration-approved drugs

PubMed Central

Ellis, BL; Hirsch, ML; Porter, SN; Samulski, RJ; Porteus, MH

2016-01-01

An emerging strategy for the treatment of monogenic diseases uses genetic engineering to precisely correct the mutation(s) at the genome level. Recent advancements in this technology have demonstrated therapeutic levels of gene correction using a zinc-finger nuclease (ZFN)-induced DNA double-strand break in conjunction with an exogenous DNA donor substrate. This strategy requires efficient nucleic acid delivery and among viral vectors, recombinant adeno-associated virus (rAAV) has demonstrated clinical success without pathology. However, a major limitation of rAAV is the small DNA packaging capacity and to date, the use of rAAV for ZFN gene delivery has yet to be reported. Theoretically, an ideal situation is to deliver both ZFNs and the repair substrate in a single vector to avoid inefficient gene targeting and unwanted mutagenesis, both complications of a rAAV co-transduction strategy. Therefore, a rAAV format was generated in which a single polypeptide encodes the ZFN monomers connected by a ribosome skipping 2A peptide and furin cleavage sequence. On the basis of this arrangement, a DNA repair substrate of 750 nucleotides was also included in this vector. Efficient polypeptide processing to discrete ZFNs is demonstrated, as well as the ability of this single vector format to stimulate efficient gene targeting in a human cell line and mouse model derived fibroblasts. Additionally, we increased rAAV-mediated gene correction up to sixfold using a combination of Food and Drug Administration-approved drugs, which act at the level of AAV vector transduction. Collectively, these experiments demonstrate the ability to deliver ZFNs and a repair substrate by a single AAV vector and offer insights for the optimization of rAAV-mediated gene correction using drug therapy. PMID:22257934

Interferon signaling in Peromyscus leucopus confers a potent and specific restriction to vector-borne flaviviruses.

PubMed

Izuogu, Adaeze O; McNally, Kristin L; Harris, Stephen E; Youseff, Brian H; Presloid, John B; Burlak, Christopher; Munshi-South, Jason; Best, Sonja M; Taylor, R Travis

2017-01-01

Tick-borne flaviviruses (TBFVs), including Powassan virus and tick-borne encephalitis virus cause encephalitis or hemorrhagic fevers in humans with case-fatality rates ranging from 1-30%. Despite severe disease in humans, TBFV infection of natural rodent hosts has little noticeable effect. Currently, the basis for resistance to disease is not known. We hypothesize that the coevolution of flaviviruses with their respective hosts has shaped the evolution of potent antiviral factors that suppress virus replication and protect the host from lethal infection. In the current study, we compared virus infection between reservoir host cells and related susceptible species. Infection of primary fibroblasts from the white-footed mouse (Peromyscus leucopus, a representative host) with a panel of vector-borne flaviviruses showed up to a 10,000-fold reduction in virus titer compared to control Mus musculus cells. Replication of vesicular stomatitis virus was equivalent in P. leucopus and M. musculus cells suggesting that restriction was flavivirus-specific. Step-wise comparison of the virus infection cycle revealed a significant block to viral RNA replication, but not virus entry, in P. leucopus cells. To understand the role of the type I interferon (IFN) response in virus restriction, we knocked down signal transducer and activator of transcription 1 (STAT1) or the type I IFN receptor (IFNAR1) by RNA interference. Loss of IFNAR1 or STAT1 significantly relieved the block in virus replication in P. leucopus cells. The major IFN antagonist encoded by TBFV, nonstructural protein 5, was functional in P. leucopus cells, thus ruling out ineffective viral antagonism of the host IFN response. Collectively, this work demonstrates that the IFN response of P. leucopus imparts a strong and virus-specific barrier to flavivirus replication. Future identification of the IFN-stimulated genes responsible for virus restriction specifically in P. leucopus will yield mechanistic insight into efficient control of virus replication and may inform the development of antiviral therapeutics.

A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize.

PubMed

Mei, Yu; Zhang, Chunquan; Kernodle, Bliss M; Hill, John H; Whitham, Steven A

2016-06-01

Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. © 2016 American Society of Plant Biologists. All Rights Reserved.

A Foxtail mosaic virus Vector for Virus-Induced Gene Silencing in Maize1[OPEN

PubMed Central

Mei, Yu; Kernodle, Bliss M.; Hill, John H.

2016-01-01

Plant viruses have been widely used as vectors for foreign gene expression and virus-induced gene silencing (VIGS). A limited number of viruses have been developed into viral vectors for the purposes of gene expression or VIGS in monocotyledonous plants, and among these, the tripartite viruses Brome mosaic virus and Cucumber mosaic virus have been shown to induce VIGS in maize (Zea mays). We describe here a new DNA-based VIGS system derived from Foxtail mosaic virus (FoMV), a monopartite virus that is able to establish systemic infection and silencing of endogenous maize genes homologous to gene fragments inserted into the FoMV genome. To demonstrate VIGS applications of this FoMV vector system, four genes, phytoene desaturase (functions in carotenoid biosynthesis), lesion mimic22 (encodes a key enzyme of the porphyrin pathway), iojap (functions in plastid development), and brown midrib3 (caffeic acid O-methyltransferase), were silenced and characterized in the sweet corn line Golden × Bantam. Furthermore, we demonstrate that the FoMV infectious clone establishes systemic infection in maize inbred lines, sorghum (Sorghum bicolor), and green foxtail (Setaria viridis), indicating the potential wide applications of this viral vector system for functional genomics studies in maize and other monocots. PMID:27208311

Antiviral effects of herpes simplex virus specific anti-sense nucleic acids.

PubMed

Cantin, E M; Podsakoff, G; Willey, D E; Openshaw, H

1992-01-01

We have targeted mRNA sequences encompassing the translation initiation codon of the essential herpes simplex virus type 1 (HSV-1) IE3 gene with three kinds of anti-sense molecule. Addition of a 15mer oligodeoxyribonucleoside methylphosphonate to tissue culture cells resulted in suppression of viral replication. HSV-1 replication was also inhibited in cultured cells containing anti-sense vectors expressing transcripts complementary to the IE3 mRNA. We have also constructed a ribozyme which upon base pairing with the target IE3 mRNA induces cleavage at the predicted GUC site. A major obstacle to anti-sense studies in animals is drug delivery of preformed antisense molecules to ganglionic neurons, the site of HSV latency and reactivation. We speculate as to how this may be accomplished through carrier compounds which are taken up by nerve terminals and transported by retrograde axoplasmic flow. By the same route, HSV itself may be used as an anti-sense vector.

Bunyavirus-Vector Interactions

PubMed Central

Horne, Kate McElroy; Vanlandingham, Dana L.

2014-01-01

The Bunyaviridae family is comprised of more than 350 viruses, of which many within the Hantavirus, Orthobunyavirus, Nairovirus, Tospovirus, and Phlebovirus genera are significant human or agricultural pathogens. The viruses within the Orthobunyavirus, Nairovirus, and Phlebovirus genera are transmitted by hematophagous arthropods, such as mosquitoes, midges, flies, and ticks, and their associated arthropods not only serve as vectors but also as virus reservoirs in many cases. This review presents an overview of several important emerging or re-emerging bunyaviruses and describes what is known about bunyavirus-vector interactions based on epidemiological, ultrastructural, and genetic studies of members of this virus family. PMID:25402172

Engineered Viruses as Genome Editing Devices.

PubMed

Chen, Xiaoyu; Gonçalves, Manuel A F V

2016-03-01

Genome editing based on sequence-specific designer nucleases, also known as programmable nucleases, seeks to modify in a targeted and precise manner the genetic information content of living cells. Delivering into cells designer nucleases alone or together with donor DNA templates, which serve as surrogate homologous recombination (HR) substrates, can result in gene knockouts or gene knock-ins, respectively. As engineered replication-defective viruses, viral vectors are having an increasingly important role as delivery vehicles for donor DNA templates and designer nucleases, namely, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 (CRISPR-Cas9) nucleases, also known as RNA-guided nucleases (RGNs). We review this dual role played by engineered viral particles on genome editing while focusing on their main scaffolds, consisting of lentiviruses, adeno-associated viruses, and adenoviruses. In addition, the coverage of the growing body of research on the repurposing of viral vectors as delivery systems for genome editing tools is complemented with information regarding their main characteristics, pros, and cons. Finally, this information is framed by a concise description of the chief principles, tools, and applications of the genome editing field as a whole.

Engineered Viruses as Genome Editing Devices

PubMed Central

Chen, Xiaoyu; Gonçalves, Manuel A F V

2016-01-01

Genome editing based on sequence-specific designer nucleases, also known as programmable nucleases, seeks to modify in a targeted and precise manner the genetic information content of living cells. Delivering into cells designer nucleases alone or together with donor DNA templates, which serve as surrogate homologous recombination (HR) substrates, can result in gene knockouts or gene knock-ins, respectively. As engineered replication-defective viruses, viral vectors are having an increasingly important role as delivery vehicles for donor DNA templates and designer nucleases, namely, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 (CRISPR−Cas9) nucleases, also known as RNA-guided nucleases (RGNs). We review this dual role played by engineered viral particles on genome editing while focusing on their main scaffolds, consisting of lentiviruses, adeno-associated viruses, and adenoviruses. In addition, the coverage of the growing body of research on the repurposing of viral vectors as delivery systems for genome editing tools is complemented with information regarding their main characteristics, pros, and cons. Finally, this information is framed by a concise description of the chief principles, tools, and applications of the genome editing field as a whole. PMID:26336974

Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lerch, Thomas F.; Chapman, Michael S., E-mail: chapmami@ohsu.edu

2012-02-05

Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites ofmore » AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.« less

Identification of the heparin binding site on adeno-associated virus serotype 3B (AAV-3B)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lerch, Thomas F.; Chapman, Michael S.

