Sample records for aav genetic vaccines

  1. Immune Responses to rAAV6: The Influence of Canine Parvovirus Vaccination and Neonatal Administration of Viral Vector.

    PubMed

    Arnett, Andrea L H; Garikipati, Dilip; Wang, Zejing; Tapscott, Stephen; Chamberlain, Jeffrey S

    2011-01-01

    Recombinant adeno-associated viral (rAAV) vectors promote long-term gene transfer in many animal species. Significant effort has focused on the evaluation of rAAV delivery and the immune response in both murine and canine models of neuromuscular disease. However, canines provided for research purposes are routinely vaccinated against canine parvovirus (CPV). rAAV and CPV possess significant homology and are both parvoviruses. Thus, any immune response generated to CPV vaccination has the potential to cross-react with rAAV vectors. In this study, we investigated the immune response to rAAV6 delivery in a cohort of CPV-vaccinated canines and evaluated multiple vaccination regimens in a mouse model of CPV-vaccination. We show that CPV-vaccination stimulates production of neutralizing antibodies with minimal cross-reactivity to rAAV6. In addition, no significant differences were observed in the magnitude of the rAAV6-directed immune response between CPV-vaccinated animals and controls. Moreover, CPV-vaccination did not inhibit rAAV6-mediated transduction. We also evaluated the immune response to early rAAV6-vaccination in neonatal mice. The influence of maternal hormones and cytokines leads to a relatively permissive state in the neonate. We hypothesized that immaturity of the immune system would permit induction of tolerance to rAAV6 when delivered during the neonatal period. Mice were vaccinated with rAAV6 at 1 or 5 days of age, and subsequently challenged with rAAV6 exposure during adulthood via two sequential IM injections, 1 month apart. All vaccinated animals generated a significant neutralizing antibody response to rAAV6-vaccination that was enhanced following IM injection in adulthood. Taken together, these data demonstrate that the immune response raised against rAAV6 is distinct from that which is elicited by the standard parvoviral vaccines and is sufficient to prevent stable tolerization in neonatal mice.

  2. Immune Responses to rAAV6: The Influence of Canine Parvovirus Vaccination and Neonatal Administration of Viral Vector

    PubMed Central

    Arnett, Andrea L. H.; Garikipati, Dilip; Wang, Zejing; Tapscott, Stephen; Chamberlain, Jeffrey S.

    2011-01-01

    Recombinant adeno-associated viral (rAAV) vectors promote long-term gene transfer in many animal species. Significant effort has focused on the evaluation of rAAV delivery and the immune response in both murine and canine models of neuromuscular disease. However, canines provided for research purposes are routinely vaccinated against canine parvovirus (CPV). rAAV and CPV possess significant homology and are both parvoviruses. Thus, any immune response generated to CPV vaccination has the potential to cross-react with rAAV vectors. In this study, we investigated the immune response to rAAV6 delivery in a cohort of CPV-vaccinated canines and evaluated multiple vaccination regimens in a mouse model of CPV-vaccination. We show that CPV-vaccination stimulates production of neutralizing antibodies with minimal cross-reactivity to rAAV6. In addition, no significant differences were observed in the magnitude of the rAAV6-directed immune response between CPV-vaccinated animals and controls. Moreover, CPV-vaccination did not inhibit rAAV6-mediated transduction. We also evaluated the immune response to early rAAV6-vaccination in neonatal mice. The influence of maternal hormones and cytokines leads to a relatively permissive state in the neonate. We hypothesized that immaturity of the immune system would permit induction of tolerance to rAAV6 when delivered during the neonatal period. Mice were vaccinated with rAAV6 at 1 or 5 days of age, and subsequently challenged with rAAV6 exposure during adulthood via two sequential IM injections, 1 month apart. All vaccinated animals generated a significant neutralizing antibody response to rAAV6-vaccination that was enhanced following IM injection in adulthood. Taken together, these data demonstrate that the immune response raised against rAAV6 is distinct from that which is elicited by the standard parvoviral vaccines and is sufficient to prevent stable tolerization in neonatal mice. PMID:22065964

  3. Next-generation AAV vectors for clinical use: an ever-accelerating race.

    PubMed

    Weinmann, Jonas; Grimm, Dirk

    2017-10-01

    During the past five decades, it has become evident that Adeno-associated virus (AAV) represents one of the most potent, most versatile, and thus most auspicious platforms available for gene delivery into cells, animals and, ultimately, humans. Particularly attractive is the ease with which the viral capsid-the major determinant of virus-host interaction including cell specificity and antibody recognition-can be modified and optimized at will. This has motivated countless researchers to develop high-throughput technologies in which genetically engineered AAV capsid libraries are subjected to a vastly hastened emulation of natural evolution, with the aim to enrich novel synthetic AAV capsids displaying superior features for clinical application. While the power and potential of these forward genetics approaches is undisputed, they are also inherently challenging as success depends on a combination of library quality, fidelity, and complexity. Here, we will describe and discuss two original, very exciting strategies that have emerged over the last three years and that promise to alleviate at least some of these concerns, namely, (i) a reverse genetics approach termed "ancestral AAV sequence reconstruction," and (ii) AAV genome barcoding as a technology that can advance both, forward and reverse genetics stratagems. Notably, despite the conceptual differences of these two technologies, they pursue the same goal which is tailored acceleration of AAV evolution and thus winning the race for the next-generation AAV vectors for clinical use.

  4. AAV Vectorization of DSB-mediated Gene Editing Technologies.

    PubMed

    Moser, Rachel J; Hirsch, Matthew L

    2016-01-01

    Recent work both at the bench and the bedside demonstrate zinc-finger nucleases (ZFNs), CRISPR/Cas9, and other programmable site-specific endonuclease technologies are being successfully utilized within and alongside AAV vectors to induce therapeutically relevant levels of directed gene editing within the human chromosome. Studies from past decades acknowledge that AAV vector genomes are enhanced substrates for homology-directed repair in the presence or absence of targeted DNA damage within the host genome. Additionally, AAV vectors are currently the most efficient format for in vivo gene delivery with no vector related complications in >100 clinical trials for diverse diseases. At the same time, advancements in the design of custom-engineered site-specific endonucleases and the utilization of elucidated endonuclease formats have resulted in efficient and facile genetic engineering for basic science and for clinical therapies. AAV vectors and gene editing technologies are an obvious marriage, using AAV for the delivery of repair substrate and/or a gene encoding a designer endonuclease; however, while efficient delivery and enhanced gene targeting by vector genomes are advantageous, other attributes of AAV vectors are less desirable for gene editing technologies. This review summarizes the various roles that AAV vectors play in gene editing technologies and provides insight into its trending applications for the treatment of genetic diseases.

  5. 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

  6. 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

  7. AAVS1-Targeted Plasmid Integration in AAV Producer Cell Lines.

    PubMed

    Luo, Yuxia; Frederick, Amy; Martin, John M; Scaria, Abraham; Cheng, Seng H; Armentano, Donna; Wadsworth, Samuel C; Vincent, Karen A

    2017-06-01

    Adeno-associated virus (AAV) producer cell lines are created via transfection of HeLaS3 cells with a single plasmid containing three components (the vector sequence, the AAV rep and cap genes, and a selectable marker gene). As this plasmid contains both the cis (Rep binding sites) and trans (Rep protein encoded by the rep gene) elements required for site-specific integration, it was predicted that plasmid integration might occur within the AAVS1 locus on human chromosome 19 (chr19). The objective of this study was to investigate whether integration in AAVS1 might be correlated with vector yield. Plasmid integration sites within several independent cell lines were assessed via Southern, fluorescence in situ hybridization (FISH) and PCR analyses. In the Southern analyses, the presence of fragments detected by both rep- and AAVS1-specific probes suggested that for several mid- and high-producing lines, plasmid DNA had integrated into the AAVS1 locus. Analysis with puroR and AAVS1-specific probes suggested that integration in AAVS1 was a more widespread phenomenon. High-producing AAV2-secreted alkaline phosphatase (SEAP) lines (masterwell 82 [MW82] and MW278) were evaluated via FISH using probes specific for the plasmid, AAVS1, and a chr19 marker. FISH analysis detected two plasmid integration sites in MW278 (neither in AAVS1), while a total of three sites were identified in MW82 (two in AAVS1). An inverse PCR assay confirmed integration within AAVS1 for several mid- and high-producing lines. In summary, the FISH, Southern, and PCR data provide evidence of site-specific integration of the plasmid within AAVS1 in several AAV producer cell lines. The data also suggest that integration in AAVS1 is a general phenomenon that is not necessarily restricted to high producers. The results also suggest that plasmid integration within the AAVS1 locus is not an absolute requirement for a high vector yield.

  8. Humoral Immunity to AAV-6, 8, and 9 in Normal and Dystrophic Dogs

    PubMed Central

    Shin, Jin-Hong; Yue, Yongping; Smith, Bruce

    2012-01-01

    Abstract Adeno-associated virus (AAV)-6, 8, and 9 are promising gene-delivery vectors for testing novel Duchenne muscular dystrophy gene therapy in the canine model. Humoral immunity greatly influences in vivo AAV transduction. However, neutralizing antibodies to AAV-6, 8, and 9 have not been systemically examined in normal and dystrophic dogs. To gain information on the seroprevalence of antibodies to AAV-6, 8, and 9, we measured neutralizing antibody titers using an in vitro transduction inhibition assay. We examined 72 naive serum samples and 26 serum samples obtained from dogs that had received AAV gene transfer. Our data demonstrated that AAV-6 neutralizing antibody was the most prevalent antibody in dogs irrespective of age, gender, disease status (dystrophic or not), and prior parvovirus vaccination history. Surprisingly, high-level anti-AAV-6 antibody was detected at birth in newborn puppies. Further, a robust antibody response was induced in affected, but not normal newborn dogs following systemic AAV gene transfer. Taken together, our data have provided an important baseline on the seroprevalence of AAV-6, 8, and 9 neutralizing antibodies in normal and Duchenne muscular dystrophy dogs. These results will help guide translational AAV gene-therapy studies in dog models of muscular dystrophy. PMID:22040468

  9. Humoral immunity to AAV-6, 8, and 9 in normal and dystrophic dogs.

    PubMed

    Shin, Jin-Hong; Yue, Yongping; Smith, Bruce; Duan, Dongsheng

    2012-03-01

    Adeno-associated virus (AAV)-6, 8, and 9 are promising gene-delivery vectors for testing novel Duchenne muscular dystrophy gene therapy in the canine model. Humoral immunity greatly influences in vivo AAV transduction. However, neutralizing antibodies to AAV-6, 8, and 9 have not been systemically examined in normal and dystrophic dogs. To gain information on the seroprevalence of antibodies to AAV-6, 8, and 9, we measured neutralizing antibody titers using an in vitro transduction inhibition assay. We examined 72 naive serum samples and 26 serum samples obtained from dogs that had received AAV gene transfer. Our data demonstrated that AAV-6 neutralizing antibody was the most prevalent antibody in dogs irrespective of age, gender, disease status (dystrophic or not), and prior parvovirus vaccination history. Surprisingly, high-level anti-AAV-6 antibody was detected at birth in newborn puppies. Further, a robust antibody response was induced in affected, but not normal newborn dogs following systemic AAV gene transfer. Taken together, our data have provided an important baseline on the seroprevalence of AAV-6, 8, and 9 neutralizing antibodies in normal and Duchenne muscular dystrophy dogs. These results will help guide translational AAV gene-therapy studies in dog models of muscular dystrophy.

  10. Syngeneic AAV pseudo-particles potentiate gene transduction of AAV vectors

    USDA-ARS?s Scientific Manuscript database

    Gene delivery vectors based on adeno-associated virus (AAV) have emerged as safe and efficient therapeutic platform for numerous diseases. Excessive empty particles were generated as impurities during AAV vector production, but their effects on clinical outcome of AAV gene therapy are unclear. Here,...

  11. Gene Delivery to Adipose Tissue Using Transcriptionally Targeted rAAV8 Vectors

    PubMed Central

    Uhrig-Schmidt, Silke; Geiger, Matthias; Luippold, Gerd; Birk, Gerald; Mennerich, Detlev; Neubauer, Heike; Grimm, Dirk; Wolfrum, Christian; Kreuz, Sebastian

    2014-01-01

    In recent years, the increasing prevalence of obesity and obesity-related co-morbidities fostered intensive research in the field of adipose tissue biology. To further unravel molecular mechanisms of adipose tissue function, genetic tools enabling functional studies in vitro and in vivo are essential. While the use of transgenic animals is well established, attempts using viral and non-viral vectors to genetically modify adipocytes in vivo are rare. Therefore, we here characterized recombinant Adeno-associated virus (rAAV) vectors regarding their potency as gene transfer vehicles for adipose tissue. Our results demonstrate that a single dose of systemically applied rAAV8-CMV-eGFP can give rise to remarkable transgene expression in murine adipose tissues. Upon transcriptional targeting of the rAAV8 vector to adipocytes using a 2.2 kb fragment of the murine adiponectin (mAP2.2) promoter, eGFP expression was significantly decreased in off-target tissues while efficient transduction was maintained in subcutaneous and visceral fat depots. Moreover, rAAV8-mAP2.2-mediated expression of perilipin A – a lipid-droplet-associated protein – resulted in significant changes in metabolic parameters only three weeks post vector administration. Taken together, our findings indicate that rAAV vector technology is applicable as a flexible tool to genetically modify adipocytes for functional proof-of-concept studies and the assessment of putative therapeutic targets in vivo. PMID:25551639

  12. Pathotyping and genetic characterization of avian avulavirus-1 from domestic and wild waterfowl, geese and black swans in Pakistan, 2014 to 2017.

    PubMed

    Wajid, Abdul; Dundon, William G; Hussain, Tanveer; Babar, Masroor Ellahi

    2018-06-02

    Twenty-nine avian avulavirus-1 viruses (AAvV-1s) from healthy domestic and wild ducks, geese and black swans collected in Pakistan between 2014-2017 have been pathotyped and genetically characterized. A phylogenetic analysis revealed that 21 of the isolates belonged to sub-genotype VIIi, whereas eight isolates were highly similar to vaccine-like viruses of genotype II. In addition to confirming the continued presence of sub-genotype VIIi AAvV-1s in Pakistan, this study identifies the probable spill-over of vaccine-like viruses from vaccinated poultry to wild and domestic waterfowl and, as such, has important implications for the control and management of Newcastle disease in Pakistan.

  13. Product-Related Impurities in Clinical-Grade Recombinant AAV Vectors: Characterization and Risk Assessment

    PubMed Central

    Wright, J. Fraser

    2014-01-01

    Adeno-associated virus (AAV)-based vectors expressing therapeutic genes continue to demonstrate great promise for the treatment of a wide variety of diseases and together with other gene transfer vectors represent an emerging new therapeutic paradigm comparable in potential impact on human health to that achieved by recombinant proteins and vaccines. A challenge for the current pipeline of AAV-based investigational products as they advance through clinical development is the identification, characterization and lot-to-lot control of the process- and product-related impurities present in even highly purified preparations. Especially challenging are AAV vector product-related impurities that closely resemble the vector itself and are, in some cases, without clear precedent in established biotherapeutic products. The determination of acceptable levels of these impurities in vectors prepared for human clinical product development, with the goal of new product licensure, requires careful risk and feasibility assessment. This review focuses primarily on the AAV product-related impurities that have been described in vectors prepared for clinical development. PMID:28548061

  14. AAV-Mediated Gene Transfer to Dorsal Root Ganglion.

    PubMed

    Yu, Hongwei; Fischer, Gregory; Hogan, Quinn H

    2016-01-01

    Transferring genetic molecules into the peripheral sensory nervous system to manipulate nociceptive pathophysiology is a powerful approach for experimental modulation of sensory signaling and potentially for translation into therapy for chronic pain. This can be efficiently achieved by the use of recombinant adeno-associated virus (rAAV) in conjunction with nociceptor-specific regulatory transgene cassettes. Among different routes of delivery, direct injection into the dorsal root ganglia (DRGs) offers the most efficient AAV-mediated gene transfer selectively into the peripheral sensory nervous system. Here, we briefly discuss the advantages and applications of intraganglionic microinjection, and then provide a detailed approach for DRG injection, including a list of the necessary materials and description of a method for performing DRG microinjection experiments. We also discuss our experience with several adeno-associated virus (AAV) options for in vivo transgene expression in DRG neurons.

  15. Small But Increasingly Mighty: Latest Advances in AAV Vector Research, Design, and Evolution.

    PubMed

    Grimm, Dirk; Büning, Hildegard

    2017-11-01

    Recombinant gene delivery vectors derived from naturally occurring or genetically engineered adeno-associated viruses (AAV) have taken center stage in human gene therapy, fueled by rapidly accumulating and highly encouraging clinical data. Nonetheless, it has also become evident that the current generation of AAV vectors will require improvements in transduction potency, antibody evasion, and cell specificity in order to realize their full potential and to widen applicability in larger patient cohorts. Fortunately, in the recent past, the field has seen a flurry of exciting new developments that enhance our understanding of AAV vector biology, including virus-host interactions, and/or that expand our arsenal of technologies for AAV capsid design and evolution. This review highlights a collection of latest advances in these areas, which, in the authors' opinion, hold particular promise to propel the AAV vector field forward in the near future, especially when applied in combination. These include fundamental novel insights into the AAV life cycle, from an unexpected role of autophagy and interactions with other viruses to the (re-)discovery of a universal AAV receptor and the function of AAV-AAP for capsid assembly. Concurrently, recent successes in the rational design of next-generation synthetic AAV capsids are pointed out, exemplified by the structure-guided derivation of AAV mutants displaying robust in vivo immune evasion. Finally, a variety of new and innovative strategies for high-throughput generation and screening of AAV capsid libraries are briefly reviewed, including Cre recombinase-based selection, ancestral AAV capsid reconstruction, and DNA barcoding of AAV genomes. All of these examples showcase the present momentum in the AAV field and, together with work by many other academic or industrial entities, raise substantial optimism that the remaining hurdles for human gene therapy with AAV vectors will (soon) be overcome.

  16. 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.

  17. [Immune response induced by HIV DNA vaccine combined with recombinant adeno-associated virus].

    PubMed

    Liu, Yan-zheng; Zhou, Ling; Wang, Qi; Ye, Shu-qing; Li, Hong-xia; Zeng, Yi

    2004-09-01

    HIV-1 DNA vaccine and recombinant adeno-associated virus (rAAV) expressing gagV3 gene of HIV-1 subtype B were constructed and BALB/c mice were immunized by vaccination regimen consisting of consecutive priming with DNA vaccine and boosting with rAAV vaccine; the CTL and antibody response were detected and compared with those induced by DNA vaccine or rAAV vaccine separately. HIV-1 subtype B gagV3 gene was inserted into the polyclonal site of plasmid pCI-neo, DNA vaccine pCI-gagV3 was thereby constructed; pCI-gagV3 was transfected into p815 cells, G-418-resistant cells were obtained through screening transfected cells with G418, the expression of HIV-1 antigen in G-418-resistant cells was detected by EIA; BALB/c mice were immunized with pCI-gagV3 and the immune response was tested; BALB/c mouse immunized with pCI-gagV3 and combined with rAAV expressing the same gagV3 genes were tested for antibody level in sera by EIA method and cytotoxicity response by LDH method. pCI-gagV3 could express HIV-1 gene in p815 cells; pCI-gagV3 could induce HIV-1 specific humoral and cell-mediated immune response in BALB/c mice. The HIV-1 specific antibody level was 1/20; when the ratio of effector cells: target cells was 50:1, the average specific cytotoxicity was 41.7%; there was no evident increase in the antibody level induced by pCI-gagV3 combined with rAAV, but there was increase in CTL response, the average specific cytotoxicity was 61.3% when effector cells: target cells ratio was 50:1. HIV-1 specific cytotoxicity in BALB/c mice can be increased by immunization of BALB/c mice with DNA vaccine combined with rAAV vaccine.

  18. Plectin-1 Targeted AAV Vector for the Molecular Imaging of Pancreatic Cancer

    PubMed Central

    Konkalmatt, Prasad R.; Deng, Defeng; Thomas, Stephanie; Wu, Michael T.; Logsdon, Craig D.; French, Brent A.; Kelly, Kimberly A.

    2013-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is highly malignant disease that is the fourth leading cause of cancer-related death in the US. Gene therapy using AAV vectors to selectively deliver genes to PDAC cells is an attractive treatment option for pancreatic cancer. However, most AAV serotypes display a broad spectrum of tissue tropism and none of the existing serotypes specifically target PDAC cells. This study tests the hypothesis that AAV2 can be genetically re-engineered to specifically target PDAC cells by modifying the capsid surface to display a peptide that has previously been shown to bind plectin-1. Toward this end, a Plectin-1 Targeting Peptide (PTP) was inserted into the loop IV region of the AAV2 capsid, and the resulting capsid (AAV-PTP) was used in a series of in vitro and in vivo experiments. In vitro, AAV-PTP was found to target all five human PDAC cell lines tested (PANC-1, MIA PaCa-2, HPAC, MPanc-96, and BxPC-3) preferentially over two non-neoplastic human pancreatic cell lines (human pancreatic ductal epithelial and human pancreatic stellate cells). In vivo, mice bearing subcutaneous tumor xenografts were generated using the PANC-1 cell line. Once tumors reached a size of ∼1–2 mm in diameter, the mice were injected intravenously with luciferase reporter vectors packaged in the either AAV-PTP or wild type AAV2 capsids. Luciferase expression was then monitored by bioluminescence imaging on days 3, 7, and 14 after vector injection. The results indicate that the AAV-PTP capsid displays a 37-fold preference for PANC-1 tumor xenographs over liver and other tissues; whereas the wild type AAV2 capsid displays a complementary preference for liver over tumors and other tissues. Together, these results establish proof-of-principle for the ability of PTP-modified AAV capsids to selectively target gene delivery to PDAC cells in vivo, which opens promising new avenues for the early detection, diagnosis, and treatment of pancreatic cancer. PMID:23616947

  19. Genetic cancer vaccines: current status and perspectives.

    PubMed

    Aurisicchio, Luigi; Ciliberto, Gennaro

    2012-08-01

    The recent approval of the first therapeutic cancer vaccine by the US Regulatory Agency represents a breakthrough event in the history of cancer treatment. The past scepticism towards this type of therapeutic intervention is now replaced by great expectations. The field is now moving towards the development of alternative vaccination technologies, which are capable of generating stronger, more durable and efficient immune responses against specific tumour-associated antigens (TAAs) in combination with cheaper and more standardised manufacturing. In this context, genetic vaccines are emerging among the most promising methodologies. Several evidences point to combinations of different genetic immunisation modalities (heterologous prime/boost) as a powerful approach to induce superior immune responses and achieve greater clinical efficacy. In this review, we provide an overview of the current status of development of genetic cancer vaccines with particular emphasis on adenoviral vector prime/DNA boost vaccination schedules. We believe that therapeutic genetic cancer vaccines have the strong potential to become an established therapeutic modality for cancer in next coming years, in a manner similar to what have now become monoclonal antibodies.

  20. Establishment of an AAV Reverse Infection-Based Array

    PubMed Central

    Wang, Gang; Dong, Zheyue; Shen, Wei; Zheng, Gang; Wu, Xiaobing; Xue, Jinglun; Wang, Yue; Chen, Jinzhong

    2010-01-01

    Background The development of a convenient high-throughput gene transduction approach is critical for biological screening. Adeno-associated virus (AAV) vectors are broadly used in gene therapy studies, yet their applications in in vitro high-throughput gene transduction are limited. Principal Findings We established an AAV reverse infection (RI)-based method in which cells were transduced by quantified recombinant AAVs (rAAVs) pre-coated onto 96-well plates. The number of pre-coated rAAV particles and number of cells loaded per well, as well as the temperature stability of the rAAVs on the plates, were evaluated. As the first application of this method, six serotypes or hybrid serotypes of rAAVs (AAV1, AAV2, AAV5/5, AAV8, AAV25 m, AAV28 m) were compared for their transduction efficiencies using various cell lines, including BHK21, HEK293, BEAS-2BS, HeLaS3, Huh7, Hepa1-6, and A549. AAV2 and AAV1 displayed high transduction efficiency; thus, they were deemed to be suitable candidate vectors for the RI-based array. We next evaluated the impact of sodium butyrate (NaB) treatment on rAAV vector-mediated reporter gene expression and found it was significantly enhanced, suggesting that our system reflected the biological response of target cells to specific treatments. Conclusions/Significance Our study provides a novel method for establishing a highly efficient gene transduction array that may be developed into a platform for cell biological assays. PMID:20976058

  1. Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

    PubMed Central

    Ding, Jian; Lin, Zhi-Qiang; Jiang, Jian-Ming; Seidman, Christine E.; Seidman, Jonathan G.; Pu, William T.; Wang, Da-Zhi

    2016-01-01

    Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimalimmunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations. PMID:28060283

  2. Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts.

    PubMed

    Ding, Jian; Lin, Zhi-Qiang; Jiang, Jian-Ming; Seidman, Christine E; Seidman, Jonathan G; Pu, William T; Wang, Da-Zhi

    2016-12-17

    Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

  3. Engineering AAV receptor footprints for gene therapy.

    PubMed

    Madigan, Victoria J; Asokan, Aravind

    2016-06-01

    Adeno-associated viruses (AAV) are currently at the forefront of human gene therapy clinical trials as recombinant vectors. Significant progress has been made in elucidating the structure, biology and tropisms of different naturally occurring AAV isolates in the past decade. In particular, a spectrum of AAV capsid interactions with host receptors have been identified and characterized. These studies have enabled a better understanding of key determinants of AAV cell recognition and entry in different hosts. This knowledge is now being applied toward engineering new, lab-derived AAV capsids with favorable transduction profiles. The current review conveys a structural perspective of capsid-glycan interactions and provides a roadmap for generating synthetic strains by engineering AAV receptor footprints. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. 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.

  5. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges.

    PubMed

    Mingozzi, Federico; High, Katherine A

    2011-05-01

    In vivo gene replacement for the treatment of inherited disease is one of the most compelling concepts in modern medicine. Adeno-associated virus (AAV) vectors have been extensively used for this purpose and have shown therapeutic efficacy in a range of animal models. Successful translation to the clinic was initially slow, but long-term expression of donated genes at therapeutic levels has now been achieved in patients with inherited retinal disorders and haemophilia B. Recent exciting results have raised hopes for the treatment of many other diseases. As we discuss here, the prospects and challenges for AAV gene therapy are to a large extent dependent on the target tissue and the specific disease.

  6. Better Targeting, Better Efficiency for Wide-Scale Neuronal Transduction with the Synapsin Promoter and AAV-PHP.B

    PubMed Central

    Jackson, Kasey L.; Dayton, Robert D.; Deverman, Benjamin E.; Klein, Ronald L.

    2016-01-01

    Widespread genetic modification of cells in the central nervous system (CNS) with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin (CBA) hybrid promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered adeno-associated virus (AAV)-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS)-related protein transactive response DNA-binding protein, 43 kDa (TDP-43) with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats. PMID:27867348

  7. Better Targeting, Better Efficiency for Wide-Scale Neuronal Transduction with the Synapsin Promoter and AAV-PHP.B.

    PubMed

    Jackson, Kasey L; Dayton, Robert D; Deverman, Benjamin E; Klein, Ronald L

    2016-01-01

    Widespread genetic modification of cells in the central nervous system (CNS) with a viral vector has become possible and increasingly more efficient. We previously applied an AAV9 vector with the cytomegalovirus/chicken beta-actin (CBA) hybrid promoter and achieved wide-scale CNS transduction in neonatal and adult rats. However, this method transduces a variety of tissues in addition to the CNS. Thus we studied intravenous AAV9 gene transfer with a synapsin promoter to better target the neurons. We noted in systematic comparisons that the synapsin promoter drives lower level expression than does the CBA promoter. The engineered adeno-associated virus (AAV)-PHP.B serotype was compared with AAV9, and AAV-PHP.B did enhance the efficiency of expression. Combining the synapsin promoter with AAV-PHP.B could therefore be advantageous in terms of combining two refinements of targeting and efficiency. Wide-scale expression was used to model a disease with widespread pathology. Vectors encoding the amyotrophic lateral sclerosis (ALS)-related protein transactive response DNA-binding protein, 43 kDa (TDP-43) with the synapsin promoter and AAV-PHP.B were used for efficient CNS-targeted TDP-43 expression. Intracerebroventricular injections were also explored to limit TDP-43 expression to the CNS. The neuron-selective promoter and the AAV-PHP.B enhanced gene transfer and ALS disease modeling in adult rats.

  8. AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges

    PubMed Central

    Gardner, Matthew R.; Kattenhorn, Lisa M.; Kondur, Hema R.; von Schaewen, Markus; Dorfman, Tatyana; Chiang, Jessica J.; Haworth, Kevin G.; Decker, Julie M.; Alpert, Michael D.; Bailey, Charles C.; Neale, Ernest S.; Fellinger, Christoph H.; Joshi, Vinita R.; Fuchs, Sebastian P.; Martinez-Navio, Jose M.; Quinlan, Brian D.; Yao, Annie Y.; Mouquet, Hugo; Gorman, Jason; Zhang, Baoshan; Poignard, Pascal; Nussenzweig, Michel C.; Burton, Dennis R.; Kwong, Peter D.; Piatak, Michael; Lifson, Jeffrey D.; Gao, Guangping; Desrosiers, Ronald C.; Evans, David T.; Hahn, Beatrice H.; Ploss, Alexander; Cannon, Paula M.; Seaman, Michael S.; Farzan, Michael

    2015-01-01

    Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs)1,2. However even the best bNAbs neutralize 10–50% of HIV-1 isolates inefficiently (IC80 > 5 μg/ml), suggesting that high concentrations of these antibodies would be necessary to achieve general protection3–6. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean IC50 < 0.05 μg/ml). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2, and SIV isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46, and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17 to 77 μg/ml of fully functional rhesus eCD4-Ig for 40 weeks, and these macaques were protected from multiple infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine. PMID:25707797

  9. Adeno-Associated Virus Type 6 (AAV6) Vectors Mediate Efficient Transduction of Airway Epithelial Cells in Mouse Lungs Compared to That of AAV2 Vectors

    PubMed Central

    Halbert, Christine L.; Allen, James M.; Miller, A. Dusty

    2001-01-01

    Although vectors derived from adeno-associated virus type 2 (AAV2) promote gene transfer and expression in many somatic tissues, studies with animal models and cultured cells show that the apical surface of airway epithelia is resistant to transduction by AAV2 vectors. Approaches to increase transduction rates include increasing the amount of vector and perturbing the integrity of the epithelia. In this study, we explored the use of vectors based on AAV6 to increase transduction rates in airways. AAV vectors were made using combinations of rep, cap, and packaged genomes from AAV2 or AAV6. The packaged genomes encoded human placental alkaline phosphatase and contained terminal repeat sequences from AAV2 or AAV6. We found that transduction efficiency was primarily dependent on the source of Cap protein, defined here as the vector pseudotype. The AAV6 and AAV2 pseudotype vectors exhibited different tropisms in tissue-cultured cells, and cell transduction by AAV6 vectors was not inhibited by heparin, nor did they compete for entry in a transduction assay, indicating that AAV6 and AAV2 capsid bind different receptors. In vivo analysis of vectors showed that AAV2 pseudotype vectors gave high transduction rates in alveolar cells but much lower rates in the airway epithelium. In contrast, the AAV6 pseudotype vectors exhibited much more efficient transduction of epithelial cells in large and small airways, showing up to 80% transduction in some airways. These results, combined with our previous results showing lower immunogenicity of AAV6 than of AAV2 vectors, indicate that AAV6 vectors may provide significant advantages over AAV2 for gene therapy of lung diseases like cystic fibrosis. PMID:11413329

  10. The genetic basis for interindividual immune response variation to measles vaccine: new understanding and new vaccine approaches

    PubMed Central

    Haralambieva, Iana H; Ovsyannikova, Inna G; Pankratz, V Shane; Kennedy, Richard B; Jacobson, Robert M; Poland, Gregory A

    2013-01-01

    The live-attenuated measles vaccine is effective, but measles outbreaks still occur in vaccinated populations. This warrants elucidation of the determinants of measles vaccine-induced protective immunity. Interindividual variability in markers of measles vaccine-induced immunity, including neutralizing antibody levels, is regulated in part by host genetic factor variations. This review summarizes recent advances in our understanding of measles vaccine immunogenetics relative to the perspective of developing better measles vaccines. Important genetic regulators of measles vaccine-induced immunity, such as HLA class I and HLA class II genotypes, single nucleotide polymorphisms in cytokine/cytokine receptor genes (IL12B, IL12RB1, IL2, IL10) and the cell surface measles virus receptor CD46 gene, have been identified and independently replicated. New technologies present many opportunities for identification of novel genetic signatures and genetic architectures. These findings help explain a variety of immune response-related phenotypes and promote a new paradigm of ‘vaccinomics’ for novel vaccine development. PMID:23256739

  11. Emergency deployment of genetically engineered veterinary vaccines in Europe.

    PubMed

    Ramezanpour, Bahar; de Foucauld, Jean; Kortekaas, Jeroen

    2016-06-24

    On the 9th of November 2015, preceding the World Veterinary Vaccine Congress, a workshop was held to discuss how veterinary vaccines can be deployed more rapidly to appropriately respond to future epizootics in Europe. Considering their potential and unprecedented suitability for surge production, the workshop focussed on vaccines based on genetically engineered viruses and replicon particles. The workshop was attended by academics and representatives from leading pharmaceutical companies, regulatory experts, the European Medicines Agency and the European Commission. We here outline the present regulatory pathways for genetically engineered vaccines in Europe and describe the incentive for the organization of the pre-congress workshop. The participants agreed that existing European regulations on the deliberate release of genetically engineered vaccines into the environment should be updated to facilitate quick deployment of these vaccines in emergency situations. Copyright © 2016.

  12. Prevalence of Neutralizing Antibodies against Adeno-Associated Virus (AAV) Types 2, 5, and 6 in Cystic Fibrosis and Normal Populations: Implications for Gene Therapy using AAV Vectors

    PubMed Central

    Halbert, Christine L.; Miller, A. Dusty; McNamara, Sharon; Emerson, Julia; Gibson, Ronald L.; Ramsey, Bonnie; Aitken, Moira L.

    2014-01-01

    Adeno-associated virus (AAV) vectors are promising candidates for gene therapy directed to the lungs, in particular for treatment of cystic fibrosis (CF). In animal models of lung gene transfer, neutralizing antibodies in serum made in response to vector exposure have been associated with a partial to complete block to repeat transduction by vectors with the same capsid type, thus transduction by AAV vectors might be inefficient in humans previously exposed to the same AAV type. AAV type 2 (AAV2) has been used in clinical trials of lung gene transfer, but AAV5 and AAV6 have been shown to mediate more efficient transduction in rodent lungs and in cultured human airway epithelia compared to that of AAV2. Here we have measured neutralizing antibodies against AAV type 2, 5, and 6 vectors in serum from children and adults with CF, and from normal adults. About 30% of adults were seropositive for AAV2, 20–30% were seropositive for AAV6, and 10–20% were seropositive for AAV5. CF children were seropositive for AAV types 2, 5, or 6 at rates of 4–15%. All individuals seropositive for AAV6 were also seropositive for AAV2, and the AAV6 titer was low compared to the AAV2 titer. AAV5-positive sera were lower both in titers and rates than those seen for AAV6. The results indicate that AAV type 2, 5 or 6 exposure is low in CF and control populations and even lower in CF children. PMID:16610931

  13. Adeno-associated Virus (AAV) Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5, and 11.

    PubMed

    Earley, Lauriel F; Powers, John M; Adachi, Kei; Baumgart, Joshua T; Meyer, Nancy L; Xie, Qing; Chapman, Michael S; Nakai, Hiroyuki

    2017-02-01

    Adeno-associated virus (AAV) vectors have made great progress in their use for gene therapy; however, fundamental aspects of AAV's capsid assembly remain poorly characterized. In this regard, the discovery of assembly-activating protein (AAP) sheds new light on this crucial part of AAV biology and vector production. Previous studies have shown that AAP is essential for assembly; however, how its mechanistic roles in assembly might differ among AAV serotypes remains uncharacterized. Here, we show that biological properties of AAPs and capsid assembly processes are surprisingly distinct among AAV serotypes 1 to 12. In the study, we investigated subcellular localizations and assembly-promoting functions of AAP1 to -12 (i.e., AAPs derived from AAV1 to -12, respectively) and examined the AAP dependence of capsid assembly processes of these 12 serotypes using combinatorial approaches that involved immunofluorescence and transmission electron microscopy, barcode-Seq (i. e., a high-throughput quantitative method using DNA barcodes and a next-generation sequencing technology), and quantitative dot blot assays. This study revealed that AAP1 to -12 are all localized in the nucleus with serotype-specific differential patterns of nucleolar association; AAPs and assembled capsids do not necessarily colocalize; AAPs are promiscuous in promoting capsid assembly of other serotypes, with the exception of AAP4, -5, -11, and -12; assembled AAV5, -8, and -9 capsids are excluded from the nucleolus, in contrast to the nucleolar enrichment of assembled AAV2 capsids; and, surprisingly, AAV4, -5, and -11 capsids are not dependent on AAP for assembly. These observations highlight the serotype-dependent heterogeneity of the capsid assembly process and challenge current notions about the role of AAP and the nucleolus in capsid assembly. Assembly-activating protein (AAP) is a recently discovered adeno-associated virus (AAV) protein that promotes capsid assembly and provides new opportunities

  14. Vaccination coverage of children with rare genetic diseases and attitudes of their parents toward vaccines

    PubMed Central

    Esposito, Susanna; Cerutti, Marta; Milani, Donatella; Menni, Francesca; Principi, Nicola

    2016-01-01

    Abstract Despite the fact that the achievement of appropriate immunization coverage for routine vaccines is a priority for health authorities worldwide, vaccination delays or missed opportunities for immunization are common in children with chronic diseases. The main aim of this cross-sectional study was to evaluate immunization coverage and the timeliness of vaccination in children suffering from 3 different rare genetic diseases: Rubinstein-Taybi syndrome (RSTS), Sotos syndrome (SS), and Beckwith-Wiedemann syndrome (BWS). A total of 57 children with genetic diseases (15 with RSTS, 14 children with SS, and 28 with BWS) and 57 healthy controls with similar characteristics were enrolled. The coverage of all the recommended vaccines in children with genetic syndromes was significantly lower than that observed in healthy controls (p < 0.05 for all the comparisons). However, when vaccinated, all of the patients, independent of the genetic syndrome from which they suffer, were administered the primary series and the booster doses at a similar time to healthy controls. In comparison with parents of healthy controls, parents of children with genetic diseases were found to more frequently have negative attitudes toward vaccination (p < 0.05 for all the comparisons), mainly for fear of the emergence of adverse events or deterioration of the underlying disease. This study shows that vaccination coverage is poor in pediatric patients with RSTS, BWS, and SS and significantly lower than that observed in healthy children. These results highlight the need for educational programs specifically aimed at both parents and pediatricians to increase immunization coverage in children with these rare genetic diseases. PMID:26337545

  15. RNA Virus Reverse Genetics and Vaccine Design

    PubMed Central

    Stobart, Christopher C.; Moore, Martin L.

    2014-01-01

    RNA viruses are capable of rapid spread and severe or potentially lethal disease in both animals and humans. The development of reverse genetics systems for manipulation and study of RNA virus genomes has provided platforms for designing and optimizing viral mutants for vaccine development. Here, we review the impact of RNA virus reverse genetics systems on past and current efforts to design effective and safe viral therapeutics and vaccines. PMID:24967693

  16. Differential effects of two MRI contrast agents on the integrity and distribution of rAAV2 and rAAV5 in the rat striatum

    PubMed Central

    Osting, Sue; Bennett, Antonette; Power, Shelby; Wackett, Jordan; Hurley, Samuel A; Alexander, Andrew L; Agbandje-Mckena, Mavis; Burger, Corinna

    2014-01-01

    Intraoperative magnetic resonance imaging (MRI) has been proposed as a method to optimize intracerebral targeting and for tracking infusate distribution in gene therapy trials for nervous system disorders. We thus investigated possible effects of two MRI contrast agents, gadoteridol (Gd) and galbumin (Gab), on the distribution and levels of transgene expression in the rat striatum and their effect on integrity and stability of recombinant adeno-associated virus (rAAV) particles. MRI studies showed that contrast agent distribution did not predict rAAV distribution. However, green fluorescent protein (GFP) immunoreactivity revealed an increase in distribution of rAAV5-GFP, but not rAAV2-GFP, in the presence of Gd when compared with viral vector injected alone. In contrast, Gab increased the distribution of rAAV2-GFP not rAAV5-GFP. These observations pointed to a direct effect of infused contrast agent on the rAAV particles. Negative-stain electron microscopy (EM), DNAase treatment, and differential scanning calorimetry (DSC) were used to monitor rAAV2 and rAAV5 particle integrity and stability following contrast agent incubation. EMs of rAAV2-GFP and rAAV5-GFP particles pretreated with Gd appear morphologically similar to the untreated sample; however, Gab treatment resulted in surface morphology changes and aggregation. A compromise of particle integrity was suggested by sensitivity of the packaged genome to DNAase treatment following Gab incubation but not Gd for both vectors. However, neither agent significantly affected particle stability when analyzed by DSC. An increase in Tm was observed for AAV2 in lactated Ringer’s buffer. These results thus highlight potential interactions between MRI contrast agents and AAV that might affect vector distribution and stability, as well as the stabilizing effect of lactated Ringer’s solution on AAV2. PMID:26015943

  17. Recent progress and considerations for AAV gene therapies targeting the central nervous system.

    PubMed

    Lykken, Erik Allen; Shyng, Charles; Edwards, Reginald James; Rozenberg, Alejandra; Gray, Steven James

    2018-05-18

    Neurodevelopmental disorders, as a class of diseases, have been particularly difficult to treat even when the underlying cause(s), such as genetic alterations, are understood. What treatments do exist are generally not curative and instead seek to improve quality of life for affected individuals. The advent of gene therapy via gene replacement offers the potential for transformative therapies to slow or even stop disease progression for current patients and perhaps minimize or prevent the appearance of symptoms in future patients. This review focuses on adeno-associated virus (AAV) gene therapies for diseases of the central nervous system. An overview of advances in AAV vector design for therapy is provided, along with a description of current strategies to develop AAV vectors with tailored tropism. Next, progress towards treatment of neurodegenerative diseases is presented at both the pre-clinical and clinical stages, focusing on a few select diseases to highlight broad categories of therapeutic parameters. Special considerations for more challenging cases are then discussed in addition to the immunological aspects of gene therapy. With the promising clinical trial results that have been observed for the latest AAV gene therapies and continued pre-clinical successes, the question is no longer whether a therapy can be developed for certain neurodevelopmental disorders, but rather, how quickly.

  18. Design and construction of functional AAV vectors.

    PubMed

    Gray, John T; Zolotukhin, Serge

    2011-01-01

    Using the basic principles of molecular biology and laboratory techniques presented in this chapter, researchers should be able to create a wide variety of AAV vectors for both clinical and basic research applications. Basic vector design concepts are covered for both protein coding gene expression and small non-coding RNA gene expression cassettes. AAV plasmid vector backbones (available via AddGene) are described, along with critical sequence details for a variety of modular expression components that can be inserted as needed for specific applications. Protocols are provided for assembling the various DNA components into AAV vector plasmids in Escherichia coli, as well as for transferring these vector sequences into baculovirus genomes for large-scale production of AAV in the insect cell production system.

  19. Intracranial AAV-IFN-β gene therapy eliminates invasive xenograft glioblastoma and improves survival in orthotopic syngeneic murine model.

    PubMed

    GuhaSarkar, Dwijit; Neiswender, James; Su, Qin; Gao, Guangping; Sena-Esteves, Miguel

    2017-02-01

    The highly invasive property of glioblastoma (GBM) cells and genetic heterogeneity are largely responsible for tumor recurrence after the current standard-of-care treatment and thus a direct cause of death. Previously, we have shown that intracranial interferon-beta (IFN-β) gene therapy by locally administered adeno-associated viral vectors (AAV) successfully treats noninvasive orthotopic glioblastoma models. Here, we extend these findings by testing this approach in invasive human GBM xenograft and syngeneic mouse models. First, we show that a single intracranial injection of AAV encoding human IFN-β eliminates invasive human GBM8 tumors and promotes long-term survival. Next, we screened five AAV-IFN-β vectors with different promoters to drive safe expression of mouse IFN-β in the brain in the context of syngeneic GL261 tumors. Two AAV-IFN-β vectors were excluded due to safety concerns, but therapeutic studies with the other three vectors showed extensive tumor cell death, activation of microglia surrounding the tumors, and a 56% increase in median survival of the animals treated with AAV/P2-Int-mIFN-β vector. We also assessed the therapeutic effect of combining AAV-IFN-β therapy with temozolomide (TMZ). As TMZ affects DNA replication, an event that is crucial for second-strand DNA synthesis of single-stranded AAV vectors before active transcription, we tested two TMZ treatment regimens. Treatment with TMZ prior to AAV-IFN-β abrogated any benefit from the latter, while the reverse order of treatment doubled the median survival compared to controls. These studies demonstrate the therapeutic potential of intracranial AAV-IFN-β therapy in a highly migratory GBM model as well as in a syngeneic mouse model and that combination with TMZ is likely to enhance its antitumor potency. © 2016 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

  20. A phase1 study of stereotactic gene delivery of AAV2-NGF for Alzheimer's disease.

    PubMed

    Rafii, Michael S; Baumann, Tiffany L; Bakay, Roy A E; Ostrove, Jeffrey M; Siffert, Joao; Fleisher, Adam S; Herzog, Christopher D; Barba, David; Pay, Mary; Salmon, David P; Chu, Yaping; Kordower, Jeffrey H; Bishop, Kathie; Keator, David; Potkin, Steven; Bartus, Raymond T

    2014-09-01

    Nerve growth factor (NGF) is an endogenous neurotrophic-factor protein with the potential to restore function and to protect degenerating cholinergic neurons in Alzheimer's disease (AD), but safe and effective delivery has proved unsuccessful. Gene transfer, combined with stereotactic surgery, offers a potential means to solve the long-standing delivery obstacles. An open-label clinical trial evaluated the safety and tolerability, and initial efficacy of three ascending doses of the genetically engineered gene-therapy vector adeno-associated virus serotype 2 delivering NGF (AAV2-NGF [CERE-110]). Ten subjects with AD received bilateral AAV2-NGF stereotactically into the nucleus basalis of Meynert. AAV2-NGF was safe and well-tolerated for 2 years. Positron emission tomographic imaging and neuropsychological testing showed no evidence of accelerated decline. Brain autopsy tissue confirmed long-term, targeted, gene-mediated NGF expression and bioactivity. This trial provides important evidence that bilateral stereotactic administration of AAV2-NGF to the nucleus basalis of Meynert is feasible, well-tolerated, and able to produce long-term, biologically active NGF expression, supporting the initiation of an ongoing multicenter, double-blind, sham-surgery-controlled trial. Copyright © 2014 The Alzheimer's Association. Published by Elsevier Inc. All rights reserved.

  1. Reverse Genetics Approaches for the Development of Influenza Vaccines

    PubMed Central

    Nogales, Aitor; Martínez-Sobrido, Luis

    2016-01-01

    Influenza viruses cause annual seasonal epidemics and occasional pandemics of human respiratory disease. Influenza virus infections represent a serious public health and economic problem, which are most effectively prevented through vaccination. However, influenza viruses undergo continual antigenic variation, which requires either the annual reformulation of seasonal influenza vaccines or the rapid generation of vaccines against potential pandemic virus strains. The segmented nature of influenza virus allows for the reassortment between two or more viruses within a co-infected cell, and this characteristic has also been harnessed in the laboratory to generate reassortant viruses for their use as either inactivated or live-attenuated influenza vaccines. With the implementation of plasmid-based reverse genetics techniques, it is now possible to engineer recombinant influenza viruses entirely from full-length complementary DNA copies of the viral genome by transfection of susceptible cells. These reverse genetics systems have provided investigators with novel and powerful approaches to answer important questions about the biology of influenza viruses, including the function of viral proteins, their interaction with cellular host factors and the mechanisms of influenza virus transmission and pathogenesis. In addition, reverse genetics techniques have allowed the generation of recombinant influenza viruses, providing a powerful technology to develop both inactivated and live-attenuated influenza vaccines. In this review, we will summarize the current knowledge of state-of-the-art, plasmid-based, influenza reverse genetics approaches and their implementation to provide rapid, convenient, safe and more effective influenza inactivated or live-attenuated vaccines. PMID:28025504

  2. AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes.

    PubMed

    Hui, Daniel J; Edmonson, Shyrie C; Podsakoff, Gregory M; Pien, Gary C; Ivanciu, Lacramioara; Camire, Rodney M; Ertl, Hildegund; Mingozzi, Federico; High, Katherine A; Basner-Tschakarjan, Etiena

    2015-01-01

    Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings.

  3. Assaying the Stability and Inactivation of AAV Serotype 1 Vectors

    PubMed Central

    Howard, Douglas B.; Harvey, Brandon K.

    2017-01-01

    Adeno-associated virus (AAV) vectors are a commonplace tool for gene delivery ranging from cell culture to human gene therapy. One feature that makes AAV a desirable vector is its stability, in regard to both the duration of transgene expression and retention of infectivity as a viral particle. This study examined the stability of AAV serotype 1 (AAV1) vectors under different conditions. First, transducibility after storage at 4°C decreased 20% over 7 weeks. Over 10 freeze–thaw cycles, the resulting transduction efficiency became variable at 60–120% of a single thaw. Using small stainless steel slugs to mimic a biosafety cabinet or metal lab bench surface, it was found that an AAV1 vector can be reconstituted after 6 days of storage at room temperature. The stability of AAV is a desired feature, but effective decontamination procedures must be available for safety and experimental integrity. Multiple disinfectants commonly used in the laboratory for ability to inactivate an AAV1 vector were tested, and it was found that autoclaving, 0.25% peracetic acid, iodine, or 10% Clorox bleach completely prevented AAV-mediated transgene expression. These data suggest that peracetic acid should be used for inactivating AAV1 vectors on metal-based surfaces or instruments in order to avoid inadvertent transgene expression in human cells or cross-contamination of instruments. PMID:28192678

  4. Genetic Susceptibility to ANCA-Associated Vasculitis: State of the Art

    PubMed Central

    Bonatti, Francesco; Reina, Michele; Neri, Tauro Maria; Martorana, Davide

    2014-01-01

    ANCA-associated vasculitis (AAV) is a group of disorders that is caused by inflammation affecting small blood vessels. Both arteries and veins are affected. AAV includes microscopic polyangiitis (MPA), granulomatosis with polyangiitis (GPA) renamed from Wegener’s granulomatosis, and eosinophilic granulomatosis with polyangiitis (EGPA), renamed from Churg–Strauss syndrome. AAV is primarily due to leukocyte migration and resultant damage. Despite decades of research, the mechanisms behind AAV disease etiology are still not fully understood, although it is clear that genetic and environmental factors are involved. To improve the understanding of the disease, the genetic component has been extensively studied by candidate association studies and two genome-wide association studies. The majority of the identified genetic AAV risk factors are common variants. These have uncovered information that still needs further investigation to clarify its importance. In this review, we summarize and discuss the results of the genetic studies in AAV. We also present the novel approaches to identifying the causal variants in complex susceptibility loci and disease mechanisms. Finally, we discuss the limitations of current methods and the challenges that we still have to face in order to incorporate genomic and epigenomic data into clinical practice. PMID:25452756

  5. The recombinant adeno-associated virus vector (rAAV2)-mediated apolipoprotein B mRNA-specific hammerhead ribozyme: a self-complementary AAV2 vector improves the gene expression

    PubMed Central

    Zhong, Shumei; Sun, Shihua; Teng, Ba-Bie

    2004-01-01

    Background In humans, overproduction of apolipoprotein B (apoB) is positively associated with premature coronary artery diseases. To reduce the levels of apoB mRNA, we have designed an apoB mRNA-specific hammerhead ribozyme targeted at nucleotide sequences GUA6679 (RB15) mediated by adenovirus, which efficiently cleaves and decreases apoB mRNA by 80% in mouse liver and attenuates the hyperlipidemic condition. In the current study, we used an adeno-associated virus vector, serotype 2 (AAV2) and a self-complementary AAV2 vector (scAAV2) to demonstrate the effect of long-term tissue-specific gene expression of RB15 on the regulation apoB mRNA in vivo. Methods We constructed a hammerhead ribozyme RB15 driven by a liver-specific transthyretin (TTR) promoter using an AAV2 vector (rAAV2-TTR-RB15). HepG2 cells and hyperlipidemic mice deficient in both the low density lipoprotein receptor and the apoB mRNA editing enzyme genes (LDLR-/-Apobec1-/-; LDb) were transduced with rAAV2-TTR-RB15 and a control vector rAAV-TTR-RB15-mutant (inactive ribozyme). The effects of ribozyme RB15 on apoB metabolism and atherosclerosis development were determined in LDb mice at 5-month after transduction. A self-complementary AAV2 vector expressing ribozyme RB15 (scAAV2-TTR-RB15) was also engineered and used to transduce HepG2 cells. Studies were designed to compare the gene expression efficiency between rAAV2-TTR-RB15 and scAAV2-TTR-RB15. Results The effect of ribozyme RB15 RNA on reducing apoB mRNA levels in HepG2 cells was observed only on day-7 after rAAV2-TTR-RB15 transduction. And, at 5-month after rAAV2-TTR-RB15 treatment, the apoB mRNA levels in LDb mice were significantly decreased by 43%, compared to LDb mice treated with control vector rAAV2-TTR-RB15-mutant. Moreover, both the rAAV2-TTR-RB15 viral DNA and ribozyme RB15 RNA were still detectable in mice livers at 5-month after treatment. However, this rAAV2-TTR-RB15 vector mediated a prolonged but low level of ribozyme RB15 gene

  6. Superior In vivo Transduction of Human Hepatocytes Using Engineered AAV3 Capsid.

    PubMed

    Vercauteren, Koen; Hoffman, Brad E; Zolotukhin, Irene; Keeler, Geoffrey D; Xiao, Jing W; Basner-Tschakarjan, Etiena; High, Katherine A; Ertl, Hildegund Cj; Rice, Charles M; Srivastava, Arun; de Jong, Ype P; Herzog, Roland W

    2016-06-01

    Adeno-associated viral (AAV) vectors are currently being tested in multiple clinical trials for liver-directed gene transfer to treat the bleeding disorders hemophilia A and B and metabolic disorders. The optimal viral capsid for transduction of human hepatocytes has been under active investigation, but results across various models are inconsistent. We tested in vivo transduction in "humanized" mice. Methods to quantitate percent AAV transduced human and murine hepatocytes in chimeric livers were optimized using flow cytometry and confocal microscopy with image analysis. Distinct transduction efficiencies were noted following peripheral vein administration of a self-complementary vector expressing a gfp reporter gene. An engineered AAV3 capsid with two amino acid changes, S663V+T492V (AAV3-ST), showed best efficiency for human hepatocytes (~3-times, ~8-times, and ~80-times higher than for AAV9, AAV8, and AAV5, respectively). AAV5, 8, and 9 were more efficient in transducing murine than human hepatocytes. AAV8 yielded the highest transduction rate of murine hepatocytes, which was 19-times higher than that for human hepatocytes. In summary, our data show substantial differences among AAV serotypes in transduction of human and mouse hepatocytes, are the first to report on AAV5 in humanized mice, and support the use of AAV3-based vectors for human liver gene transfer.

  7. High-Throughput Dissection of AAV-Host Interactions: The Fast and the Curious.

    PubMed

    Herrmann, Anne-Kathrin; Grimm, Dirk

    2018-05-18

    Over fifty years after its initial description, Adeno-associated virus (AAV) remains a most exciting but also most elusive study object in basic or applied virology. On the one hand, its simple structure not only facilitates investigations into virus biology, but combined with the availability of numerous natural AAV variants with distinct infection efficiency and specificity also makes AAV a preferred substrate for engineering of gene delivery vectors. On the other hand, it is striking to witness a recent flurry of reports that highlight and partially close persistent gaps in our understanding of AAV virus and vector biology. This is all the more perplexing considering that recombinant AAVs have already been used in >160 clinical trials and recently been commercialized as gene therapeutics. Here, we discuss a reason for these advances in AAV research, namely, the advent and application of powerful high-throughput technology for dissection of AAV-host interactions and optimization of AAV gene therapy vectors. As relevant examples, we focus on the discovery of (i) a "new" cellular AAV receptor, AAVR, (ii) host restriction factors for AAV entry, and (iii) AAV capsid determinants that mediate trafficking through the blood-brain barrier. While (i)/(ii) are prototypes of extra- or intracellular AAV host factors that were identified via high-throughput screenings, (iii) exemplifies the power of molecular evolution to investigate the virus itself. In the future, we anticipate that these and other key technologies will continue to accelerate the dissection of AAV biology and will yield a wealth of new designer viruses for clinical use. Copyright © 2018. Published by Elsevier Ltd.

  8. Comparative biology of rAAV transduction in ferret, pig and human airway epithelia.

    PubMed

    Liu, X; Luo, M; Guo, C; Yan, Z; Wang, Y; Engelhardt, J F

    2007-11-01

    Differences between rodent and human airway cell biology have made it difficult to translate recombinant adeno-associated virus (rAAV)-mediated gene therapies to the lung for cystic fibrosis (CF). As new ferret and pig models for CF become available, knowledge about host cell/vector interactions in these species will become increasingly important for testing potential gene therapies. To this end, we have compared the transduction biology of three rAAV serotypes (AAV1, 2 and 5) in human, ferret, pig and mouse-polarized airway epithelia. Our results indicate that apical transduction of ferret and pig airway epithelia with these rAAV serotypes closely mirrors that observed in human epithelia (rAAV1>rAAV2 congruent withrAAV5), while transduction of mouse epithelia was significantly different (rAAV1>rAAV5>rAAV2). Similarly, ferret, pig and human epithelia also shared serotype-specific differences in the polarity (apical vs basolateral) and proteasome dependence of rAAV transduction. Despite these parallels, N-linked sialic acid receptors were required for rAAV1 and rAAV5 transduction of human and mouse airway epithelia, but not ferret or pig airway epithelia. Hence, although the airway tropisms of rAAV serotypes 1, 2 and 5 are conserved better among ferret, pig and human as compared to mouse, viral receptors/co-receptors appear to maintain considerable species diversity.

  9. Biological safety concepts of genetically modified live bacterial vaccines.

    PubMed

    Frey, Joachim

    2007-07-26

    Live vaccines possess the advantage of having access to induce cell-mediated and antibody-mediated immunity; thus in certain cases they are able to prevent infection, and not only disease. Furthermore, live vaccines, particularly bacterial live vaccines, are relatively cheap to produce and easy to apply. Hence they are suitable to immunize large communities or herds. The induction of both cell-mediated immunity as well as antibody-mediated immunity, which is particularly beneficial in inducing mucosal immune responses, is obtained by the vaccine-strain's ability to colonize and multiply in the host without causing disease. For this reason, live vaccines require attenuation of virulence of the bacterium to which immunity must be induced. Traditionally attenuation was achieved simply by multiple passages of the microorganism on growth medium, in animals, eggs or cell cultures or by chemical or physical mutagenesis, which resulted in random mutations that lead to attenuation. In contrast, novel molecular methods enable the development of genetically modified organisms (GMOs) targeted to specific genes that are particularly suited to induce attenuation or to reduce undesirable effects in the tissue in which the vaccine strains can multiply and survive. Since live vaccine strains (attenuated by natural selection or genetic engineering) are potentially released into the environment by the vaccinees, safety issues concerning the medical as well as environmental aspects must be considered. These involve (i) changes in cell, tissue and host tropism, (ii) virulence of the carrier through the incorporation of foreign genes, (iii) reversion to virulence by acquisition of complementation genes, (iv) exchange of genetic information with other vaccine or wild-type strains of the carrier organism and (v) spread of undesired genes such as antibiotic resistance genes. Before live vaccines are applied, the safety issues must be thoroughly evaluated case-by-case. Safety assessment

  10. Differential Effects of AAV.BDNF and AAV.Ntf3 in the Deafened Adult Guinea Pig Ear

    PubMed Central

    Budenz, Cameron L.; Wong, Hiu Tung; Swiderski, Donald L.; Shibata, Seiji B.; Pfingst, Bryan E.; Raphael, Yehoash

    2015-01-01

    Cochlear hair cell loss results in secondary regression of peripheral auditory fibers (PAFs) and loss of spiral ganglion neurons (SGNs). The performance of cochlear implants (CI) in rehabilitating hearing depends on survival of SGNs. Here we compare the effects of adeno-associated virus vectors with neurotrophin gene inserts, AAV.BDNF and AAV.Ntf3, on guinea pig ears deafened systemically (kanamycin and furosemide) or locally (neomycin). AAV.BDNF or AAV.Ntf3 was delivered to the guinea pig cochlea one week following deafening and ears were assessed morphologically 3 months later. At that time, neurotrophins levels were not significantly elevated in the cochlear fluids, even though in vitro and shorter term in vivo experiments demonstrate robust elevation of neurotrophins with these viral vectors. Nevertheless, animals receiving these vectors exhibited considerable re-growth of PAFs in the basilar membrane area. In systemically deafened animals there was a negative correlation between the presence of differentiated supporting cells and PAFs, suggesting that supporting cells influence the outcome of neurotrophin over-expression aimed at enhancing the cochlear neural substrate. Counts of SGN in Rosenthal's canal indicate that BDNF was more effective than NT-3 in preserving SGNs. The results demonstrate that a transient elevation in neurotrophin levels can sustain the cochlear neural substrate in the long term. PMID:25726967

  11. AAV capsid CD8+ T-cell epitopes are highly conserved across AAV serotypes

    PubMed Central

    Hui, Daniel J; Edmonson, Shyrie C; Podsakoff, Gregory M; Pien, Gary C; Ivanciu, Lacramioara; Camire, Rodney M; Ertl, Hildegund; Mingozzi, Federico; High, Katherine A; Basner-Tschakarjan, Etiena

    2015-01-01

    Adeno-associated virus (AAV) has become one of the most promising vectors in gene transfer in the last 10 years with successful translation to clinical trials in humans and even market approval for a first gene therapy product in Europe. Administration to humans, however, revealed that adaptive immune responses against the vector capsid can present an obstacle to sustained transgene expression due to the activation and expansion of capsid-specific T cells. The limited number of peripheral blood mononuclear cells (PBMCs) obtained from samples within clinical trials allows for little more than monitoring of T-cell responses. We were able to identify immunodominant major histocompatibility complex (MHC) class I epitopes for common human leukocyte antigen (HLA) types by using spleens isolated from subjects undergoing splenectomy for non-malignant indications as a source of large numbers of lymphocytes and restimulating them with single AAV capsid peptides in vitro. Further experiments confirmed that these epitopes are naturally processed and functionally relevant. The design of more effective and less immunogenic AAV vectors, and precise immune monitoring of vector-infused subjects, are facilitated by these findings. PMID:26445723

  12. 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

  13. Enhanced genetic characterization of influenza A(H3N2) viruses and vaccine effectiveness by genetic group, 2014–2015

    PubMed Central

    Flannery, Brendan; Zimmerman, Richard K.; Gubareva, Larisa V.; Garten, Rebecca J.; Chung, Jessie R.; Nowalk, Mary Patricia; Jackson, Michael L.; Jackson, Lisa A.; Monto, Arnold S.; Ohmit, Suzanne E.; Belongia, Edward A.; McLean, Huong Q.; Gaglani, Manjusha; Piedra, Pedro A.; Mishin, Vasiliy P.; Chesnokov, Anton P.; Spencer, Sarah; Thaker, Swathi N.; Barnes, John R.; Foust, Angie; Sessions, Wendy; Xu, Xiyan; Katz, Jacqueline; Fry, Alicia M.

    2018-01-01

    Background During the 2014–15 US influenza season, expanded genetic characterization of circulating influenza A(H3N2) viruses was used to assess the impact of genetic variability of influenza A(H3N2) viruses on influenza vaccine effectiveness (VE). Methods A novel pyrosequencing assay was used to determine genetic group based on hemagglutinin (HA) gene sequences of influenza A(H3N2) viruses from patients enrolled US Flu Vaccine Effectiveness network sites. Vaccine effectiveness was estimated using a test-negative design comparing vaccination among patients infected with influenza A(H3N2) viruses and uninfected patients. Results Among 9710 enrollees, 1868 (19%) tested positive for influenza A(H3N2); genetic characterization of 1397 viruses showed 1134 (81%) belonged to one HA genetic group (3C.2a) of antigenically drifted H3N2 viruses. Effectiveness of 2014–15 influenza vaccination varied by A(H3N2) genetic group from 1% (95% confidence interval [CI], −14% to 14%) against illness caused by antigenically drifted A(H3N2) group 3C.2a viruses versus 44% (95% CI, 16% to 63%) against illness caused by vaccine-like A(H3N2) group 3C.3b viruses. Conclusion Effectiveness of 2014–15 influenza vaccination varied by genetic group of influenza A(H3N2) virus. Changes in hemagglutinin genes related to antigenic drift were associated with reduced vaccine effectiveness. PMID:27190176

  14. Arenavirus reverse genetics for vaccine development

    PubMed Central

    Ortiz-Riaño, Emilio; Cheng, Benson Yee Hin; Carlos de la Torre, Juan

    2013-01-01

    Arenaviruses are important human pathogens with no Food and Drug Administration (FDA)-licensed vaccines available and current antiviral therapy being limited to an off-label use of the nucleoside analogue ribavirin of limited prophylactic efficacy. The development of reverse genetics systems represented a major breakthrough in arenavirus research. However, rescue of recombinant arenaviruses using current reverse genetics systems has been restricted to rodent cells. In this study, we describe the rescue of recombinant arenaviruses from human 293T cells and Vero cells, an FDA-approved line for vaccine development. We also describe the generation of novel vectors that mediate synthesis of both negative-sense genome RNA and positive-sense mRNA species of lymphocytic choriomeningitis virus (LCMV) directed by the human RNA polymerases I and II, respectively, within the same plasmid. This approach reduces by half the number of vectors required for arenavirus rescue, which could facilitate virus rescue in cell lines approved for human vaccine production but that cannot be transfected at high efficiencies. We have shown the feasibility of this approach by rescuing both the Old World prototypic arenavirus LCMV and the live-attenuated vaccine Candid#1 strain of the New World arenavirus Junín. Moreover, we show the feasibility of using these novel strategies for efficient rescue of recombinant tri-segmented both LCMV and Candid#1. PMID:23364194

  15. Evaluation of a genetically modified foot-and-mouth disease virus vaccine candidate generated by reverse genetics

    PubMed Central

    2012-01-01

    Background Foot-and-mouth disease (FMD) is the most economically important and highly contagious disease of cloven-hoofed animals worldwide. Control of the disease has been mainly based on large-scale vaccinations with whole-virus inactivated vaccines. In recent years, a series of outbreaks of type O FMD occurred in China (including Chinese Taipei, Chinese Hong Kong) posed a tremendous threat to Chinese animal husbandry. Its causative agent, type O FMDV, has evolved into three topotypes (East–South Asia (ME-SA), Southeast Asia (SEA), Cathay (CHY)) in these regions, which represents an important obstacle to disease control. The available FMD vaccine in China shows generally good protection against ME-SA and SEA topotype viruses infection, but affords insufficient protection against some variants of the CHY topotype. Therefore, the choice of a new vaccine strain is of fundamental importance. Results The present study describes the generation of a full-length infectious cDNA clone of FMDV vaccine strain and a genetically modified virus with some amino acid substitutions in antigenic sites 1, 3, and 4, based on the established infectious clone. The recombinant viruses had similar growth properties to the wild O/HN/CHA/93 virus. All swine immunized with inactivated vaccine prepared from the O/HN/CHA/93 were fully protected from challenge with the viruses of ME-SA and SEA topotypes and partially protected against challenge with the virus of CHY topotype at 28 days post-immunization. In contrast, the swine inoculated with the genetically modified vaccine were completely protected from the infection of viruses of the three topotypes. Conclusions Some amino acid substitutions in the FMDV vaccine strain genome did not have an effect on the ability of viral replication in vitro. The vaccine prepared from genetically modified FMDV by reverse genetics significantly improved the protective efficacy to the variant of the CHY topotype, compared with the wild O/HN/CHA/93 virus

  16. Assessing the potential for AAV vector genotoxicity in a murine model

    PubMed Central

    Li, Hojun; Malani, Nirav; Hamilton, Shari R.; Schlachterman, Alexander; Bussadori, Giulio; Edmonson, Shyrie E.; Shah, Rachel; Arruda, Valder R.; Mingozzi, Federico; Fraser Wright, J.; Bushman, Frederic D.

    2011-01-01

    Gene transfer using adeno-associated virus (AAV) vectors has great potential for treating human disease. Recently, questions have arisen about the safety of AAV vectors, specifically, whether integration of vector DNA in transduced cell genomes promotes tumor formation. This study addresses these questions with high-dose liver-directed AAV-mediated gene transfer in the adult mouse as a model (80 AAV-injected mice and 52 controls). After 18 months of follow-up, AAV-injected mice did not show a significantly higher rate of hepatocellular carcinoma compared with controls. Tumors in mice treated with AAV vectors did not have significantly different amounts of vector DNA compared with adjacent normal tissue. A novel high-throughput method for identifying AAV vector integration sites was developed and used to clone 1029 integrants. Integration patterns in tumor tissue and adjacent normal tissue were similar to each other, showing preferences for active genes, cytosine-phosphate-guanosine islands, and guanosine/cysteine-rich regions. Gene expression data showed that genes near integration sites did not show significant changes in expression patterns compared with genes more distal to integration sites. No integration events were identified as causing increased oncogene expression. Thus, we did not find evidence that AAV vectors cause insertional activation of oncogenes and subsequent tumor formation. PMID:21106988

  17. Prolonged Expression of an Anti-HIV-1 gp120 Minibody to the Female Rhesus Macaque Lower Genital Tract by AAV Gene Transfer

    PubMed Central

    Abdel-Motal, Ussama M.; Harbison, Carole; Han, Thomas; Pudney, Jeffrey; Anderson, Deborah J.; Zhu, Quan; Westmoreland, Susan; Marasco, Wayne A.

    2014-01-01

    Topical microbicides are a leading strategy for prevention of HIV mucosal infection to women, however, numerous pharmacokinetic limitations associated with coitally-related dosing strategy have contributed to their limited success. Here we test the hypothesis that adeno-associated virus (AAV) mediated delivery of the b12 human anti-HIV-1 gp120 minibody gene to the lower genital tract of female rhesus macaques (Rh) can provide prolonged expression of b12 minibodies in the cervical-vaginal secretions. Gene transfer studies demonstrated that, of various GFP-expressing AAV serotypes, AAV-6 most efficiently transduced freshly immortalized and primary genital epithelial cells (PGECs) of female Rh in vitro. In addition, AAV-6-b12 minibody transduction of Rh PGECs led to inhibition of SHIV162p4 transmigration and virus infectivity in vitro. AAV-6-GFP could also successfully transduce vaginal epithelial cells of Rh when applied intra-vaginally, including p63+ epithelial stem cells. Moreover, intra-vaginal application of AAV-6-b12 to female Rh resulted in prolonged minibody detection in their vaginal secretions throughout the 79 day study period. These data provide proof-of-principle that AAV-6-mediated delivery of anti-HIV broadly neutralizing antibody (BnAb) genes to the lower genital tract of female Rh results in persistent minibody detection for several months. This strategy offers promise that an anti-HIV-1 genetic microbicide strategy may be possible in which topical application of AAV vector, with periodic reapplication as needed, may provide sustained local BnAb expression and protection. PMID:24965083

  18. Prolonged expression of an anti-HIV-1 gp120 minibody to the female rhesus macaque lower genital tract by AAV gene transfer.

    PubMed

    Abdel-Motal, U M; Harbison, C; Han, T; Pudney, J; Anderson, D J; Zhu, Q; Westmoreland, S; Marasco, W A

    2014-09-01

    Topical microbicides are a leading strategy for prevention of HIV mucosal infection to women; however, numerous pharmacokinetic limitations associated with coitally related dosing strategy have contributed to their limited success. Here we test the hypothesis that adeno-associated virus (AAV) mediated delivery of the b12 human anti-HIV-1 gp120 minibody gene to the lower genital tract of female rhesus macaques (Rh) can provide prolonged expression of b12 minibodies in the cervical-vaginal secretions. Gene transfer studies demonstrated that, of various green fluorescent protein (GFP)-expressing AAV serotypes, AAV-6 most efficiently transduced freshly immortalized and primary genital epithelial cells (PGECs) of female Rh in vitro. In addition, AAV-6-b12 minibody transduction of Rh PGECs led to inhibition of SHIV162p4 transmigration and virus infectivity in vitro. AAV-6-GFP could also successfully transduce vaginal epithelial cells of Rh when applied intravaginally, including p63+ epithelial stem cells. Moreover, intravaginal application of AAV-6-b12 to female Rh resulted in prolonged minibody detection in their vaginal secretions throughout the 79-day study period. These data provide proof of principle that AAV-6-mediated delivery of anti-HIV broadly neutralizing antibody (BnAb) genes to the lower genital tract of female Rh results in persistent minibody detection for several months. This strategy offers promise that an anti-HIV-1 genetic microbicide strategy may be possible in which topical application of AAV vector, with periodic reapplication as needed, may provide sustained local BnAb expression and protection.