2012-05-24

Adeno-associated virus is a promising vector for gene therapy. In the current study, the binding site on AAV serotype 3B for the heparan sulfate proteoglycan (HSPG) receptor has been characterized. X-ray diffraction identified a disaccharide binding site at the most positively charged region on the virus surface. The contributions of basic amino acids at this and other sites were characterized using site-directed mutagenesis. Both heparin and cell binding are correlated to positive charge at the disaccharide binding site, and transduction is significantly decreased in AAV-3B vectors mutated at this site to reduce heparin binding. While the receptor attachment sites ofmore » AAV-3B and AAV-2 are both in the general vicinity of the viral spikes, the exact amino acids that participate in electrostatic interactions are distinct. Diversity in the mechanisms of cell attachment by AAV serotypes will be an important consideration for the rational design of improved gene therapy vectors.« less

Use of a tandem affinity purification assay to detect interactions between West Nile and dengue viral proteins and proteins of the mosquito vector

PubMed Central

Colpitts, Tonya M.; Cox, Jonathan; Nguyen, Annie; Feitosa, Fabiana; Krishnan, Manoj N.; Fikrig, Erol

2011-01-01

West Nile and dengue viruses are (re)emerging mosquito-borne flaviviruses that cause significant morbidity and mortality in man. The identification of mosquito proteins that associate with flaviviruses may provide novel targets to inhibit infection of the vector or block transmission to humans. Here, a tandem affinity purification (TAP) assay was used to identify 18 mosquito proteins that interact with dengue and West Nile capsid, envelope, NS2A or NS2B proteins. We further analyzed the interaction of mosquito cadherin with dengue and West Nile virus envelope protein using co-immunoprecipitation and immunofluorescence. Blocking the function of select mosquito factors, including actin, myosin, PI3-kinase and myosin light chain kinase, reduced both dengue and West Nile virus infection in mosquito cells. We show that the TAP method may be used in insect cells to accurately identify flaviviral-host protein interactions. Our data also provides several targets for interrupting flavivirus infection in mosquito vectors. PMID:21700306

Adenoviral Vectors Armed with Cell Fusion-Inducing Proteins as Anti-Cancer Agents

PubMed Central

Del Papa, Joshua; Parks, Robin J.

2017-01-01

Cancer is a devastating disease that affects millions of patients every year, and causes an enormous economic burden on the health care system and emotional burden on affected families. The first line of defense against solid tumors is usually extraction of the tumor, when possible, by surgical methods. In cases where solid tumors can not be safely removed, chemotherapy is often the first line of treatment. As metastatic cancers often become vigorously resistant to treatments, the development of novel, more potent and selective anti-cancer strategies is of great importance. Adenovirus (Ad) is the most commonly used virus in cancer clinical trials, however, regardless of the nature of the Ad-based therapeutic, complete responses to treatment remain rare. A number of pre-clinical studies have shown that, for all vector systems, viral spread throughout the tumor mass can be a major limiting factor for complete tumor elimination. By expressing exogenous cell-fusion proteins, many groups have shown improved spread of Ad-based vectors. This review summarizes the research done to examine the potency of Ad vectors expressing fusogenic proteins as anti-cancer therapeutics. PMID:28106842

Transmission of Rift Valley fever virus from European-breed lambs to Culex pipiens mosquitoes.

PubMed

Vloet, Rianka P M; Vogels, Chantal B F; Koenraadt, Constantianus J M; Pijlman, Gorben P; Eiden, Martin; Gonzales, Jose L; van Keulen, Lucien J M; Wichgers Schreur, Paul J; Kortekaas, Jeroen

2017-12-01

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus of the genus Phlebovirus that is highly pathogenic to ruminants and humans. The disease is currently confined to Africa and the Arabian Peninsula, but globalization and climate change may facilitate introductions of the virus into currently unaffected areas via infected animals or mosquitoes. The consequences of such an introduction will depend on environmental factors, the availability of susceptible ruminants and the capacity of local mosquitoes to transmit the virus. We have previously demonstrated that lambs native to the Netherlands are highly susceptible to RVFV and we here report the vector competence of Culex (Cx.) pipiens, the most abundant and widespread mosquito species in the country. Vector competence was first determined after artificial blood feeding of laboratory-reared mosquitoes using the attenuated Clone 13 strain. Subsequently, experiments with wild-type RVFV and mosquitoes hatched from field-collected eggs were performed. Finally, the transmission of RVFV from viremic lambs to mosquitoes was studied. Artificial feeding experiments using Clone 13 demonstrated that indigenous, laboratory-reared Cx. pipiens mosquitoes are susceptible to RVFV and that the virus can be transmitted via their saliva. Experiments with wild-type RVFV and mosquitoes hatched from field-collected eggs confirmed the vector competence of Cx. pipiens mosquitoes from the Netherlands. To subsequently investigate transmission of the virus under more natural conditions, mosquitoes were allowed to feed on RVFV-infected lambs during the viremic period. We found that RVFV is efficiently transmitted from lambs to mosquitoes, although transmission was restricted to peak viremia. Interestingly, in the mosquito-exposed skin samples, replication of RVFV was detected in previously unrecognized target cells. We here report the vector competence of Cx. pipiens mosquitoes from the Netherlands for RVFV. Both laboratory-reared mosquitoes and well as those hatched from field-collected eggs were found to be competent vectors. Moreover, RVFV was transmitted efficiently from indigenous lambs to mosquitoes, although the duration of host infectivity was found to be shorter than previously assumed. Interestingly, analysis of mosquito-exposed skin samples revealed previously unidentified target cells of the virus. Our findings underscore the value of including natural target species in vector competence experiments.

Apoptin towards safe and efficient anticancer therapies.

PubMed

Backendorf, Claude; Noteborn, Mathieu H M

2014-01-01

The chicken anemia virus derived protein apoptin harbors cancer-selective cell killing characteristics, essentially based on phosphorylation-mediated nuclear transfer in cancer cells and efficient cytoplasmic degradation in normal cells. Here, we describe a growing set of preclinical experiments underlying the promises of the anti-cancer potential of apoptin. Various non-replicative oncolytic viral vector systems have revealed the safety and efficacy of apoptin. In addition, apoptin enhanced the oncolytic potential of adenovirus, parvovirus and Newcastle disease virus vectors. Intratumoral injection of attenuated Salmonella typhimurium bacterial strains and plasmid-based systems expressing apoptin resulted in significant tumor regression. In-vitro and in-vivo experiments showed that recombinant membrane-transferring PTD4- or TAT-apoptin proteins have potential as a future anticancer therapeutics. In xenografted hepatoma and melanoma mouse models PTD4-apoptin protein entered both cancer and normal cells, but only killed cancer cells. Combinatorial treatment of PTD4-apoptin with various (chemo)therapeutic compounds revealed an additive or even synergistic effect, reducing the side effects of the single (chemo)therapeutic treatment. Degradable polymeric nanocapsules harboring MBP-apoptin fusion-protein induced tumor-selective cell killing in-vitro and in-vivo and revealed the potential of polymer-apoptin protein vehicles as an anticancer agent.Besides its direct use as an anticancer therapeutic, apoptin research has also generated novel possibilities for drug design. The nuclear location domains of apoptin are attractive tools for targeting therapeutic compounds into the nucleus of cancer cells. Identification of cancer-related processes targeted by apoptin can potentially generate novel drug targets. Recent breakthroughs important for clinical applications are reported inferring apoptin-based clinical trials as a feasible reality.

Deletion of Specific Immune-Modulatory Genes from Modified Vaccinia Virus Ankara-Based HIV Vaccines Engenders Improved Immunogenicity in Rhesus Macaques

PubMed Central

O'Mara, Leigh A.; Gangadhara, Sailaja; McQuoid, Monica; Zhang, Xiugen; Zheng, Rui; Gill, Kiran; Verma, Meena; Yu, Tianwei; Johnson, Brent; Li, Bing; Derdeyn, Cynthia A.; Ibegbu, Chris; Altman, John D.; Hunter, Eric; Feinberg, Mark B.