  19. Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain

    PubMed Central

    Deverman, Benjamin E.; Pravdo, Piers L.; Simpson, Bryan P.; Kumar, Sripriya Ravindra; Chan, Ken Y.; Banerjee, Abhik; Wu, Wei-Li; Yang, Bin; Huber, Nina; Pasca, Sergiu P.; Gradinaru, Viviana

    2015-01-01

    Recombinant adeno-associated viruses (rAAVs) are commonly used vehicles for in vivo gene transfer1-6. However, the tropism repertoire of naturally occurring AAVs is limited, prompting a search for novel AAV capsids with desired characteristics7-13. Here we describe a capsid selection method, called Cre-recombination-based AAV targeted evolution (CREATE), that enables the development of AAV capsids that more efficiently transduce defined Cre-expressing cell populations in vivo. We use CREATE to generate AAV variants that efficiently and widely transduce the adult mouse central nervous system (CNS) after intravenous injection. One variant, AAV-PHP.B, transfers genes throughout the CNS with an efficiency that is at least 40-fold greater than that of the current standard, AAV914-17, and transduces the majority of astrocytes and neurons across multiple CNS regions. In vitro, it transduces human neurons and astrocytes more efficiently than does AAV9, demonstrating the potential of CREATE to produce customized AAV vectors for biomedical applications. PMID:26829320

  20. Phase 1 Gene Therapy for Duchenne Muscular Dystrophy Using a Translational Optimized AAV Vector

    PubMed Central

    Bowles, Dawn E; McPhee, Scott WJ; Li, Chengwen; Gray, Steven J; Samulski, Jade J; Camp, Angelique S; Li, Juan; Wang, Bing; Monahan, Paul E; Rabinowitz, Joseph E; Grieger, Joshua C; Govindasamy, Lakshmanan; Agbandje-McKenna, Mavis; Xiao, Xiao; Samulski, R Jude

    2012-01-01

    Efficient and widespread gene transfer is required for successful treatment of Duchenne muscular dystrophy (DMD). Here, we performed the first clinical trial using a chimeric adeno-associated virus (AAV) capsid variant (designated AAV2.5) derived from a rational design strategy. AAV2.5 was generated from the AAV2 capsid with five mutations from AAV1. The novel chimeric vector combines the improved muscle transduction capacity of AAV1 with reduced antigenic crossreactivity against both parental serotypes, while keeping the AAV2 receptor binding. In a randomized double-blind placebo-controlled phase I clinical study in DMD boys, AAV2.5 vector was injected into the bicep muscle in one arm, with saline control in the contralateral arm. A subset of patients received AAV empty capsid instead of saline in an effort to distinguish an immune response to vector versus minidystrophin transgene. Recombinant AAV genomes were detected in all patients with up to 2.56 vector copies per diploid genome. There was no cellular immune response to AAV2.5 capsid. This trial established that rationally designed AAV2.5 vector was safe and well tolerated, lays the foundation of customizing AAV vectors that best suit the clinical objective (e.g., limb infusion gene delivery) and should usher in the next generation of viral delivery systems for human gene transfer. PMID:22068425

  1. Molecular design for recombinant adeno-associated virus (rAAV) vector production.

    PubMed

    Aponte-Ubillus, Juan Jose; Barajas, Daniel; Peltier, Joseph; Bardliving, Cameron; Shamlou, Parviz; Gold, Daniel

    2018-02-01

    Recombinant adeno-associated virus (rAAV) vectors are increasingly popular tools for gene therapy applications. Their non-pathogenic status, low inflammatory potential, availability of viral serotypes with different tissue tropisms, and prospective long-lasting gene expression are important attributes that make rAAVs safe and efficient therapeutic options. Over the last three decades, several groups have engineered recombinant AAV-producing platforms, yielding high titers of transducing vector particles. Current specific productivity yields from different platforms range from 10 3 to 10 5 vector genomes (vg) per cell, and there is an ongoing effort to improve vector yields in order to satisfy high product demands required for clinical trials and future commercialization.Crucial aspects of vector production include the molecular design of the rAAV-producing host cell line along with the design of AAV genes, promoters, and regulatory elements. Appropriately, configuring and balancing the expression of these elements not only contributes toward high productivity, it also improves process robustness and product quality. In this mini-review, the rational design of rAAV-producing expression systems is discussed, with special attention to molecular strategies that contribute to high-yielding, biomanufacturing-amenable rAAV production processes. Details on molecular optimization from four rAAV expression systems are covered: adenovirus, herpesvirus, and baculovirus complementation systems, as well as a recently explored yeast expression system.

  2. Syngeneic AAV pseudo-vectors potentiates full vector transduction

    USDA-ARS?s Scientific Manuscript database

    An excessive amount of empty capsids are generated during regular AAV vector production process. These pseudo-vectors often remain in final vectors used for animal studies or clinical trials. The potential effects of these pseudo-vectors on AAV transduction have been a major concern. In the current ...

  3. Naturally enveloped AAV vectors for shielding neutralizing antibodies and robust gene delivery in vivo

    PubMed Central

    György, Bence; Fitzpatrick, Zachary; Crommentuijn, Matheus HW; Mu, Dakai; Maguire, Casey A.

    2014-01-01

    Recently adeno-associated virus (AAV) became the first clinically approved gene therapy product in the western world. To develop AAV for future clinical application in a widespread patient base, particularly in therapies which require intravenous (i.v.) administration of vector, the virus must be able to evade pre-existing antibodies to the wild type virus. Here we demonstrate that in mice, AAV vectors associated with extracellular vesicles (EVs) can evade human anti-AAV neutralizing antibodies. We observed different antibody evasion and gene transfer abilities with populations of EVs isolated by different centrifugal forces. EV-associated AAV vector (ev-AAV) was up to 136-fold more resistant over a range of neutralizing antibody concentrations relative to standard AAV vector in vitro. Importantly in mice, at a concentration of passively transferred human antibodies which decreased i.v. administered standard AAV transduction of brain by 80%, transduction of ev-AAV transduction was not reduced and was 4,000-fold higher. Finally, we show that expressing a brain targeting peptide on the EV surface allowed significant enhancement of transduction compared to untargeted ev-AAV. Using ev-AAV represents an effective, clinically relevant approach to evade human neutralizing anti-AAV antibodies after systemic administration of vector. PMID:24917028

  4. Tyrosine Mutation in AAV9 Capsid Improves Gene Transfer to the Mouse Lung.

    PubMed

    Martini, Sabrina V; Silva, Adriana L; Ferreira, Debora; Rabelo, Rafael; Ornellas, Felipe M; Gomes, Karina; Rocco, Patricia R M; Petrs-Silva, Hilda; Morales, Marcelo M

    2016-01-01

    Adeno-associated virus (AAV) vectors are being increasingly used as the vector of choice for in vivo gene delivery and gene therapy for many pulmonary diseases. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV capsid targets the viral particles for ubiquitination and proteasome-mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo in different organs. In this study, we evaluated the pulmonary transgene expression efficacy of AAV9 vectors containing point mutations in surface-exposed capsid tyrosine residues. Eighteen C57BL/6 mice were randomly assigned into three groups: (1) a control group (CTRL) animals underwent intratracheal (i.t.) instillation of saline, (2) the wild-type AAV9 group (WT-AAV9, 1010 vg), and (3) the tyrosine-mutant Y731F AAV9 group (M-AAV9, 1010 vg), which received (i.t.) self-complementary AAV9 vectors containing the DNA sequence of enhanced green fluorescence protein (eGFP). Four weeks after instillation, lung mechanics, morphometry, tissue cellularity, gene expression, inflammatory cytokines, and growth factor expression were analyzed. No significant differences were observed in lung mechanics and morphometry among the experimental groups. However, the number of polymorphonuclear cells was higher in the WT-AAV9 group than in the CTRL and M-AAV9 groups, suggesting that the administration of tyrosine-mutant AAV9 vectors was better tolerated. Tyrosine-mutant AAV9 vectors significantly improved transgene delivery to the lung (30%) compared with their wild-type counterparts, without eliciting an inflammatory response. Our results provide the impetus for further studies to exploit the use of AAV9 vectors as a tool for pulmonary gene therapy. © 2016 The Author(s) Published by S. Karger AG, Basel.

  5. Intraspinal AAV Injections Immediately Rostral to a Thoracic Spinal Cord Injury Site Efficiently Transduces Neurons in Spinal Cord and Brain

    PubMed Central

    Klaw, Michelle C; Xu, Chen; Tom, Veronica J

    2013-01-01

    In the vast majority of studies utilizing adeno-associated virus (AAV) in central nervous system applications, including those published with spinal cord injury (SCI) models, AAV has been administered at the level of the cell body of neurons targeted for genetic modification, resulting in transduction of neurons in the vicinity of the injection site. However, as SCI interrupts many axon tracts, it may be more beneficial to transduce a diverse pool of supraspinal neurons. We determined if descending axons severed by SCI are capable of retrogradely transporting AAV to remotely transduce a variety of brain regions. Different AAV serotypes encoding the reporter green fluorescent protein (GFP) were injected into gray and white matter immediately rostral to a spinal transection site. This resulted in the transduction of thousands of neurons within the spinal cord and in multiple regions within the brainstem that project to spinal cord. In addition, we established that different serotypes had disparate regional specificity and that AAV5 transduced the most brain and spinal cord neurons. This is the first demonstration that retrograde transport of AAV by axons severed by SCI is an effective means to transduce a collection of supraspinal neurons. Thus, we identify a novel, minimally invasive means to transduce a variety of neuronal populations within both the spinal cord and the brain following SCI. This paradigm to broadly distribute viral vectors has the potential to be an important component of a combinatorial strategy to promote functional axonal regeneration. PMID:23881451

  6. Blood-brain barrier shuttle peptides enhance AAV transduction in the brain after systemic administration.

    PubMed

    Zhang, Xintao; He, Ting; Chai, Zheng; Samulski, R Jude; Li, Chengwen

    2018-09-01

    The adeno-associated virus (AAV) vector has been used in preclinical and clinical trials of gene therapy for central nervous system (CNS) diseases. One of the biggest challenges of effectively delivering AAV to the brain is to surmount the blood-brain barrier (BBB). Herein, we identified several potential BBB shuttle peptides that significantly enhanced AAV8 transduction in the brain after a systemic administration, the best of which was the THR peptide. The enhancement of AAV8 brain transduction by THR is dose-dependent, and neurons are the primary THR targets. Mechanism studies revealed that THR directly bound to the AAV8 virion, increasing its ability to cross the endothelial cell barrier. Further experiments showed that binding of THR to the AAV virion did not interfere with AAV8 infection biology, and that THR competitively blocked transferrin from binding to AAV8. Taken together, our results demonstrate, for the first time, that BBB shuttle peptides are able to directly interact with AAV and increase the ability of the AAV vectors to cross the BBB for transduction enhancement in the brain. These results will shed important light on the potential applications of BBB shuttle peptides for enhancing brain transduction with systemic administration of AAV vectors. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Overcoming preexisting humoral immunity to AAV using capsid decoys.

    PubMed

    Mingozzi, Federico; Anguela, Xavier M; Pavani, Giulia; Chen, Yifeng; Davidson, Robert J; Hui, Daniel J; Yazicioglu, Mustafa; Elkouby, Liron; Hinderer, Christian J; Faella, Armida; Howard, Carolann; Tai, Alex; Podsakoff, Gregory M; Zhou, Shangzhen; Basner-Tschakarjan, Etiena; Wright, John Fraser; High, Katherine A

    2013-07-17

    Adeno-associated virus (AAV) vectors delivered through the systemic circulation successfully transduce various target tissues in animal models. However, similar attempts in humans have been hampered by the high prevalence of neutralizing antibodies to AAV, which completely block vector transduction. We show in both mouse and nonhuman primate models that addition of empty capsid to the final vector formulation can, in a dose-dependent manner, adsorb these antibodies, even at high titers, thus overcoming their inhibitory effect. To further enhance the safety of the approach, we mutated the receptor binding site of AAV2 to generate an empty capsid mutant that can adsorb antibodies but cannot enter a target cell. Our work suggests that optimizing the ratio of full/empty capsids in the final formulation of vector, based on a patient's anti-AAV titers, will maximize the efficacy of gene transfer after systemic vector delivery.

  8. Overcoming Preexisting Humoral Immunity to AAV Using Capsid Decoys

    PubMed Central

    Anguela, Xavier M.; Pavani, Giulia; Chen, Yifeng; Davidson, Robert J.; Hui, Daniel J.; Yazicioglu, Mustafa; Elkouby, Liron; Hinderer, Christian J.; Faella, Armida; Howard, Carolann; Tai, Alex; Podsakoff, Gregory M.; Zhou, Shangzhen; Basner-Tschakarjan, Etiena; Wright, John Fraser

    2014-01-01

    Adeno-associated virus (AAV) vectors delivered through the systemic circulation successfully transduce various target tissues in animal models. However, similar attempts in humans have been hampered by the high prevalence of neutralizing antibodies to AAV, which completely block vector transduction. We show in both mouse and nonhuman primate models that addition of empty capsid to the final vector formulation can, in a dose-dependent manner, adsorb these antibodies, even at high titers, thus overcoming their inhibitory effect. To further enhance the safety of the approach, we mutated the receptor binding site of AAV2 to generate an empty capsid mutant that can adsorb antibodies but cannot enter a target cell. Our work suggests that optimizing the ratio of full/empty capsids in the final formulation of vector, based on a patient's anti-AAV titers, will maximize the efficacy of gene transfer after systemic vector delivery. PMID:23863832

  9. Effective delivery of large genes to the retina by dual AAV vectors

    PubMed Central

    Trapani, Ivana; Colella, Pasqualina; Sommella, Andrea; Iodice, Carolina; Cesi, Giulia; de Simone, Sonia; Marrocco, Elena; Rossi, Settimio; Giunti, Massimo; Palfi, Arpad; Farrar, Gwyneth J; Polishchuk, Roman; Auricchio, Alberto

    2014-01-01

    Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on ‘forced’ packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes. PMID:24150896

  10. The delicate balance in genetically engineering live vaccines

    PubMed Central

    Galen, James E.; Curtiss, Roy

    2014-01-01

    Contemporary vaccine development relies less on empirical methods of vaccine construction, and now employs a powerful array of precise engineering strategies to construct immunogenic live vaccines. In this review, we will survey various engineering techniques used to create attenuated vaccines, with an emphasis on recent advances and insights. We will further explore the adaptation of attenuated strains to create multivalent vaccine platforms for immunization against multiple unrelated pathogens. These carrier vaccines are engineered to deliver sufficient levels of protective antigens to appropriate lymphoid inductive sites to elicit both carrier-specific and foreign antigen-specific immunity. Although many of these technologies were originally developed for use in Salmonella vaccines, application of the essential logic of these approaches will be extended to development of other enteric vaccines where possible. A central theme driving our discussion will stress that the ultimate success of an engineered vaccine rests on achieving the proper balance between attenuation and immunogenicity. Achieving this balance will avoid over-activation of inflammatory responses, which results in unacceptable reactogenicity, but will retain sufficient metabolic fitness to enable the live vaccine to reach deep tissue inductive sites and trigger protective immunity. The breadth of examples presented herein will clearly demonstrate that genetic engineering offers the potential for rapidly propelling vaccine development forward into novel applications and therapies which will significantly expand the role of vaccines in public health. PMID:24370705

  11. Tyrosine-phosphorylation of AAV2 vectors and its consequences on viral intracellular trafficking and transgene expression

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

    Zhong Li; Powell Gene Therapy Center, University of Florida College of Medicine, Gainesville, FL; Genetics Institute, University of Florida College of Medicine, Gainesville, FL

    2008-11-25

    We have documented that epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK) signaling negatively affects intracellular trafficking and transduction efficiency of recombinant adeno-associated virus 2 (AAV2) vectors. Specifically, inhibition of EGFR-PTK signaling leads to decreased ubiquitination of AAV2 capsid proteins, which in turn, facilitates viral nuclear transport by limiting proteasome-mediated degradation of AAV2 vectors. In the present studies, we observed that AAV capsids can indeed be phosphorylated at tyrosine residues by EGFR-PTK in in vitro phosphorylation assays and that phosphorylated AAV capsids retain their structural integrity. However, although phosphorylated AAV vectors enter cells as efficiently as their unphosphorylated counterparts, theirmore » transduction efficiency is significantly reduced. This reduction is not due to impaired viral second-strand DNA synthesis since transduction efficiency of both single-stranded AAV (ssAAV) and self-complementary AAV (scAAV) vectors is decreased by {approx} 68% and {approx} 74%, respectively. We also observed that intracellular trafficking of tyrosine-phosphorylated AAV vectors from cytoplasm to nucleus is significantly decreased, which results from ubiquitination of AAV capsids followed by proteasome-mediated degradation, although downstream consequences of capsid ubiquitination may also be affected by tyrosine-phosphorylation. These studies provide new insights into the role of tyrosine-phosphorylation of AAV capsids in various steps in the virus life cycle, which has implications in the optimal use of recombinant AAV vectors in human gene therapy.« less

  12. AAV Gene Therapy for MPS1-associated Corneal Blindness.

    PubMed

    Vance, Melisa; Llanga, Telmo; Bennett, Will; Woodard, Kenton; Murlidharan, Giridhar; Chungfat, Neil; Asokan, Aravind; Gilger, Brian; Kurtzberg, Joanne; Samulski, R Jude; Hirsch, Matthew L

    2016-02-22

    Although cord blood transplantation has significantly extended the lifespan of mucopolysaccharidosis type 1 (MPS1) patients, over 95% manifest cornea clouding with about 50% progressing to blindness. As corneal transplants are met with high rejection rates in MPS1 children, there remains no treatment to prevent blindness or restore vision in MPS1 children. Since MPS1 is caused by mutations in idua, which encodes alpha-L-iduronidase, a gene addition strategy to prevent, and potentially reverse, MPS1-associated corneal blindness was investigated. Initially, a codon optimized idua cDNA expression cassette (opt-IDUA) was validated for IDUA production and function following adeno-associated virus (AAV) vector transduction of MPS1 patient fibroblasts. Then, an AAV serotype evaluation in human cornea explants identified an AAV8 and 9 chimeric capsid (8G9) as most efficient for transduction. AAV8G9-opt-IDUA administered to human corneas via intrastromal injection demonstrated widespread transduction, which included cells that naturally produce IDUA, and resulted in a >10-fold supraphysiological increase in IDUA activity. No significant apoptosis related to AAV vectors or IDUA was observed under any conditions in both human corneas and MPS1 patient fibroblasts. The collective preclinical data demonstrate safe and efficient IDUA delivery to human corneas, which may prevent and potentially reverse MPS1-associated cornea blindness.

  13. AAV Gene Therapy for MPS1-associated Corneal Blindness

    PubMed Central

    Vance, Melisa; Llanga, Telmo; Bennett, Will; Woodard, Kenton; Murlidharan, Giridhar; Chungfat, Neil; Asokan, Aravind; Gilger, Brian; Kurtzberg, Joanne; Samulski, R. Jude; Hirsch, Matthew L.

    2016-01-01

    Although cord blood transplantation has significantly extended the lifespan of mucopolysaccharidosis type 1 (MPS1) patients, over 95% manifest cornea clouding with about 50% progressing to blindness. As corneal transplants are met with high rejection rates in MPS1 children, there remains no treatment to prevent blindness or restore vision in MPS1 children. Since MPS1 is caused by mutations in idua, which encodes alpha-L-iduronidase, a gene addition strategy to prevent, and potentially reverse, MPS1-associated corneal blindness was investigated. Initially, a codon optimized idua cDNA expression cassette (opt-IDUA) was validated for IDUA production and function following adeno-associated virus (AAV) vector transduction of MPS1 patient fibroblasts. Then, an AAV serotype evaluation in human cornea explants identified an AAV8 and 9 chimeric capsid (8G9) as most efficient for transduction. AAV8G9-opt-IDUA administered to human corneas via intrastromal injection demonstrated widespread transduction, which included cells that naturally produce IDUA, and resulted in a >10-fold supraphysiological increase in IDUA activity. No significant apoptosis related to AAV vectors or IDUA was observed under any conditions in both human corneas and MPS1 patient fibroblasts. The collective preclinical data demonstrate safe and efficient IDUA delivery to human corneas, which may prevent and potentially reverse MPS1-associated cornea blindness. PMID:26899286

  14. Reverse Genetics for Newcastle Disease Virus as a Vaccine Vector.

    PubMed

    Kim, Shin-Hee; Samal, Siba K

    2018-02-22

    Newcastle disease virus (NDV) is an economically important pathogen in the poultry industry worldwide. Recovery of infectious NDV from cDNA using reverse genetics has made it possible to manipulate the genome of NDV. This has greatly contributed to our understanding of the molecular biology and pathogenesis of NDV. Furthermore, NDV has modular genome and accommodates insertion of a foreign gene as a transcriptional unit, thus enabling NDV as a vaccine vector against diseases of humans and animals. Avirulent NDV strains (e.g., LaSota and B1) have been commonly used as vaccine vectors. In this protocol, we have described reverse genetics of NDV to be used as a vaccine vector by exemplifying the recovery of NDV vectored avian influenza virus vaccine. Specifically, cloning and recovery of NDV expressing the hemagglutinin protein of highly pathogenic influenza virus were explained. © 2018 by John Wiley & Sons, Inc. Copyright © 2018 John Wiley & Sons, Inc.

  15. Cellular selectivity of AAV serotypes for gene delivery in neurons and astrocytes by neonatal intracerebroventricular injection

    PubMed Central

    Hammond, Sean L.; Leek, Ashley N.; Richman, Evan H.

    2017-01-01

    The non-pathogenic parvovirus, adeno-associated virus (AAV), is an efficient vector for transgene expression in vivo and shows promise for treatment of brain disorders in clinical trials. Currently, there are more than 100 AAV serotypes identified that differ in the binding capacity of capsid proteins to specific cell surface receptors that can transduce different cell types and brain regions in the CNS. In the current study, multiple AAV serotypes expressing a GFP reporter (AAV1, AAV2/1, AAVDJ, AAV8, AAVDJ8, AAV9, AAVDJ9) were screened for their infectivity in both primary murine astrocyte and neuronal cell cultures. AAV2/1, AAVDJ8 and AAV9 were selected for further investigation of their tropism throughout different brain regions and cell types. Each AAV was administered to P0-neonatal mice via intracerebroventricular injections (ICV). Brains were then systematically analyzed for GFP expression at 3 or 6 weeks post-infection in various regions, including the olfactory bulb, striatum, cortex, hippocampus, substantia nigra (SN) and cerebellum. Cell counting data revealed that AAV2/1 infections were more prevalent in the cortical layers but penetrated to the midbrain less than AAVDJ8 and AAV9. Additionally, there were differences in the persistence of viral transgene expression amongst the three serotypes examined in vivo at 3 and 6 weeks post-infection. Because AAV-mediated transgene expression is of interest in neurodegenerative diseases such as Parkinson’s Disease, we examined the SN with microscopy techniques, such as CLARITY tissue transmutation, to identify AAV serotypes that resulted in optimal transgene expression in either astrocytes or dopaminergic neurons. AAVDJ8 displayed more tropism in astrocytes compared to AAV9 in the SN region. We conclude that ICV injection results in lasting expression of virally encoded transgene when using AAV vectors and that specific AAV serotypes are required to selectively deliver transgenes of interest to different brain

  16. Direct comparison of administration routes for AAV8-mediated ocular gene therapy.

    PubMed

    Igarashi, Tsutomu; Miyake, Koichi; Asakawa, Nagisa; Miyake, Noriko; Shimada, Takashi; Takahashi, Hiroshi

    2013-05-01

    We recently demonstrated that direct subretinal (SR) injection of adeno-associated virus (AAV) type 8 (AAV8) into photoreceptor cells and retinal pigment epithelium (RPE) is a highly efficient model of gene delivery. The current study compared transduction efficiency and expression patterns associated with various routes of vector administration. The efficacy of intravitreal (VT), SR and subconjunctival (SC) injections for delivery of AAV8-derived vectors, i.e. those expressing luciferase (Luc) and enhanced green fluorescent protein (GFP) - AAV8/Luc and AAV8/GFP, respectively - were compared in an animal (mouse) model (n = 8 mice/group). Transduction efficiency and expression patterns were examined at post-injection weeks 1 and 2, and months 1, 3, 6 and 12 via in vivo imaging. One year after AAV injection, AAV8/Luc-treated mice exhibited stable and sustained high expression of vector in the VT and SR groups, but not in the SC group (VT:SR:SC = 3,218:2,923:115; 1 × 10(5 )photons/s). Histological analysis showed that GFP expression was observed in the inner retina of VT group mice, and in photoreceptor cells and RPE of SR group mice, whereas no GFP expression was noted in the SC group. Electroretinography (ERG) revealed adverse effects following SR delivery. Results suggest that both SR and VT injections of AAV8 vectors are useful routes for administering ocular gene therapy, and stress the importance of selecting an appropriate administration route, i.e. one that targets specific cells, for treating ocular disorders.

  17. AAV-CRISPR/Cas9-Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro.

    PubMed

    Wu, Wenyi; Duan, Yajian; Ma, Gaoen; Zhou, Guohong; Park-Windhol, Cindy; D'Amore, Patricia A; Lei, Hetian

    2017-12-01

    Pathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)-mediated CRISPR (clustered regularly interspaced short palindromic repeats)-associated endonuclease (Cas)9 can be used to deplete expression of VEGF receptor 2 (VEGFR2) in human vascular endothelial cells in vitro and thus suppress its downstream signaling events. The dual AAV system of CRISPR/Cas9 from Streptococcus pyogenes (AAV-SpGuide and -SpCas9) was adapted to edit genomic VEGFR2 in primary human retinal microvascular endothelial cells (HRECs). In this system, the endothelial-specific promoter for intercellular adhesion molecule 2 (ICAM2) was cloned into the dual AAV vectors of SpGuide and SpCas9 for driving expression of green fluorescence protein (GFP) and SpCas9, respectively. These two AAV vectors were applied to production of recombinant AAV serotype 5 (rAAV5), which were used to infect HRECs for depletion of VEGFR2. Protein expression was determined by Western blot; and cell proliferation, migration, as well as tube formation were examined. AAV5 effectively infected vascular endothelial cells (ECs) and retinal pigment epithelial (RPE) cells; the ICAM2 promoter drove expression of GFP and SpCas9 in HRECs, but not in RPE cells. The results showed that the rAAV5-CRISPR/Cas9 depleted VEGFR2 by 80% and completely blocked VEGF-induced activation of Akt, and proliferation, migration as well as tube formation of HRECs. AAV-CRISRP/Cas9-mediated depletion of VEGFR2 is a potential therapeutic strategy for pathologic angiogenesis.

  18. Intravitreal delivery of AAV-NDI1 provides functional benefit in a murine model of Leber hereditary optic neuropathy.

    PubMed

    Chadderton, Naomi; Palfi, Arpad; Millington-Ward, Sophia; Gobbo, Oliverio; Overlack, Nora; Carrigan, Matthew; O'Reilly, Mary; Campbell, Matthew; Ehrhardt, Carsten; Wolfrum, Uwe; Humphries, Peter; Kenna, Paul F; Farrar, G Jane

    2013-01-01

    Leber hereditary optic neuropathy (LHON) is a mitochondrially inherited form of visual dysfunction caused by mutations in several genes encoding subunits of the mitochondrial respiratory NADH-ubiquinone oxidoreductase complex (complex I). Development of gene therapies for LHON has been impeded by genetic heterogeneity and the need to deliver therapies to the mitochondria of retinal ganglion cells (RGCs), the cells primarily affected in LHON. The therapy under development entails intraocular injection of a nuclear yeast gene NADH-quinone oxidoreductase (NDI1) that encodes a single subunit complex I equivalent and as such is mutation independent. NDI1 is imported into mitochondria due to an endogenous mitochondrial localisation signal. Intravitreal injection represents a clinically relevant route of delivery to RGCs not previously used for NDI1. In this study, recombinant adenoassociated virus (AAV) serotype 2 expressing NDI1 (AAV-NDI1) was shown to protect RGCs in a rotenone-induced murine model of LHON. AAV-NDI1 significantly reduced RGC death by 1.5-fold and optic nerve atrophy by 1.4-fold. This led to a significant preservation of retinal function as assessed by manganese enhanced magnetic resonance imaging and optokinetic responses. Intraocular injection of AAV-NDI1 overcomes many barriers previously associated with developing therapies for LHON and holds great therapeutic promise for a mitochondrial disorder for which there are no effective therapies.

  19. Glymphatic fluid transport controls paravascular clearance of AAV vectors from the brain

    PubMed Central

    Murlidharan, Giridhar; Crowther, Andrew; Reardon, Rebecca A.; Song, Juan

    2016-01-01

    Adeno-associated viruses (AAV) are currently being evaluated in clinical trials for gene therapy of CNS disorders. However, host factors that influence the spread, clearance, and transduction efficiency of AAV vectors in the brain are not well understood. Recent studies have demonstrated that fluid flow mediated by aquaporin-4 (AQP4) channels located on astroglial end feet is essential for exchange of solutes between interstitial and cerebrospinal fluid. This phenomenon, which is essential for interstitial clearance of solutes from the CNS, has been termed glial-associated lymphatic transport or glymphatic transport. In the current study, we demonstrate that glymphatic transport profoundly affects various aspects of AAV gene transfer in the CNS. Altered localization of AQP4 in aged mouse brains correlated with significantly increased retention of AAV vectors in the parenchyma and reduced systemic leakage following ventricular administration. We observed a similar increase in AAV retention and transgene expression upon i.c.v. administration in AQP4–/– mice. Consistent with this observation, fluorophore-labeled AAV vectors showed markedly reduced flux from the ventricles of AQP4–/– mice compared with WT mice. These results were further corroborated by reduced AAV clearance from the AQP4-null brain, as demonstrated by reduced transgene expression and vector genome accumulation in systemic organs. We postulate that deregulation of glymphatic transport in aged and diseased brains could markedly affect the parenchymal spread, clearance, and gene transfer efficiency of AAV vectors. Assessment of biomarkers that report the kinetics of CSF flux in prospective gene therapy patients might inform variable treatment outcomes and guide future clinical trial design. PMID:27699236

  20. Development of Intrathecal AAV9 Gene Therapy for Giant Axonal Neuropathy.

    PubMed

    Bailey, Rachel M; Armao, Diane; Nagabhushan Kalburgi, Sahana; Gray, Steven J

    2018-06-15

    An NIH-sponsored phase I clinical trial is underway to test a potential treatment for giant axonal neuropathy (GAN) using viral-mediated GAN gene replacement (https://clinicaltrials.gov/ct2/show/NCT02362438). This trial marks the first instance of intrathecal (IT) adeno-associated viral (AAV) gene transfer in humans. GAN is a rare pediatric neurodegenerative disorder caused by autosomal recessive loss-of-function mutations in the GAN gene, which encodes the gigaxonin protein. Gigaxonin is involved in the regulation, turnover, and degradation of intermediate filaments (IFs). The pathologic signature of GAN is giant axonal swellings filled with disorganized accumulations of IFs. Herein, we describe the development and characterization of the AAV vector carrying a normal copy of the human GAN transgene (AAV9/JeT-GAN) currently employed in the clinical trial. Treatment with AAV/JeT-GAN restored the normal configuration of IFs in patient fibroblasts within days in cell culture and by 4 weeks in GAN KO mice. IT delivery of AAV9/JeT-GAN in aged GAN KO mice preserved sciatic nerve ultrastructure, reduced neuronal IF accumulations and attenuated rotarod dysfunction. This strategy conferred sustained wild-type gigaxonin expression across the PNS and CNS for at least 1 year in mice. These results support the clinical evaluation of AAV9/JeT-GAN for potential therapeutic outcomes and treatment for GAN patients.

  1. Recombinant AAV-directed gene therapy for type I glycogen storage diseases

    PubMed Central

    Chou, JY; Mansfield, BC

    2011-01-01

    Introduction Glycogen storage disease (GSD) type Ia and Ib are disorders of impaired glucose homeostasis affecting the liver and kidney. GSD-Ib also affects neutrophils. Current dietary therapies cannot prevent long-term complications. In animal studies, recombinant adeno-associated virus (rAAV) vector-mediated gene therapy can correct or minimize multiple aspects of the disorders, offering hope for human gene therapy. Areas covered A summary of recent progress in rAAV-mediated gene therapy for GSD-I; strategies to improve rAAV-mediated gene delivery, transduction efficiency and immune avoidance; and vector refinements that improve expression. Expert opinion rAAV-mediated gene delivery to the liver can restore glucose homeostasis in preclinical models of GSD-I, but some long-term complications of the liver and kidney remain. Gene therapy for GSD-Ib is less advanced than for GSD-Ia and only transient correction of myeloid dysfunction has been achieved. A question remains whether a single rAAV vector can meet the expression efficiency and tropism required to treat all aspects of GSD-I, or if a multi-prong approach is needed. An understanding of the strengths and weaknesses of rAAV vectors in the context of strategies to achieve efficient transduction of the liver, kidney, and hematopoietic stem cells is required for treating GSD-I. PMID:21504389

  2. Cell Cycle-Dependent Expression of Adeno-Associated Virus 2 (AAV2) Rep in Coinfections with Herpes Simplex Virus 1 (HSV-1) Gives Rise to a Mosaic of Cells Replicating either AAV2 or HSV-1

    PubMed Central

    Franzoso, Francesca D.; Seyffert, Michael; Vogel, Rebecca; Yakimovich, Artur; de Andrade Pereira, Bruna; Meier, Anita F.; Sutter, Sereina O.; Tobler, Kurt; Vogt, Bernd; Greber, Urs F.; Büning, Hildegard; Ackermann, Mathias

    2017-01-01

    ABSTRACT Adeno-associated virus 2 (AAV2) depends on the simultaneous presence of a helper virus such as herpes simplex virus 1 (HSV-1) for productive replication. At the same time, AAV2 efficiently blocks the replication of HSV-1, which would eventually limit its own replication by diminishing the helper virus reservoir. This discrepancy begs the question of how AAV2 and HSV-1 can coexist in a cell population. Here we show that in coinfected cultures, AAV2 DNA replication takes place almost exclusively in S/G2-phase cells, while HSV-1 DNA replication is restricted to G1 phase. Live microscopy revealed that not only wild-type AAV2 (wtAAV2) replication but also reporter gene expression from both single-stranded and double-stranded (self-complementary) recombinant AAV2 vectors preferentially occurs in S/G2-phase cells, suggesting that the preference for S/G2 phase is independent of the nature of the viral genome. Interestingly, however, a substantial proportion of S/G2-phase cells transduced by the double-stranded but not the single-stranded recombinant AAV2 vectors progressed through mitosis in the absence of the helper virus. We conclude that cell cycle-dependent AAV2 rep expression facilitates cell cycle-dependent AAV2 DNA replication and inhibits HSV-1 DNA replication. This may limit competition for cellular and viral helper factors and, hence, creates a biological niche for either virus to replicate. IMPORTANCE Adeno-associated virus 2 (AAV2) differs from most other viruses, as it requires not only a host cell for replication but also a helper virus such as an adenovirus or a herpesvirus. This situation inevitably leads to competition for cellular resources. AAV2 has been shown to efficiently inhibit the replication of helper viruses. Here we present a new facet of the interaction between AAV2 and one of its helper viruses, herpes simplex virus 1 (HSV-1). We observed that AAV2 rep gene expression is cell cycle dependent and gives rise to distinct time

  3. Intraperitoneal AAV9-shRNA inhibits target expression in neonatal skeletal and cardiac muscles.

    PubMed

    Mayra, Azat; Tomimitsu, Hiroyuki; Kubodera, Takayuki; Kobayashi, Masaki; Piao, Wenying; Sunaga, Fumiko; Hirai, Yukihiko; Shimada, Takashi; Mizusawa, Hidehiro; Yokota, Takanori

    2011-02-11

    Systemic injections of AAV vectors generally transduce to the liver more effectively than to cardiac and skeletal muscles. The short hairpin RNA (shRNA)-expressing AAV9 (shRNA-AAV9) can also reduce target gene expression in the liver, but not enough in cardiac or skeletal muscles. Higher doses of shRNA-AAV9 required for inhibiting target genes in cardiac and skeletal muscles often results in shRNA-related toxicity including microRNA oversaturation that can induce fetal liver failure. In this study, we injected high-dose shRNA-AAV9 to neonates and efficiently silenced genes in cardiac and skeletal muscles without inducing liver toxicity. This is because AAV is most likely diluted or degraded in the liver than in cardiac or skeletal muscle during cell division after birth. We report that this systemically injected shRNA-AAV method does not induce any major side effects, such as liver dysfunction, and the dose of shRNA-AAV is sufficient for gene silencing in skeletal and cardiac muscle tissues. This novel method may be useful for generating gene knockdown in skeletal and cardiac mouse tissues, thus providing mouse models useful for analyzing diseases caused by loss-of-function of target genes. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Formation of AAV Single Stranded DNA Genome from a Circular Plasmid in Saccharomyces cerevisiae

    PubMed Central

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3+ clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway. PMID:21853137

  5. Formation of AAV single stranded DNA genome from a circular plasmid in Saccharomyces cerevisiae.

    PubMed

    Cervelli, Tiziana; Backovic, Ana; Galli, Alvaro

    2011-01-01

    Adeno-associated virus (AAV)-based vectors are promising tools for targeted transfer in gene therapy studies. Many efforts have been accomplished to improve production and purification methods. We thought to develop a simple eukaryotic system allowing AAV replication which could provide an excellent opportunity for studying AAV biology and, more importantly, for AAV vector production. It has been shown that yeast Saccharomyces cerevisiae is able to replicate and form the capsid of many viruses. We investigated the ability of the yeast Saccharomyces cerevisiae to carry out the replication of a recombinant AAV (rAAV). When a plasmid containing a rAAV genome in which the cap gene was replaced with the S. cerevisiae URA3 gene, was co-transformed in yeast with a plasmid expressing Rep68, a significant number of URA3(+) clones were scored (more than 30-fold over controls). Molecular analysis of low molecular weight DNA by Southern blotting revealed that single stranded DNA is formed and that the plasmid is entirely replicated. The ssDNA contains the ITRs, URA3 gene and also vector sequences suggesting the presence of two distinct molecules. Its formation was dependent on Rep68 expression and ITR. These data indicate that DNA is not obtained by the canonical AAV replication pathway.