2012-01-01

Modified vaccinia virus Ankara (MVA) is a safe, attenuated orthopoxvirus that is being developed as a vaccine vector but has demonstrated limited immunogenicity in several early-phase clinical trials. Our objective was to rationally improve the immunogenicity of MVA-based HIV/AIDS vaccines via the targeted deletion of specific poxvirus immune-modulatory genes. Vaccines expressing codon-optimized HIV subtype C consensus Env and Gag antigens were generated from MVA vector backbones that (i) harbor simultaneous deletions of four viral immune-modulatory genes, encoding an interleukin-18 (IL-18) binding protein, an IL-1β receptor, a dominant negative Toll/IL-1 signaling adapter, and CC-chemokine binding protein (MVAΔ4-HIV); (ii) harbor a deletion of an additional (fifth) viral gene, encoding uracil-DNA glycosylase (MVAΔ5-HIV); or (iii) represent the parental MVA backbone as a control (MVA-HIV). We performed head-to-head comparisons of the cellular and humoral immune responses that were elicited by these vectors during homologous prime-boost immunization regimens utilizing either high-dose (2 × 108 PFU) or low-dose (1 × 107 PFU) intramuscular immunization of rhesus macaques. At all time points, a majority of the HIV-specific T cell responses, elicited by all vectors, were directed against Env, rather than Gag, determinants, as previously observed with other vector systems. Both modified vectors elicited up to 6-fold-higher frequencies of HIV-specific CD8 and CD4 T cell responses and up to 25-fold-higher titers of Env (gp120)-specific binding (nonneutralizing) antibody responses that were relatively transient in nature. While the correlates of protection against HIV infection remain incompletely defined, our results indicate that the rational deletion of specific genes from MVA vectors can positively alter their cellular and humoral immunogenicity profiles in nonhuman primates. PMID:22973033

Adventitious viruses in insect cell lines used for recombinant protein expression.

PubMed

Geisler, Christoph; Jarvis, Donald L

2018-04-01

Insect cells are widely used for recombinant protein expression, typically as hosts for recombinant baculovirus vectors, but also for plasmid-mediated transient transfection or stable genetic transformation. Insect cells are used to express proteins for research, as well as to manufacture biologicals for human and veterinary medicine. Recently, several insect cell lines used for recombinant protein expression were found to be persistently infected with adventitious viruses. This has raised questions about how these infections might affect research performed using those cell lines. Furthermore, these findings raised serious concerns about the safety of biologicals produced using those cell lines. In response, new insect cell lines lacking adventitious viruses have been isolated for use as improved research tools and safer biological manufacturing platforms. Here, we review the scientific and patent literature on adventitious viruses found in insect cell lines, affected cell lines, and new virus-free cell lines. Copyright © 2017 Elsevier Inc. All rights reserved.

The small non-coding RNA response to virus infection in the Leishmania vector Lutzomyia longipalpis.

PubMed

Ferreira, Flávia Viana; Aguiar, Eric Roberto Guimarães Rocha; Olmo, Roenick Proveti; de Oliveira, Karla Pollyanna Vieira; Silva, Emanuele Guimarães; Sant'Anna, Maurício Roberto Viana; Gontijo, Nelder de Figueiredo; Kroon, Erna Geessien; Imler, Jean Luc; Marques, João Trindade

2018-06-01

Sandflies are well known vectors for Leishmania but also transmit a number of arthropod-borne viruses (arboviruses). Few studies have addressed the interaction between sandflies and arboviruses. RNA interference (RNAi) mechanisms utilize small non-coding RNAs to regulate different aspects of host-pathogen interactions. The small interfering RNA (siRNA) pathway is a broad antiviral mechanism in insects. In addition, at least in mosquitoes, another RNAi mechanism mediated by PIWI interacting RNAs (piRNAs) is activated by viral infection. Finally, endogenous microRNAs (miRNA) may also regulate host immune responses. Here, we analyzed the small non-coding RNA response to Vesicular stomatitis virus (VSV) infection in the sandfly Lutzoymia longipalpis. We detected abundant production of virus-derived siRNAs after VSV infection in adult sandflies. However, there was no production of virus-derived piRNAs and only mild changes in the expression of vector miRNAs in response to infection. We also observed abundant production of virus-derived siRNAs against two other viruses in Lutzomyia Lulo cells. Together, our results suggest that the siRNA but not the piRNA pathway mediates an antiviral response in sandflies. In agreement with this hypothesis, pre-treatment of cells with dsRNA against VSV was able to inhibit viral replication while knock-down of the central siRNA component, Argonaute-2, led to increased virus levels. Our work begins to elucidate the role of RNAi mechanisms in the interaction between L. longipalpis and viruses and should also open the way for studies with other sandfly-borne pathogens.

T-Cell Gene Therapy to Eradicate Disseminated Breast Cancers

DTIC Science & Technology

2011-05-01

reactivation of cytomegalovirus infection following successful rituximab therapy for Epstein - Barr virus - associated posttransplantation lymphoproliferative...improve virus transduction b. New vector supernatants with higher viral titer 2. Development of procedures for improved expansions (>10^6 fold) a...nude mice by passaging cell line. Development of novel dendritic cell vaccine that harnesses the G250-Fc’s capability to elicit both CTL and Th

A defective retroviral vector encoding human interferon-alpha2 can transduce human leukemic cell lines.

PubMed

Austruy, E; Bagnis, C; Carbuccia, N; Maroc, C; Birg, F; Dubreuil, P; Mannoni, P; Chabannon, C

1998-01-01

Using the LXSN backbone, a defective retroviral vector (LISN) was constructed that encodes the human interferon (IFN)-alpha2 (hIFN-alpha2) gene and the neomycin resistance gene; the hIFN-alpha2 gene was cloned from human placental genomic DNA. High titers of the LISN retrovirus were produced by the amphotropic packaging cell line GP+envAM12. LISN is able to infect three human hematopoietic and leukemic cell lines: K562, LAMA-84, and TF-1. G418-resistant cells were detected in a similar proportion after infection with either the LISN retroviral vector or the LnLSN retroviral vector (encoding the nlsLacZ gene instead of hIFN-alpha2), suggesting that hIFN-alpha2 does not inhibit (or only partially inhibits) the production of retroviral particles by the packaging cell line and the infection of human cells. LISN-infected cells express and secrete hIFN-alpha2 as demonstrated by Northern blot analysis of poly(A)+ RNA, detection of the intracellular protein by fluorescence-activated cell sorter analysis, and detection of secreted hIFN-alpha in cell supernatants using an enzyme-linked immunosorbent assay. Retrovirally produced hIFN-alpha2 is biologically active, as demonstrated by the partial inhibition of the growth of K562 and TF-1, the modulation of the expression of cell surface antigens, the induction of the (2'-5') oligoadenylate synthetase, and, for LAMA-84, the down-modulation of the BCR-ABL protein. We conclude that the infection of human leukemic cell lines with a retroviral vector encoding hIFN-alpha2 is feasible and induces the expected biological effects. This experimental model will be useful in investigating the possibility of transducing normal and leukemic cells and hematopoietic progenitors and in determining the consequences of the autocrine production of hIFN-alpha2 on the behavior of these cells.

The FACT Complex Promotes Avian Leukosis Virus DNA Integration.

PubMed

Winans, Shelby; Larue, Ross C; Abraham, Carly M; Shkriabai, Nikolozi; Skopp, Amelie; Winkler, Duane; Kvaratskhelia, Mamuka; Beemon, Karen L

2017-04-01

All retroviruses need to integrate a DNA copy of their genome into the host chromatin. Cellular proteins regulating and targeting lentiviral and gammaretroviral integration in infected cells have been discovered, but the factors that mediate alpharetroviral avian leukosis virus (ALV) integration are unknown. In this study, we have identified the FACT protein complex, which consists of SSRP1 and Spt16, as a principal cellular binding partner of ALV integrase (IN). Biochemical experiments with purified recombinant proteins show that SSRP1 and Spt16 are able to individually bind ALV IN, but only the FACT complex effectively stimulates ALV integration activity in vitro Likewise, in infected cells, the FACT complex promotes ALV integration activity, with proviral integration frequency varying directly with cellular expression levels of the FACT complex. An increase in 2-long-terminal-repeat (2-LTR) circles in the depleted FACT complex cell line indicates that this complex regulates the ALV life cycle at the level of integration. This regulation is shown to be specific to ALV, as disruption of the FACT complex did not inhibit either lentiviral or gammaretroviral integration in infected cells. IMPORTANCE The majority of human gene therapy approaches utilize HIV-1- or murine leukemia virus (MLV)-based vectors, which preferentially integrate near genes and regulatory regions; thus, insertional mutagenesis is a substantial risk. In contrast, ALV integrates more randomly throughout the genome, which decreases the risks of deleterious integration. Understanding how ALV integration is regulated could facilitate the development of ALV-based vectors for use in human gene therapy. Here we show that the FACT complex directly binds and regulates ALV integration efficiency in vitro and in infected cells. Copyright © 2017 American Society for Microbiology.

Electric cell-substrate impedance sensing (ECIS) based real-time measurement of titer dependent cytotoxicity induced by adenoviral vectors in an IPI-2I cell culture model.

PubMed

Müller, Jakob; Thirion, Christian; Pfaffl, Michael W

2011-01-15

Recombinant viral vectors are widespread tools for transfer of genetic material in various modern biotechnological applications like for example RNA interference (RNAi). However, an accurate and reproducible titer assignment represents the basic step for most downstream applications regarding a precise multiplicity of infection (MOI) adjustment. As necessary scaffold for the studies described in this work we introduce a quantitative real-time PCR (qPCR) based approach for viral particle measurement. Still an implicated problem concerning physiological effects is that the appliance of viral vectors is often attended by toxic effects on the individual target. To determine the critical viral dose leading to cell death we developed an electric cell-substrate impedance sensing (ECIS) based assay. With ECIS technology the impedance change of a current flow through the cell culture medium in an array plate is measured in a non-invasive manner, visualizing effects like cell attachment, cell-cell contacts or proliferation. Here we describe the potential of this online measurement technique in an in vitro model using the porcine ileal epithelial cell line IPI-2I in combination with an adenoviral transfection vector (Ad5-derivate). This approach shows a clear dose-depending toxic effect, as the amount of applied virus highly correlates (p<0.001) with the level of cell death. Thus this assay offers the possibility to discriminate the minimal non-toxic dose of the individual transfection method. In addition this work suggests that the ECIS-device bears the feasibility to transfer this assay to multiple other cytotoxicological questions. Copyright © 2010 Elsevier B.V. All rights reserved.

A Simple Method to Increase the Transduction Efficiency of Single-Stranded Adeno-Associated Virus Vectors In Vitro and In Vivo

PubMed Central

Ma, Wenqin; Li, Baozheng; Ling, Chen; Jayandharan, Giridhara R.; Byrne, Barry J.

2011-01-01

Abstract We have recently shown that co-administration of conventional single-stranded adeno-associated virus 2 (ssAAV2) vectors with self-complementary (sc) AAV2-protein phosphatase 5 (PP5) vectors leads to a significant increase in the transduction efficiency of ssAAV2 vectors in human cells in vitro as well as in murine hepatocytes in vivo. In the present study, this strategy has been further optimized by generating a mixed population of ssAAV2-EGFP and scAAV2-PP5 vectors at a 10:1 ratio to achieve enhanced green fluorescent protein (EGFP) transgene expression at approximately 5- to 10-fold higher efficiency, both in vitro and in vivo. This simple coproduction method should be adaptable to any ssAAV serotype vector containing transgene cassettes that are too large to be encapsidated in scAAV vectors. PMID:21219084

Immunomodulatory effects of exosomes produced by virus-infected cells.

PubMed

Petrik, Juraj

2016-08-01

Viruses have developed a spectrum of ways to modify cellular pathways to hijack the cell machinery for the synthesis of their nucleic acid and proteins. Similarly, they use intracellular vesicular mechanisms of trafficking for their assembly and eventual release, with a number of viruses acquiring their envelope from internal or plasma cell membranes. There is an increasing number of reports on viral exploitation of cell secretome pathways to avoid recognition and stimulation of the immune response. Extracellular vesicles (EV) containing viral particles have been shown to shield viruses after exiting the host cell, in some cases challenging the boundaries between viral groups traditionally characterised as enveloped and non-enveloped. Apart from viral particles, EV can spread the virus also carrying viral genome and can modify the target cells through their cargo of virus-coded miRNAs and proteins as well as selectively packaged cellular mRNAs, miRNAs, proteins and lipids, differing in composition and quantities from the cell of origin. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

A helper virus-free HSV-1 vector containing the vesicular glutamate transporter-1 promoter supports expression preferentially in VGLUT1-containing glutamatergic neurons.

PubMed

Zhang, Guo-rong; Geller, Alfred I

2010-05-17

Multiple potential uses of direct gene transfer into neurons require restricting expression to specific classes of glutamatergic neurons. Thus, it is desirable to develop vectors containing glutamatergic class-specific promoters. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. We previously reported a plasmid (amplicon) Herpes Simplex Virus (HSV-1) vector that placed the Lac Z gene under the regulation of the VGLUT1 promoter (pVGLUT1lac). Using helper virus-free vector stocks, we showed that this vector supported approximately 90% glutamatergic neuron-specific expression in postrhinal (POR) cortex, in rats sacrificed at either 4 days or 2 months after gene transfer. We now show that pVGLUT1lac supports expression preferentially in VGLUT1-containing glutamatergic neurons. pVGLUT1lac vector stock was injected into either POR cortex, which contains primarily VGLUT1-containing glutamatergic neurons, or into the ventral medial hypothalamus (VMH), which contains predominantly VGLUT2-containing glutamatergic neurons. Rats were sacrificed at 4 days after gene transfer, and the types of cells expressing ss-galactosidase were determined by immunofluorescent costaining. Cell counts showed that pVGLUT1lac supported expression in approximately 10-fold more cells in POR cortex than in the VMH, whereas a control vector supported expression in similar numbers of cells in these two areas. Further, in POR cortex, pVGLUT1lac supported expression predominately in VGLUT1-containing neurons, and, in the VMH, pVGLUT1lac showed an approximately 10-fold preference for the rare VGLUT1-containing neurons. VGLUT1-specific expression may benefit specific experiments on learning or specific gene therapy approaches, particularly in the neocortex. Copyright 2010 Elsevier B.V. All rights reserved.

A Roadmap for Tick-Borne Flavivirus Research in the "Omics" Era.

PubMed

Grabowski, Jeffrey M; Hill, Catherine A

2017-01-01

Tick-borne flaviviruses (TBFs) affect human health globally. Human vaccines provide protection against some TBFs, and antivirals are available, yet TBF-specific control strategies are limited. Advances in genomics offer hope to understand the viral complement transmitted by ticks, and to develop disruptive, data-driven technologies for virus detection, treatment, and control. The genome assemblies of Ixodes scapularis , the North American tick vector of the TBF, Powassan virus, and other tick vectors, are providing insights into tick biology and pathogen transmission and serve as nucleation points for expanded genomic research. Systems biology has yielded insights to the response of tick cells to viral infection at the transcript and protein level, and new protein targets for vaccines to limit virus transmission. Reverse vaccinology approaches have moved candidate tick antigenic epitopes into vaccine development pipelines. Traditional drug and in silico screening have identified candidate antivirals, and target-based approaches have been developed to identify novel acaricides. Yet, additional genomic resources are required to expand TBF research. Priorities include genome assemblies for tick vectors, "omic" studies involving high consequence pathogens and vectors, and emphasizing viral metagenomics, tick-virus metabolomics, and structural genomics of TBF and tick proteins. Also required are resources for forward genetics, including the development of tick strains with quantifiable traits, genetic markers and linkage maps. Here we review the current state of genomic research on ticks and tick-borne viruses with an emphasis on TBFs. We outline an ambitious 10-year roadmap for research in the "omics era," and explore key milestones needed to accomplish the goal of delivering three new vaccines, antivirals and acaricides for TBF control by 2030.

A Roadmap for Tick-Borne Flavivirus Research in the “Omics” Era

PubMed Central

Grabowski, Jeffrey M.; Hill, Catherine A.

2017-01-01

Tick-borne flaviviruses (TBFs) affect human health globally. Human vaccines provide protection against some TBFs, and antivirals are available, yet TBF-specific control strategies are limited. Advances in genomics offer hope to understand the viral complement transmitted by ticks, and to develop disruptive, data-driven technologies for virus detection, treatment, and control. The genome assemblies of Ixodes scapularis, the North American tick vector of the TBF, Powassan virus, and other tick vectors, are providing insights into tick biology and pathogen transmission and serve as nucleation points for expanded genomic research. Systems biology has yielded insights to the response of tick cells to viral infection at the transcript and protein level, and new protein targets for vaccines to limit virus transmission. Reverse vaccinology approaches have moved candidate tick antigenic epitopes into vaccine development pipelines. Traditional drug and in silico screening have identified candidate antivirals, and target-based approaches have been developed to identify novel acaricides. Yet, additional genomic resources are required to expand TBF research. Priorities include genome assemblies for tick vectors, “omic” studies involving high consequence pathogens and vectors, and emphasizing viral metagenomics, tick-virus metabolomics, and structural genomics of TBF and tick proteins. Also required are resources for forward genetics, including the development of tick strains with quantifiable traits, genetic markers and linkage maps. Here we review the current state of genomic research on ticks and tick-borne viruses with an emphasis on TBFs. We outline an ambitious 10-year roadmap for research in the “omics era,” and explore key milestones needed to accomplish the goal of delivering three new vaccines, antivirals and acaricides for TBF control by 2030. PMID:29312896

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

PubMed Central

2009-01-01

Background Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. Conclusion These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency. PMID:20042112

The Consequences of Reconfiguring the Ambisense S Genome Segment of Rift Valley Fever Virus on Viral Replication in Mammalian and Mosquito Cells and for Genome Packaging

PubMed Central

Elliott, Richard M.

2014-01-01

Rift Valley fever virus (RVFV, family Bunyaviridae) is a mosquito-borne pathogen of both livestock and humans, found primarily in Sub-Saharan Africa and the Arabian Peninsula. The viral genome comprises two negative-sense (L and M segments) and one ambisense (S segment) RNAs that encode seven proteins. The S segment encodes the nucleocapsid (N) protein in the negative-sense and a nonstructural (NSs) protein in the positive-sense, though NSs cannot be translated directly from the S segment but rather from a specific subgenomic mRNA. Using reverse genetics we generated a virus, designated rMP12:S-Swap, in which the N protein is expressed from the NSs locus and NSs from the N locus within the genomic S RNA. In cells infected with rMP12:S-Swap NSs is expressed at higher levels with respect to N than in cells infected with the parental rMP12 virus. Despite NSs being the main interferon antagonist and determinant of virulence, growth of rMP12:S-Swap was attenuated in mammalian cells and gave a small plaque phenotype. The increased abundance of the NSs protein did not lead to faster inhibition of host cell protein synthesis or host cell transcription in infected mammalian cells. In cultured mosquito cells, however, infection with rMP12:S-Swap resulted in cell death rather than establishment of persistence as seen with rMP12. Finally, altering the composition of the S segment led to a differential packaging ratio of genomic to antigenomic RNA into rMP12:S-Swap virions. Our results highlight the plasticity of the RVFV genome and provide a useful experimental tool to investigate further the packaging mechanism of the segmented genome. PMID:24550727

Detection of receptor-specific murine leukemia virus binding to cells by immunofluorescence analysis.