  6. Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems

    PubMed Central

    Chan, Ken Y; Jang, Min J; Yoo, Bryan B; Greenbaum, Alon; Ravi, Namita; Wu, Wei-Li; Sánchez-Guardado, Luis; Lois, Carlos; Mazmanian, Sarkis K; Deverman, Benjamin E; Gradinaru, Viviana

    2017-01-01

    Adeno-associated viruses (AAVs) are commonly used for in vivo gene transfer. Nevertheless, AAVs that provide efficient transduction across specific organs or cell populations are needed. Here, we describe AAV-PHP.eB and AAV-PHP.S, capsids that efficiently transduce the central and peripheral nervous systems, respectively. In the adult mouse, intravenous administration of 1×1011 vector genomes (vg) of AAV-PHP.eB transduced 69% of cortical and 55% of striatal neurons, while 1×1012 vg AAV-PHP.S transduced 82% of dorsal root ganglion neurons, as well as cardiac and enteric neurons. The efficiency of these vectors facilitates robust co-transduction and stochastic, multicolor labeling for individual cell morphology studies. To support such efforts, we provide methods for labeling a tunable fraction of cells without compromising color diversity. Furthermore, when used with cell type-specific promoters, these AAVs provide targeted gene expression across the nervous system and enable efficient and versatile gene manipulation throughout the nervous system of transgenic and non-transgenic animals. PMID:28671695

  7. Modular adeno-associated virus (rAAV) vectors used for cellular virus-directed enzyme prodrug therapy

    PubMed Central

    Hagen, Sven; Baumann, Tobias; Wagner, Hanna J.; Morath, Volker; Kaufmann, Beate; Fischer, Adrian; Bergmann, Stefan; Schindler, Patrick; Arndt, Katja M.; Müller, Kristian M.

    2014-01-01

    The pre-clinical and clinical development of viral vehicles for gene transfer increased in recent years, and a recombinant adeno-associated virus (rAAV) drug took center stage upon approval in the European Union. However, lack of standardization, inefficient purification methods and complicated retargeting limit general usability. We address these obstacles by fusing rAAV-2 capsids with two modular targeting molecules (DARPin or Affibody) specific for a cancer cell-surface marker (EGFR) while simultaneously including an affinity tag (His-tag) in a surface-exposed loop. Equipping these particles with genes coding for prodrug converting enzymes (thymidine kinase or cytosine deaminase) we demonstrate tumor marker specific transduction and prodrug-dependent apoptosis of cancer cells. Coding terminal and loop modifications in one gene enabled specific and scalable purification. Our genetic parts for viral production adhere to a standardized cloning strategy facilitating rapid prototyping of virus directed enzyme prodrug therapy (VDEPT). PMID:24457557

  8. Genetically defined race, but not sex, is associated with higher humoral and cellular immune responses to measles vaccination

    PubMed Central

    Voigt, Emily A.; Ovsyannikova, Inna G.; Haralambieva, Iana H.; Kennedy, Richard B.; Larrabee, Beth R.; Schaid, Daniel J.; Poland, Gregory A.

    2017-01-01

    In addition to host genetic and environmental factors, variations in immune responses to vaccination are influenced by demographic variables, such as race and sex. The influence of genetic race and sex on measles vaccine responses is not well understood, yet important for the development of much-needed improved measles vaccines with lower failure rates. We assessed associations between genetically defined race and sex with measles humoral and cellular immunity after measles vaccination in three independent and geographically distinct cohorts totaling 2,872 healthy racially diverse children, older adolescents, and young adults. We found no associations between biological sex and either humoral or cellular immunity to measles vaccine, and no correlation between humoral and cellular immunity in these study subjects. Genetically defined race was, however, significantly associated with both measles vaccine-induced humoral and cellular immune responses, with subjects genetically classified as having African-American ancestry demonstrating significantly higher antibody and cell-mediated immune responses relative to subjects of Caucasian ancestry. This information may be useful in designing novel measles vaccines that are optimally effective across human genetic backgrounds. PMID:27591105

  9. Vector-based genetically modified vaccines: Exploiting Jenner's legacy.

    PubMed

    Ramezanpour, Bahar; Haan, Ingrid; Osterhaus, Ab; Claassen, Eric

    2016-12-07

    The global vaccine market is diverse while facing a plethora of novel developments. Genetic modification (GM) techniques facilitate the design of 'smarter' vaccines. For many of the major infectious diseases of humans, like AIDS and malaria, but also for most human neoplastic disorders, still no vaccines are available. It may be speculated that novel GM technologies will significantly contribute to their development. While a promising number of studies is conducted on GM vaccines and GM vaccine technologies, the contribution of GM technology to newly introduced vaccines on the market is disappointingly limited. In this study, the field of vector-based GM vaccines is explored. Data on currently available, actually applied, and newly developed vectors is retrieved from various sources, synthesised and analysed, in order to provide an overview on the use of vector-based technology in the field of GM vaccine development. While still there are only two vector-based vaccines on the human vaccine market, there is ample activity in the fields of patenting, preclinical research, and different stages of clinical research. Results of this study revealed that vector-based vaccines comprise a significant part of all GM vaccines in the pipeline. This study further highlights that poxviruses and adenoviruses are among the most prominent vectors in GM vaccine development. After the approval of the first vectored human vaccine, based on a flavivirus vector, vaccine vector technology, especially based on poxviruses and adenoviruses, holds great promise for future vaccine development. It may lead to cheaper methods for the production of safe vaccines against diseases for which no or less perfect vaccines exist today, thus catering for an unmet medical need. After the introduction of Jenner's vaccinia virus as the first vaccine more than two centuries ago, which eventually led to the recent eradication of smallpox, this and other viruses may now be the basis for constructing vectors

  10. Efficient mouse airway transduction following recombination between AAV vectors carrying parts of a larger gene.

    PubMed

    Halbert, Christine L; Allen, James M; Miller, A Dusty

    2002-07-01

    The small packaging capacity of adeno-associated virus (AAV) vectors limits the utility of this promising vector system for transfer of large genes. We explored the possibility that larger genes could be reconstituted following homologous recombination between AAV vectors carrying overlapping gene fragments. An alkaline phosphatase (AP) gene was split between two such AAV vectors (rec vectors) and packaged using AAV2 or AAV6 capsid proteins. Rec vectors having either capsid protein recombined to express AP in cultured cells at about 1-2% of the rate observed for an intact vector. Surprisingly, the AAV6 rec vectors transduced lung cells in mice almost as efficiently as did an intact vector, with 10% of airway epithelial cells, the target for treatment of cystic fibrosis (CF), being positive. Thus AAV rec vectors may be useful for diseases such as CF that require transfer of large genes.

  11. The ANCA Vasculitis Questionnaire (AAV-PRO©)

    ClinicalTrials.gov

    2017-05-01

    Eosinophilic Granulomatosis With Polyangiitis (Churg-Strauss) (EGPA); Churg-Strauss Syndrome (CSS); Granulomatosis With Polyangiitis (Wegener's) (GPA); Wegener Granulomatosis (WG); Microscopic Polyangiitis (MPA); ANCA-Associated Vasculitis (AAV); Vasculitis

  12. Nephron segment-specific gene expression using AAV vectors.

    PubMed

    Asico, Laureano D; Cuevas, Santiago; Ma, Xiaobo; Jose, Pedro A; Armando, Ines; Konkalmatt, Prasad R

    2018-02-26

    AAV9 vector provides efficient gene transfer in all segments of the renal nephron, with minimum expression in non-renal cells, when administered retrogradely via the ureter. It is important to restrict the transgene expression to the desired cell type within the kidney, so that the physiological endpoints represent the function of the transgene expressed in that specific cell type within kidney. We hypothesized that segment-specific gene expression within the kidney can be accomplished using the highly efficient AAV9 vectors carrying the promoters of genes that are expressed exclusively in the desired segment of the nephron in combination with administration by retrograde infusion into the kidney via the ureter. We constructed AAV vectors carrying eGFP under the control of: kidney-specific cadherin (KSPC) gene promoter for expression in the entire nephron; Na + /glucose co-transporter (SGLT2) gene promoter for expression in the S1 and S2 segments of the proximal tubule; sodium, potassium, 2 chloride co-transporter (NKCC2) gene promoter for expression in the thick ascending limb of Henle's loop (TALH); E-cadherin (ECAD) gene promoter for expression in the collecting duct (CD); and cytomegalovirus (CMV) early promoter that provides expression in most of the mammalian cells, as control. We tested the specificity of the promoter constructs in vitro for cell type-specific expression in mouse kidney cells in primary culture, followed by retrograde infusion of the AAV vectors via the ureter in the mouse. Our data show that AAV9 vector, in combination with the segment-specific promoters administered by retrograde infusion via the ureter, provides renal nephron segment-specific gene expression. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Genetic stability of a dengue vaccine based on chimeric yellow fever/dengue viruses.

    PubMed

    Mantel, N; Girerd, Y; Geny, C; Bernard, I; Pontvianne, J; Lang, J; Barban, V

    2011-09-02

    A tetravalent dengue vaccine based on four live, attenuated, chimeric viruses (CYD1-4), constructed by replacing the genes coding for premembrane (prM) and envelope (E) proteins of the yellow fever (YF)-17D vaccine strain with those of the four serotypes of dengue virus, is in clinical phase III evaluation. We assessed the vaccine's genetic stability by fully sequencing each vaccine virus throughout the development and manufacturing process. The four viruses displayed complete genetic stability, with no change from premaster seed lots to bulk lots. When pursuing the virus growth beyond bulk lots, a few genetic variations were observed. Usually both the initial nucleotide and the new one persisted, and mutations appeared after a relatively high number of virus duplication cycles (65-200, depending on position). Variations were concentrated in the prM-E and non-structural (NS)4B regions. PrM-E variations had no impact on lysis-plaque size or neurovirulence in mice. None of the variations located in the YF-17D-derived genes corresponded with reversion to the wild-type Yellow Fever sequence. Variations in NS4B likely reflect virus adaptation to Vero cells growth. A low to undetectable viremia has been reported previously [1-3] in vaccinated non-human and human primates. Combined with the data reported here about the genetic stability of the vaccine strains, the probability of in vivo emergence of mutant viruses appears very low. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. 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

  15. Genetically attenuated Trypanosoma cruzi parasites as a potential vaccination tool

    PubMed Central

    Brandan, Cecilia Pérez; Basombrío, Miguel Ángel

    2012-01-01

    Chagas disease is the clinical manifestation of the infection produced by the parasite Trypanosoma cruzi. Currently there is no vaccine to prevent this disease and the protection attained with vaccines containing non-replicating parasites is limited. Genetically attenuated trypanosomatid parasites can be obtained by deletion of selected genes. Gene deletion takes advantage of the fact that this parasite can undergo homologous recombination between endogenous and foreign DNA sequences artificially introduced in the cells. This approach facilitated the discovery of several unknown gene functions, as well as allowing us to speculate about the potential for genetically attenuated live organisms as experimental immunogens. Vaccination with live attenuated parasites has been used effectively in mice to reduce parasitemia and histological damage, and in dogs, to prevent vector-delivered infection in the field. However, the use of live parasites as immunogens is controversial due to the risk of reversion to a virulent phenotype. Herein, we present our results from experiments on genetic manipulation of two T. cruzi strains to produce parasites with impaired replication and infectivity, and using the mutation of the dhfr-ts gene as a safety device against reversion to virulence. PMID:22705838

  16. Genetic modification of adeno-associated viral vector type 2 capsid enhances gene transfer efficiency in polarized human airway epithelial cells.

    PubMed

    White, April F; Mazur, Marina; Sorscher, Eric J; Zinn, Kurt R; Ponnazhagan, Selvarangan

    2008-12-01

    Cystic fibrosis (CF) is a common genetic disease characterized by defects in the expression of the CF transmembrane conductance regulator (CFTR) gene. Gene therapy offers better hope for the treatment of CF. Adeno-associated viral (AAV) vectors are capable of stable expression with low immunogenicity. Despite their potential in CF gene therapy, gene transfer efficiency by AAV is limited because of pathophysiological barriers in these patients. Although a few AAV serotypes have shown better transduction compared with the AAV2-based vectors, gene transfer efficiency in human airway epithelium has still not reached therapeutic levels. To engineer better AAV vectors for enhanced gene delivery in human airway epithelium, we developed and characterized mutant AAV vectors by genetic capsid modification, modeling the well-characterized AAV2 serotype. We genetically incorporated putative high-affinity peptide ligands to human airway epithelium on the GH loop region of AAV2 capsid protein. Six independent mutant AAV were constructed, containing peptide ligands previously reported to bind with high affinity for known and unknown receptors on human airway epithelial cells. The vectors were tested on nonairway cells and nonpolarized and polarized human airway epithelial cells for enhanced infectivity. One of the mutant vectors, with the peptide sequence THALWHT, not only showed the highest transduction in undifferentiated human airway epithelial cells but also indicated significant transduction in polarized cells. Interestingly, this modified vector was also able to infect cells independently of the heparan sulfate proteoglycan receptor. Incorporation of this ligand on other AAV serotypes, which have shown improved gene transfer efficiency in the human airway epithelium, may enhance the application of AAV vectors in CF gene therapy.

  17. Intraganglionic AAV6 results in efficient and long-term gene transfer to peripheral sensory nervous system in adult rats.

    PubMed

    Yu, Hongwei; Fischer, Gregory; Ferhatovic, Lejla; Fan, Fan; Light, Alan R; Weihrauch, Dorothee; Sapunar, Damir; Nakai, Hiroyuki; Park, Frank; Hogan, Quinn H

    2013-01-01

    We previously demonstrated safe and reliable gene transfer to the dorsal root ganglion (DRG) using a direct microinjection procedure to deliver recombinant adeno-associated virus (AAV) vector. In this study, we proceed to compare the in vivo transduction patterns of self-complementary (sc) AAV6 and AAV8 in the peripheral sensory pathway. A single, direct microinjection of either AAV6 or AAV8 expressing EGFP, at the adjusted titer of 2×10(9) viral particle per DRG, into the lumbar (L) 4 and L5 DRGs of adult rats resulted in efficient EGFP expression (48±20% for AAV6 and 25±4% for AAV8, mean ± SD) selectively in sensory neurons and their axonal projections 3 weeks after injection, which remained stable for up to 3 months. AAV6 efficiently transfers EGFP to all neuronal size groups without differential neurotropism, while AAV8 predominantly targets large-sized neurons. Neurons transduced with AAV6 penetrate into the spinal dorsal horn (DH) and terminate predominantly in superficial DH laminae, as well as in the dorsal columns and deeper laminae III-V. Only few AAV8-transduced afferents were evident in the superficial laminae, and spinal EGFP was mostly present in the deeper dorsal horn (lamina III-V) and dorsal columns, with substantial projections to the ventral horn. AAV6-mediated EGFP-positive nerve fibers were widely observed in the medial plantar skin of ipsilateral hindpaws. No apparent inflammation, tissue damage, or major pain behaviors were observed for either AAV serotype. Taken together, both AAV6 and AAV8 are efficient and safe vectors for transgene delivery to primary sensory neurons, but they exhibit distinct functional features. Intraganglionic delivery of AAV6 is more uniform and efficient compared to AAV8 in gene transfer to peripheral sensory neurons and their axonal processes.

  18. 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

  19. Safety and Efficacy of AAV Retrograde Pancreatic Ductal Gene Delivery in Normal and Pancreatic Cancer Mice.

    PubMed

    Quirin, Kayla A; Kwon, Jason J; Alioufi, Arafat; Factora, Tricia; Temm, Constance J; Jacobsen, Max; Sandusky, George E; Shontz, Kim; Chicoine, Louis G; Clark, K Reed; Mendell, Joshua T; Korc, Murray; Kota, Janaiah

    2018-03-16

    Recombinant adeno-associated virus (rAAV)-mediated gene delivery shows promise to transduce the pancreas, but safety/efficacy in a neoplastic context is not well established. To identify an ideal AAV serotype, route, and vector dose and assess safety, we have investigated the use of three AAV serotypes (6, 8, and 9) expressing GFP in a self-complementary (sc) AAV vector under an EF1α promoter (scAAV.GFP) following systemic or retrograde pancreatic intraductal delivery. Systemic delivery of scAAV9.GFP transduced the pancreas with high efficiency, but gene expression did not exceed >45% with the highest dose, 5 × 10 12 viral genomes (vg). Intraductal delivery of 1 × 10 11 vg scAAV6.GFP transduced acini, ductal cells, and islet cells with >50%, ∼48%, and >80% efficiency, respectively, and >80% pancreatic transduction was achieved with 5 × 10 11 vg. In a Kras G12D -driven pancreatic cancer mouse model, intraductal delivery of scAAV6.GFP targeted acini, epithelial, and stromal cells and exhibited persistent gene expression 5 months post-delivery. In normal mice, intraductal delivery induced a transient increase in serum amylase/lipase that resolved within a day of infusion with no sustained pancreatic inflammation or fibrosis. Similarly, in PDAC mice, intraductal delivery did not increase pancreatic intraepithelial neoplasia progression/fibrosis. Our study demonstrates that scAAV6 targets the pancreas/neoplasm efficiently and safely via retrograde pancreatic intraductal delivery.

  20. Direct and Retrograde Transduction of Nigral Neurons with AAV6, 8, and 9 and Intraneuronal Persistence of Viral Particles

    PubMed Central

    Aebischer, Patrick

    2013-01-01

    Abstract Recombinant adeno-associated viral (AAV) vectors of serotypes 6, 8, and 9 were characterized as tools for gene delivery to dopaminergic neurons in the substantia nigra for future gene therapeutic applications in Parkinson's disease. While vectors of all three serotypes transduced nigral dopaminergic neurons with equal efficiency when directly injected to the substantia nigra, AAV6 was clearly superior to AAV8 and AAV9 for retrograde transduction of nigral neurons after striatal delivery. For sequential transduction of nigral dopaminergic neurons, the combination of AAV9 with AAV6 proved to be more powerful than AAV8 with AAV6 or repeated AAV6 administration. Surprisingly, single-stranded viral genomes persisted in nigral dopaminergic neurons within cell bodies and axon terminals in the striatum, and intact assembled AAV capsid was enriched in nuclei of nigral neurons, 4 weeks after virus injections to the substantia nigra. 6-Hydroxydopamine (6-OHDA)–induced degeneration of dopaminergic neurons in the substantia nigra reduced the number of viral genomes in the striatum, in line with viral genome persistence in axon terminals. However, 6-OHDA–induced axonal degeneration did not induce any transsynaptic spread of AAV infection in the striatum. Therefore, the potential presence of viral particles in axons may not represent an important safety issue for AAV gene therapy applications in neurodegenerative diseases. PMID:23600720

  1. Creating an arsenal of Adeno-associated virus (AAV) gene delivery stealth vehicles.

    PubMed

    Smith, J Kennon; Agbandje-McKenna, Mavis

    2018-05-01

    The Adeno-associated virus (AAV) gene delivery system is ushering in a new and exciting era in the United States; following the first approved gene therapy (Glybera) in Europe, the FDA has approved a second therapy, Luxturna [1]. However, challenges to this system remain. In viral gene therapy, the surface of the capsid is an important determinant of tissue tropism, impacts gene transfer efficiency, and is targeted by the human immune system. Preexisting immunity is a significant challenge to this approach, and the ability to visualize areas of antibody binding ("footprints") can inform efforts to improve the efficacy of viral vectors. Atomic resolution, smaller proteins, and asymmetric structures are the goals to attain in cryo-electron microscopy and image reconstruction (cryo-EM) as of late. The versatility of the technique and the ability to vitrify a wide range of heterogeneous molecules in solution allow structural biologists to characterize a variety of protein-DNA and protein-protein interactions at lower resolution. Cryo-EM has served as an important means to study key surface areas of the AAV gene delivery vehicle-specifically, those involved with binding neutralizing antibodies (NAbs) [2-4]. This method offers a unique opportunity for visualizing antibody binding "hotspots" on the surface of these and other viral vectors. When combined with mutagenesis, one can eliminate these hotspots to create viral vectors with the ability to avoid preexisting host immune recognition during gene delivery and genetic defect correction in disease treatment. Here, we discuss the use of structure-guided site-directed mutagenesis and directed evolution to create "stealth" AAV vectors with modified surface amino acid sequences that allow NAb avoidance while maintaining natural capsid functions or gaining desired novel tropisms.

  2. Creating an arsenal of Adeno-associated virus (AAV) gene delivery stealth vehicles

    PubMed Central

    Agbandje-McKenna, Mavis

    2018-01-01

    The Adeno-associated virus (AAV) gene delivery system is ushering in a new and exciting era in the United States; following the first approved gene therapy (Glybera) in Europe, the FDA has approved a second therapy, Luxturna [1]. However, challenges to this system remain. In viral gene therapy, the surface of the capsid is an important determinant of tissue tropism, impacts gene transfer efficiency, and is targeted by the human immune system. Preexisting immunity is a significant challenge to this approach, and the ability to visualize areas of antibody binding (“footprints”) can inform efforts to improve the efficacy of viral vectors. Atomic resolution, smaller proteins, and asymmetric structures are the goals to attain in cryo-electron microscopy and image reconstruction (cryo-EM) as of late. The versatility of the technique and the ability to vitrify a wide range of heterogeneous molecules in solution allow structural biologists to characterize a variety of protein–DNA and protein–protein interactions at lower resolution. Cryo-EM has served as an important means to study key surface areas of the AAV gene delivery vehicle—specifically, those involved with binding neutralizing antibodies (NAbs) [2–4]. This method offers a unique opportunity for visualizing antibody binding “hotspots” on the surface of these and other viral vectors. When combined with mutagenesis, one can eliminate these hotspots to create viral vectors with the ability to avoid preexisting host immune recognition during gene delivery and genetic defect correction in disease treatment. Here, we discuss the use of structure-guided site-directed mutagenesis and directed evolution to create “stealth” AAV vectors with modified surface amino acid sequences that allow NAb avoidance while maintaining natural capsid functions or gaining desired novel tropisms. PMID:29723270

  3. Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery

    PubMed Central

    Towne, Chris; Pertin, Marie; Beggah, Ahmed T; Aebischer, Patrick; Decosterd, Isabelle

    2009-01-01

    Background Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. Results Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependant manner. More than 90% of transduced cells were small and medium sized neurons (< 700 μm2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (≈ 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell

  4. High density recombinant AAV particles are competent vectors for in vivo transduction

    USDA-ARS?s Scientific Manuscript database

    Recombinant adeno-associated viral (rAAV) vectors have recently achieved clinical successes in human gene therapy. However, the commonly observed heavier particles found in AAV preparations have traditionally been ignored due to its low in vitro infectivity. In this study, we systemically compared t...

  5. Systemic Correction of Murine Glycogen Storage Disease Type IV by an AAV-Mediated Gene Therapy.

    PubMed

    Yi, Haiqing; Zhang, Quan; Brooks, Elizabeth D; Yang, Chunyu; Thurberg, Beth L; Kishnani, Priya S; Sun, Baodong

    2017-03-01

    Deficiency of glycogen branching enzyme (GBE) causes glycogen storage disease type IV (GSD IV), which is characterized by the accumulation of a less branched, poorly soluble form of glycogen called polyglucosan (PG) in multiple tissues. This study evaluates the efficacy of gene therapy with an adeno-associated viral (AAV) vector in a mouse model of adult form of GSD IV (Gbe1 ys/ys ). An AAV serotype 9 (AAV9) vector containing a human GBE expression cassette (AAV-GBE) was intravenously injected into 14-day-old Gbe1 ys/ys mice at a dose of 5 × 10 11 vector genomes per mouse. Mice were euthanized at 3 and 9 months of age. In the AAV-treated mice at 3 months of age, GBE enzyme activity was highly elevated in heart, which is consistent with the high copy number of the viral vector genome detected. GBE activity also increased significantly in skeletal muscles and the brain, but not in the liver. The glycogen content was reduced to wild-type levels in muscles and significantly reduced in the liver and brain. At 9 months of age, though GBE activity was only significantly elevated in the heart, glycogen levels were significantly reduced in the liver, brain, and skeletal muscles of the AAV-treated mice. In addition, the AAV treatment resulted in an overall decrease in plasma activities of alanine transaminase, aspartate transaminase, and creatine kinase, and a significant increase in fasting plasma glucose concentration at 9 months of age. This suggests an alleviation of damage and improvement of function in the liver and muscles by the AAV treatment. This study demonstrated a long-term benefit of a systemic injection of an AAV-GBE vector in Gbe1 ys/ys mice.

  6. Engineering of Genetically Arrested Parasites (GAPs) For a Precision Malaria Vaccine.

    PubMed

    Kreutzfeld, Oriana; Müller, Katja; Matuschewski, Kai

    2017-01-01

    Continuous stage conversion and swift changes in the antigenic repertoire in response to acquired immunity are hallmarks of complex eukaryotic pathogens, including Plasmodium species, the causative agents of malaria. Efficient elimination of Plasmodium liver stages prior to blood infection is one of the most promising malaria vaccine strategies. Here, we describe different genetically arrested parasites (GAPs) that have been engineered in Plasmodium berghei, P. yoelii and P. falciparum and compare their vaccine potential. A better understanding of the immunological mechanisms of prime and boost by arrested sporozoites and experimental strategies to enhance vaccine efficacy by further engineering existing GAPs into a more immunogenic form hold promise for continuous improvements of GAP-based vaccines. A critical hurdle for vaccines that elicit long-lasting protection against malaria, such as GAPs, is safety and efficacy in vulnerable populations. Vaccine research should focus on solutions toward turning malaria into a vaccine-preventable disease, which would offer an exciting new path of malaria control.

  7. Tyrosine triple mutated AAV2-BDNF gene therapy in a rat model of transient IOP elevation

    PubMed Central

    Igarashi, Tsutomu; Kobayashi, Maika; Kameya, Shuhei; Fujimoto, Chiaki; Nakamoto, Kenji; Takahashi, Hisatomo; Igarashi, Toru; Miyake, Noriko; Iijima, Osamu; Hirai, Yukihiko; Shimada, Takashi; Okada, Takashi; Takahashi, Hiroshi

    2016-01-01

    Purpose We examined the neuroprotective effects of exogenous brain-derived neurotrophic factor (BDNF), which provides protection to retinal ganglion cells (RGCs) in rodents, in a model of transient intraocular pressure (IOP) elevation using a mutant (triple Y-F) self-complementary adeno-associated virus type 2 vector encoding BDNF (tm-scAAV2-BDNF). Methods The tm-scAAV2-BDNF or control vector encoding green fluorescent protein (GFP; tm-scAAV2-GFP) was intravitreally administered to rats, which were then divided into four groups: control, ischemia/reperfusion (I/R) injury only, I/R injury with tm-scAAV2-GFP, and tm-scAAV2-BDNF. I/R injury was then induced by transiently increasing IOP, after which the rats were euthanized to measure the inner retinal thickness and cell counts in the RGC layer. Results Intravitreous injection of tm-scAAV2-BDNF resulted in high levels of BDNF expression in the neural retina. Histological analysis showed that the inner retinal thickness and cell numbers in the RGC layer were preserved after transient IOP elevation in eyes treated with tm-scAAV2-BDNF but not in the other I/R groups. Significantly reduced glial fibrillary acidic protein (GFAP) immunostaining after I/R injury in the rats that received tm-scAAV2-BDNF indicated reduced retinal stress, and electroretinogram (ERG) analysis confirmed preservation of retinal function in the tm-scAAV2-BDNF group. Conclusions These results demonstrate the feasibility and effectiveness of neuroprotective gene therapy using tm-scAAV2-BDNF to protect the inner retina from transiently high intraocular pressure. An in vivo gene therapeutic approach to the clinical management of retinal diseases in conditions such as glaucoma, retinal artery occlusion, hypertensive retinopathy, and diabetic retinopathy thus appears feasible. PMID:27440998

  8. Finding optimal vaccination strategies for pandemic influenza using genetic algorithms.

    PubMed

    Patel, Rajan; Longini, Ira M; Halloran, M Elizabeth

    2005-05-21

    In the event of pandemic influenza, only limited supplies of vaccine may be available. We use stochastic epidemic simulations, genetic algorithms (GA), and random mutation hill climbing (RMHC) to find optimal vaccine distributions to minimize the number of illnesses or deaths in the population, given limited quantities of vaccine. Due to the non-linearity, complexity and stochasticity of the epidemic process, it is not possible to solve for optimal vaccine distributions mathematically. However, we use GA and RMHC to find near optimal vaccine distributions. We model an influenza pandemic that has age-specific illness attack rates similar to the Asian pandemic in 1957-1958 caused by influenza A(H2N2), as well as a distribution similar to the Hong Kong pandemic in 1968-1969 caused by influenza A(H3N2). We find the optimal vaccine distributions given that the number of doses is limited over the range of 10-90% of the population. While GA and RMHC work well in finding optimal vaccine distributions, GA is significantly more efficient than RMHC. We show that the optimal vaccine distribution found by GA and RMHC is up to 84% more effective than random mass vaccination in the mid range of vaccine availability. GA is generalizable to the optimization of stochastic model parameters for other infectious diseases and population structures.

  9. Limited genetic and antigenic diversity within parasite isolates used in a live vaccine against Theileria parva.

    PubMed

    Hemmink, Johanneke D; Weir, William; MacHugh, Niall D; Graham, Simon P; Patel, Ekta; Paxton, Edith; Shiels, Brian; Toye, Philip G; Morrison, W Ivan; Pelle, Roger

    2016-07-01

    An infection and treatment protocol is used to vaccinate cattle against Theileria parva infection. Due to incomplete cross-protection between different parasite isolates, a mixture of three isolates, termed the Muguga cocktail, is used for vaccination. While vaccination of cattle in some regions provides high levels of protection, some animals are not protected against challenge with buffalo-derived T. parva. Knowledge of the genetic composition of the Muguga cocktail vaccine is required to understand how vaccination is able to protect against field challenge and to identify the potential limitations of the vaccine. The aim of the current study was to determine the extent of genetic and antigenic diversity within the parasite isolates that constitute the Muguga cocktail. High throughput multi-locus sequencing of antigen-encoding loci was performed in parallel with typing using a panel of micro- and mini-satellite loci. The former focused on genes encoding CD8(+) T cell antigens, believed to be relevant to protective immunity. The results demonstrate that each of the three component stocks of the cocktail contains limited parasite genotypic diversity, with single alleles detected at many gene/satellite loci and, moreover, that two of the components show a very high level of similarity. Thus, the vaccine incorporates very little of the genetic and antigenic diversity observed in field populations of T. parva. The presence of alleles at low frequency (<10%) within vaccine component populations also points to the possibility of variability in the content of vaccine doses and the potential for loss of allelic diversity during tick passage. The results demonstrate that there is scope to modify the content of the vaccine in order to enhance its diversity and thus its potential for providing broad protection. The ability to accurately quantify genetic diversity in vaccine component stocks will facilitate improved quality control procedures designed to ensure the long

  10. Development of a Genetically Engineered Venezuelan Equine Encephalitis Virus Vaccine

    DTIC Science & Technology

    1988-12-20

    immunization, the horses will be returned to the large animal biocontainment facility to be challenged with equine virulent VEE virus. The animals will be...AD £IT FiLE C p DEVELOPMENT OF A GENETICALLY ENGINEERED VENEZUELAN EQUINE ENCEPHALITIS VIRUS VACCINE ANNUAL REPORT to DENNIS W. TRENT 0DECEMBER 20...Engineered Venezuelan Equine Encephalitis Virus Vaccine 12. PERSONAL AUTHOR(S) Dennis W. Trent 13a. TYPE OF REPORT 13b. TIME COVERED 14. DATE OF REPORT

  11. Caspase Inhibition with XIAP as an Adjunct to AAV Vector Gene-Replacement Therapy: Improving Efficacy and Prolonging the Treatment Window

    PubMed Central

    Yao, Jingyu; Jia, Lin; Khan, Naheed; Zheng, Qiong-Duan; Moncrief, Ashley; Hauswirth, William W.; Thompson, Debra A.; Zacks, David N.

    2012-01-01

    Purpose AAV-mediated gene therapy in the rd10 mouse, with retinal degeneration caused by mutation in the rod cyclic guanosine monophosphate phosphodiesterase β-subunit (PDEβ) gene, produces significant, but transient, rescue of photoreceptor structure and function. This study evaluates the ability of AAV-mediated delivery of X-linked inhibitor of apoptosis (XIAP) to enhance and prolong the efficacy of PDEβ gene-replacement therapy. Methods Rd10 mice were bred and housed in darkness. Two groups of animals were generated: Group 1 received sub-retinal AAV5-XIAP or AAV5-GFP at postnatal age (P) 4 or 21 days; Group 2 received sub-retinal AAV5-XIAP plus AAV5- PDEβ, AAV5-GFP plus AAV5- PDEβ, or AAV- PDEβ alone at age P4 or P21. Animals were maintained for an additional 4 weeks in darkness before being moved to a cyclic-light environment. A subset of animals from Group 1 received a second sub-retinal injection of AAV8-733-PDEβ two weeks after being moved to the light. Histology, immunohistochemistry, Western blots, and electroretinograms were performed at different times after moving to the light. Results Injection of AAV5-XIAP alone at P4 and 21 resulted in significant slowing of light-induced retinal degeneration, as measured by outer nuclear thickness and cell counts, but did not result in improved outer segment structure and rhodopsin localization. In contrast, co-injection of AAV5-XIAP and AAV5-PDEβ resulted in increased levels of rescue and decreased rates of retinal degeneration compared to treatment with AAV5-PDEβ alone. Mice treated with AAV5-XIAP at P4, but not P21, remained responsive to subsequent rescue by AAV8-733-PDEβ when injected two weeks after moving to a light-cycling environment. Conclusions Adjunctive treatment with the anti-apoptotic gene XIAP confers additive protective effect to gene-replacement therapy with AAV5-PDEβ in the rd10 mouse. In addition, AAV5-XIAP, when given early, can increase the age at which gene-replacement therapy

  12. Genetic characterization of L-Zagreb mumps vaccine strain.

    PubMed

    Ivancic, Jelena; Gulija, Tanja Kosutic; Forcic, Dubravko; Baricevic, Marijana; Jug, Renata; Mesko-Prejac, Majda; Mazuran, Renata

    2005-04-01

    Eleven mumps vaccine strains, all containing live attenuated virus, have been used throughout the world. Although L-Zagreb mumps vaccine has been licensed since 1972, only its partial nucleotide sequence was previously determined (accession numbers , and ). Therefore, we sequenced the entire genome of L-Zagreb vaccine strain (Institute of Immunology Inc., Zagreb, Croatia). In order to investigate the genetic stability of the vaccine, sequences of both L-Zagreb master seed and currently produced vaccine batch were determined and no difference between them was observed. A phylogenetic analysis based on SH gene sequence has shown that L-Zagreb strain does not belong to any of established mumps genotypes and that it is most similar to old, laboratory preserved European strains (1950s-1970s). L-Zagreb nucleotide and deduced protein sequences were compared with other mumps virus sequences obtained from the GenBank. Emphasis was put on functionally important protein regions and known antigenic epitopes. The extensive comparisons of nucleotide and deduced protein sequences between L-Zagreb vaccine strain and other previously determined mumps virus sequences have shown that while the functional regions of HN, V, and L proteins are well conserved among various mumps strains, there can be a substantial amino acid difference in antigenic epitopes of all proteins and in functional regions of F protein. No molecular pattern was identified that can be used as a distinction marker between virulent and attenuated strains.

  13. Ribosomal DNA Integrating rAAV-rDNA Vectors Allow for Stable Transgene Expression

    PubMed Central

    Lisowski, Leszek; Lau, Ashley; Wang, Zhongya; Zhang, Yue; Zhang, Feijie; Grompe, Markus; Kay, Mark A

    2012-01-01

    Although recombinant adeno-associated virus (rAAV) vectors are proving to be efficacious in clinical trials, the episomal character of the delivered transgene restricts their effectiveness to use in quiescent tissues, and may not provide lifelong expression. In contrast, integrating vectors enhance the risk of insertional mutagenesis. In an attempt to overcome both of these limitations, we created new rAAV-rDNA vectors, with an expression cassette flanked by ribosomal DNA (rDNA) sequences capable of homologous recombination into genomic rDNA. We show that after in vivo delivery the rAAV-rDNA vectors integrated into the genomic rDNA locus 8–13 times more frequently than control vectors, providing an estimate that 23–39% of the integrations were specific to the rDNA locus. Moreover, a rAAV-rDNA vector containing a human factor IX (hFIX) expression cassette resulted in sustained therapeutic levels of serum hFIX even after repeated manipulations to induce liver regeneration. Because of the relative safety of integration in the rDNA locus, these vectors expand the usage of rAAV for therapeutics requiring long-term gene transfer into dividing cells. PMID:22990671

  14. Live vaccines for human metapneumovirus designed by reverse genetics.

    PubMed

    Buchholz, Ursula J; Nagashima, Kunio; Murphy, Brian R; Collins, Peter L

    2006-10-01

    Human metapneumovirus (HMPV) was first described in 2001 and has quickly become recognized as an important cause of respiratory tract disease worldwide, especially in the pediatric population. A vaccine against HMPV is required to prevent severe disease associated with infection in infancy. The primary strategy is to develop a live-attenuated virus for intranasal immunization, which is particularly well suited against a respiratory virus. Reverse genetics provides a means of developing highly characterized 'designer' attenuated vaccine candidates. To date, several promising vaccine candidates have been developed, each using a different mode of attenuation. One candidate involves deletion of the G glycoprotein, providing attenuation that is probably based on reduced efficiency of attachment. A second candidate involves deletion of the M2-2 protein, which participates in regulating RNA synthesis and whose deletion has the advantageous property of upregulating transcription and increasing antigen synthesis. A third candidate involves replacing the P protein gene of HMPV with its counterpart from the related avian metapneumovirus, thereby introducing attenuation owing to its chimeric nature and host range restriction. Another live vaccine strategy involves using an attenuated parainfluenza virus as a vector to express HMPV protective antigens, providing a bivalent pediatric vaccine. Additional modifications to provide improved vaccines will also be discussed.

  15. Co-overexpression of TGF-β and SOX9 via rAAV gene transfer modulates the metabolic and chondrogenic activities of human bone marrow-derived mesenchymal stem cells.

    PubMed

    Tao, Ke; Frisch, Janina; Rey-Rico, Ana; Venkatesan, Jagadeesh K; Schmitt, Gertrud; Madry, Henning; Lin, Jianhao; Cucchiarini, Magali

    2016-02-01

    Articular cartilage has a limited potential for self-healing. Transplantation of genetically modified progenitor cells like bone marrow-derived mesenchymal stem cells (MSCs) is an attractive strategy to improve the intrinsic repair capacities of damaged articular cartilage. In this study, we examined the potential benefits of co-overexpressing the pleiotropic transformation growth factor beta (TGF-β) with the cartilage-specific transcription factor SOX9 via gene transfer with recombinant adeno-associated virus (rAAV) vectors upon the biological activities of human MSCs (hMSCs). Freshly isolated hMSCs were transduced over time with separate rAAV vectors carrying either TGF-β or sox9 in chondrogenically-induced aggregate cultures to evaluate the efficacy and duration of transgene expression and to monitor the effects of rAAV-mediated genetic modification upon the cellular activities (proliferation, matrix synthesis) and chondrogenic differentiation potency compared with control conditions (lacZ treatment, sequential transductions). Significant, prolonged TGF-β/sox9 co-overexpression was achieved in chondrogenically-induced hMSCs upon co-transduction via rAAV for up to 21 days, leading to enhanced proliferative, biosynthetic, and chondrogenic activities relative to control treatments, especially when co-applying the candidate vectors at the highest vector doses tested. Optimal co-administration of TGF-β with sox9 also advantageously reduced hypertrophic differentiation of the cells in the conditions applied here. The present findings demonstrate the possibility of modifying MSCs by combined therapeutic gene transfer as potent future strategies for implantation in clinically relevant animal models of cartilage defects in vivo.

  16. AAV9-mediated engineering of autotransplanted kidney of non-human primates.

    PubMed

    Tomasoni, S; Trionfini, P; Azzollini, N; Zentilin, L; Giacca, M; Aiello, S; Longaretti, L; Cozzi, E; Baldan, N; Remuzzi, G; Benigni, A

    2017-05-01

    Ex vivo gene transfer to the graft before transplantation is an attractive option for circumventing systemic side effects of chronic antirejection therapy. Gene delivery of the immunomodulatory protein cytotoxic T-lymphocyte-associated protein 4-immunoglobulin (CTLA4-Ig) prevented chronic kidney rejection in a rat model of allotransplantation without the need for systemic immunosuppression. Here we generated adeno-associated virus type 2 (AAV2) and AAV9 vectors encoding for LEA29Y, an optimized version of CTLA4-Ig. Both LEA29Y vectors were equally efficient for reducing T-cell proliferation in vitro. Serotype 9 was chosen for in vivo experiments owing to a lower frequency of preformed antibodies against the AAV9 capsid in 16 non-human primate tested sera. AAV9-LEA29Y was able to transduce the kidney of non-human primates in an autotransplantation model. Expression of LEA29Y mRNA by renal cells translated into the production of the corresponding protein, which was confined to the graft but not detected in serum. Results in non-human primates represent a step forward in maintaining the portability of this strategy into clinics.

  17. AAV Gene Therapy for Alcoholism: Inhibition of Mitochondrial Aldehyde Dehydrogenase Enzyme Expression in Hepatoma Cells.

    PubMed

    Sanchez, Anamaria C; Li, Chengwen; Andrews, Barbara; Asenjo, Juan A; Samulski, R Jude

    2017-09-01

    Most ethanol is broken down in the liver in two steps by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH2) enzymes, which metabolize down ethanol into acetaldehyde and then acetate. Some individuals from the Asian population who carry a mutation in the aldehyde dehydrogenase gene (ALDH2*2) cannot metabolize acetaldehyde as efficiently, producing strong effects, including facial flushing, dizziness, hypotension, and palpitations. This results in an aversion to alcohol intake and protection against alcoholism. The large prevalence of this mutation in the human population strongly suggests that modulation of ALDH2 expression by genetic technologies could result in a similar phenotype. scAAV2 vectors encoding ALDH2 small hairpin RNA (shRNA) were utilized to validate this hypothesis by silencing ALDH2 gene expression in human cell lines. Human cell lines HEK-293 and HepG2 were transduced with scAAV2/shRNA, showing a reduction in ALDH2 RNA and protein expression with the two viral concentration assayed (1 × 10 4 and 1 × 10 5 vg/cell) at two different time points. In both cell lines, ALDH2 RNA levels were reduced by 90% and protein expression was inhibited by 90% and 52%, respectively, 5 days post infection. Transduced HepG2 VL17A cells (ADH+) exposed to ethanol resulted in a 50% increase in acetaldehyde levels. These results suggest that gene therapy could be a useful tool for the treatment of alcoholism by knocking down ALDH2 expression using shRNA technology delivered by AAV vectors.

  18. Genetic heterogeneity of L-Zagreb mumps virus vaccine strain.

    PubMed

    Kosutic-Gulija, Tanja; Forcic, Dubravko; Santak, Maja; Ramljak, Ana; Mateljak-Lukacevic, Sanja; Mazuran, Renata

    2008-07-10

    The most often used mumps vaccine strains Jeryl Lynn (JL), RIT4385, Urabe-AM9, L-Zagreb and L-3 differ in immunogenicity and reactogenicity. Previous analyses showed that JL, Urabe-AM9 and L-3 are genetically heterogeneous. We identified the heterogeneity of L-Zagreb throughout the entire genome. Two major variants were defined: variant A being identical to the consensus sequence of viral seeds and vaccine(s) and variant B which differs from variant A in three nucleotide positions. The difference between viral variants in L-Zagreb strain is insufficient for distinct viral strains to be defined. We demonstrated that proportion of variants in L-Zagreb viral population depends on cell substrate used for viral replication in vitro and in vivo. L-Zagreb strain should be considered as a single strain composed of at least two variant viral genomes.

  19. AAV viral vector delivery to the brain by shape-conforming MR-guided infusions.

    PubMed

    Bankiewicz, Krystof S; Sudhakar, Vivek; Samaranch, Lluis; San Sebastian, Waldy; Bringas, John; Forsayeth, John

    2016-10-28

    Gene transfer technology offers great promise as a potential therapeutic approach to the brain but has to be viewed as a very complex technology. Success of ongoing clinical gene therapy trials depends on many factors such as selection of the correct genetic and anatomical target in the brain. In addition, selection of the viral vector capable of transfer of therapeutic gene into target cells, along with long-term expression that avoids immunotoxicity has to be established. As with any drug development strategy, delivery of gene therapy has to be consistent and predictable in each study subject. Failed drug and vector delivery will lead to failed clinical trials. In this article, we describe our experience with AAV viral vector delivery system, that allows us to optimize and monitor in real time viral vector administration into affected regions of the brain. In addition to discussing MRI-guided technology for administration of AAV vectors we have developed and now employ in current clinical trials, we also describe ways in which infusion cannula design and stereotactic trajectory may be used to maximize the anatomical coverage by using fluid backflow. This innovative approach enables more precise coverage by fitting the shape of the infusion to the shape of the anatomical target. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Assessment of tropism and effectiveness of new primate-derived hybrid recombinant AAV serotypes in the mouse and primate retina.

    PubMed

    Charbel Issa, Peter; De Silva, Samantha R; Lipinski, Daniel M; Singh, Mandeep S; Mouravlev, Alexandre; You, Qisheng; Barnard, Alun R; Hankins, Mark W; During, Matthew J; Maclaren, Robert E

    2013-01-01

    Adeno-associated viral vectors (AAV) have been shown to be safe in the treatment of retinal degenerations in clinical trials. Thus, improving the efficiency of viral gene delivery has become increasingly important to increase the success of clinical trials. In this study, structural domains of different rAAV serotypes isolated from primate brain were combined to create novel hybrid recombinant AAV serotypes, rAAV2/rec2 and rAAV2/rec3. The efficacy of these novel serotypes were assessed in wild type mice and in two models of retinal degeneration (the Abca4(-/-) mouse which is a model for Stargardt disease and in the Pde6b(rd1/rd1) mouse) in vivo, in primate tissue ex-vivo, and in the human-derived SH-SY5Y cell line, using an identical AAV2 expression cassette. We show that these novel hybrid serotypes can transduce retinal tissue in mice and primates efficiently, although no more than AAV2/2 and rAAV2/5 serotypes. Transduction efficiency appeared lower in the Abca4(-/-) mouse compared to wild type with all vectors tested, suggesting an effect of specific retinal diseases on the efficiency of gene delivery. Shuffling of AAV capsid domains may have clinical applications for patients who develop T-cell immune responses following AAV gene therapy, as specific peptide antigen sequences could be substituted using this technique prior to vector re-treatments.

  1. Synergistic cardioprotective effects of rAAV9-CyclinA2 combined with fibrin glue in rats after myocardial infarction.

    PubMed

    Cao, Wen; Chang, Ya-Fei; Zhao, Ai-Chao; Chen, Bang-Dang; Liu, Fen; Ma, Yi-Tong; Ma, Xiang

    2017-08-01

    The present study aimed to investigate the protective effects of rAAV9-CyclinA2 combined with fibrin glue (FG) in vivo in rats after myocardial infarction (MI). Ninety male Sprague-Dawley rats were randomized into 6 groups (15 in each group): sham, MI, rAAV9-green fluorescent protein (GFP) + MI, rAAV9-CyclinA2 + MI, FG + MI, and rAAV9-CyclinA2 + FG + MI. Packed virus (5 × 10 11 vg/ml) in 150 µl of normal saline or FG was injected into the infarcted myocardium at five locations in rAAV9-GFP + MI, rAAV9-CyclinA2 + MI, and rAAV9-CyclinA2 + FG + MI groups. The sham, MI, and FG + MI groups were injected with an equal volume of normal saline or FG at the same sites. Five weeks after injection, echocardiography was performed to evaluate the left ventricular function. The expressions of CyclinA2, proliferating cell nuclear antigen (PCNA), and phospho-histone-H3 (H3P), vascular density, and infarct area were assessed by Western blot, immunohistochemistry, immunofluorescence, and Masson staining. As a result, the combination of rAAV9-CyclinA2 and FG increased ejection fraction and fractional shortening compared with FG or rAAV9-CyclinA2 alone. The expression level of CyclinA2 was significantly higher in the rAAV9-CyclinA2 + FG + MI group compared with the rAAV9-CyclinA2 + MI and FG + MI groups (70.1 ± 1.86% vs. 14.74 ± 2.02%, P < 0.01; or vs. 50.13 ± 3.80%; P < 0.01). A higher expression level of PCNA and H3P was found in the rAAV9-CyclinA2 + FG + MI group compared with other groups. Comparing with other experiment groups, collagen deposition and the infarct size significantly decreased in rAAV9-CyclinA2 + Fibrin + MI group. The vascular density was much higher in the rAAV9-CyclinA2 + FG + MI group compared with the rAAV9-CyclinA2 + MI group. We concluded that fibrin glue combined with rAAV9-CyclinA2 was found to be effective in cardiac remodeling and improving

  2. Construction of PR39 recombinant AAV under control of the HRE promoter and the effect of recombinant AAV on gene therapy of ischemic heart disease

    PubMed Central

    SUN, LIJUN; HAO, YUEWEN; NIE, XIAOWEI; ZHANG, XUEXIN; YANG, GUANGXIAO; WANG, QUANYING

    2012-01-01

    The objective of this study was to investigate the effect of the PR39 recombinant adeno-associated virus (AAV) controlled by the hypoxia-responsive element (HRE) on gene therapy of ischemic heart disease. The minimal HRE was artificially synthesized and the AAV vector controlled by HRE was introduced with NT4-TAT-His-PR39 to investigate the expression of AAV-PR39 in hypoxic vascular endothelial cells (VEC) of human umbilical vein (CRL-1730 cell line) and the angiogenesis-promoting effect in pigs with acute myocardial infraction (AMI). The minimal HRE/CMV was designed and artificially synthesized using the PCR method and cloned with the T vector cloning method. The pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV plasmid was constructed. Using the calcium phosphate precipitation method, HEK-293 cells were co-transfected with three plasmids to produce the recombinant virus. An equal volume of pSS-HRE-CMV-NT4-6His-PR39-PolyAAAV and enterovirus (EV, blank virus) was transfected into CRL-1730 cell lines, respectively. The immunohistochemical method was used to assay the expression of 6xHis in CRL-1730 cell lines and the expression of PR39 under hypoxia. Eighteen AMI miniature pigs were randomized into the experimental group (HRE-AAV-PR39 group), control group 1 (physical saline group) and control group 2 (EV group). The area of ischemia was assessed with conventional MRI and myocardium perfusion MRI. Pigs were sacrificed at preset time-points to obtain samples of ischemic myocardium. Morphological and pathological data were collected. According to data in the literature and databases, the minimal HRE was designed and synthesized with the PCR method. A large number of HREs were connected to modified pSSHGAAV (pSSV9int-/XbaI) vector followed by insertion of the NT4-6His-PR39 gene segment and, thus, the recombinant plasmid pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV was successfully constructed. The expression of 6xHis in CRL-1730 cells under the regulation of HRE was assayed using the

  3. Construction of PR39 recombinant AAV under control of the HRE promoter and the effect of recombinant AAV on gene therapy of ischemic heart disease.

    PubMed

    Sun, Lijun; Hao, Yuewen; Nie, Xiaowei; Zhang, Xuexin; Yang, Guangxiao; Wang, Quanying

    2012-11-01

    The objective of this study was to investigate the effect of the PR39 recombinant adeno-associated virus (AAV) controlled by the hypoxia-responsive element (HRE) on gene therapy of ischemic heart disease. The minimal HRE was artificially synthesized and the AAV vector controlled by HRE was introduced with NT4-TAT-His-PR39 to investigate the expression of AAV-PR39 in hypoxic vascular endothelial cells (VEC) of human umbilical vein (CRL-1730 cell line) and the angiogenesis-promoting effect in pigs with acute myocardial infraction (AMI). The minimal HRE/CMV was designed and artificially synthesized using the PCR method and cloned with the T vector cloning method. The pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV plasmid was constructed. Using the calcium phosphate precipitation method, HEK-293 cells were co-transfected with three plasmids to produce the recombinant virus. An equal volume of pSS-HRE-CMV-NT4-6His-PR39-PolyAAAV and enterovirus (EV, blank virus) was transfected into CRL-1730 cell lines, respectively. The immunohistochemical method was used to assay the expression of 6xHis in CRL-1730 cell lines and the expression of PR39 under hypoxia. Eighteen AMI miniature pigs were randomized into the experimental group (HRE-AAV-PR39 group), control group 1 (physical saline group) and control group 2 (EV group). The area of ischemia was assessed with conventional MRI and myocardium perfusion MRI. Pigs were sacrificed at preset time-points to obtain samples of ischemic myocardium. Morphological and pathological data were collected. According to data in the literature and databases, the minimal HRE was designed and synthesized with the PCR method. A large number of HREs were connected to modified pSSHGAAV (pSSV9int-/XbaI) vector followed by insertion of the NT4-6His-PR39 gene segment and, thus, the recombinant plasmid pSS-HRE-CMV-NT4-6His-PR39-PolyA-AAV was successfully constructed. The expression of 6xHis in CRL-1730 cells under the regulation of HRE was assayed using the

  4. Application of a haematopoetic progenitor cell-targeted adeno-associated viral (AAV) vector established by selection of an AAV random peptide library on a leukaemia cell line

    PubMed Central

    Stiefelhagen, Marius; Sellner, Leopold; Kleinschmidt, Jürgen A; Jauch, Anna; Laufs, Stephanie; Wenz, Frederik; Zeller, W Jens; Fruehauf, Stefan; Veldwijk, Marlon R

    2008-01-01

    Background For many promising target cells (e.g.: haematopoeitic progenitors), the susceptibility to standard adeno-associated viral (AAV) vectors is low. Advancements in vector development now allows the generation of target cell-selected AAV capsid mutants. Methods To determine its suitability, the method was applied on a chronic myelogenous leukaemia (CML) cell line (K562) to obtain a CML-targeted vector and the resulting vectors tested on leukaemia, non-leukaemia, primary human CML and CD34+ peripheral blood progenitor cells (PBPC); standard AAV2 and a random capsid mutant vector served as controls. Results Transduction of CML (BV173, EM3, K562 and Lama84) and AML (HL60 and KG1a) cell lines with the capsid mutants resulted in an up to 36-fold increase in CML transduction efficiency (K562: 2-fold, 60% ± 2% green fluorescent protein (GFP)+ cells; BV173: 9-fold, 37% ± 2% GFP+ cells; Lama84: 36-fold, 29% ± 2% GFP+ cells) compared to controls. For AML (KG1a, HL60) and one CML cell line (EM3), no significant transduction (<1% GFP+ cells) was observed for any vector. Although the capsid mutant clone was established on a cell line, proof-of-principle experiments using primary human cells were performed. For CML (3.2-fold, mutant: 1.75% ± 0.45% GFP+ cells, p = 0.03) and PBPC (3.5-fold, mutant: 4.21% ± 3.40% GFP+ cells) a moderate increase in gene transfer of the capsid mutant compared to control vectors was observed. Conclusion Using an AAV random peptide library on a CML cell line, we were able to generate a capsid mutant, which transduced CML cell lines and primary human haematopoietic progenitor cells with higher efficiency than standard recombinant AAV vectors. PMID:18789140

  5. Intramuscular Adeno-Associated Virus-Mediated Expression of Monoclonal Antibodies Provides 100% Protection Against Ebola Virus Infection in Mice.

    PubMed

    van Lieshout, Laura P; Soule, Geoff; Sorensen, Debra; Frost, Kathy L; He, Shihua; Tierney, Kevin; Safronetz, David; Booth, Stephanie A; Kobinger, Gary P; Qiu, Xiangguo; Wootton, Sarah K

    2018-03-05

    The 2013-2016 West Africa outbreak demonstrated the epidemic potential of Ebola virus and highlighted the need for counter strategies. Monoclonal antibody (mAb)-based therapies hold promise as treatment options for Ebola virus infections. However, production of clinical-grade mAbs is labor intensive, and immunity is short lived. Conversely, adeno-associated virus (AAV)-mediated mAb gene transfer provides the host with a genetic blueprint to manufacture mAbs in vivo, leading to steady release of antibody over many months. Here we demonstrate that AAV-mediated expression of nonneutralizing mAb 5D2 or 7C9 confers 100% protection against mouse-adapted Ebola virus infection, while neutralizing mAb 2G4 was 83% protective. A 2-component cocktail, AAV-2G4/AAV-5D2, provided complete protection when administered 7 days prior to challenge and was partially protective with a 3-day lead time. Finally, AAV-mAb therapies provided sustained protection from challenge 5 months following AAV administration. AAV-mAb may be a viable alternative strategy for vaccination against emerging infectious diseases.

  6. Genetic appraisal of serological response post vaccination against enterotoxaemia (ET) in Malpura and Avikalin sheep.

    PubMed

    Gowane, G R; Akram, Najif; Prince, L L L; Prakash, Ved; Kumar, Arun

    2017-04-01

    Enterotoxaemia (ET) is a fatal enteric disease of small ruminants attributable to a toxigenic type of Clostridium perfringens. The key strategy for prevention of ET is the management and vaccination. Present study aimed at identifying the sources of variation for ET vaccine response especially against epsilon toxin in 173 sheep that included 83 Avikalin and 90 Malpura lambs raised at the institute flock in the semi-arid region of India. The mean age at vaccination was 90 days. Sera were tested by blocking ELISA. Study showed significant variability for response to ET vaccine. 5.2% animals had + positivity, 20.8% animals had ++ positivity, 51.4% animals had +++ positivity and 22.5% animals had ++++ positivity. Amongst environmental determinants, breed, season, sex and age at vaccination proved to be non-significant sources of variation (P > 0.05). MHC genotypes with DRB1 gene and DQA2 genes also revealed non-significant association with ET vaccine response; however, a trend of decreasing PI values with increasing ranks was observed. Study revealed strong response of epsilon toxin along with complexity of the ET vaccine response as phenotype to be explained by genetic and non-genetic factors. The importance of better management practices and vaccination is suggested for preventive measures.

  7. Genetic heterogeneity of L-Zagreb mumps virus vaccine strain

    PubMed Central

    Kosutic-Gulija, Tanja; Forcic, Dubravko; Šantak, Maja; Ramljak, Ana; Mateljak-Lukacevic, Sanja; Mazuran, Renata

    2008-01-01

    Background The most often used mumps vaccine strains Jeryl Lynn (JL), RIT4385, Urabe-AM9, L-Zagreb and L-3 differ in immunogenicity and reactogenicity. Previous analyses showed that JL, Urabe-AM9 and L-3 are genetically heterogeneous. Results We identified the heterogeneity of L-Zagreb throughout the entire genome. Two major variants were defined: variant A being identical to the consensus sequence of viral seeds and vaccine(s) and variant B which differs from variant A in three nucleotide positions. The difference between viral variants in L-Zagreb strain is insufficient for distinct viral strains to be defined. We demonstrated that proportion of variants in L-Zagreb viral population depends on cell substrate used for viral replication in vitro and in vivo. Conclusion L-Zagreb strain should be considered as a single strain composed of at least two variant viral genomes. PMID:18616793

  8. The complete genomic sequence of egg drop syndrome virus strain AAV-2.

    PubMed

    Jin, Q; Zeng, L; Yang, F; Li, M; Hou, Y

    1999-12-01

    In the search for the genome of egg drop syndrome virus (EDSV-76) Chinese strain AAV-2, part of restriction endonuclease physical map is analyzed, the complete genomic library is organized. On basis of this, the complete genome nucleotide sequences (32 838 bp in length, including terminal structures) are determined. The data analysis shows: compared with the other Adenoviruses, strain AAV-2 has more disparity on genomic structure and the distribution of open reading frame (ORF). There are no clear E1, E3 and E4 regions in AAV-2 genome. Two segments located at both ends of genome (1.1 kb and 8.3 kb in length respectively) have no homology with the other adenovirus genomes. In addition, strain AAV-2 genome lacks ORFs encoding ElA, pV and pIX, which are common ORFs encoding early, lately proteins in Adenovirus. This reveals differences between EDSA-76, the sole standard strain of group III Avian Adenoviruses, and the other Avian Adenoviruses for the first time. It will help the search for Avian Adenovirus and will also help the search of all Adenoviruses.

  9. [Construction of a general AAV vector regulated by minimal and artificial hypoxic-responsive element].

    PubMed

    Nie, Xiao-wei; Sun, Li-jun; Hao, Yue-wen; Yang, Guang-xiao; Wang, Quan-ying

    2011-03-01

    To synthesize the minimal and artificial HRE, and to insert it into the anterior extremity of CMV promoter of a AAV plasmid, and then to construct the AAV regulated by hypoxic-responsive element which was introduced into 293 cell by method of Ca3(PO4)2 using three plasmids. Thus obtaining the adenoassociated virus vector regulated by hypoxic-responsive element was possibly used for gene therapy in ischemia angiocardiopathy and cerebrovascular disease. Artificially synthesize the 36 bp nucleotide sequences of four connection in series HIF-binding sites A/GCGTG(4×HBS)and a 35 bp nucleotide sequences spacing inserted into anterior extremity of CMV promoter TATA Box, then amplified by PCR. The cDNA fragment was confirmed to be right by DNA sequencing. Molecular biology routine method was used to construct a AAV vector regulated by minimal hypoxic-responsive element after the normal CMV promoter in AAV vector was replaced by the CMV promoter included minimal hypoxic-responsive element. Then, NT4-6His-PR39 fusogenic peptide was inserted into MCS of the plasmid, the recombinant AAV vector was obtained by three plasmid co-transfection in 293 cells, in which we can also investigate the expression of 6×His using immunochemistry in hypoxia environment. Artificial HRE was inserted into anterior extremity of CMV promoter and there was a correct spacing between the HRE and the TATA-box. The DNA sequencing and restriction enzyme digestion results indicated that the AAV regulated by hypoxic-responsive element was successfully constructed. Compared to the control group, the expressions of 6×His was significantly increased in the experimental groups in hypoxia environment, which confirmed that the AAV effectually regulated by the minimal HRE was inserted into anterior extremity of CMV promoter. The HRE is inserted into anterior extremity of CMV promoter to lack incision enzyme recognition site by PCR. And eukaryotic expression vector regulated by hypoxic-responsive is constructed

  10. Correlation of genetic variability with safety of mumps vaccine Urabe AM9 strain.

    PubMed

    Amexis, G; Fineschi, N; Chumakov, K

    2001-08-15

    The Urabe AM9 strain of mumps vaccine live is known for its genetic instability and some vaccines derived from this strain were withdrawn from the market due to an excessive number of vaccine-associated parotitis and meningitis cases. To identify the molecular basis of this instability, we determined complete nucleotide sequences of several stocks of the Urabe strain used for vaccine production by different manufacturers and of two clinical isolates from cases of vaccine-associated meningitis. In contrast to previously published studies relating the Lys335 --> Glu mutation in the viral HN gene with neurovirulence of mumps virus, we could not confirm any association of this mutation with the safety of mumps vaccine. Each of the three vaccine stocks studied had its own characteristic profile of mutations that was identified by cDNA sequencing and quantitated by mutant analysis by PCR and restriction enzyme cleavage. Determination of the mutational profile of mumps vaccine lots could allow vaccine manufacturers to characterize seed viruses and monitor the consistency of vaccine production to prevent emergence of virulent revertants.

  11. An outbreak of mumps with genetic strain variation in a highly vaccinated student population in Scotland.

    PubMed

    Willocks, L J; Guerendiain, D; Austin, H I; Morrison, K E; Cameron, R L; Templeton, K E; DE Lima, V R F; Ewing, R; Donovan, W; Pollock, K G J

    2017-11-01

    An outbreak of mumps within a student population in Scotland was investigated to assess the effect of previous vaccination on infection and clinical presentation, and any genotypic variation. Of the 341 cases, 79% were aged 18-24. Vaccination status was available for 278 cases of whom 84% had received at least one dose of mumps containing vaccine and 62% had received two. The complication rate was 5·3% (mainly orchitis), and 1·2% were admitted to hospital. Genetic sequencing of mumps virus isolated from cases across Scotland classified 97% of the samples as genotype G. Two distinct clusters of genotype G were identified, one circulating before the outbreak and the other thereafter, suggesting the virus that caused this outbreak was genetically different from the previously circulating virus. Whilst the poor vaccine effectiveness we found may be due to waning immunity over time, a contributing factor may be that the current mumps vaccine is less effective against some genotypes. Although the general benefits of the measles-mumps-rubella (MMR) vaccine should continue to be promoted, there may be value in reassessing the UK vaccination schedule and the current mumps component of the MMR vaccine.

  12. 75 FR 55808 - Prospective Grant of Exclusive License: Development of AAV5 Based Therapeutics To Treat Human...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-14

    .... Patent 6, 855, 314 entitled ``AAV5 Vector for Transducing Brain Cells and Lung Cells'' [HHS Ref. No. E... sale of AAV5 based therapeutic products to be delivered to the brain, eyes and liver for treatment of... vectors and particles. The specific brain cells that are targeted by AAV5 belong to both non-neuronal...

  13. 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

  14. 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

  15. New Generation Live Vaccines against Human Respiratory Syncytial Virus Designed by Reverse Genetics

    PubMed Central

    Collins, Peter L.; Murphy, Brian R.

    2005-01-01

    Development of a live pediatric vaccine against human respiratory syncytial virus (RSV) is complicated by the need to immunize young infants and the difficulty in balancing attenuation and immunogenicity. The ability to introduce desired mutations into infectious virus by reverse genetics provides a method for identifying and designing highly defined attenuating mutations. These can be introduced in combinations as desired to achieve gradations of attenuation. Attenuation is based on several strategies: multiple independent temperature-sensitive point mutations in the polymerase, a temperature-sensitive point mutation in a transcription signal, a set of non–temperature-sensitive mutations involving several genes, deletion of a viral RNA synthesis regulatory protein, and deletion of viral IFN α/β antagonists. The genetic stability of the live vaccine can be increased by judicious choice of mutations. The virus also can be engineered to increase the level of expression of the protective antigens. Protective antigens from antigenically distinct RSV strains can be added or swapped to increase the breadth of coverage. Alternatively, the major RSV protective antigens can be expressed from transcription units added to an attenuated parainfluenza vaccine virus, making a bivalent vaccine. This would obviate the difficulties inherent in the fragility and inefficient in vitro growth of RSV, simplifying vaccine design and use. PMID:16113487

  16. Copackaged AAV9 Vectors Promote Simultaneous Immune Tolerance and Phenotypic Correction of Pompe Disease

    PubMed Central

    Doerfler, Phillip A.; Todd, Adrian G.; Clément, Nathalie; Falk, Darin J.; Nayak, Sushrusha; Herzog, Roland W.; Byrne, Barry J.

    2016-01-01

    Pompe disease is a progressive neuromuscular disorder caused by lysosomal accumulation of glycogen from a deficiency in acid alpha-glucosidase (GAA). Replacement of the missing enzyme is available by repeated protein infusions; however, efficacy is limited by immune response and inability to restore enzymatic function in the central nervous system. An alternative therapeutic option is adeno-associated virus (AAV)-mediated gene therapy, which results in widespread gene transfer and prolonged transgene expression. Both enzyme replacement therapy (ERT) and gene therapy can elicit anti-GAA immune reactions that dampen their effectiveness and pose life-threatening risks to patient safety. To modulate the immune responses related to gene therapy, we show that a human codon-optimized GAA (coGAA) driven by a liver-specific promoter (LSP) using AAV9 is capable of promoting immune tolerance in a Gaa−/− mouse model. Copackaging AAV9-LSP-coGAA with the tissue-restricted desmin promoter (AAV9-DES-coGAA) demonstrates the necessary cell autonomous expression in cardiac muscle, skeletal muscle, peripheral nerve, and the spinal cord. Simultaneous high-level expression in liver led to the expansion of GAA-specific regulatory T-cells (Tregs) and induction of immune tolerance. Transfer of Tregs into naïve recipients prevented pathogenic allergic reactions after repeated ERT challenges. Copackaged AAV9 also attenuated preexisting humoral and cellular immune responses, which enhanced the biochemical correction. Our data present a therapeutic design in which simultaneous administration of two copackaged AAV constructs may provide therapeutic benefit and resolve immune reactions in the treatment of multisystem disorders. PMID:26603344

  17. Comparison of Serum rAAV Serotype-Specific Antibodies in Patients with Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, Inclusion Body Myositis, or GNE Myopathy.

    PubMed

    Zygmunt, Deborah A; Crowe, Kelly E; Flanigan, Kevin M; Martin, Paul T

    2017-09-01

    Recombinant adeno-associated virus (rAAV) is a commonly used gene therapy vector for the delivery of therapeutic transgenes in a variety of human diseases, but pre-existing serum antibodies to viral capsid proteins can greatly inhibit rAAV transduction of tissues. Serum was assayed from patients with Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), inclusion body myositis (IBM), and GNE myopathy (GNE). These were compared to serum from otherwise normal human subjects to determine the extent of pre-existing serum antibodies to rAAVrh74, rAAV1, rAAV2, rAAV6, rAAV8, and rAAV9. In almost all cases, patients with measurable titers to one rAAV serotype showed titers to all other serotypes tested, with average titers to rAAV2 being highest in all instances. Twenty-six percent of all young normal subjects (<18 years old) had measurable rAAV titers to all serotypes tested, and this percentage increased to almost 50% in adult normal subjects (>18 years old). Fifty percent of all IBM and GNE patients also had antibody titers to all rAAV serotypes, while only 18% of DMD and 0% of BMD patients did. In addition, serum-naïve macaques treated systemically with rAAVrh74 could develop cross-reactive antibodies to all other serotypes tested at 24 weeks post treatment. These data demonstrate that most DMD and BMD patients should be amenable to vascular rAAV-mediated treatment without the concern of treatment blockage by pre-existing serum rAAV antibodies, and that serum antibodies to rAAVrh74 are no more common than those for rAAV6, rAAV8, or rAAV9.

  18. Distribution of AAV-TK following intracranial convection-enhanced delivery into rats.

    PubMed

    Cunningham, J; Oiwa, Y; Nagy, D; Podsakoff, G; Colosi, P; Bankiewicz, K S

    2000-01-01

    Adeno-associated virus (AAV)-based vectors are being tested in animal models as viable treatments for glioma and neurodegenerative disease and could potentially be employed to target a variety of central nervous system disorders. The relationship between dose of injected vector and its resulting distribution in brain tissue has not been previously reported nor has the most efficient method of delivery been determined. Here we report that convection-enhanced delivery (CED) of 2.5 x 10(8), 2.5 x 10(9), or 2.5 x 10(10) particles of AAV-thymidine kinase (AAV-TK) into rat brain revealed a clear dose response. In the high-dose group, a volume of 300 mm3 of brain tissue was partially transduced. Results showed that infusion pump and subcutaneous osmotic pumps were both capable of delivering vector via CED and that total particle number was the most important determining factor in obtaining efficient expression. Results further showed differences in histopathology between the delivery groups. While administration of vector using infusion pump had relatively benign effects, the use of osmotic pumps resulted in notable toxicity to the surrounding brain tissue. To determine tissue distribution of vector following intracranial delivery, PCR analysis was performed on tissues from rats that received high doses of AAV-TK. Three weeks following CED, vector could be detected in both hemispheres of the brain, spinal cord, spleen, and kidney.