PubMed Central

Kadan, M J; Sturm, S; Anderson, W F; Eglitis, M A

1992-01-01

Four classes of murine leukemia virus (MuLV) which display distinct cellular tropisms and bind to different retrovirus receptors to initiate virus infection have been described. In the present study, we describe a rapid, sensitive immunofluorescence assay useful for characterizing the initial binding of MuLV to cells. By using the rat monoclonal antibody 83A25 (L. H. Evans, R. P. Morrison, F. G. Malik, J. Portis, and W. J. Britt, J. Virol. 64:6176-6183, 1990), which recognizes an epitope of the envelope gp70 molecule common to the different classes of MuLV, it is possible to analyse the binding of ecotropic, amphotropic, or xenotropic MuLV by using only a single combination of primary and secondary antibodies. The MuLV binding detected by this assay is envelope receptor specific and matches the susceptibility to infection determined for cells from a variety of species. The binding of amphotropic MuLV to NIH 3T3 cells was shown to be rapid, saturable, and temperature dependent. Chinese hamster ovary (CHO-K1) cells normally lack the ability to bind ecotropic virus and are not infectible by ecotropic vectors. Expression of the cloned ecotropic retrovirus receptor gene (Rec) in CHO-K1 cells confers high levels of ecotropic virus-specific binding and confers susceptibility to infection. Characterization of MuLV binding to primary cells may provide insight into the infectibility of cells by retroviruses and aid in the selection of appropriate vectors for gene transfer experiments. PMID:1312632

Alteration of intersubunit acid–base pair interactions at the quasi-threefold axis of symmetry of Cucumber mosaic virus disrupts aphid vector transmission

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bricault, Christine A.; Perry, Keith L., E-mail: KLP3@cornell.edu

2013-06-05

In the atomic model of Cucumber mosaic virus (CMV), six amino acid residues form stabilizing salt bridges between subunits of the asymmetric unit at the quasi-threefold axis of symmetry. To evaluate the effects of these positions on virion stability and aphid vector transmissibility, six charged amino acid residues were individually mutated to alanine. All of the six engineered viruses were viable and exhibited near wild type levels of virion stability in the presence of urea. Aphid vector transmissibility was nearly or completely eliminated in the case of four of the mutants; two mutants demonstrated intermediate aphid transmissibility. For the majoritymore » of the engineered mutants, second-site mutations were observed following aphid transmission and/or mechanical passaging, and one restored transmission rates to that of the wild type. CMV capsids tolerate disruption of acid–base pairing interactions at the quasi-threefold axis of symmetry, but these interactions are essential for maintaining aphid vector transmissibility. - Highlights: ► Amino acids between structural subunits of Cucumber mosaic virus affect vector transmission. ► Mutant structural stability was retained, while aphid vector transmissibility was disrupted. ► Spontaneous, second-site mutations restored aphid vector transmissibility.« less

Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression.

PubMed

Lim, Hyoun-Sub; Vaira, Anna Maria; Domier, Leslie L; Lee, Sung Chul; Kim, Hong Gi; Hammond, John

2010-06-20

We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5' and 3' non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS. Published by Elsevier Inc.

Genetic and Phenotypic Characterization of Manufacturing Seeds for a Tetravalent Dengue Vaccine (DENVax)

PubMed Central

Huang, Claire Y.-H.; Kinney, Richard M.; Livengood, Jill A.; Bolling, Bethany; Arguello, John J.; Luy, Betty E.; Silengo, Shawn J.; Boroughs, Karen L.; Stovall, Janae L.; Kalanidhi, Akundi P.; Brault, Aaron C.; Osorio, Jorge E.; Stinchcomb, Dan T.

2013-01-01

Background We have developed a manufacturing strategy that can improve the safety and genetic stability of recombinant live-attenuated chimeric dengue vaccine (DENVax) viruses. These viruses, containing the pre-membrane (prM) and envelope (E) genes of dengue serotypes 1–4 in the replicative background of the attenuated dengue-2 PDK-53 vaccine virus candidate, were manufactured under cGMP. Methodology/Principal Findings After deriving vaccine viruses from RNA-transfected Vero cells, six plaque-purified viruses for each serotype were produced. The plaque-purified strains were then analyzed to select one stock for generation of the master seed. Full genetic and phenotypic characterizations of the master virus seeds were conducted to ensure these viruses retained the previously identified attenuating determinants and phenotypes of the vaccine viruses. We also assessed vector competence of the vaccine viruses in sympatric (Thai) Aedes aegypti mosquito vectors. Conclusion/Significance All four serotypes of master vaccine seeds retained the previously defined safety features, including all three major genetic loci of attenuation, small plaques, temperature sensitivity in mammalian cells, reduced replication in mosquito cell cultures, and reduced neurovirulence in new-born mice. In addition, the candidate vaccine viruses demonstrated greatly reduced infection and dissemination in Aedes aegypti mosquitoes, and are not likely to be transmissible by these mosquitoes. This manufacturing strategy has successfully been used to produce the candidate tetravalent vaccine, which is currently being tested in human clinical trials in the United States, Central and South America, and Asia. PMID:23738026

DNA and modified vaccinia virus Ankara vaccines encoding multiple cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) are safe but weakly immunogenic in HIV-1-uninfected, vaccinia virus-naive adults.

PubMed

Gorse, Geoffrey J; Newman, Mark J; deCamp, Allan; Hay, Christine Mhorag; De Rosa, Stephen C; Noonan, Elizabeth; Livingston, Brian D; Fuchs, Jonathan D; Kalams, Spyros A; Cassis-Ghavami, Farah L

2012-05-01

We evaluated a DNA plasmid-vectored vaccine and a recombinant modified vaccinia virus Ankara vaccine (MVA-mBN32), each encoding cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) in a randomized, double-blinded, placebo-controlled trial in 36 HIV-1-uninfected adults using a heterologous prime-boost schedule. HIV-1-specific cellular immune responses, measured as interleukin-2 and/or gamma interferon production, were induced in 1 (4%) of 28 subjects after the first MVA-mBN32 immunization and in 3 (12%) of 25 subjects after the second MVA-mBN32 immunization. Among these responders, polyfunctional T-cell responses, including the production of tumor necrosis factor alpha and perforin, were detected. Vaccinia virus-specific antibodies were induced to the MVA vector in 27 (93%) of 29 and 26 (93%) of 28 subjects after the first and second immunizations with MVA-mBN32. These peptide-based vaccines were safe but were ineffective at inducing HIV-1-specific immune responses and induced much weaker responses than MVA vaccines expressing the entire open reading frames of HIV-1 proteins.

Predictive Models for Tomato Spotted Wilt Virus Spread Dynamics, Considering Frankliniella occidentalis Specific Life Processes as Influenced by the Virus.

PubMed

Ogada, Pamella Akoth; Moualeu, Dany Pascal; Poehling, Hans-Michael

2016-01-01

Several models have been studied on predictive epidemics of arthropod vectored plant viruses in an attempt to bring understanding to the complex but specific relationship between the three cornered pathosystem (virus, vector and host plant), as well as their interactions with the environment. A large body of studies mainly focuses on weather based models as management tool for monitoring pests and diseases, with very few incorporating the contribution of vector's life processes in the disease dynamics, which is an essential aspect when mitigating virus incidences in a crop stand. In this study, we hypothesized that the multiplication and spread of tomato spotted wilt virus (TSWV) in a crop stand is strongly related to its influences on Frankliniella occidentalis preferential behavior and life expectancy. Model dynamics of important aspects in disease development within TSWV-F. occidentalis-host plant interactions were developed, focusing on F. occidentalis' life processes as influenced by TSWV. The results show that the influence of TSWV on F. occidentalis preferential behaviour leads to an estimated increase in relative acquisition rate of the virus, and up to 33% increase in transmission rate to healthy plants. Also, increased life expectancy; which relates to improved fitness, is dependent on the virus induced preferential behaviour, consequently promoting multiplication and spread of the virus in a crop stand. The development of vector-based models could further help in elucidating the role of tri-trophic interactions in agricultural disease systems. Use of the model to examine the components of the disease process could also boost our understanding on how specific epidemiological characteristics interact to cause diseases in crops. With this level of understanding we can efficiently develop more precise control strategies for the virus and the vector.

A potyvirus vector efficiently targets recombinant proteins to chloroplasts, mitochondria and nuclei in plant cells when expressed at the amino terminus of the polyprotein.