  19. Restoration of visual response in aged dystrophic RCS rats using AAV-mediated channelopsin-2 gene transfer.

    PubMed

    Tomita, Hiroshi; Sugano, Eriko; Yawo, Hiromu; Ishizuka, Toru; Isago, Hitomi; Narikawa, Satoko; Kügler, Sebastian; Tamai, Makoto

    2007-08-01

    To investigate whether the channelopsin-2 (Chop2) gene would restore visual responses in 10-month-old dystrophic Royal College of Surgeons (aged RCS; rdy/rdy) rats, the authors transferred the Chop2 gene into the retinal cells of aged RCS rats using the adenoassociated virus (AAV) vector. The N-terminal fragment (residues 1-315) of Chop2 was fused to a fluorescent protein, Venus, in frame at the end of the Chop2 coding fragment. The viral vector construct (AAV-Chop2V) for the expression of the Chop2V in the retina was made by subcloning into an adenoassociated virus vector, including the CAG promoter. To evaluate the expression profile of Chop2V in the retina, the rats were killed and the eyes were removed and fixed with 4% paraformaldehyde in 0.1 M phosphate-buffered saline. Retinal wholemount specimens and cryosections were made. Under anesthetized conditions, electrodes for the recording of visually evoked potentials (VEPs) were implanted onto the visual cortex in aged-RCS (rdy/rdy) rats. AAV-Chop2V vectors were then injected into the vitreous cavity of the left eyes. As a control, AAV-Venus vectors were applied to the right eyes. VEPs were evoked by the flash of a blue, white, or red light-emitting diode (LED) and were recorded from the visual cortex of the rats at various time points after the AAV vector injection. Chop2V fluorescence was predominantly observed in retinal ganglion cells (RGCs). Some fluorescence was observed in the inner nuclear layer and the inner plexiform layer neurites. A tendency of recovery was observed in the VEPs of aged RCS (rdy/rdy) rats after the AAV-Chop2V injection but not after the AAV-Venus injection. The visual response of AAV-Chop2V-injected aged RCS (rdy/rdy) rats was less sensitive to the blue LED flash than that of nondystrophic RCS (+/+) rats. The AAV-Chop2V-injected aged RCS (rdy/rdy) rats were insensitive to the red LED flash, which evoked a robust VEP in the RCS (+/+) rats. The visual response of aged RCS (rdy/rdy) rats

  20. In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease

    PubMed Central

    Lau, Cia-Hin; Suh, Yousin

    2017-01-01

    Adeno-associated virus (AAV) has shown promising therapeutic efficacy with a good safety profile in a wide range of animal models and human clinical trials. With the advent of clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome-editing technologies, AAV provides one of the most suitable viral vectors to package, deliver, and express CRISPR components for targeted gene editing. Recent discoveries of smaller Cas9 orthologues have enabled the packaging of Cas9 nuclease and its chimeric guide RNA into a single AAV delivery vehicle for robust in vivo genome editing. Here, we discuss how the combined use of small Cas9 orthologues, tissue-specific minimal promoters, AAV serotypes, and different routes of administration has advanced the development of efficient and precise in vivo genome editing and comprehensively review the various AAV-CRISPR systems that have been effectively used in animals. We then discuss the clinical implications and potential strategies to overcome off-target effects, immunogenicity, and toxicity associated with CRISPR components and AAV delivery vehicles. Finally, we discuss ongoing non-viral-based ex vivo gene therapy clinical trials to underscore the current challenges and future prospects of CRISPR/Cas9 delivery for human therapeutics. PMID:29333255

  1. Genetically Engineered Poxviruses for Recombinant Gene Expression, Vaccination, and Safety

    NASA Astrophysics Data System (ADS)

    Moss, Bernard

    1996-10-01

    Vaccinia virus, no longer required for immunization against smallpox, now serves as a unique vector for expressing genes within the cytoplasm of mammalian cells. As a research tool, recombinant vaccinia viruses are used to synthesize and analyze the structure--function relationships of proteins, determine the targets of humoral and cell-mediated immunity, and investigate the types of immune response needed for protection against specific infectious diseases and cancer. The vaccine potential of recombinant vaccinia virus has been realized in the form of an effective oral wild-life rabies vaccine, although no product for humans has been licensed. A genetically altered vaccinia virus that is unable to replicate in mammalian cells and produces diminished cytopathic effects retains the capacity for high-level gene expression and immunogenicity while promising exceptional safety for laboratory workers and potential vaccine recipients.

  2. Polymers for Improving the In Vivo Transduction Efficiency of AAV2 Vectors

    PubMed Central

    Moulay, Gilles; Boutin, Sylvie; Masurier, Carole; Scherman, Daniel; Kichler, Antoine

    2010-01-01

    Background Adeno-associated virus has attracted great attention as vehicle for body-wide gene delivery. However, for the successful treatment of a disease such as Duchenne muscular dystrophy infusion of very large amounts of vectors is required. This not only raises questions about the technical feasibility of the large scale production but also about the overall safety of the approach. One way to overcome these problems would be to find strategies able to increase the in vivo efficiency. Methodology Here, we investigated whether polymers can act as adjuvants to increase the in vivo efficiency of AAV2. Our strategy consisted in the pre-injection of polymers before intravenous administration of mice with AAV2 encoding a murine secreted alkaline phosphatase (mSeAP). The transgene expression, vector biodistribution and tissue transduction were studied by quantification of the mSeAP protein and real time PCR. The injection of polyinosinic acid and polylysine resulted in an increase of plasmatic mSeAP of 2- and 12-fold, respectively. Interestingly, polyinosinic acid pre-injection significantly reduced the neutralizing antibody titer raised against AAV2. Conclusions Our results show that the pre-injection of polymers can improve the overall transduction efficiency of systemically administered AAV2 and reduce the humoral response against the capsid proteins. PMID:21203395

  3. The Assembly-Activating Protein Promotes Stability and Interactions between AAV's Viral Proteins to Nucleate Capsid Assembly.

    PubMed

    Maurer, Anna C; Pacouret, Simon; Cepeda Diaz, Ana Karla; Blake, Jessica; Andres-Mateos, Eva; Vandenberghe, Luk H

    2018-05-08

    The adeno-associated virus (AAV) vector is a preferred delivery platform for in vivo gene therapy. Natural and engineered variations of the AAV capsid affect a plurality of phenotypes relevant to gene therapy, including vector production and host tropism. Fundamental to these aspects is the mechanism of AAV capsid assembly. Here, the role of the viral co-factor assembly-activating protein (AAP) was evaluated in 12 naturally occurring AAVs and 9 putative ancestral capsid intermediates. The results demonstrate increased capsid protein stability and VP-VP interactions in the presence of AAP. The capsid's dependence on AAP can be partly overcome by strengthening interactions between monomers within the assembly, as illustrated by the transfer of a minimal motif defined by a phenotype-to-phylogeny mapping method. These findings suggest that the emergence of AAP within the Dependovirus genus relaxes structural constraints on AAV assembly in favor of increasing the degrees of freedom for the capsid to evolve. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Intra-Amniotic rAAV-Mediated Microdystrophin Gene Transfer Improves Canine X-Linked Muscular Dystrophy and May Induce Immune Tolerance

    PubMed Central

    Hayashita-Kinoh, Hiromi; Yugeta, Naoko; Okada, Hironori; Nitahara-Kasahara, Yuko; Chiyo, Tomoko; Okada, Takashi; Takeda, Shin'ichi

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a severe congenital disease due to mutations in the dystrophin gene. Supplementation of dystrophin using recombinant adenoassociated virus vector has promise as a treatment of DMD, although therapeutic benefit of the truncated dystrophin still remains to be elucidated. Besides, host immune responses against the vector as well as transgene products have been denoted in the clinical gene therapy studies. Here, we transduced dystrophic dogs fetuses to investigate the therapeutic effects of an AAV vector expressing microdystrophin under conditions of immune tolerance. rAAV-CMV-microdystrophin and a rAAV-CAG-luciferase were injected into the amniotic fluid surrounding fetuses. We also reinjected rAAV9-CMV-microdystrophin into the jugular vein of an infant dystrophic dog to induce systemic expression of microdystrophin. Gait and cardiac function significantly improved in the rAAV-microdystrophin-injected dystrophic dog, suggesting that an adequate treatment of rAAV-microdystrophin with immune modulation induces successful long-term transgene expression to analyze improved dystrophic phenotype. PMID:25586688

  5. From The Cover: Poly- amino ester-containing microparticles enhance the activity of nonviral genetic vaccines

    NASA Astrophysics Data System (ADS)

    Little, Steven R.; Lynn, David M.; Ge, Qing; Anderson, Daniel G.; Puram, Sidharth V.; Chen, Jianzhu; Eisen, Herman N.; Langer, Robert

    2004-06-01

    Current nonviral genetic vaccine systems are less effective than viral vaccines, particularly in cancer systems where epitopes can be weakly immunogenic and antigen-presenting cell processing and presentation to T cells is down-regulated. A promising nonviral delivery method for genetic vaccines involves microencapsulation of antigen-encoding DNA, because such particles protect plasmid payloads and target them to phagocytic antigen-presenting cells. However, conventional microparticle formulations composed of poly lactic-co-glycolic acid take too long to release encapsulated payload and fail to induce high levels of target gene expression. Here, we describe a microparticle-based DNA delivery system composed of a degradable, pH-sensitive poly- amino ester and poly lactic-co-glycolic acid. These formulations generate an increase of 3-5 orders of magnitude in transfection efficiency and are potent activators of dendritic cells in vitro. When used as vaccines in vivo, these microparticle formulations, unlike conventional formulations, induce antigen-specific rejection of transplanted syngenic tumor cells.

  6. AAV vector-mediated secretion of chondroitinase provides a sensitive tracer for axonal arborisations.

    PubMed

    Alves, João Nuno; Muir, Elizabeth M; Andrews, Melissa R; Ward, Anneliese; Michelmore, Nicholas; Dasgupta, Debayan; Verhaagen, Joost; Moloney, Elizabeth B; Keynes, Roger J; Fawcett, James W; Rogers, John H

    2014-04-30

    As part of a project to express chondroitinase ABC (ChABC) in neurons of the central nervous system, we have inserted a modified ChABC gene into an adeno-associated viral (AAV) vector and injected it into the vibrissal motor cortex in adult rats to determine the extent and distribution of expression of the enzyme. A similar vector for expression of green fluorescent protein (GFP) was injected into the same location. For each vector, two versions with minor differences were used, giving similar results. After 4 weeks, the brains were stained to show GFP and products of chondroitinase digestion. Chondroitinase was widely expressed, and the AAV-ChABC and AAV-GFP vectors gave similar expression patterns in many respects, consistent with the known projections from the directly transduced neurons in vibrissal motor cortex and adjacent cingulate cortex. In addition, diffusion of vector to deeper neuronal populations led to labelling of remote projection fields which was much more extensive with AAV-ChABC than with AAV-GFP. The most notable of these populations are inferred to be neurons of cortical layer 6, projecting widely in the thalamus, and neurons of the anterior pole of the hippocampus, projecting through most of the hippocampus. We conclude that, whereas GFP does not label the thinnest axonal branches of some neuronal types, chondroitinase is efficiently secreted from these arborisations and enables their extent to be sensitively visualised. After 12 weeks, chondroitinase expression was undiminished. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Immune and Genetic Correlates of Vaccine Protection Against Mucosal Infection by SIV in Monkeys

    PubMed Central

    Letvin, Norman L.; Rao, Srinivas S.; Montefiori, David C.; Seaman, Michael S.; Sun, Yue; Lim, So-Yon; Yeh, Wendy W.; Asmal, Mohammed; Gelman, Rebecca S.; Shen, Ling; Whitney, James B.; Seoighe, Cathal; Lacerda, Miguel; Keating, Sheila; Norris, Philip J.; Hudgens, Michael G.; Gilbert, Peter B.; Buzby, Adam P.; Mach, Linh V.; Zhang, Jinrong; Balachandran, Harikrishnan; Shaw, George M.; Schmidt, Stephen D.; Todd, John-Paul; Dodson, Alan; Mascola, John R.; Nabel, Gary J.

    2013-01-01

    The RV144 vaccine trial in Thailand demonstrated that an HIV vaccine could prevent infection in humans and highlights the importance of understanding protective immunity against HIV. We used a nonhuman primate model to define immune and genetic mechanisms of protection against mucosal infection by the simian immunodeficiency virus (SIV). A plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen was evaluated for its ability to protect monkeys from infection by SIVmac251 or SIVsmE660 isolates after repeat intrarectal challenges. Although this prime-boost vaccine regimen failed to protect against SIVmac251 infection, 50% of vaccinated monkeys were protected from infection with SIVsmE660. Among SIVsmE660-infected animals, there was an about one-log reduction in peak plasma virus RNA in monkeys expressing the major histocompatibility complex class I allele Mamu-A*01, implicating cytotoxic T lymphocytes in the control of SIV replication once infection is established. Among Mamu-A*01–negative monkeys challenged with SIVsmE660, no CD8+ T cell response or innate immune response was associated with protection against virus acquisition. However, low levels of neutralizing antibodies and an envelope-specific CD4+ T cell response were associated with vaccine protection in these monkeys. Moreover, monkeys that expressed two TRIM5 alleles that restrict SIV replication were more likely to be protected from infection than monkeys that expressed at least one permissive TRIM5 allele. This study begins to elucidate the mechanism of vaccine protection against immunodeficiency viruses and highlights the need to analyze these immune and genetic correlates of protection in future trials of HIV vaccine strategies. PMID:21543722

  8. Immune and Genetic Correlates of Vaccine Protection Against Mucosal Infection by SIV in Monkeys.

    PubMed

    Letvin, Norman L; Rao, Srinivas S; Montefiori, David C; Seaman, Michael S; Sun, Yue; Lim, So-Yon; Yeh, Wendy W; Asmal, Mohammed; Gelman, Rebecca S; Shen, Ling; Whitney, James B; Seoighe, Cathal; Lacerda, Miguel; Keating, Sheila; Norris, Philip J; Hudgens, Michael G; Gilbert, Peter B; Buzby, Adam P; Mach, Linh V; Zhang, Jinrong; Balachandran, Harikrishnan; Shaw, George M; Schmidt, Stephen D; Todd, John-Paul; Dodson, Alan; Mascola, John R; Nabel, Gary J

    2011-05-04

    The RV144 vaccine trial in Thailand demonstrated that an HIV vaccine could prevent infection in humans and highlights the importance of understanding protective immunity against HIV. We used a nonhuman primate model to define immune and genetic mechanisms of protection against mucosal infection by the simian immunodeficiency virus (SIV). A plasmid DNA prime/recombinant adenovirus serotype 5 (rAd5) boost vaccine regimen was evaluated for its ability to protect monkeys from infection by SIVmac251 or SIVsmE660 isolates after repeat intrarectal challenges. Although this prime-boost vaccine regimen failed to protect against SIVmac251 infection, 50% of vaccinated monkeys were protected from infection with SIVsmE660. Among SIVsmE660-infected animals, there was about a one-log reduction in peak plasma virus RNA in monkeys expressing the major histocompatibility complex class I allele Mamu-A*01, implicating cytotoxic T lymphocytes in the control of SIV replication once infection is established. Among Mamu-A*01-negative monkeys challenged with SIVsmE660, no CD8(+) T cell response or innate immune response was associated with protection against virus acquisition. However, low levels of neutralizing antibodies and an envelope-specific CD4(+) T cell response were associated with vaccine protection in these monkeys. Moreover, monkeys that expressed two TRIM5 alleles that restrict SIV replication were more likely to be protected from infection than monkeys that expressed at least one permissive TRIM5 allele. This study begins to elucidate the mechanisms of vaccine protection against immunodeficiency viruses and highlights the need to analyze these immune and genetic correlates of protection in future trials of HIV vaccine strategies.

  9. Long-Term Correction of Sandhoff Disease Following Intravenous Delivery of rAAV9 to Mouse Neonates

    PubMed Central

    Walia, Jagdeep S; Altaleb, Naderah; Bello, Alexander; Kruck, Christa; LaFave, Matthew C; Varshney, Gaurav K; Burgess, Shawn M; Chowdhury, Biswajit; Hurlbut, David; Hemming, Richard; Kobinger, Gary P; Triggs-Raine, Barbara

    2015-01-01

    GM2 gangliosidoses are severe neurodegenerative disorders resulting from a deficiency in β-hexosaminidase A activity and lacking effective therapies. Using a Sandhoff disease (SD) mouse model (Hexb−/−) of the GM2 gangliosidoses, we tested the potential of systemically delivered adeno-associated virus 9 (AAV9) expressing Hexb cDNA to correct the neurological phenotype. Neonatal or adult SD and normal mice were intravenously injected with AAV9-HexB or –LacZ and monitored for serum β-hexosaminidase activity, motor function, and survival. Brain GM2 ganglioside, β-hexosaminidase activity, and inflammation were assessed at experimental week 43, or an earlier humane end point. SD mice injected with AAV9-LacZ died by 17 weeks of age, whereas all neonatal AAV9-HexB–treated SD mice survived until 43 weeks (P < 0.0001) with only three exhibiting neurological dysfunction. SD mice treated as adults with AAV9-HexB died between 17 and 35 weeks. Neonatal SD-HexB–treated mice had a significant increase in brain β-hexosaminidase activity, and a reduction in GM2 ganglioside storage and neuroinflammation compared to adult SD-HexB– and SD-LacZ–treated groups. However, at 43 weeks, 8 of 10 neonatal-HexB injected control and SD mice exhibited liver or lung tumors. This study demonstrates the potential for long-term correction of SD and other GM2 gangliosidoses through early rAAV9 based systemic gene therapy. PMID:25515709

  10. Reverse genetics of measles virus and resulting multivalent recombinant vaccines: applications of recombinant measles viruses.

    PubMed

    Billeter, M A; Naim, H Y; Udem, S A

    2009-01-01

    An overview is given on the development of technologies to allow reverse genetics of RNA viruses, i.e., the rescue of viruses from cDNA, with emphasis on nonsegmented negative-strand RNA viruses (Mononegavirales), as exemplified for measles virus (MV). Primarily, these technologies allowed site-directed mutagenesis, enabling important insights into a variety of aspects of the biology of these viruses. Concomitantly, foreign coding sequences were inserted to (a) allow localization of virus replication in vivo through marker gene expression, (b) develop candidate multivalent vaccines against measles and other pathogens, and (c) create candidate oncolytic viruses. The vector use of these viruses was experimentally encouraged by the pronounced genetic stability of the recombinants unexpected for RNA viruses, and by the high load of insertable genetic material, in excess of 6 kb. The known assets, such as the small genome size of the vector in comparison to DNA viruses proposed as vectors, the extensive clinical experience of attenuated MV as vaccine with a proven record of high safety and efficacy, and the low production cost per vaccination dose are thus favorably complemented.

  11. 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.

  12. 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

  13. Intramuscular injection of AAV8 in mice and macaques is associated with substantial hepatic targeting and transgene expression.

    PubMed

    Greig, Jenny A; Peng, Hui; Ohlstein, Jason; Medina-Jaszek, C Angelica; Ahonkhai, Omua; Mentzinger, Anne; Grant, Rebecca L; Roy, Soumitra; Chen, Shu-Jen; Bell, Peter; Tretiakova, Anna P; Wilson, James M

    2014-01-01

    Intramuscular (IM) administration of adeno-associated viral (AAV) vectors has entered the early stages of clinical development with some success, including the first approved gene therapy product in the West called Glybera. In preparation for broader clinical development of IM AAV vector gene therapy, we conducted detailed pre-clinical studies in mice and macaques evaluating aspects of delivery that could affect performance. We found that following IM administration of AAV8 vectors in mice, a portion of the vector reached the liver and hepatic gene expression contributed significantly to total expression of secreted transgenes. The contribution from liver could be controlled by altering injection volume and by the use of traditional (promoter) and non-traditional (tissue-specific microRNA target sites) expression control elements. Hepatic distribution of vector following IM injection was also noted in rhesus macaques. These pre-clinical data on AAV delivery should inform safe and efficient development of future AAV products.

  14. Contrasting genetic association of IL2RA with SLE and ANCA-associated vasculitis.

    PubMed

    Carr, Edward J; Clatworthy, Menna R; Lowe, Christopher E; Todd, John A; Wong, Andrew; Vyse, Timothy J; Kamesh, Lavanya; Watts, Richard A; Lyons, Paul A; Smith, Kenneth G C

    2009-03-05

    Autoimmune diseases are complex and have genetic and environmental susceptibility factors. The objective was to test the genetic association of systemic lupus erythematosus (SLE) and anti-neutrophil cytoplasmic antibody (ANCA) - associated systemic vasculitis (AAV) with SNPs in the IL2RA region and to correlate genotype with serum levels of IL-2RA. Using a cohort of over 700 AAV patients, two SLE case-control studies and an SLE trio collection (totalling over 1000 SLE patients), and a TaqMan genotyping approach, we tested 3 SNPs in the IL2RA locus, rs11594656, rs2104286 & rs41295061, each with a prior association with autoimmune disease; rs11594656 and rs41295061 with type 1 diabetes (T1D) and rs2104286 with multiple sclerosis (MS) and T1D. We show that SLE is associated with rs11594656 (P = 3.87 x 10-7) and there is some evidence of association of rs41295061 with AAV (P = 0.0122), which both have prior association with T1D. rs2104286, an MS and T1D - associated SNP in the IL2RA locus, is not associated with either SLE or AAV. We have confirmed a previous suggestion that the IL2RA locus is associated with SLE and showed some evidence of association with AAV. Soluble IL-2RA concentrations correlate with rs11594656 genotype in quiescent disease in both AAV and SLE. Differential association of autoimmune diseases and SNPs within the IL2RA locus suggests that the IL2RA pathway may prove to play differing, as yet undefined, roles in each disease.

  15. Pathology Associated with AAV Mediated Expression of Beta Amyloid or C100 in Adult Mouse Hippocampus and Cerebellum

    PubMed Central

    Drummond, Eleanor S.; Muhling, Jill; Martins, Ralph N.; Wijaya, Linda K.; Ehlert, Erich M.; Harvey, Alan R.

    2013-01-01

    Accumulation of beta amyloid (Aβ) in the brain is a primary feature of Alzheimer’s disease (AD) but the exact molecular mechanisms by which Aβ exerts its toxic actions are not yet entirely clear. We documented pathological changes 3 and 6 months after localised injection of recombinant, bi-cistronic adeno-associated viral vectors (rAAV2) expressing human Aβ40-GFP, Aβ42-GFP, C100-GFP or C100V717F-GFP into the hippocampus and cerebellum of 8 week old male mice. Injection of all rAAV2 vectors resulted in wide-spread transduction within the hippocampus and cerebellum, as shown by expression of transgene mRNA and GFP protein. Despite the lack of accumulation of Aβ protein after injection with AAV vectors, injection of rAAV2-Aβ42-GFP and rAAV2- C100V717F-GFP into the hippocampus resulted in significantly increased microgliosis and altered permeability of the blood brain barrier, the latter revealed by high levels of immunoglobulin G (IgG) around the injection site and the presence of IgG positive cells. In comparison, injection of rAAV2-Aβ40-GFP and rAAV2-C100-GFP into the hippocampus resulted in substantially less neuropathology. Injection of rAAV2 vectors into the cerebellum resulted in similar types of pathological changes, but to a lesser degree. The use of viral vectors to express different types of Aβ and C100 is a powerful technique with which to examine the direct in vivo consequences of Aβ expression in different regions of the mature nervous system and will allow experimentation and analysis of pathological AD-like changes in a broader range of species other than mouse. PMID:23516609

  16. Widespread transduction of astrocytes and neurons in the mouse central nervous system after systemic delivery of a self-complementary AAV-PHP.B vector.

    PubMed

    Rincon, Melvin Y; de Vin, Filip; Duqué, Sandra I; Fripont, Shelly; Castaldo, Stephanie A; Bouhuijzen-Wenger, Jessica; Holt, Matthew G

    2018-04-01

    Until recently, adeno-associated virus 9 (AAV9) was considered the AAV serotype most effective in crossing the blood-brain barrier (BBB) and transducing cells of the central nervous system (CNS), following systemic injection. However, a newly engineered capsid, AAV-PHP.B, is reported to cross the BBB at even higher efficiency. We investigated how much we could boost CNS transgene expression by using AAV-PHP.B carrying a self-complementary (sc) genome. To allow comparison, 6 weeks old C57BL/6 mice received intravenous injections of scAAV2/9-GFP or scAAV2/PHP.B-GFP at equivalent doses. Three weeks postinjection, transgene expression was assessed in brain and spinal cord. We consistently observed more widespread CNS transduction and higher levels of transgene expression when using the scAAV2/PHP.B-GFP vector. In particular, we observed an unprecedented level of astrocyte transduction in the cortex, when using a ubiquitous CBA promoter. In comparison, neuronal transduction was much lower than previously reported. However, strong neuronal expression (including spinal motor neurons) was observed when the human synapsin promoter was used. These findings constitute the first reported use of an AAV-PHP.B capsid, encapsulating a scAAV genome, for gene transfer in adult mice. Our results underscore the potential of this AAV construct as a platform for safer and more efficacious gene therapy vectors for the CNS.

  17. Efficient CNS targeting in adult mice by intrathecal infusion of single-stranded AAV9-GFP for gene therapy of neurological disorders.

    PubMed

    Bey, K; Ciron, C; Dubreil, L; Deniaud, J; Ledevin, M; Cristini, J; Blouin, V; Aubourg, P; Colle, M-A

    2017-05-01

    Adeno-associated virus (AAV) gene therapy constitutes a powerful tool for the treatment of neurodegenerative diseases. While AAVs are generally administered systemically to newborns in preclinical studies of neurological disorders, in adults the maturity of the blood-brain barrier (BBB) must be considered when selecting the route of administration. Delivery of AAVs into the cerebrospinal fluid (CSF) represents an attractive approach to target the central nervous system (CNS) and bypass the BBB. In this study, we investigated the efficacy of intra-CSF delivery of a single-stranded (ss) AAV9-CAG-GFP vector in adult mice via intracisternal (iCist) or intralumbar (it-Lumb) administration. It-Lumb ssAAV9 delivery resulted in greater diffusion throughout the entire spinal cord and green fluorescent protein (GFP) expression mainly in the cerebellum, cortex and olfactory bulb. By contrast, iCist delivery led to strong GFP expression throughout the entire brain. Comparison of the transduction efficiency of ssAAV9-CAG-GFP versus ssAAV9-SYN1-GFP following it-Lumb administration revealed widespread and specific GFP expression in neurons and motoneurons of the spinal cord and brain when the neuron-specific synapsin 1 (SYN1) promoter was used. Our findings demonstrate that it-Lumb ssAAV9 delivery is a safe and highly efficient means of targeting the CNS in adult mice.

  18. AAV-mediated pancreatic overexpression of Igf1 counteracts progression to autoimmune diabetes in mice.

    PubMed

    Mallol, Cristina; Casana, Estefania; Jimenez, Veronica; Casellas, Alba; Haurigot, Virginia; Jambrina, Claudia; Sacristan, Victor; Morró, Meritxell; Agudo, Judith; Vilà, Laia; Bosch, Fatima

    2017-07-01

    Type 1 diabetes is characterized by autoimmune destruction of β-cells leading to severe insulin deficiency. Although many improvements have been made in recent years, exogenous insulin therapy is still imperfect; new therapeutic approaches, focusing on preserving/expanding β-cell mass and/or blocking the autoimmune process that destroys islets, should be developed. The main objective of this work was to test in non-obese diabetic (NOD) mice, which spontaneously develop autoimmune diabetes, the effects of local expression of Insulin-like growth factor 1 (IGF1), a potent mitogenic and pro-survival factor for β-cells with immunomodulatory properties. Transgenic NOD mice overexpressing IGF1 specifically in β-cells (NOD-IGF1) were generated and phenotyped. In addition, miRT-containing, IGF1-encoding adeno-associated viruses (AAV) of serotype 8 (AAV8-IGF1-dmiRT) were produced and administered to 4- or 11-week-old non-transgenic NOD females through intraductal delivery. Several histological, immunological, and metabolic parameters were measured to monitor disease over a period of 28-30 weeks. In transgenic mice, local IGF1 expression led to long-term suppression of diabetes onset and robust protection of β-cell mass from the autoimmune insult. AAV-mediated pancreatic-specific overexpression of IGF1 in adult animals also dramatically reduced diabetes incidence, both when vectors were delivered before pathology onset or once insulitis was established. Transgenic NOD-IGF1 and AAV8-IGF1-dmiRT-treated NOD animals had much less islet infiltration than controls, preserved β-cell mass, and normal insulinemia. Transgenic and AAV-treated islets showed less expression of antigen-presenting molecules, inflammatory cytokines, and chemokines important for tissue-specific homing of effector T cells, suggesting IGF1 modulated islet autoimmunity in NOD mice. Local expression of Igf1 by AAV-mediated gene transfer counteracts progression to diabetes in NOD mice. This study suggests a

  19. AAV-mediated RLBP1 gene therapy improves the rate of dark adaptation in Rlbp1 knockout mice

    PubMed Central

    Choi, Vivian W; Bigelow, Chad E; McGee, Terri L; Gujar, Akshata N; Li, Hui; Hanks, Shawn M; Vrouvlianis, Joanna; Maker, Michael; Leehy, Barrett; Zhang, Yiqin; Aranda, Jorge; Bounoutas, George; Demirs, John T; Yang, Junzheng; Ornberg, Richard; Wang, Yu; Martin, Wendy; Stout, Kelly R; Argentieri, Gregory; Grosenstein, Paul; Diaz, Danielle; Turner, Oliver; Jaffee, Bruce D; Police, Seshidhar R; Dryja, Thaddeus P

    2015-01-01

    Recessive mutations in RLBP1 cause a form of retinitis pigmentosa in which the retina, before its degeneration leads to blindness, abnormally slowly recovers sensitivity after exposure to light. To develop a potential gene therapy for this condition, we tested multiple recombinant adeno-associated vectors (rAAVs) composed of different promoters, capsid serotypes, and genome conformations. We generated rAAVs in which sequences from the promoters of the human RLBP1, RPE65, or BEST1 genes drove the expression of a reporter gene (green fluorescent protein). A promoter derived from the RLBP1 gene mediated expression in the retinal pigment epithelium and Müller cells (the intended target cell types) at qualitatively higher levels than in other retinal cell types in wild-type mice and monkeys. With this promoter upstream of the coding sequence of the human RLBP1 gene, we compared the potencies of vectors with an AAV2 versus an AAV8 capsid in transducing mouse retinas, and we compared vectors with a self-complementary versus a single-stranded genome. The optimal vector (scAAV8-pRLBP1-hRLBP1) had serotype 8 capsid and a self-complementary genome. Subretinal injection of scAAV8-pRLBP1-hRLBP1 in Rlbp1 nullizygous mice improved the rate of dark adaptation based on scotopic (rod-plus-cone) and photopic (cone) electroretinograms (ERGs). The effect was still present after 1 year. PMID:26199951

  20. Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice.

    PubMed

    Tadokoro, Takahiro; Miyanohara, Atsushi; Navarro, Michael; Kamizato, Kota; Juhas, Stefan; Juhasova, Jana; Marsala, Silvia; Platoshyn, Oleksandr; Curtis, Erik; Gabel, Brandon; Ciacci, Joseph; Lukacova, Nada; Bimbova, Katarina; Marsala, Martin

    2017-07-13

    The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 µL; 1.2 x 10 13 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

  1. IL-17 and IL-22 genetic polymorphisms in HBV vaccine non- and low-responders among healthcare workers

    PubMed Central

    Borzooy, Zohreh; Streinu-Cercel, Adrian; Mirshafiey, Abbass; Khamseh, Azam; Mahmoudie, Masoud Karkhaneh; Navabi, Shadi Sadat; Nosrati, Marjan; Najafi, Zahra; Hosseini, Mostafa; Jazayeri, Seyed Mohammad

    2016-01-01

    Background Healthcare workers constitute a population at high risk for HBV infection. Efficient vaccination options are available; however, the individual response to HBV vaccination may vary widely between subjects, potentially due to cytokine profiles and genetic variations. In the present study, we investigated the relationship between IL-17 and IL-22 gene polymorphisms versus non- and low-responsiveness to HBV vaccination in healthcare workers. Methods We selected the following IL-17 and IL-22 polymorphisms: rs4711998 (A/G) from IL-17 and rs2227501 (A/T), rs2227503 (A/G), rs1026786 (A/G) from IL-22 sequences genes. These were determined by polymerase chain reaction restriction fragment length polymorphisms. Results The IL-17 rs4711998 GG genotype had a significantly lower frequency in non-responders compared to low-responders (p=0.025). However, we did not identify a relationship between IL-22 rs1026780, rs2227501 and rs2227503 genotypes and the anti-HBs response following HBV vaccination. Conclusion These data suggest that genetic variation in rs4711998 polymorphisms in the IL-17 cytokine may influence vaccine-induced immune responses to HBV vaccine in healthcare workers. PMID:27019828

  2. Genetic Variation among Plasmodium vivax Isolates Adapted to Non-Human Primates and the Implication for Vaccine Development

    PubMed Central

    Ntumngia, Francis B.; McHenry, Amy M.; Barnwel, John W.; Cole-Tobian, Jennifer; King, Christopher L.; Adams, John H.

    2009-01-01

    Plasmodium vivax Duffy binding protein (DBP) is vital for parasite development, thereby making this molecule a good vaccine candidate. Preclinical development of a P. vivax vaccine often involves use of primate models prior to testing efficacy in humans, but primate isolates are poorly characterized. We analyzed the complete gene coding for the DBP in several P. vivax isolates that are used for experimental primate infections and compared these sequences with the Salvador I DBP isolate, which is being used for vaccine development. Our results affirm that primate-adapted isolates are genetically similar to P. vivax circulating in humans, but variability is greatest in the putative target of protective antibodies. In addition, some P. vivax isolates contain multiple genetically different clones. Testing a DBP vaccine may therefore be complicated by heterogeneity and diversity of the P. vivax isolates available for in vivo challenge. PMID:19190217

  3. Synthetic virus seeds for improved vaccine safety: Genetic reconstruction of poliovirus seeds for a PER.C6 cell based inactivated poliovirus vaccine.

    PubMed

    Sanders, Barbara P; Edo-Matas, Diana; Papic, Natasa; Schuitemaker, Hanneke; Custers, Jerome H H V

    2015-10-13

    Safety of vaccines can be compromised by contamination with adventitious agents. One potential source of adventitious agents is a vaccine seed, typically derived from historic clinical isolates with poorly defined origins. Here we generated synthetic poliovirus seeds derived from chemically synthesized DNA plasmids encoding the sequence of wild-type poliovirus strains used in marketed inactivated poliovirus vaccines. The synthetic strains were phenotypically identical to wild-type polioviruses as shown by equivalent infectious titers in culture supernatant and antigenic content, even when infection cultures are scaled up to 10-25L bioreactors. Moreover, the synthetic seeds were genetically stable upon extended passaging on the PER.C6 cell culture platform. Use of synthetic seeds produced on the serum-free PER.C6 cell platform ensures a perfectly documented seed history and maximum control over starting materials. It provides an opportunity to maximize vaccine safety which increases the prospect of a vaccine end product that is free from adventitious agents. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Genetic and antigenic characterisation of serotype A FMD viruses from East Africa to select new vaccine strains

    PubMed Central

    Bari, Fufa D.; Parida, Satya; Tekleghiorghis, Tesfaalem; Dekker, Aldo; Sangula, Abraham; Reeve, Richard; Haydon, Daniel T.; Paton, David J.; Mahapatra, Mana

    2014-01-01

    Vaccine strain selection for emerging foot-and-mouth disease virus (FMDV) outbreaks in enzootic countries can be addressed through antigenic and genetic characterisation of recently circulating viruses. A total of 56 serotype A FMDVs isolated between 1998 and 2012, from Central, East and North African countries were characterised antigenically by virus neutralisation test using antisera to three existing and four candidate vaccine strains and, genetically by characterising the full capsid sequence data. A Bayesian analysis of the capsid sequence data revealed the viruses to be of either African or Asian topotypes with subdivision of the African topotype viruses into four genotypes (Genotypes I, II, IV and VII). The existing vaccine strains were found to be least cross-reactive (good matches observed for only 5.4–46.4% of the sampled viruses). Three bovine antisera, raised against A-EA-2007, A-EA-1981 and A-EA-1984 viruses, exhibited broad cross-neutralisation, towards more than 85% of the circulating viruses. Of the three vaccines, A-EA-2007 was the best showing more than 90% in-vitro cross-protection, as well as being the most recent amongst the vaccine strains used in this study. It therefore appears antigenically suitable as a vaccine strain to be used in the region in FMD control programmes. PMID:25171846

  5. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR.

    PubMed

    D'Costa, Susan; Blouin, Veronique; Broucque, Frederic; Penaud-Budloo, Magalie; François, Achille; Perez, Irene C; Le Bec, Christine; Moullier, Philippe; Snyder, Richard O; Ayuso, Eduard

    2016-01-01

    Clinical trials using recombinant adeno-associated virus (rAAV) vectors have demonstrated efficacy and a good safety profile. Although the field is advancing quickly, vector analytics and harmonization of dosage units are still a limitation for commercialization. AAV reference standard materials (RSMs) can help ensure product safety by controlling the consistency of assays used to characterize rAAV stocks. The most widely utilized unit of vector dosing is based on the encapsidated vector genome. Quantitative polymerase chain reaction (qPCR) is now the most common method to titer vector genomes (vg); however, significant inter- and intralaboratory variations have been documented using this technique. Here, RSMs and rAAV stocks were titered on the basis of an inverted terminal repeats (ITRs) sequence-specific qPCR and we found an artificial increase in vg titers using a widely utilized approach. The PCR error was introduced by using single-cut linearized plasmid as the standard curve. This bias was eliminated using plasmid standards linearized just outside the ITR region on each end to facilitate the melting of the palindromic ITR sequences during PCR. This new "Free-ITR" qPCR delivers vg titers that are consistent with titers obtained with transgene-specific qPCR and could be used to normalize in-house product-specific AAV vector standards and controls to the rAAV RSMs. The free-ITR method, including well-characterized controls, will help to calibrate doses to compare preclinical and clinical data in the field.

  6. A Translational Pathway Toward a Clinical Trial Using the Second-Generation AAV Micro-Dystrophin Vector

    DTIC Science & Technology

    2015-09-01

    injection. We also showed that systemic delivery of a canine micro-dystrophin AAV vector is safe in young adult affected dogs. These results...In addition, we have performed a comprehensive review on the current status of DMD gene therapy in the canine model. We also contributed another...micro-dystrophin, adeno-associated virus, AAV, muscle, gene therapy, systemic gene delivery, canine model 16. SECURITY CLASSIFICATION OF: 17

  7. Hepatitis virus protein X-Phenylalanine Hydroxylase fusion proteins identified in PKU mice treated with AAV-WPRE vectors

    USDA-ARS?s Scientific Manuscript database

    Utilizing the Pahenu2 mouse model for phenylketonuria (PKU), we developed an improved expression vector containing the Woodchuck Hepatitis Virus post-transcriptional regulatory element inserted into a rAAV-mPAH construct (rAAV-mPAH-WPRE) for treatment of PKU. Following portal vein delivery of these ...

  8. A 5′ Noncoding Exon Containing Engineered Intron Enhances Transgene Expression from Recombinant AAV Vectors in vivo

    PubMed Central

    Lu, Jiamiao; Williams, James A.; Luke, Jeremy; Zhang, Feijie; Chu, Kirk; Kay, Mark A.

    2017-01-01

    We previously developed a mini-intronic plasmid (MIP) expression system in which the essential bacterial elements for plasmid replication and selection are placed within an engineered intron contained within a universal 5′ UTR noncoding exon. Like minicircle DNA plasmids (devoid of bacterial backbone sequences), MIP plasmids overcome transcriptional silencing of the transgene. However, in addition MIP plasmids increase transgene expression by 2 and often >10 times higher than minicircle vectors in vivo and in vitro. Based on these findings, we examined the effects of the MIP intronic sequences in a recombinant adeno-associated virus (AAV) vector system. Recombinant AAV vectors containing an intron with a bacterial replication origin and bacterial selectable marker increased transgene expression by 40 to 100 times in vivo when compared with conventional AAV vectors. Therefore, inclusion of this noncoding exon/intron sequence upstream of the coding region can substantially enhance AAV-mediated gene expression in vivo. PMID:27903072

  9. Chondrogenic Differentiation Processes in Human Bone Marrow Aspirates Seeded in Three-Dimensional Woven Poly(ε-Caprolactone) Scaffolds Enhanced by rAAV-Mediated SOX9 Gene Transfer.

    PubMed

    Venkatesan, Jagadeesh Kumar; Moutos, Franklin T; Rey-Rico, Ana; Estes, Bradley T; Frisch, Janina; Schmitt, Gertrud; Madry, Henning; Guilak, Farshid; Cucchiarini, Magali

    2018-05-02

    Combining gene therapy approaches with tissue engineering procedures is an active area of translational research for the effective treatment of articular cartilage lesions, especially to target chondrogenic progenitor cells such as those derived from the bone marrow. Here, we evaluated the effect of genetically modifying concentrated human mesenchymal stem cells from bone marrow to induce chondrogenesis by recombinant adeno-associated viral (rAAV) vector gene transfer of the sex-determining region Y-type high-mobility group box 9 (SOX9) factor upon seeding in three-dimensional (3D) woven poly(ε-caprolactone) (PCL) scaffolds that provide mechanical properties mimicking those of native articular cartilage. Prolonged, effective SOX9 expression was reported in the constructs for at least 21 days, the longest time point evaluated, leading to enhanced metabolic and chondrogenic activities relative to the control conditions (reporter lacZ gene transfer or absence of vector treatment) but without affecting the proliferative activities in the samples. The application of the rAAV SOX9 vector also prevented undesirable hypertrophic and terminal differentiation in the seeded concentrates. As bone marrow is readily accessible during surgery, such findings reveal the therapeutic potential of providing rAAV-modified marrow concentrates within 3D woven PCL scaffolds for repair of focal cartilage lesions.

  10. 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

  11. Development and validation of novel AAV2 random libraries displaying peptides of diverse lengths and at diverse capsid positions.

    PubMed

    Naumer, Matthias; Ying, Ying; Michelfelder, Stefan; Reuter, Antje; Trepel, Martin; Müller, Oliver J; Kleinschmidt, Jürgen A

    2012-05-01

    Libraries based on the insertion of random peptide ligands into the capsid of adeno-associated virus type 2 (AAV2) have been widely used to improve the efficiency and selectivity of the AAV vector system. However, so far only libraries of 7-mer peptide ligands have been inserted at one well-characterized capsid position. Here, we expanded the combinatorial AAV2 display system to a panel of novel AAV libraries, displaying peptides of 5, 7, 12, 19, or 26 amino acids in length at capsid position 588 or displaying 7-mer peptides at position 453, the most prominently exposed region of the viral capsid. Library selections on two unrelated cell types-human coronary artery endothelial cells and rat cardiomyoblasts-revealed the isolation of cell type-characteristic peptides of different lengths mediating strongly improved target-cell transduction, except for the 26-mer peptide ligands. Characterization of vector selectivity by transduction of nontarget cells and comparative gene-transduction analysis using a panel of 44 human tumor cell lines revealed that insertion of different-length peptides allows targeting of distinct cellular receptors for cell entry with similar efficiency, but with different selectivity. The application of such novel AAV2 libraries broadens the spectrum of targetable receptors by capsid-modified AAV vectors and provides the opportunity to choose the best suited targeting ligand for a certain application from a number of different candidates.

  12. 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

  13. How can plant genetic engineering contribute to cost-effective fish vaccine development for promoting sustainable aquaculture?

    PubMed

    Clarke, Jihong Liu; Waheed, Mohammad Tahir; Lössl, Andreas G; Martinussen, Inger; Daniell, Henry

    2013-09-01

    Aquaculture, the fastest growing food-producing sector, now accounts for nearly 50 % of the world's food fish (FAO in The state of world fisheries and aquaculture. FAO, Rome, 2010). The global aquaculture production of food fish reached 62.7 million tonnes in 2011 and is continuously increasing with an estimated production of food fish of 66.5 million tonnes in 2012 (a 9.4 % increase in 1 year, FAO, www.fao.org/fishery/topic/16140 ). Aquaculture is not only important for sustainable protein-based food fish production but also for the aquaculture industry and economy worldwide. Disease prevention is the key issue to maintain a sustainable development of aquaculture. Widespread use of antibiotics in aquaculture has led to the development of antibiotic-resistant bacteria and the accumulation of antibiotics in the environment, resulting in water and soil pollution. Thus, vaccination is the most effective and environmentally-friendly approach to combat diseases in aquaculture to manage fish health. Furthermore, when compared to >760 vaccines against human diseases, there are only about 30 fish vaccines commercially available, suggesting the urgent need for development and cost-effective production of fish vaccines for managing fish health, especially in the fast growing fish farming in Asia where profit is minimal and therefore given high priority. Plant genetic engineering has made significant contributions to production of biotech crops for food, feed, valuable recombinant proteins etc. in the past three decades. The use of plants for vaccine production offers several advantages such as low cost, safety and easy scaling up. To date a large number of plant-derived vaccines, antibodies and therapeutic proteins have been produced for human health, of which a few have been made commercially available. However, the development of animal vaccines in plants, especially fish vaccines by genetic engineering, has not yet been addressed. Therefore, there is a need to exploit

  14. Molecular sequence data of hepatitis B virus and genetic diversity after vaccination.

    PubMed

    van Ballegooijen, W Marijn; van Houdt, Robin; Bruisten, Sylvia M; Boot, Hein J; Coutinho, Roel A; Wallinga, Jacco

    2009-12-15

    The effect of vaccination programs on transmission of infectious disease is usually assessed by monitoring programs that rely on notifications of symptomatic illness. For monitoring of infectious diseases with a high proportion of asymptomatic cases or a low reporting rate, molecular sequence data combined with modern coalescent-based techniques offer a complementary tool to assess transmission. Here, the authors investigate the added value of using viral sequence data to monitor a vaccination program that was started in 1998 and was targeted against hepatitis B virus in men who have sex with men in Amsterdam, the Netherlands. The incidence in this target group, as estimated from the notifications of acute infections with hepatitis B virus, was low; therefore, there was insufficient power to show a significant change in incidence. In contrast, the genetic diversity, as estimated from the viral sequence collected from the target group, revealed a marked decrease after vaccination was introduced. Taken together, the findings suggest that introduction of vaccination coincided with a change in the target group toward behavior with a higher risk of infection. The authors argue that molecular sequence data provide a powerful additional monitoring instrument, next to conventional case registration, for assessing the impact of vaccination.

  15. Biomaterials at the interface of nano- and micro-scale vector-cellular interactions in genetic vaccine design.

    PubMed

    Jones, Charles H; Hakansson, Anders P; Pfeifer, Blaine A

    2014-01-01

    The development of safe and effective vaccines for the prevention of elusive infectious diseases remains a public health priority. Immunization, characterized by adaptive immune responses to specific antigens, can be raised by an array of delivery vectors. However, current commercial vaccination strategies are predicated on the retooling of archaic technology. This review will discuss current and emerging strategies designed to elicit immune responses in the context of genetic vaccination. Selected strategies at the biomaterial-biological interface will be emphasized to illustrate the potential of coupling both fields towards a common goal.

  16. Structure-function Analysis of Receptor-binding in Adeno-Associated Virus Serotype 6 (AAV-6)

    PubMed Central

    Xie, Qing; Lerch, Thomas F.; Meyer, Nancy L.; Chapman, Michael S.

    2011-01-01

    Crystal structures of the AAV-6 capsid at 3 Å reveal a subunit fold homologous to other parvoviruses with greatest differences in two external loops. The electrostatic potential suggests that receptor-attachment is mediated by four residues: Arg576, Lys493, Lys459 and Lys531, defining a positively charged region curving up from the valley between adjacent spikes. It overlaps only partially with the receptor-binding site of AAV-2, and the residues endowing the electrostatic character are not homologous. Mutational substitution of each residue decreases heparin affinity, particularly Lys531 and Lys459. Neither is conserved among heparin-binding serotypes, indicating that diverse modes of receptor attachment have been selected in different serotypes. Surface topology and charge are also distinct at the shoulder of the spike, where linear epitopes for AAV-2’s neutralizing monoclonal antibody A20 come together. Evolutionarily, selection of changed side-chain charge may have offered a conservative means to evade immune neutralization while preserving other essential functionality. PMID:21917284

  17. Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1*

    PubMed Central

    Musayev, Faik N.; Zarate-Perez, Francisco; Bishop, Clayton; Burgner, John W.; Escalante, Carlos R.

    2015-01-01

    Adeno-associated virus (AAV) is the only eukaryotic virus with the property of establishing latency by integrating site-specifically into the human genome. The integration site known as AAVS1 is located in chromosome 19 and contains multiple GCTC repeats that are recognized by the AAV non-structural Rep proteins. These proteins are multifunctional, with an N-terminal origin-binding domain (OBD) and a helicase domain joined together by a short linker. As a first step to understand the process of site-specific integration, we proceeded to characterize the recognition and assembly of Rep68 onto the AAVS1 site. We first determined the x-ray structure of AAV-2 Rep68 OBD in complex with the AAVS1 DNA site. Specificity is achieved through the interaction of a glycine-rich loop that binds the major groove and an α-helix that interacts with a downstream minor groove on the same face of the DNA. Although the structure shows a complex with three OBD molecules bound to the AAVS1 site, we show by using analytical centrifugation and electron microscopy that the full-length Rep68 forms a heptameric complex. Moreover, we determined that a minimum of two direct repeats is required to form a stable complex and to melt DNA. Finally, we show that although the individual domains bind DNA poorly, complex assembly requires oligomerization and cooperation between its OBD, helicase, and the linker domains. PMID:26370092

  18. Genetic Polymorphisms Associated with Rubella Virus-Specific Cellular Immunity Following MMR Vaccination

    PubMed Central

    Kennedy, Richard B.; Ovsyannikova, Inna G.; Haralambieva, Iana H.; Lambert, Nathaniel D.; Pankratz, V. Shane; Poland, Gregory A.

    2014-01-01

    Rubella virus causes a relatively benign disease in most cases, although infection during pregnancy can result in serious birth defects. An effective vaccine has been available since the early 1970s and outbreaks typically do not occur among highly vaccinated (≥2 doses) populations. Nevertheless, considerable inter-individual variation in immune response to rubella immunization does exist, with single dose seroconversion rates ~95%. Understanding the mechanisms behind this variability may provide important insights into rubella immunity. In the current study, we examined associations between single nucleotide polymorphisms (SNPs) in selected cytokine, cytokine receptor, and innate/antiviral genes and immune responses following rubella vaccination in order to understand genetic influences on vaccine response. Our approach consisted of a discovery cohort of 887 subjects ages 11–22 at the time of enrollment and a replication cohort of 542 older adolescents and young adults (ages 18–40). Our data indicate that SNPs near the butyrophilin genes (BTN3A3/BTN2A1) and cytokine receptors (IL10RB/IFNAR1) are associated with variations in IFNγ secretion and that multiple SNPs in the PVR gene, as well as SNPs located in the ADAR gene, exhibit significant associations with rubella virus-specific IL-6 secretion. This information may be useful, not only in furthering our understanding immune responses to rubella vaccine, but also in identifying key pathways for targeted adjuvant use to boost immunity in those with weak or absent immunity following vaccination. PMID:25098560

  19. Genetic polymorphisms associated with rubella virus-specific cellular immunity following MMR vaccination.

    PubMed

    Kennedy, Richard B; Ovsyannikova, Inna G; Haralambieva, Iana H; Lambert, Nathaniel D; Pankratz, V Shane; Poland, Gregory A

    2014-11-01

    Rubella virus causes a relatively benign disease in most cases, although infection during pregnancy can result in serious birth defects. An effective vaccine has been available since the early 1970s and outbreaks typically do not occur among highly vaccinated (≥2 doses) populations. Nevertheless, considerable inter-individual variation in immune response to rubella immunization does exist, with single-dose seroconversion rates ~95 %. Understanding the mechanisms behind this variability may provide important insights into rubella immunity. In the current study, we examined associations between single nucleotide polymorphisms (SNPs) in selected cytokine, cytokine receptor, and innate/antiviral genes and immune responses following rubella vaccination in order to understand genetic influences on vaccine response. Our approach consisted of a discovery cohort of 887 subjects aged 11-22 at the time of enrollment and a replication cohort of 542 older adolescents and young adults (age 18-40). Our data indicate that SNPs near the butyrophilin genes (BTN3A3/BTN2A1) and cytokine receptors (IL10RB/IFNAR1) are associated with variations in IFNγ secretion and that multiple SNPs in the PVR gene, as well as SNPs located in the ADAR gene, exhibit significant associations with rubella virus-specific IL-6 secretion. This information may be useful, not only in furthering our understanding immune responses to rubella vaccine, but also in identifying key pathways for targeted adjuvant use to boost immunity in those with weak or absent immunity following vaccination.

  20. AAV Delivery of Endothelin-1 shRNA Attenuates Cold-Induced Hypertension.

    PubMed

    Chen, Peter Gin-Fu; Sun, Zhongjie

    2017-02-01

    Cold temperatures are associated with increased prevalence of hypertension. Cold exposure increases endothelin-1 (ET1) production. The purpose of this study is to determine whether upregulation of ET1 contributes to cold-induced hypertension (CIH). In vivo RNAi silencing of the ET1 gene was achieved by adeno-associated virus 2 (AAV2) delivery of ET1 short-hairpin small interfering RNA (ET1-shRNA). Four groups of male rats were used. Three groups were given AAV.ET1-shRNA, AAV.SC-shRNA (scrambled shRNA), and phosphate-buffered saline (PBS), respectively, before exposure to a moderately cold environment (6.7 ± 2°C), while the last group was given PBS and kept at room temperature (warm, 24 ± 2°C) and served as a control. We found that systolic blood pressure of the PBS-treated and SC-shRNA-treated groups increased significantly within 2 weeks of exposure to cold, reached a peak level (145 ± 4.8 mmHg) by 6 weeks, and remained elevated thereafter. By contrast, blood pressure of the ET1-shRNA-treated group did not increase, suggesting that silencing of ET1 prevented the development of CIH. Animals were euthanized after 10 weeks of exposure to cold. Cold exposure significantly increased the left ventricle (LV) surface area and LV weight in cold-exposed rats, suggesting LV hypertrophy. Superoxide production in the heart was increased by cold exposure. Interestingly, ET1-shRNA prevented cold-induced superoxide production and cardiac hypertrophy. ELISA assay indicated that ET1-shRNA abolished the cold-induced upregulation of ET1 levels, indicating effective silencing of ET1. In conclusion, upregulation of ET1 plays a critical role in the pathogenesis of CIH and cardiac hypertrophy. AAV delivery of ET1-shRNA is an effective therapeutic strategy for cold-related cardiovascular disease.

  1. AAV1/2-induced overexpression of A53T-α-synuclein in the substantia nigra results in degeneration of the nigrostriatal system with Lewy-like pathology and motor impairment: a new mouse model for Parkinson's disease.

    PubMed

    Ip, Chi Wang; Klaus, Laura-Christin; Karikari, Akua A; Visanji, Naomi P; Brotchie, Jonathan M; Lang, Anthony E; Volkmann, Jens; Koprich, James B

    2017-02-01

    α-Synuclein is a protein implicated in the etiopathogenesis of Parkinson's disease (PD). AAV1/2-driven overexpression of human mutated A53T-α-synuclein in rat and monkey substantia nigra (SN) induces degeneration of nigral dopaminergic neurons and decreases striatal dopamine and tyrosine hydroxylase (TH). Given certain advantages of the mouse, especially it being amendable to genetic manipulation, translating the AAV1/2-A53T α-synuclein model to mice would be of significant value. AAV1/2-A53T α-synuclein or AAV1/2 empty vector (EV) at a concentration of 5.16 x 10 12 gp/ml were unilaterally injected into the right SN of male adult C57BL/6 mice. Post-mortem examinations included immunohistochemistry to analyze nigral α-synuclein, Ser129 phosphorylated α-synuclein and TH expression, striatal dopamine transporter (DAT) levels by autoradiography and dopamine levels by high performance liquid chromatography. At 10 weeks, in AAV1/2-A53T α-synuclein mice there was a 33% reduction in TH+ dopaminergic nigral neurons (P < 0.001), 29% deficit in striatal DAT binding (P < 0.05), 38% and 33% reductions in dopamine (P < 0.001) and DOPAC (P < 0.01) levels and a 60% increase in dopamine turnover (homovanilic acid/dopamine ratio; P < 0.001). Immunofluorescence showed that the AAV1/2-A53T α-synuclein injected mice had widespread nigral and striatal expression of vector-delivered A53T-α-synuclein. Concurrent staining with human PD SN samples using gold standard histological methodology for Lewy pathology detection by proteinase K digestion and application of specific antibody raised against human Lewy body α-synuclein (LB509) and Ser129 phosphorylated α-synuclein (81A) revealed insoluble α-synuclein aggregates in AAV1/2-A53T α-synuclein mice resembling Lewy-like neurites and bodies. In the cylinder test, we observed significant paw use asymmetry in the AAV1/2-A53T α-synuclein group when compared to EV controls at 5 and 9 weeks post injection (P

  2. Impact of Pre-exposure History and Host Genetics on Antibody Avidity Following Norovirus Vaccination.

    PubMed

    Lindesmith, Lisa C; Mallory, Michael L; Jones, Taylor A; Richardson, Charles; Goodwin, Robert R; Baehner, Frank; Mendelman, Paul M; Bargatze, Robert F; Baric, Ralph S

    2017-03-15

    Development of high avidity, broadly neutralizing antibodies (Abs) is a priority after vaccination against rapidly evolving, widely disseminated viruses like human norovirus. After vaccination with a multivalent GI.1 and GII.4c norovirus virus-like particle (VLP) vaccine candidate adjuvanted with alum and monophosphoryl lipid A (MPL), blockade Ab titers peaked early, with no increase in titer following a second vaccine dose. Blockade Ab relative avidity was evaluated by measuring the slope of blockade Ab neutralization curves. Blockade Ab avidity to the GI.1 vaccine component peaked at day 35 (7 days after dose 2). Avidities to heterotypic genogroup I VLPs were not sustained at day 35 after vaccination or GI.1 infection, as measured from archived sera. Only secretor-positive participants maintained high avidity blockade Ab to GI.1 at day 180. Avidity to the GII.4c vaccine component peaked at day 7, remained elevated through day 180, and was not secretor dependent. Avidity to an immunologically novel GII.4 strain VLP correlated with preexisting Ab titer to an ancestral strain Epitope A. Host genetics and pre-exposure history shape norovirus vaccine Ab responses, including blockade Ab avidity. Avidity of potentially neutralizing Ab may be an important metric for evaluating vaccine responses to highly penetrant viruses with cross-reactive serotypes. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

  3. Therapeutic effect of adeno-associated virus (AAV)-mediated ADNF-9 expression on cochlea of kanamycin-deafened guinea pigs.

    PubMed

    Zheng, Guoxi; Zhu, Zhu; Zhu, Kang; Wei, Junrong; Jing, Yang; Duan, Maoli

    2013-10-01

    rAAV-NT4-ADNF-9 could ameliorate the damage to auditory function and repair previous impairment of cochlear hair cell loss induced by kanamycin. To investigate the therapeutic effect of ADNF-9 on cochlear hair cells using the recombinant adeno-associated virus (AAV) carrying fusion gene NT4-ADNF-9 and the kanamycin-deafened guinea pig model. Forty white guinea pigs with normal auricle reflex and normal auditory brainstem responses (ABRs) were randomly divided into four groups. Kanamycin was administered to the animals in groups A, B, and C to establish the deafened guinea pig model. rAAV-NT4-ADNF-9, vector only, and artificial perilymph were then delivered to the cochlear tissue of animals in groups A, B, and C, respectively, through the round window membrane. Animals in group D did not receive any treatment and acted as normal controls. The hearing thresholds on the surgery side were recorded before and after the transfection treatment. Fourteen days after treatment, cochleae were removed for paraffin slide preparation and cochlear surface preparation. A phase contrast microscope was used to observe the protective effect of ADNF-9 on hair cells. Significant reduction of the ABR threshold was observed after rAAV-NT4-ADNF-9 treatment (p < 0.05). After 14 days of treatment, the ABR threshold was also significantly different between the rAAV-NT4-ADNF-9-infected group and the non-infected group. Moreover, phase contrast microscopy showed significantly less hair cell damage or hair cell loss in the group treated with rAAV-NT4-ADNF-9 than in the groups treated with vector only or artificial perilymph (p < 0.05).

  4. Widespread spinal cord transduction by intrathecal injection of rAAV delivers efficacious RNAi therapy for amyotrophic lateral sclerosis.

    PubMed

    Wang, Hongyan; Yang, Bin; Qiu, Linghua; Yang, Chunxing; Kramer, Joshua; Su, Qin; Guo, Yansu; Brown, Robert H; Gao, Guangping; Xu, Zuoshang

    2014-02-01

    Amyotrophic lateral sclerosis (ALS) causes motor neuron degeneration and paralysis. No treatment can significantly slow or arrest the disease progression. Mutations in the SOD1 gene cause a subset of familial ALS by a gain of toxicity. In principle, these cases could be treated with RNAi that destroys the mutant mRNA, thereby abolishing the toxic protein. However, no system is available to efficiently deliver the RNAi therapy. Recombinant adenoassociated virus (rAAV) is a promising vehicle due to its long-lasting gene expression and low toxicity. However, ALS afflicts broad areas of the central nervous system (CNS). A lack of practical means to spread rAAV broadly has hindered its application in treatment of ALS. To overcome this barrier, we injected several rAAV serotypes into the cerebrospinal fluid. We found that some rAAV serotypes such as rAAVrh10 and rAAV9 transduced cells throughout the length of the spinal cord following a single intrathecal injection and in the broad forebrain following a single injection into the third ventricle. Furthermore, a single intrathecal injection of rAAVrh10 robustly transduced motor neurons throughout the spinal cord in a non-human primate. These results suggested a therapeutic potential of this vector for ALS. To test this, we injected a rAAVrh10 vector that expressed an artificial miRNA targeting SOD1 into the SOD1G93A mice. This treatment knocked down the mutant SOD1 expression and slowed the disease progression. Our results demonstrate the potential of rAAVs for delivering gene therapy to treat ALS and other diseases that afflict broad areas of the CNS.

  5. Long-term correction of obesity and diabetes in genetically obese mice by a single intramuscular injection of recombinant adeno-associated virus encoding mouse leptin

    PubMed Central

    Murphy, John E.; Zhou, Shangzhen; Giese, Klaus; Williams, Lewis T.; Escobedo, Jaime A.; Dwarki, Varavani J.

    1997-01-01

    The ob/ob mouse is genetically deficient in leptin and exhibits a phenotype that includes obesity and non-insulin-dependent diabetes melitus. This phenotype closely resembles the morbid obesity seen in humans. In this study, we demonstrate that a single intramuscular injection of a recombinant adeno-associated virus (AAV) vector encoding mouse leptin (rAAV-leptin) in ob/ob mice leads to prevention of obesity and diabetes. The treated animals show normalization of metabolic abnormalities including hyperglycemia, insulin resistance, impaired glucose tolerance, and lethargy. The effects of a single injection have lasted through the 6-month course of the study. At all time points measured the circulating levels of leptin in the serum were similar to age-matched control C57 mice. These results demonstrate that maintenance of normal levels of leptin (2–5 ng/ml) in the circulation can prevent both the onset of obesity and associated non-insulin-dependent diabetes. Thus a single injection of a rAAV vector expressing a therapeutic gene can lead to complete and long-term correction of a genetic disorder. Our study demonstrates the long-term correction of a disease caused by a genetic defect and proves the feasibility of using rAAV-based vectors for the treatment of chronic disorders like obesity. PMID:9391128

  6. [Vaccine application of recombinant herpesviruses].

    PubMed

    Yokoyama, N; Xuan, X; Mikami, T

    2000-04-01

    Recently, genetic engineering using recombinant DNA techniques has been applied to design new viral vaccines in order to reduce some problems which the present viral vaccines have. Up to now, many viruses have been investigated for development of recombinant attenuated vaccines or live viral vectors for delivery of foreign genes coding immunogenic antigens. In this article, we introduced the new vaccine strategy using genetically engineered herpesviruses.

  7. AAV-Mediated Administration of Myostatin Pro-Peptide Mutant in Adult Ldlr Null Mice Reduces Diet-Induced Hepatosteatosis and Arteriosclerosis

    PubMed Central

    Guo, Wen; Wong, Siu; Bhasin, Shalender

    2013-01-01

    Genetic disruption of myostatin or its related signaling is known to cause strong protection against diet-induced metabolic disorders. The translational value of these prior findings, however, is dependent on whether such metabolically favorable phenotype can be reproduced when myostatin blockade begins at an adult age. Here, we reported that AAV-mediated delivery of a myostatin pro-peptide D76A mutant in adult mice attenuates the development of hepatic steatosis and arteriosclerosis, two common diet-induced metabolic diseases. A single dose of AAV-D76A in adult Ldlr null mice resulted in sustained expression of myostatin pro-peptide in the liver. Compared to vehicle-treated mice, D76A-treated mice gained similar amount of lean and fat mass when fed a high fat diet. However, D76A-treated mice displayed significantly reduced aortic lesions and liver fat, in association with a reduction in hepatic expression of lipogenic genes and improvement in liver insulin sensitivity. This suggests that muscle and fat may not be the primary targets of treatment under our experimental condition. In support to this argument, we show that myostatin directly up-regulated lipogenic genes and increased fat accumulation in cultured liver cells. We also show that both myostatin and its receptor were abundantly expressed in mouse aorta. Cultured aortic endothelial cells responded to myostatin with a reduction in eNOS phosphorylation and an increase in ICAM-1 and VCAM-1 expression. Conclusions: AAV-mediated expression of myostatin pro-peptide D76A mutant in adult Ldlr null mice sustained metabolic protection without remarkable impacts on body lean and fat mass. Further investigations are needed to determine whether direct impact of myostatin on liver and aortic endothelium may contribute to the related metabolic phenotypes. PMID:23936482

  8. Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer

    PubMed Central

    Bortolussi, Giulia; Zentilin, Lorena; Baj, Gabriele; Giraudi, Pablo; Bellarosa, Cristina; Giacca, Mauro; Tiribelli, Claudio; Muro, Andrés F.

    2012-01-01

    Crigler-Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11 d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno-associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV-treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene-addition-based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.—Bortolussi, G., Zentilin, L., Baj, G., Giraudi, P., Bellarosa, C., Giacca, M., Tiribelli, C., Muro, A. F. Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer. PMID:22094718

  9. Synergistic inhibition of PARP-1 and NF-κB signaling downregulates immune response against recombinant AAV2 vectors during hepatic gene therapy.

    PubMed

    Hareendran, Sangeetha; Ramakrishna, Banumathi; Jayandharan, Giridhara R

    2016-01-01

    Host immune response remains a key obstacle to widespread application of adeno-associated virus (AAV) based gene therapy. Thus, targeted inhibition of the signaling pathways that trigger such immune responses will be beneficial. Previous studies have reported that DNA damage response proteins such as poly(ADP-ribose) polymerase-1 (PARP-1) negatively affect the integration of AAV in the host genome. However, the role of PARP-1 in regulating AAV transduction and the immune response against these vectors has not been elucidated. In this study, we demonstrate that repression of PARP-1 improves the transduction of single-stranded AAV vectors both in vitro (∼174%) and in vivo (two- to 3.4-fold). Inhibition of PARP-1, also significantly downregulated the expression of several proinflammatory and cytokine markers such as TLRs, ILs, NF-κB subunit proteins associated with the host innate response against self-complementary AAV2 vectors. The suppression of the inflammatory response targeted against these vectors was more effective upon combined inhibition of PARP-1 and NF-κB signaling. This strategy also effectively attenuated the AAV capsid-specific cytotoxic T-cell response, with minimal effect on vector transduction, as demonstrated in normal C57BL/6 and hemophilia B mice. These data suggest that targeting specific host cellular proteins could be useful to attenuate the immune barriers to AAV-mediated gene therapy. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Longevity of rAAV vector and plasmid DNA in blood after intramuscular injection in nonhuman primates: implications for gene doping.

    PubMed

    Ni, W; Le Guiner, C; Gernoux, G; Penaud-Budloo, M; Moullier, P; Snyder, R O

    2011-07-01

    Legitimate uses of gene transfer technology can benefit from sensitive detection methods to determine vector biodistribution in pre-clinical studies and in human clinical trials, and similar methods can detect illegitimate gene transfer to provide sports-governing bodies with the ability to maintain fairness. Real-time PCR assays were developed to detect a performance-enhancing transgene (erythropoietin, EPO) and backbone sequences in the presence of endogenous cellular sequences. In addition to developing real-time PCR assays, the steps involved in DNA extraction, storage and transport were investigated. By real-time PCR, the vector transgene is distinguishable from the genomic DNA sequence because of the absence of introns, and the vector backbone can be identified by heterologous gene expression control elements. After performance of the assays was optimized, cynomolgus macaques received a single dose by intramuscular (IM) injection of plasmid DNA, a recombinant adeno-associated viral vector serotype 1 (rAAV1) or a rAAV8 vector expressing cynomolgus macaque EPO. Macaques received a high plasmid dose intended to achieve a significant, but not life-threatening, increase in hematocrit. rAAV vectors were used at low doses to achieve a small increase in hematocrit and to determine the limit of sensitivity for detecting rAAV sequences by single-step PCR. DNA extracted from white blood cells (WBCs) was tested to determine whether WBCs can be collaterally transfected by plasmid or transduced by rAAV vectors in this context, and can be used as a surrogate marker for gene doping. We demonstrate that IM injection of a conventional plasmid and rAAV vectors results in the presence of DNA that can be detected at high levels in blood before rapid elimination, and that rAAV genomes can persist for several months in WBCs.

  11. Adenovirus-based genetic vaccines for biodefense.

    PubMed

    Boyer, Julie L; Kobinger, Gary; Wilson, James M; Crystal, Ronald G

    2005-02-01

    The robust host responses elicited against transgenes encoded by (E1-)(E3-) adenovirus (Ad) gene transfer vectors can be used to develop Ad-based vectors as platform technologies for vaccines against potential bioterror pathogens. This review focuses on pathogens of major concern as bioterror agents and why Ad vectors are ideal as anti-bioterror vaccine platforms, providing examples from our laboratories of using Ad vectors as vaccines against potential bioterror pathogens and how Ad vectors can be developed to enhance vaccine efficacy in the bioterror war.

  12. The Neurotropic Properties of AAV-PHP.B Are Limited to C57BL/6J Mice.

    PubMed

    Hordeaux, Juliette; Wang, Qiang; Katz, Nathan; Buza, Elizabeth L; Bell, Peter; Wilson, James M

    2018-03-07

    Improved delivery of adeno-associated virus (AAV) vectors to the CNS will greatly enhance their clinical utility. Selection of AAV9 variants in a mouse model led to the isolation of a capsid called PHP.B, which resulted in remarkable transduction of the CNS following intravenous infusion. However, we now show here that this enhanced CNS tropism is restricted to the model in which it was selected, i.e., a Cre transgenic mouse in a C57BL/6J background, and was not found in nonhuman primates or the other commonly used mouse strain BALB/cJ. We also report the potential for serious acute toxicity in NHP after systemic administration of high dose of AAV. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  13. Genetic stability of genome-scale deoptimized RNA virus vaccine candidates under selective pressure

    PubMed Central

    Le Nouën, Cyril; McCarty, Thomas; Brown, Michael; Smith, Melissa Laird; Lleras, Roberto; Dolan, Michael A.; Mehedi, Masfique; Yang, Lijuan; Luongo, Cindy; Liang, Bo; Munir, Shirin; DiNapoli, Joshua M.; Mueller, Steffen; Wimmer, Eckard; Collins, Peter L.; Buchholz, Ursula J.