PubMed

Majer, Eszter; Navarro, José-Antonio; Daròs, José-Antonio

2015-09-01

Plant virus-based expression systems allow quick and efficient production of recombinant proteins in plant biofactories. Among them, a system derived from tobacco etch virus (TEV; genus potyvirus) permits coexpression of equimolar amounts of several recombinant proteins. This work analyzed how to target recombinant proteins to different subcellular localizations in the plant cell using this system. We constructed TEV clones in which green fluorescent protein (GFP), with a chloroplast transit peptide (cTP), a nuclear localization signal (NLS) or a mitochondrial targeting peptide (mTP) was expressed either as the most amino-terminal product or embedded in the viral polyprotein. Results showed that cTP and mTP mediated efficient translocation of GFP to the corresponding organelle only when present at the amino terminus of the viral polyprotein. In contrast, the NLS worked efficiently at both positions. Viruses expressing GFP in the amino terminus of the viral polyprotein produced milder symptoms. Untagged GFPs and cTP and NLS tagged amino-terminal GFPs accumulated to higher amounts in infected tissues. Finally, viral progeny from clones with internal GFPs maintained the extra gene better. These observations will help in the design of potyvirus-based vectors able to coexpress several proteins while targeting different subcellular localizations, as required in plant metabolic engineering. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Genetically Modified Adenoviral Vector with a Phage Display-Derived Peptide Incorporated into Fiber Fibritin Chimera Prolongs Survival in Experimental Glioma.

PubMed

Kim, Julius W; Kane, J Robert; Young, Jacob S; Chang, Alan L; Kanojia, Deepak; Morshed, Ramin A; Miska, Jason; Ahmed, Atique U; Balyasnikova, Irina V; Han, Yu; Zhang, Lingjiao; Curiel, David T; Lesniak, Maciej S

2015-09-01

The dismal clinical context of advanced-grade glioma demands the development of novel therapeutic strategies with direct patient impact. Adenovirus-mediated virotherapy represents a potentially effective approach for glioma therapy. In this research, we generated a novel glioma-specific adenovirus by instituting more advanced genetic modifications that can maximize the efficiency and safety of therapeutic adenoviral vectors. In this regard, a glioma-specific targeted fiber was developed through the incorporation of previously published glioma-specific, phage-panned peptide (VWT peptide) on a fiber fibritin-based chimeric fiber, designated as "GliomaFF." We showed that the entry of this virus was highly restricted to glioma cells, supporting the specificity imparted by the phage-panned peptide. In addition, the stability of the targeting moiety presented by fiber fibritin structure permitted greatly enhanced infectivity. Furthermore, the replication of this virus was restricted in glioma cells by controlling expression of the E1 gene under the activity of the tumor-specific survivin promoter. Using this approach, we were able to explore the combinatorial efficacy of various adenoviral modifications that could amplify the specificity, infectivity, and exclusive replication of this therapeutic adenovirus in glioma. Finally, virotherapy with this modified virus resulted in up to 70% extended survival in an in vivo murine glioma model. These data demonstrate that this novel adenoviral vector is a safe and efficient treatment for this difficult malignancy.

Chikungunya Virus Vaccines: Viral Vector-Based Approaches.

PubMed

Ramsauer, Katrin; Tangy, Frédéric

2016-12-15

In 2013, a major chikungunya virus (CHIKV) epidemic reached the Americas. In the past 2 years, >1.7 million people have been infected. In light of the current epidemic, with millions of people in North and South America at risk, efforts to rapidly develop effective vaccines have increased. Here, we focus on CHIKV vaccines that use viral-vector technologies. This group of vaccine candidates shares an ability to potently induce humoral and cellular immune responses by use of highly attenuated and safe vaccine backbones. So far, well-described vectors such as modified vaccinia virus Ankara, complex adenovirus, vesicular stomatitis virus, alphavirus-based chimeras, and measles vaccine Schwarz strain (MV/Schw) have been described as potential vaccines. We summarize here the recent data on these experimental vaccines, with a focus on the preclinical and clinical activities on the MV/Schw-based candidate, which is the first CHIKV-vectored vaccine that has completed a clinical trial. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Single-Molecule FISH Reveals Non-selective Packaging of Rift Valley Fever Virus Genome Segments

PubMed Central

Wichgers Schreur, Paul J.; Kortekaas, Jeroen

2016-01-01

The bunyavirus genome comprises a small (S), medium (M), and large (L) RNA segment of negative polarity. Although genome segmentation confers evolutionary advantages by enabling genome reassortment events with related viruses, genome segmentation also complicates genome replication and packaging. Accumulating evidence suggests that genomes of viruses with eight or more genome segments are incorporated into virions by highly selective processes. Remarkably, little is known about the genome packaging process of the tri-segmented bunyaviruses. Here, we evaluated, by single-molecule RNA fluorescence in situ hybridization (FISH), the intracellular spatio-temporal distribution and replication kinetics of the Rift Valley fever virus (RVFV) genome and determined the segment composition of mature virions. The results reveal that the RVFV genome segments start to replicate near the site of infection before spreading and replicating throughout the cytoplasm followed by translocation to the virion assembly site at the Golgi network. Despite the average intracellular S, M and L genome segments approached a 1:1:1 ratio, major differences in genome segment ratios were observed among cells. We also observed a significant amount of cells lacking evidence of M-segment replication. Analysis of two-segmented replicons and four-segmented viruses subsequently confirmed the previous notion that Golgi recruitment is mediated by the Gn glycoprotein. The absence of colocalization of the different segments in the cytoplasm and the successful rescue of a tri-segmented variant with a codon shuffled M-segment suggested that inter-segment interactions are unlikely to drive the copackaging of the different segments into a single virion. The latter was confirmed by direct visualization of RNPs inside mature virions which showed that the majority of virions lack one or more genome segments. Altogether, this study suggests that RVFV genome packaging is a non-selective process. PMID:27548280

Recovery of infectious virus from full-length cowpox virus (CPXV) DNA cloned as a bacterial artificial chromosome (BAC)

PubMed Central

2011-01-01

Transmission from pet rats and cats to humans as well as severe infection in felids and other animal species have recently drawn increasing attention to cowpox virus (CPXV). We report the cloning of the entire genome of cowpox virus strain Brighton Red (BR) as a bacterial artificial chromosome (BAC) in Escherichia coli and the recovery of infectious virus from cloned DNA. Generation of a full-length CPXV DNA clone was achieved by first introducing a mini-F vector, which allows maintenance of large circular DNA in E. coli, into the thymidine kinase locus of CPXV by homologous recombination. Circular replication intermediates were then electroporated into E. coli DH10B cells. Upon successful establishment of the infectious BR clone, we modified the full-length clone such that recombination-mediated excision of bacterial sequences can occur upon transfection in eukaryotic cells. This self-excision of the bacterial replicon is made possible by a sequence duplication within mini-F sequences and allows recovery of recombinant virus progeny without remaining marker or vector sequences. The in vitro growth properties of viruses derived from both BAC clones were determined and found to be virtually indistinguishable from those of parental, wild-type BR. Finally, the complete genomic sequence of the infectious clone was determined and the cloned viral genome was shown to be identical to that of the parental virus. In summary, the generated infectious clone will greatly facilitate studies on individual genes and pathogenesis of CPXV. Moreover, the vector potential of CPXV can now be more systematically explored using this newly generated tool. PMID:21314965

In planta expression of HIV-1 p24 protein using an RNA plant virus-based expression vector.

PubMed

Zhang, G; Leung, C; Murdin, L; Rovinski, B; White, K A

2000-02-01

Plant viruses show significant potential as expression vectors for the production of foreign proteins (e.g., antigens) in plants. The HIV-1 p24 nucleocapsid protein is an important early marker of HIV infection and has been used as an antigen in the development of HIV vaccines. Toward developing a plant-based expression system for the production of p24, we have investigated the use of a (positive)-strand RNA plant virus, tomato bushy stunt virus (TBSV), as an expression vector. The HIV p24 open reading frame (ORF) was introduced into a cloned cDNA copy of the TBSV genome as an in-frame fusion with a 5'-terminal portion of the TBSV coat protein ORF. In vitro-generated RNA transcripts corresponding to the engineered virus vector were infectious when inoculated into plant protoplasts; Northern and Western blot analyses verified the accumulation of a predicted p24-encoding viral subgenomic mRNA and the production of p24 fusion product. Whole-plant infections with the viral vector led to the accumulation of p24 fusion protein in inoculated leaves, which cross-reacted with p24-specific antibodies, thus confirming the maintenance of key antigenic determinants. This study is the first to demonstrate that TBSV can be engineered to express a complete foreign protein of clinical importance. Strategies for optimizing protein yield from this viral vector are discussed.

The chestnut blight fungus for studies on virus/host and virus/virus interactions: from a natural to a model host.

PubMed

Eusebio-Cope, Ana; Sun, Liying; Tanaka, Toru; Chiba, Sotaro; Kasahara, Shin; Suzuki, Nobuhiro

2015-03-01

The chestnut blight fungus, Cryphonectria parasitica, is an important plant pathogenic ascomycete. The fungus hosts a wide range of viruses and now has been established as a model filamentous fungus for studying virus/host and virus/virus interactions. This is based on the development of methods for artificial virus introduction and elimination, host genome manipulability, available host genome sequence with annotations, host mutant strains, and molecular tools. Molecular tools include sub-cellular distribution markers, gene expression reporters, and vectors with regulatable promoters that have been long available for unicellular organisms, cultured cells, individuals of animals and plants, and certain filamentous fungi. A comparison with other filamentous fungi such as Neurospora crassa has been made to establish clear advantages and disadvantages of C. parasitica as a virus host. In addition, a few recent studies on RNA silencing vs. viruses in this fungus are introduced. Copyright © 2014 Elsevier Inc. All rights reserved.

Development of apple latent spherical virus-based vaccines against three tospoviruses.

PubMed

Taki, Ayano; Yamagishi, Noriko; Yoshikawa, Nobuyuki

2013-09-01

Apple latent spherical virus (ALSV) is characterized by its relatively broad host range, latency in most host plants, and ability to induce gene silencing in host plants. Herein, we focus on the above characteristic of ALSV and describe our development of ALSV vector vaccines against three tospoviruses, namely, Impatiens necrotic spot virus (INSV), Iris yellow spot virus (IYSV), and Tomato spotted wilt virus (TSWV). DNA fragments of 201 nt of three tospovirus S-RNAs (silencing suppressor (NSS) and nucleocapsid protein (N) coding regions for each tospovirus) were inserted into an ALSV-RNA2 vector to obtain six types of ALSV vector vaccines. Nicotiana benthamiana plants at the five-leaf stage were inoculated with each ALSV vector vaccine and challenged with the corresponding tospovirus species. Tospovirus-induced symptoms and tospovirus replication after challenge were significantly suppressed in plants preinoculated with all ALSV vector vaccines having the N region fragment, indicating that strong resistance was acquired after infection with ALSV vector vaccines. On the other hand, cross protection was not significant in plants preinoculated with ALSV vectors having the NSs region fragment. Similarly, inoculation with an ALSV-RNA1 vector having the N region fragment in the 3'-noncoding region, but not the NSs region fragment, induced cross protection, indicating that cross protection is via RNA silencing, not via the function of the protein derived from the N region fragment. Our approach, wherein ALSV vectors and selected target inserts are used, enables rapid establishment of ALSV vector vaccines against many pathogenic RNA viruses with known sequences. Copyright © 2013 Elsevier B.V. All rights reserved.

Viral Determinants of Integration Site Preferences of Simian Immunodeficiency Virus-Based Vectors

PubMed Central

Monse, Hella; Laufs, Stephanie; Kuate, Seraphin; Zeller, W. Jens; Fruehauf, Stefan; Überla, Klaus

2006-01-01

Preferential integration into transcriptionally active regions of genomes has been observed for retroviral vectors based on gamma-retroviruses and lentiviruses. However, differences in the integration site preferences were detected, which might be explained by differences in viral components of the preintegration complexes. Viral determinants of integration site preferences have not been defined. Therefore, integration sites of simian immunodeficiency virus (SIV)-based vectors produced in the absence of accessory genes or lacking promoter and enhancer elements were compared. Similar integration patterns for the different SIV vectors indicate that vif, vpr, vpx, nef, env, and promoter or enhancer elements are not required for preferential integration of SIV into transcriptionally active regions of genomes. PMID:16873270

Induction and Maintenance of CX3CR1-Intermediate Peripheral Memory CD8+ T Cells by Persistent Viruses and Vaccines.

PubMed

Gordon, Claire Louse; Lee, Lian Ni; Swadling, Leo; Hutchings, Claire; Zinser, Madeleine; Highton, Andrew John; Capone, Stefania; Folgori, Antonella; Barnes, Eleanor; Klenerman, Paul

2018-04-17

The induction and maintenance of T cell memory is critical to the success of vaccines. A recently described subset of memory CD8 + T cells defined by intermediate expression of the chemokine receptor CX3CR1 was shown to have self-renewal, proliferative, and tissue-surveillance properties relevant to vaccine-induced memory. We tracked these cells when memory is sustained at high levels: memory inflation induced by cytomegalovirus (CMV) and adenovirus-vectored vaccines. In mice, both CMV and vaccine-induced inflationary T cells showed sustained high levels of CX3R1 int cells exhibiting an effector-memory phenotype, characteristic of inflationary pools, in early memory. In humans, CX3CR1 int CD8 + T cells were strongly induced following adenovirus-vectored vaccination for hepatitis C virus (HCV) (ChAd3-NSmut) and during natural CMV infection and were associated with a memory phenotype similar to that in mice. These data indicate that CX3CR1 int cells form an important component of the memory pool in response to persistent viruses and vaccines in both mice and humans. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Generation of Recombinant Modified Vaccinia Virus Ankara Encoding VP2, NS1, and VP7 Proteins of Bluetongue Virus.

PubMed

Marín-López, Alejandro; Ortego, Javier

2016-01-01

Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination, and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV), an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of recombinant MVAs, the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification.

Using HSV-TK/GCV suicide gene therapy to inhibit lens epithelial cell proliferation for treatment of posterior capsular opacification

PubMed Central

Jiang, Yong-Xiang; Liu, Tian-Jing; Yang, Jin; Chen, Yan; Fang, Yan-Wen

2011-01-01

Purpose To establish a novel, targeted lentivirus-based HSV-tk (herpes simplex virus thymidine kinase)/GCV (ganciclovir) gene therapy system to inhibit lens epithelial cell proliferation for treatment of posterior capsular opacification (PCO) after cataract surgery. Methods An enhanced Cre recombinase (Cre/loxP) system with a lentiviral vector expressing Cre under the control of the lens-specific promoter LEP503 (Lenti-LEP503-HSVtk-Cre [LTKCRE]) was constructed, as well as another lentiviral vector containing a switching unit. The latter vector contains a stuffer sequence encoding EGFP (Lenti-hPGK-Loxp-EGFP-pA-Loxp-HSVtk [PGFPTK]) with a functional polyadenylation signal between two loxP sites, followed by the herpes simplex virus thymidine kinase (HSV-tk) gene, both under the control of the human posphoglycerate kinase (hPGK) promoter. Expression of the downstream gene (HSV-tk) is activated by co-expression of Cre. Human lens epithelial cells (HLECs) or retinal pigmental epithelial cells (RPECs) were co-infected with LTKCRE and PGFPTK. The inhibitory effects on HLECs and RPECs infected by the enhanced specific lentiviral vector combination at the concentration of 20 µg/ml GCV were assayed and compared. Results The specific gene expression of Cre and HSV-tk in HLECs is activated by the LEP503 promoter. LTKCRE and PGFPTK co-infected HLECs, but not RPECs, expressed high levels of the HSV-tk protein. After 96 h of GCV treatment, the percentage of apoptotic HLECs infected by the enhanced specific lentiviral vector combination was 87.23%, whereas that of apoptotic RPECs was only 10.12%. Electron microscopy showed that GCV induced apoptosis and necrosis of the infected HLECs. Conclusions The enhanced specific lentiviral vector combination selectively and effectively expressed HSV-tk in HLECs. A concentration of 20 µg/ml, GCV is effective against the proliferation of HLECs in vitro. This cell-type-specific gene therapy using a Cre/loxP lentivirus system may be a feasible treatment strategy to prevent PCO. PMID:21283526

VP7: an attachment protein of bluetongue virus for cellular receptors in Culicoides variipennis.

PubMed

Xu, G; Wilson, W; Mecham, J; Murphy, K; Zhou, E M; Tabachnick, W

1997-07-01

The importance of VP7 of bluetongue virus (BTV) in the binding of BTV to membrane proteins of the BTV vector Culicoides variipennis was investigated. Core BTV particles, prepared from whole viruses, lacked outer proteins VP2 and VP5 and had VP7 exposed. More core particles and whole viruses bound to membrane preparations of adults of C. variipennis and KC cells, which were cultured from this vector insect, than to membrane preparations of Manduca sexta larvae. More core particles than whole viruses bound to membrane preparations of adults of C. variipennis and KC cells. Polyclonal anti-idiotypic antibodies (anti-Id), which were made against an antigen-combining region of an anti-BTV-10 VP7 antibody and functionally mimicked VP7, bound more to the membrane preparations of adults of C. variipennis and KC cells, and less to cytosol preparations. In Western overalay analysis, the Culicoides plasma membrane preparation reduced binding of an anti-VP7 monoclonal antibody to VP7. Whole and core BTV particles and the anti-Id bound to a membrane protein with a molecular mass of 23 kDa that was present predominantly in membrane preparations of adults of C. variipennis and KC cells. This protein was present in much lower concentrations in membrane preparations of C6/36 and DM-2 insect cells.

T-cell receptor transfer into human T cells with ecotropic retroviral vectors.

PubMed

Koste, L; Beissert, T; Hoff, H; Pretsch, L; Türeci, Ö; Sahin, U

2014-05-01

Adoptive T-cell transfer for cancer immunotherapy requires genetic modification of T cells with recombinant T-cell receptors (TCRs). Amphotropic retroviral vectors (RVs) used for TCR transduction for this purpose are considered safe in principle. Despite this, TCR-coding and packaging vectors could theoretically recombine to produce replication competent vectors (RCVs), and transduced T-cell preparations must be proven free of RCV. To eliminate the need for RCV testing, we transduced human T cells with ecotropic RVs so potential RCV would be non-infectious for human cells. We show that transfection of synthetic messenger RNA encoding murine cationic amino-acid transporter 1 (mCAT-1), the receptor for murine retroviruses, enables efficient transient ecotropic transduction of human T cells. mCAT-1-dependent transduction was more efficient than amphotropic transduction performed in parallel, and preferentially targeted naive T cells. Moreover, we demonstrate that ecotropic TCR transduction results in antigen-specific restimulation of primary human T cells. Thus, ecotropic RVs represent a versatile, safe and potent tool to prepare T cells for the adoptive transfer.

Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent

PubMed Central

Lévy, Camille; Amirache, Fouzia; Girard-Gagnepain, Anais; Frecha, Cecilia; Roman-Rodríguez, Francisco J.; Bernadin, Ornellie; Costa, Caroline; Nègre, Didier; Gutierrez-Guerrero, Alejandra; Vranckx, Lenard S.; Clerc, Isabelle; Taylor, Naomi; Thielecke, Lars; Cornils, Kerstin; Bueren, Juan A.; Rio, Paula; Gijsbers, Rik; Cosset, François-Loïc

2017-01-01

Hematopoietic stem cell (HSC)–based gene therapy trials are now moving toward the use of lentiviral vectors (LVs) with success. However, one challenge in the field remains: efficient transduction of HSCs without compromising their stem cell potential. Here we showed that measles virus glycoprotein–displaying LVs (hemagglutinin and fusion protein LVs [H/F-LVs]) were capable of transducing 100% of early-acting cytokine-stimulated human CD34+ (hCD34+) progenitor cells upon a single application. Strikingly, these H/F-LVs also allowed transduction of up to 70% of nonstimulated quiescent hCD34+ cells, whereas conventional vesicular stomatitis virus G (VSV-G)–LVs reached 5% at the most with H/F-LV entry occurring exclusively through the CD46 complement receptor. Importantly, reconstitution of NOD/SCIDγc−/− (NSG) mice with H/F-LV transduced prestimulated or resting hCD34+ cells confirmed these high transduction levels in all myeloid and lymphoid lineages. Remarkably, for resting CD34+ cells, secondary recipients exhibited increasing transduction levels of up to 100%, emphasizing that H/F-LVs efficiently gene-marked HSCs in the resting state. Because H/F-LVs promoted ex vivo gene modification of minimally manipulated CD34+ progenitors that maintained stemness, we assessed their applicability in Fanconi anemia, a bone marrow (BM) failure with chromosomal fragility. Notably, only H/F-LVs efficiently gene-corrected minimally stimulated hCD34+ cells in unfractionated BM from these patients. These H/F-LVs improved HSC gene delivery in the absence of cytokine stimulation while maintaining their stem cell potential. Thus, H/F-LVs will facilitate future clinical applications requiring HSC gene modification, including BM failure syndromes, for which treatment has been very challenging up to now. PMID:29296856

Vaccines against Botulism.

PubMed

Sundeen, Grace; Barbieri, Joseph T

2017-09-02

Botulinum neurotoxins (BoNT) cause the flaccid paralysis of botulism by inhibiting the release of acetylcholine from motor neurons. There are seven serotypes of BoNT (A-G), with limited therapies, and no FDA approved vaccine for botulism. An investigational formalin-inactivated penta-serotype-BoNT/A-E toxoid vaccine was used to vaccinate people who are at high risk of contracting botulism. However, this formalin-inactivated penta-serotype-BoNT/A-E toxoid vaccine was losing potency and was discontinued. This article reviews the different vaccines being developed to replace the discontinued toxoid vaccine. These vaccines include DNA-based, viral vector-based, and recombinant protein-based vaccines. DNA-based vaccines include plasmids or viral vectors containing the gene encoding one of the BoNT heavy chain receptor binding domains (HC). Viral vectors reviewed are adenovirus, influenza virus, rabies virus, Semliki Forest virus, and Venezuelan Equine Encephalitis virus. Among the potential recombinant protein vaccines reviewed are HC, light chain-heavy chain translocation domain, and chemically or genetically inactivated holotoxin.

Vaccines against Botulism

PubMed Central

Sundeen, Grace; Barbieri, Joseph T.

2017-01-01

Botulinum neurotoxins (BoNT) cause the flaccid paralysis of botulism by inhibiting the release of acetylcholine from motor neurons. There are seven serotypes of BoNT (A-G), with limited therapies, and no FDA approved vaccine for botulism. An investigational formalin-inactivated penta-serotype-BoNT/A-E toxoid vaccine was used to vaccinate people who are at high risk of contracting botulism. However, this formalin-inactivated penta-serotype-BoNT/A-E toxoid vaccine was losing potency and was discontinued. This article reviews the different vaccines being developed to replace the discontinued toxoid vaccine. These vaccines include DNA-based, viral vector-based, and recombinant protein-based vaccines. DNA-based vaccines include plasmids or viral vectors containing the gene encoding one of the BoNT heavy chain receptor binding domains (HC). Viral vectors reviewed are adenovirus, influenza virus, rabies virus, Semliki Forest virus, and Venezuelan Equine Encephalitis virus. Among the potential recombinant protein vaccines reviewed are HC, light chain-heavy chain translocation domain, and chemically or genetically inactivated holotoxin. PMID:28869493

Interleukin 10 mediated by herpes simplex virus vectors suppresses neuropathic pain induced by human immunodeficiency virus gp120 in rats.

PubMed

Zheng, Wenwen; Huang, Wan; Liu, Shue; Levitt, Roy C; Candiotti, Keith A; Lubarsky, David A; Hao, Shuanglin

2014-09-01

Human immunodeficiency virus (HIV)-associated sensory neuropathy is a common neurological complication of HIV infection affecting up to 30% of HIV-positive individuals. However, the exact neuropathological mechanisms remain unknown, which hinders our ability to develop effective treatments for HIV-related neuropathic pain (NP). In this study, we tested the hypothesis that inhibition of proinflammatory factors with overexpression of interleukin (IL)-10 reduces HIV-related NP in a rat model. NP was induced by the application of recombinant HIV-1 envelope protein gp120 into the sciatic nerve. The hindpaws of rats were inoculated with nonreplicating herpes simplex virus (HSV) vectors expressing anti-inflammatory cytokine IL-10 or control vector. Mechanical threshold was tested using von Frey filaments before and after treatments with the vectors. The mechanical threshold response was assessed over time using the area under curves. The expression of phosphorylated p38 mitogen-activated kinase, tumor necrosis factor-α, stromal cell-derived factor-1α, and C-X-C chemokine receptor type 4 in both the lumbar spinal cord and the L4/5 dorsal root ganglia (DRG), was examined at 14 and 28 days after vector inoculation using Western blots. We found that in the gp120-induced NP model, IL-10 overexpression mediated by the HSV vector resulted in a significant elevation of the mechanical threshold that was apparent on day 3 after vector inoculation compared with the control vector (P < 0.001). The antiallodynic effect of the single HSV vector inoculation expressing IL-10 lasted >28 days. The area under curve in the HSV vector expressing IL-10 was increased compared with that in the control vector (P < 0.0001). HSV vectors expressing IL-10 reversed the upregulation of phosphorylated p38 mitogen-activated kinase, tumor necrosis factor-α, stromal cell-derived factor-1α, and C-X-C chemokine receptor type 4 expression at 14 and/or 28 days in the DRG and/or the spinal dorsal horn. Our studies demonstrate that blocking the signaling of these proinflammatory molecules in the DRG and/or the spinal cord using the HSV vector expressing IL-10 is able to reduce HIV-related NP. These results provide new insights on the potential mechanisms of HIV-associated NP and a proof of concept for treating painful HIV sensory neuropathy with this type of gene therapy.

Recombinant rabies virus expressing the H protein of canine distemper virus protects dogs from the lethal distemper challenge.

PubMed

Wang, Feng-Xue; Zhang, Shu-Qin; Zhu, Hong-Wei; Yang, Yong; Sun, Na; Tan, Bin; Li, Zhen-Guang; Cheng, Shi-Peng; Fu, Zhen F; Wen, Yong-Jun

2014-12-05

The rabies virus (RV) vector LBNSE expressing foreign antigens have shown considerable promise as vaccines against viral and bacteria diseases, which is effective and safe. We produced a new RV-based vaccine vehicle expressing 1.824 kb hemagglutinin (H) gene of the canine distemper virus (CDV) by reverse genetics technology. The recombinant virus LBNSE-CDV-H retained growth properties similar to those of vector LBNSE both in BSR and mNA cell culture. The H gene of CDV was expressed and detected by immunostaining. To compare the immunogenicity of LBNSE-CDV-H, dogs were immunized with each of these recombinant viruses by intramuscular (i.m.). The dogs were bled at third weeks after the immunization for the measurement of virus neutralizing antibody (VNA) and then challenged with virulent virus (ZJ 7) at fourth weeks. The parent virus (LBNSE) without expression of any foreign molecules was included for comparison. Dogs inoculated with LBNSE-CDV-H showed no any signs of disease and exhibited seroconversion against both RV and CDV H protein. The LBNSE-CDV-H did not cause disease in dogs and conferred protection from challenge with a lethal wild type CDV strain, demonstrating its potential value for wildlife conservation efforts. Together, these studies suggest that recombinant RV expressing H protein from CDV stimulated high levels of adaptive immune responses (VNA), and protected all dogs challenge infection. Copyright © 2014 Elsevier B.V. All rights reserved.