    2017-01-01

    Recoding viral genomes by numerous synonymous but suboptimal substitutions provides live attenuated vaccine candidates. These vaccine candidates should have a low risk of deattenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. We evaluated phenotypic reversion of deoptimized human respiratory syncytial virus (RSV) vaccine candidates in the context of strong selective pressure. Codon pair deoptimized (CPD) versions of RSV were attenuated and temperature-sensitive. During serial passage at progressively increasing temperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11 ORFs did not lose temperature sensitivity, remained genetically stable, and was restricted at temperatures of 34 °C/35 °C and above. However, a CPD RSV containing 1,378 synonymous mutations solely in the polymerase L ORF quickly lost substantial attenuation. Comprehensive sequence analysis of virus populations identified many different potentially deattenuating mutations in the L ORF as well as, surprisingly, many appearing in other ORFs. Phenotypic analysis revealed that either of two competing mutations in the virus transcription antitermination factor M2-1, outside of the CPD area, substantially reversed defective transcription of the CPD L gene and substantially restored virus fitness in vitro and in case of one of these two mutations, also in vivo. Paradoxically, the introduction into Min L of one mutation each in the M2-1, N, P, and L proteins resulted in a virus with increased attenuation in vivo but increased immunogenicity. Thus, in addition to providing insights on the adaptability of genome-scale deoptimized RNA viruses, stability studies can yield improved synthetic RNA virus vaccine candidates. PMID:28049853

  14. The Skeletal Muscle Environment and Its Role in Immunity and Tolerance to AAV Vector-Mediated Gene Transfer

    PubMed Central

    Boisgérault, Florence; Mingozzi, Federico

    2015-01-01

    Since the early days of gene therapy, muscle has been one the most studied tissue targets for the correction of enzyme deficiencies and myopathies. Several preclinical and clinical studies have been conducted using adeno-associated virus (AAV) vectors. Exciting progress has been made in the gene delivery technologies, from the identification of novel AAV serotypes to the development of novel vector delivery techniques. In parallel, significant knowledge has been generated on the host immune system and its interaction with both the vector and the transgene at the muscle level. In particular, the role of underlying muscle inflammation, characteristic of several diseases affecting the muscle, has been defined in terms of its potential detrimental impact on gene transfer with AAV vectors. At the same time, feedback immunomodulatory mechanisms peculiar of skeletal muscle involving resident regulatory T cells have been identified, which seem to play an important role in maintaining, at least to some extent, muscle homeostasis during inflammation and regenerative processes. Devising strategies to tip this balance towards unresponsiveness may represent an avenue to improve the safety and efficacy of muscle gene transfer with AAV vectors. PMID:26122097

  15. The behavioural and neuropathological impact of intranigral AAV-α-synuclein is exacerbated by systemic infusion of the Parkinson's disease-associated pesticide, rotenone, in rats.

    PubMed

    Mulcahy, Pádraig; O'Doherty, Aideen; Paucard, Alexia; O'Brien, Timothy; Kirik, Deniz; Dowd, Eilís

    2013-04-15

    Despite the widely held belief that Parkinson's disease is caused by both underlying genetics and exposure to environmental risk factors, it is still widely modelled in preclinical models using a single genetic or neurotoxic insult. This single-insult approach has resulted in a variety of models that are limited with respect to their aetiological, construct, face and/or predictive validity. Thus, the aim of the current study was to investigate the interplay between genes and the environment as an alternative approach to modelling Parkinson's disease. To do so, rats underwent stereotaxic surgery for unilateral delivery of the Parkinson's disease-associated gene, α-synuclein, into the substantia nigra (using AAV vectors). This was followed 13 weeks later by subcutaneous implantation of an osmotic minipump delivering the Parkinson's disease-associated pesticide, rotenone (2.5mgkg(-1)day(-1) for 4 weeks). The effect of the genetic and environmental insults alone or in combination on lateralised motor performance (Corridor, Stepping and Whisker Tests), nigrostriatal integrity (tyrosine hydroxylase immunohistochemistry) and α-synucleinopathy (α-synuclein immunohistochemistry) was assessed. We found that exposing AAV-α-synuclein-treated rats to rotenone led to a model in which the classical Parkinson's disease triad of progressive motor dysfunction, nigrostriatal neurodegeneration and α-synucleinopathy was evident. However, delivering rotenone systemically was also associated with bilateral motor dysfunction and loss of body weight. Thus, although we have shown that Parkinson's disease can be modelled in experimental animals by combined exposure to both genetic and environmental risk factors, this approach is limited by systemic toxicity of the pesticide rotenone. Direct intracerebral delivery of rotenone may be more useful in longer-term studies as we have previously shown that it overcomes this limitation. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Genetic targeting of the active transcription factor XBP1s to dendritic cells potentiates vaccine-induced prophylactic and therapeutic antitumor immunity.

    PubMed

    Tian, Shenghe; Liu, Zuqiang; Donahue, Cara; Falo, Louis D; You, Zhaoyang

    2012-02-01

    In vivo dendritic cells (DC) targeting is an attractive approach with potential advantages in vaccine efficacy, cost, and availability. Identification of molecular adjuvants to in vivo "modulate " DC to coordinately render improved Th1 and CD8 T cell immunity, and attenuated deleterious Treg effects, is a critical challenge. Here, we report that in vivo genetic targeting of the active transcription factor XBP1s to DC (XBP1s/DC) potentiated vaccine-induced prophylactic and therapeutic antitumor immunity in multiple tumor models. This immunization strategy is based on a genetic vaccine encoding both cytomegalovirus (CMV)-driven vaccine Aghsp70 and DC-specific CD11c-driven XBP1s. The novel targeted vaccine induced durable Th1 and CD8 T cell responses to poorly immunogenic self/tumor antigen (Ag) and attenuated tumor-associated Treg suppressive function. Bone marrow (BM)-derived DC genetically modified to simultaneously overexpress XBP1s and express Aghsp70 upregulated CD40, CD70, CD86, interleukin (IL)-15, IL-15Rα, and CCR7 expression, and increased IL-6, IL-12, and tumor necrosis factor (TNF)-α production in vitro. XBP1s/DC elevated functional DEC205(+)CD8α(+)DC in the draining lymph nodes (DLN). The data suggest a novel role for XBP1s in modulating DC to potentiate tumor vaccine efficacy via overcoming two major obstacles to tumor vaccines (i.e., T cell hyporesponsiveness against poorly immunologic self/tumor Ag and tumor-associated Treg-mediated suppression) and improving DEC205(+)CD8α(+)DC.

  17. Dual AAV therapy ameliorates exercise-induced muscle injury and functional ischemia in murine models of Duchenne muscular dystrophy.

    PubMed

    Zhang, Yadong; Yue, Yongping; Li, Liang; Hakim, Chady H; Zhang, Keqing; Thomas, Gail D; Duan, Dongsheng

    2013-09-15

    Neuronal nitric oxide synthase (nNOS) membrane delocalization contributes to the pathogenesis of Duchenne muscular dystrophy (DMD) by promoting functional muscle ischemia and exacerbating muscle injury during exercise. We have previously shown that supra-physiological expression of nNOS-binding mini-dystrophin restores normal blood flow regulation and prevents functional ischemia in transgenic mdx mice, a DMD model. A critical next issue is whether systemic dual adeno-associated virus (AAV) gene therapy can restore nNOS-binding mini-dystrophin expression and mitigate muscle activity-related functional ischemia and injury. Here, we performed systemic gene transfer in mdx and mdx4cv mice using a pair of dual AAV vectors that expressed a 6 kb nNOS-binding mini-dystrophin gene. Vectors were packaged in tyrosine mutant AAV-9 and co-injected (5 × 10(12) viral genome particles/vector/mouse) via the tail vein to 1-month-old dystrophin-null mice. Four months later, we observed 30-50% mini-dystrophin positive myofibers in limb muscles. Treatment ameliorated histopathology, increased muscle force and protected against eccentric contraction-induced injury. Importantly, dual AAV therapy successfully prevented chronic exercise-induced muscle force drop. Doppler hemodynamic assay further showed that therapy attenuated adrenergic vasoconstriction in contracting muscle. Our results suggest that partial transduction can still ameliorate nNOS delocalization-associated functional deficiency. Further evaluation of nNOS binding mini-dystrophin dual AAV vectors is warranted in dystrophic dogs and eventually in human patients.

  18. Concomitant Intravenous Nitroglycerin With Intracoronary Delivery of AAV1.SERCA2a Enhances Gene Transfer in Porcine Hearts

    PubMed Central

    Karakikes, Ioannis; Hadri, Lahouaria; Rapti, Kleopatra; Ladage, Dennis; Ishikawa, Kiyotake; Tilemann, Lisa; Yi, Geng-Hua; Morel, Charlotte; Gwathmey, Judith K; Zsebo, Krisztina; Weber, Thomas; Kawase, Yoshiaki; Hajjar, Roger J

    2012-01-01

    SERCA2a gene therapy improves contractile and energetic function of failing hearts and has been shown to be associated with benefits in clinical outcomes, symptoms, functional status, biomarkers, and cardiac structure in a phase 2 clinical trial. In an effort to enhance the efficiency and homogeneity of gene uptake in cardiac tissue, we examined the effects of nitroglycerin (NTG) in a porcine model following AAV1.SERCA2a gene delivery. Three groups of Göttingen minipigs were assessed: (i) group A: control intracoronary (IC) AAV1.SERCA2a (n = 6); (ii) group B: a single bolus IC injection of NTG (50 µg) immediately before administration of intravenous (IV) AAV1.SERCA2a (n = 6); and (iii) group C: continuous IV NTG (1 µg/kg/minute) during the 10 minutes of AAV1.SERCA2a infusion (n = 6). We found that simultaneous IV infusion of NTG and AAV1.SERCA2a resulted in increased viral transduction efficiency, both in terms of messenger RNA (mRNA) as well as SERCA2a protein levels in the whole left ventricle (LV) compared to control animals. On the other hand, IC NTG pretreatment did not result in enhanced gene transfer efficiency, mRNA or protein levels when compared to control animals. Importantly, the transgene expression was restricted to the heart tissue. In conclusion, we have demonstrated that IV infusion of NTG significantly improves cardiac gene transfer efficiency in porcine hearts. PMID:22215018

  19. AAV-mediated netrin-1 overexpression increases peri-infarct blood vessel density and improves motor function recovery after experimental stroke.

    PubMed

    Sun, Hui; Le, Thang; Chang, Tiffany T J; Habib, Aisha; Wu, Steven; Shen, Fanxia; Young, William L; Su, Hua; Liu, Jialing

    2011-10-01

    Apart from its role in axon guidance, netrin-1 is also known to be pro-angiogenic. The aim of this study is to determine whether adeno-associated viral (AAV) mediated overexpression of netrin-1 improves post-stroke neurovascular structure and recovery of function. AAV-Netrin-1 or AAV-LacZ of 1×10(10) genome copies each was injected medial and posterior to ischemic lesion at one hour following reperfusion using the distal middle cerebral artery occlusion (MCAO) method. Quantitative RT-PCR revealed that the expression of netrin-1 transgene began as early as one day and increased dramatically about 3 weeks following vector injection. Western blot analysis and confocal microscopy suggested that both the endogenous and transduced netrin-1 were expressed in the neurons of the peri-infarct cortex after MCAO. AAV-mediated netrin-1 overexpression significantly increased vascular density in the peri-infarct cortex and promoted the migration of immature neurons into the peri-infarct white matter, but it did not significantly reduce infarct size. Netrin-1 overexpression also enhanced post-stroke locomotor activity, improved exploratory behavior, and reduced ischemia-induced motor asymmetry in forelimb usage. However, it had little effect on post-stroke spatial learning and memory. Our results suggest that AAV mediated netrin-1 overexpression improves peri-infarct vascular density and post stroke motor function. Published by Elsevier Inc.

  20. Effective genetic modification and differentiation of hMSCs upon controlled release of rAAV vectors using alginate/poloxamer composite systems.

    PubMed

    Díaz-Rodríguez, P; Rey-Rico, A; Madry, H; Landin, M; Cucchiarini, M

    2015-12-30

    Viral vectors are common tools in gene therapy to deliver foreign therapeutic sequences in a specific target population via their natural cellular entry mechanisms. Incorporating such vectors in implantable systems may provide strong alternatives to conventional gene transfer procedures. The goal of the present study was to generate different hydrogel structures based on alginate (AlgPH155) and poloxamer PF127 as new systems to encapsulate and release recombinant adeno-associated viral (rAAV) vectors. Inclusion of rAAV in such polymeric capsules revealed an influence of the hydrogel composition and crosslinking temperature upon the vector release profiles, with alginate (AlgPH155) structures showing the fastest release profiles early on while over time vector release was more effective from AlgPH155+PF127 [H] capsules crosslinked at a high temperature (50°C). Systems prepared at room temperature (AlgPH155+PF127 [C]) allowed instead to achieve a more controlled release profile. When tested for their ability to target human mesenchymal stem cells, the different systems led to high transduction efficiencies over time and to gene expression levels in the range of those achieved upon direct vector application, especially when using AlgPH155+PF127 [H]. No detrimental effects were reported on either cell viability or on the potential for chondrogenic differentiation. Inclusion of PF127 in the capsules was also capable of delaying undesirable hypertrophic cell differentiation. These findings are of promising value for the further development of viral vector controlled release strategies. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Genetic and antigenic characterization of serotype O FMD viruses from East Africa for the selection of suitable vaccine strain.

    PubMed

    Lloyd-Jones, Katie; Mahapatra, Mana; Upadhyaya, Sasmita; Paton, David J; Babu, Aravindh; Hutchings, Geoff; Parida, Satya

    2017-12-14

    Foot-and-mouth disease (FMD) is endemic in Eastern Africa with circulation of multiple serotypes of the virus in the region. Most of the outbreaks are caused by serotype O followed by serotype A. The lack of concerted FMD control programmes in Africa has provided little incentive for vaccine producers to select vaccines that are tailored to circulating regional isolates creating further negative feedback to deter the introduction of vaccine-based control schemes. In this study a total of 80 serotype O FMD viruses (FMDV) isolated from 1993 to 2012 from East and North Africa were characterized by virus neutralisation tests using bovine antisera to three existing (O/KEN/77/78, O/Manisa and O/PanAsia-2) and three putative (O/EA/2002, O/EA/2009 and O/EA/2010) vaccine strains and by capsid sequencing. Genetically, these viruses were grouped as either of East African origin with subdivision into four topotypes (EA-1, 2, 3 and 4) or of Middle-East South Asian (ME-SA) topotype. The ME-SA topotype viruses were mainly detected in Egypt and Libya reflecting the trade links with the Middle East countries. There was good serological cross-reactivity between the vaccine strains and most of the field isolates analysed, indicating that vaccine selection should not be a major constraint for control of serotype O FMD by vaccination, and that both local and internationally available commercial vaccines could be used. The O/KEN/77/78 vaccine, commonly used in the region, exhibited comparatively lower percent in vitro match against the predominant topotypes (EA-2 and EA-3) circulating in the region whereas O/PanAsia-2 and O/Manisa vaccines revealed broader protection against East African serotype O viruses, even though they genetically belong to the ME-SA topotype. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

  2. Ultramicroscopy as a novel tool to unravel the tropism of AAV gene therapy vectors in the brain.

    PubMed

    Alves, Sandro; Bode, Julia; Bemelmans, Alexis-Pierre; von Kalle, Christof; Cartier, Nathalie; Tews, Björn

    2016-06-20

    Recombinant adeno-associated viral (AAV) vectors have advanced to the vanguard of gene therapy. Numerous naturally occurring serotypes have been used to target cells in various tissues. There is a strong need for fast and dynamic methods which efficiently unravel viral tropism in whole organs. Ultramicroscopy (UM) is a novel fluorescence microscopy technique that images optically cleared undissected specimens, achieving good resolutions at high penetration depths while being non-destructive. UM was applied to obtain high-resolution 3D analysis of AAV transduction in adult mouse brains, especially in the hippocampus, a region of interest for Alzheimer's disease therapy. We separately or simultaneously compared transduction efficacies for commonly used serotypes (AAV9 and AAVrh10) using fluorescent reporter expression. We provide a detailed comparative and quantitative analysis of the transduction profiles. UM allowed a rapid analysis of marker fluorescence expression in neurons with intact projections deep inside the brain, in defined anatomical structures. Major hippocampal neuronal transduction was observed with both vectors, with slightly better efficacy for AAV9 in UM. Glial response and synaptic marker expression did not change post transduction.We propose UM as a novel valuable complementary tool to efficiently and simultaneously unravel tropism of different viruses in a single non-dissected adult rodent brain.

  3. Comparison of immune responses to different foot-and-mouth disease genetically engineered vaccines in guinea pigs.

    PubMed

    Yao, Qingxia; Qian, Ping; Huang, Qinfeng; Cao, Yi; Chen, Huanchun

    2008-01-01

    The P12A3C gene from FMDV (serotype O) encoding the capsid precursor protein, and the highly immunogenic gene FHG, which encodes multiple epitopes of FMDV capsid proteins, were inserted into eukaryotic expression vectors to compare different candidate genetically engineered vaccines for foot-and-mouth disease (FMD). A modified live pseudorabies virus (MLPRV) was also used to deliver P12A3C. Guinea pigs were inoculated intramuscularly with the candidate vaccines to compare the ability to elicit immunity of the DNA vector and a live viral vector. An indirect enzyme-linked immunosorbent assay (iELISA), virus-neutralization test and lymphoproliferation assay were used to detect antibody and cellular responses. The group immunized with P12A3C delivered by MLPRV produced significantly greater antibody and cellular responses indicating that MLPRV has a greater ability to mediate exogenous gene delivery than the plasmid DNA vector. Comparison of the immune responses induced by P12A3C and FHG, which were both mediated by DNA plasmids, showed that FHG and P12A3C elicited similar cellular responses, while P12A3C induced higher antibody levels, suggesting that P12A3C is a more powerful immunogen than FHG. In challenge experiments, guinea pigs vaccinated with P12A3C delivered by MLPRV were protected fully from FMDV challenge, whereas guinea pigs vaccinated with P12A3C or FHG delivered by DNA plasmid were only protected partially. This study provides a basis for future construction of a genetically engineered vaccine for FMDV.

  4. Increase in Genetic Diversity of Haemophilus influenzae Serotype b (Hib) Strains after Introduction of Hib Vaccination in The Netherlands

    PubMed Central

    Schouls, Leo M.; van der Ende, Arie; van de Pol, Ingrid; Schot, Corrie; Spanjaard, Lodewijk; Vauterin, Paul; Wilderbeek, Dorus; Witteveen, Sandra

    2005-01-01

    Recently, there has been an increase in The Netherlands in the number of cases of invasive disease caused by Haemophilus influenzae serotype b (Hib). To study a possible change in the Hib population that could explain the rise in incidence, a multiple-locus variable number tandem repeats analysis (MLVA) was developed to genotype H. influenzae isolates. The MLVA enabled the differentiation of H. influenzae serotype b strains with higher discriminatory power than multilocus sequence typing (MLST). MLVA profiles of noncapsulated H. influenzae and H. influenzae serotype f strains were more heterogeneous than serotype b strains and were distinct from Hib, although some overlap occurred. The MLVA was used to genotype a collection of 520 H. influenzae serotype b strains isolated from patients in The Netherlands with invasive disease. The strains were collected from 1983 from 2002, covering a time period of 10 years before and 9 years after the introduction of the Hib vaccine in the Dutch national vaccination program. MLVA revealed a sharp increase in genetic diversity of Hib strains isolated from neonates to 4-year-old patients after 1993, when the Hib vaccine was introduced. Hib strains isolated from patients older than 4 years in age were genetically diverse, and no significant change in diversity was seen after the introduction of the vaccine. These observations suggest that after the introduction of the Hib vaccine young children no longer constitute the reservoir for Hib and that they are infected by adults carrying genetically diverse Hib strains. PMID:15956392

  5. Genetic Polymorphisms in Host Antiviral Genes: Associations with Humoral and Cellular Immunity to Measles Vaccine

    PubMed Central

    Haralambieva, Iana H.; Ovsyannikova, Inna G.; Umlauf, Benjamin J.; Vierkant, Robert A.; Pankratz, V. Shane; Jacobson, Robert M.; Poland, Gregory A.

    2014-01-01

    Host antiviral genes are important regulators of antiviral immunity and plausible genetic determinants of immune response heterogeneity after vaccination. We genotyped and analyzed 307 common candidate tagSNPs from 12 antiviral genes in a cohort of 745 schoolchildren immunized with two doses of measles-mumps-rubella vaccine. Associations between SNPs/haplotypes and measles virus-specific immune outcomes were assessed using linear regression methodologies in Caucasians and African-Americans. Genetic variants within the DDX58/RIG-I gene, including a coding polymorphism (rs3205166/Val800Val), were associated as single-SNPs (p≤0.017; although these SNPs did not remain significant after correction for false discovery rate/FDR) and in haplotype-level analysis, with measles-specific antibody variations in Caucasians (haplotype allele p-value=0.021; haplotype global p-value=0.076). Four DDX58 polymorphisms, in high LD, demonstrated also associations (after correction for FDR) with variations in both measles-specific IFN-γ and IL-2 secretion in Caucasians (p≤0.001, q=0.193). Two intronic OAS1 polymorphisms, including the functional OAS1 SNP rs10774671 (p=0.003), demonstrated evidence of association with a significant allele-dose-related increase in neutralizing antibody levels in African-Americans. Genotype and haplotype-level associations demonstrated the role of ADAR genetic variants, including a non-synonymous SNP (rs2229857/Arg384Lys; p=0.01), in regulating measles virus-specific IFN-γ Elispot responses in Caucasians (haplotype global p-value=0.017). After correction FDR, 15 single-SNP associations (11 SNPs in Caucasians and 4 SNPs in African-Americans) still remained significant at the q-value<0.20. In conclusion, our findings strongly point to genetic variants/genes, involved in antiviral sensing and antiviral control, as critical determinants, differentially modulating the adaptive immune responses to live attenuated measles vaccine in Caucasians and African

  6. Genetically detoxified pertussis toxin (PT-9K/129G): implications for immunization and vaccines.

    PubMed

    Seubert, Anja; D'Oro, Ugo; Scarselli, Maria; Pizza, Mariagrazia

    2014-10-01

    Pertussis toxin (PT) is one of the major virulence factors of Bordetella pertussis and the primary component of all pertussis vaccines available to date. Because of its various noxious effects the toxin needs to be detoxified. In all currently available vaccines, detoxification is achieved by treatment with high quantity of chemical agents such as formaldehyde, glutaraldehyde or hydrogen peroxide. Although effective in detoxification, this chemical treatment alters dramatically the immunological properties of the toxin. In contrast, PT genetically detoxified through the substitution of two residues necessary for its enzymatic activity maintains all functional and immunological properties. This review describes in detail the characteristics of this PT-9K/129G mutant and shows that it is non-toxic and a superior immunogen compared with chemically detoxified PT. Importantly, data from an efficacy trial show that the PT-9K/129G-based vaccine induces earlier and longer-lasting protection, further supporting the hypothesis that PT-9K/129G represents an ideal candidate for future pertussis vaccine formulations.

  7. Gene therapy using self-complementary Y733F capsid mutant AAV2/8 restores vision in a model of early onset Leber congenital amaurosis.

    PubMed

    Ku, Cristy A; Chiodo, Vince A; Boye, Sanford L; Goldberg, Andrew F X; Li, Tiansen; Hauswirth, William W; Ramamurthy, Visvanathan

    2011-12-01

    Defects in the photoreceptor-specific gene aryl hydrocarbon receptor interacting protein-like 1 (Aipl1) are associated with Leber congenital amaurosis (LCA), a childhood blinding disease with early-onset retinal degeneration and vision loss. Furthermore, Aipl1 defects are characterized at the most severe end of the LCA spectrum. The rapid photoreceptor degeneration and vision loss observed in the LCA patient population are mimicked in a mouse model lacking AIPL1. Using this model, we evaluated if gene replacement therapy using recent advancements in adeno-associated viral vectors (AAV) provides advantages in preventing rapid retinal degeneration. Specifically, we demonstrated that the novel self-complementary Y733F capsid mutant AAV2/8 (sc-Y733F-AAV) provided greater preservation of photoreceptors and functional vision in Aipl1 null mice compared with single-stranded AAV2/8. The benefits of sc-Y733F-AAV were evident following viral administration during the active phase of retinal degeneration, where only sc-Y733F-AAV treatment achieved functional vision rescue. This result was likely due to higher and earlier onset of Aipl1 expression. Based on our studies, we conclude that the sc-Y733F-AAV2/8 viral vector, to date, achieves the best rescue for rapid retinal degeneration in Aipl1 null mice. Our results provide important considerations for viral vectors to be used in future gene therapy clinical trials targeting a wider severity spectrum of inherited retinal dystrophies.

  8. Generation by Reverse Genetics of an Effective, Stable, Live-Attenuated Newcastle Disease Virus Vaccine Based on a Currently Circulating, Highly Virulent Indonesian Strain

    PubMed Central

    Xiao, Sa; Nayak, Baibaswata; Samuel, Arthur; Paldurai, Anandan; Kanabagattebasavarajappa, Mallikarjuna; Prajitno, Teguh Y.; Bharoto, Eny E.; Collins, Peter L.; Samal, Siba K.

    2012-01-01

    Newcastle disease virus (NDV) can cause severe disease in chickens. Although NDV vaccines exist, there are frequent reports of outbreaks in vaccinated chickens. During 2009–2010, despite intense vaccination, NDV caused major outbreaks among commercial poultry farms in Indonesia. These outbreaks raised concern regarding the protective immunity of current vaccines against circulating virulent strains in Indonesia. In this study, we investigated whether a recombinant attenuated Indonesian NDV strain could provide better protection against prevalent Indonesian viruses. A reverse genetics system for the highly virulent NDV strain Banjarmasin/010/10 (Ban/010) isolated in Indonesia in 2010 was constructed. The Ban/010 virus is classified in genotype VII of class II NDV, which is genetically distinct from the commercial vaccine strains B1 and LaSota, which belong to genotype II, and shares only 89 and 87% amino acid identity for the protective antigens F and HN, respectively. A mutant virus, named Ban/AF, was developed in which the virulent F protein cleavage site motif “RRQKR↓F” was modified to an avirulent motif “GRQGR↓L” by three amino acid substitutions (underlined). The Ban/AF vaccine virus did not produce syncytia or plaques in cell culture, even in the presence of added protease. Pathogenicity tests showed that Ban/AF was completely avirulent. Ban/AF replicated efficiently during 10 consecutive passages in chickens and remained genetically stable. Serological analysis showed that Ban/AF induced higher neutralization and hemagglutination inhibition antibody titers against the prevalent viruses than the commercial vaccines B1 or LaSota. Both Ban/AF and commercial vaccines provided protection against clinical disease and mortality after challenge with virulent NDV strain Ban/010 (genotype VII) or GB Texas (genotype II). However, Ban/AF significantly reduced challenge virus shedding from the vaccinated birds compared to B1 vaccine. These results suggest

  9. Application of pharmacogenomics to vaccines

    PubMed Central

    Poland, Gregory A; Ovsyannikova, Inna G; Jacobson, Robert M

    2009-01-01

    The field of pharmacogenomics and pharmacogenetics provides a promising science base for vaccine research and development. A broad range of phenotype/genotype data combined with high-throughput genetic sequencing and bioinformatics are increasingly being integrated into this emerging field of vaccinomics. This paper discusses the hypothesis of the ‘immune response gene network’ and genetic (and bioinformatic) strategies to study associations between immune response gene polymorphisms and variations in humoral and cellular immune responses to prophylactic viral vaccines, such as measles–mumps–rubella, influenza, HIV, hepatitis B and smallpox. Immunogenetic studies reveal promising new vaccine targets by providing a better understanding of the mechanisms by which gene polymorphisms may influence innate and adaptive immune responses to vaccines, including vaccine failure and vaccine-associated adverse events. Additional benefits from vaccinomic studies include the development of personalized vaccines, the development of novel vaccines and the development of novel vaccine adjuvants. PMID:19450131

  10. A Single Intravenous rAAV Injection as Late as P20 Achieves Efficacious and Sustained CNS Gene Therapy in Canavan Mice

    PubMed Central

    Ahmed, Seemin Seher; Li, Huapeng; Cao, Chunyan; Sikoglu, Elif M; Denninger, Andrew R; Su, Qin; Eaton, Samuel; Liso Navarro, Ana A; Xie, Jun; Szucs, Sylvia; Zhang, Hongwei; Moore, Constance; Kirschner, Daniel A; Seyfried, Thomas N; Flotte, Terence R; Matalon, Reuben; Gao, Guangping

    2013-01-01

    Canavan's disease (CD) is a fatal pediatric leukodystrophy caused by mutations in aspartoacylase (AspA) gene. Currently, there is no effective treatment for CD; however, gene therapy is an attractive approach to ameliorate the disease. Here, we studied progressive neuropathology and gene therapy in short-lived (≤1 month) AspA−/− mice, a bona-fide animal model for the severest form of CD. Single intravenous (IV) injections of several primate-derived recombinant adeno-associated viruses (rAAVs) as late as postnatal day 20 (P20) completely rescued their early lethality and alleviated the major disease symptoms, extending survival in P0-injected rAAV9 and rAAVrh8 groups to as long as 2 years thus far. We successfully used microRNA (miRNA)-mediated post-transcriptional detargeting for the first time to restrict therapeutic rAAV expression in the central nervous system (CNS) and minimize potentially deleterious effects of transgene overexpression in peripheral tissues. rAAV treatment globally improved CNS myelination, although some abnormalities persisted in the content and distribution of myelin-specific and -enriched lipids. We demonstrate that systemically delivered and CNS-restricted rAAVs can serve as efficacious and sustained gene therapeutics in a model of a severe neurodegenerative disorder even when administered as late as P20. PMID:23817205

  11. Biological characteristics of genetic variants of Urabe AM9 mumps vaccine virus.

    PubMed

    Wright, K E; Dimock, K; Brown, E G

    2000-03-01

    The Urabe AM9 mumps vaccine is composed of a mixture of variants distinguishable by a difference at nucleotide (nt) 1081 of the hemagglutinin-neuraminidase (HN) gene (Brown, E.G., Dimock, K., Wright, K.E., 1996. The Urabe AM9 mumps vaccine is a mixture of viruses differing at amino acid (aa) 335 of the hemagglutinin-neuraminidase gene with one form associated with disease. J. Infect. Dis. 174, 619-622.). Further genetic and biological variation was detected in plaque purified viruses from the Urabe AM9 vaccine by examining the HN gene sequence, plaque morphology, cytopathic effects and growth in Vero cells, and temperature sensitivity (ts). Infection of Vero cells with plaque purified viruses with a G at nt 1081 of the HN gene produced large, clear plaques, caused significant CPE early after infection but yielded lower titres of virus than other purified viruses. None of these viruses were ts. In contrast, half of the plaque purified viruses with an A at nt 1081 were sensitive to a temperature of 39.5 degrees C. These viruses produced small plaques, caused significant CPE and grew to low titres. Two ts viruses possessed a unique aa substitution at aa 468 of HN. The remaining A(1081) viruses were not ts, produced large plaques but little CPE, and grew to titres 10-fold higher than the G(1081) viruses. Isolates of Urabe AM9 associated with post-vaccination illness were similar to these non-ts A(1081) viruses, but could be further sub-divided into two groups on the basis of a difference at aa 464 of HN. The post-vaccination isolates may represent insufficiently attenuated components of the vaccine, while the G(1081) and ts subset of A(1081) viruses may be more fully attenuated.

  12. Promise and problems associated with the use of recombinant AAV for the delivery of anti-HIV antibodies

    PubMed Central

    Fuchs, Sebastian P; Desrosiers, Ronald C

    2016-01-01

    Attempts to elicit antibodies with potent neutralizing activity against a broad range of human immunodeficiency virus (HIV) isolates have so far proven unsuccessful. Long-term delivery of monoclonal antibodies (mAbs) with such activity is a creative alternative that circumvents the need for an immune response and has the potential for creating a long-lasting sterilizing barrier against HIV. This approach is made possible by an incredible array of potent broadly neutralizing antibodies (bnAbs) that have been identified over the last several years. Recombinant adeno-associated virus (rAAV) vectors are ideally suited for long-term delivery for a variety of reasons. The only products made from rAAV are derived from the transgenes that are put into it; as long as those products are not viewed as foreign, expression from muscle tissue may continue for decades. Thus, use of rAAV to achieve long-term delivery of anti-HIV mAbs with potent neutralizing activity against a broad range of HIV-1 isolates is emerging as a promising concept for the prevention or treatment of HIV-1 infection in humans. Experiments in mice and monkeys that have demonstrated protective efficacy against AIDS virus infection have raised hopes for the promise of this approach. However, all published experiments in monkeys have encountered unwanted immune responses to the AAV-delivered antibody, and these immune responses appear to limit the levels of delivered antibody that can be achieved. In this review, we highlight the promise of rAAV-mediated antibody delivery for the prevention or treatment of HIV infection in humans, but we also discuss the obstacles that will need to be understood and solved in order for the promise of this approach to be realized. PMID:28197421

  13. Efficient CRISPR-rAAV engineering of endogenous genes to study protein function by allele-specific RNAi.

    PubMed

    Kaulich, Manuel; Lee, Yeon J; Lönn, Peter; Springer, Aaron D; Meade, Bryan R; Dowdy, Steven F

    2015-04-20

    Gene knockout strategies, RNAi and rescue experiments are all employed to study mammalian gene function. However, the disadvantages of these approaches include: loss of function adaptation, reduced viability and gene overexpression that rarely matches endogenous levels. Here, we developed an endogenous gene knockdown/rescue strategy that combines RNAi selectivity with a highly efficient CRISPR directed recombinant Adeno-Associated Virus (rAAV) mediated gene targeting approach to introduce allele-specific mutations plus an allele-selective siRNA Sensitive (siSN) site that allows for studying gene mutations while maintaining endogenous expression and regulation of the gene of interest. CRISPR/Cas9 plus rAAV targeted gene-replacement and introduction of allele-specific RNAi sensitivity mutations in the CDK2 and CDK1 genes resulted in a >85% site-specific recombination of Neo-resistant clones versus ∼8% for rAAV alone. RNAi knockdown of wild type (WT) Cdk2 with siWT in heterozygotic knockin cells resulted in the mutant Cdk2 phenotype cell cycle arrest, whereas allele specific knockdown of mutant CDK2 with siSN resulted in a wild type phenotype. Together, these observations demonstrate the ability of CRISPR plus rAAV to efficiently recombine a genomic locus and tag it with a selective siRNA sequence that allows for allele-selective phenotypic assays of the gene of interest while it remains expressed and regulated under endogenous control mechanisms. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. AAV-Mediated Clarin-1 Expression in the Mouse Retina: Implications for USH3A Gene Therapy.

    PubMed

    Dinculescu, Astra; Stupay, Rachel M; Deng, Wen-Tao; Dyka, Frank M; Min, Seok-Hong; Boye, Sanford L; Chiodo, Vince A; Abrahan, Carolina E; Zhu, Ping; Li, Qiuhong; Strettoi, Enrica; Novelli, Elena; Nagel-Wolfrum, Kerstin; Wolfrum, Uwe; Smith, W Clay; Hauswirth, William W

    2016-01-01

    Usher syndrome type III (USH3A) is an autosomal recessive disorder caused by mutations in clarin-1 (CLRN1) gene, leading to progressive retinal degeneration and sensorineural deafness. Efforts to develop therapies for preventing photoreceptor cell loss are hampered by the lack of a retinal phenotype in the existing USH3 mouse models and by conflicting reports regarding the endogenous retinal localization of clarin-1, a transmembrane protein of unknown function. In this study, we used an AAV-based approach to express CLRN1 in the mouse retina in order to determine the pattern of its subcellular localization in different cell types. We found that all major classes of retinal cells express AAV-delivered CLRN1 driven by the ubiquitous, constitutive small chicken β-actin promoter, which has important implications for the design of future USH3 gene therapy studies. Within photoreceptor cells, AAV-expressed CLRN1 is mainly localized at the inner segment region and outer plexiform layer, similar to the endogenous expression of other usher proteins. Subretinal delivery using a full strength viral titer led to significant loss of retinal function as evidenced by ERG analysis, suggesting that there is a critical limit for CLRN1 expression in photoreceptor cells. Taken together, these results suggest that CLRN1 expression is potentially supported by a variety of retinal cells, and the right combination of AAV vector dose, promoter, and delivery method needs to be selected to develop safe therapies for USH3 disorder.

  15. [Preventive and therapeutic effect of genetic vaccine based on recombinant alpha virus against mouse mastocytoma P815].

    PubMed

    Ni, Bing; Yang, Ri-gao; Li, Yan-qiu; Wu, Yu-zhang

    2004-01-01

    To explore the immunological effect of genetic vaccine based on alpha-virus and to seek out better forms of gene vaccines. Expression plasmid P1A/pSMART2a and packaging plasmid helper were cotransfected into mammalian 293 cells by calcium phosphate precipitation method and high level of recombinant alpha-virus P1A/SFV was prepared. Following identification of rSFV and its expression, BALB/c mice were inoculated with rSFV, and the production of antigen-specific antibody and the cytotoxic effect of CTLs were determined. In the preventive and therapeutic experiments, the percents of tumor-free and of survival mice immunized with rSFV were observed. The recombinant SFV could express correctly in cultured cells. After being inoculated into the mice, rSFV could prime stronger CTL response than that in control mice. When the ratio of E/T cells was 100:1, the (51)Cr release rate reached 75%. No antibody could be detected in mice from all groups. The immunological effect of P1A/SFV among all groups was the best in both preventive and therapeutic experiment within experimental deadline. On 60th day in preventive experiment, the percent of tumor-free animal in P1A/SFV group reached 60%, whereas that was only 20% in P1A/pCI-neogroup. On 60th day in therapeutic experiment, survival rate of mice in P1A/SFV group reached 50%, but only 10% mice could survive in all control groups. Compared with common gene vaccines, the genetic vaccine based on recombinant SFV has the best immunological effect, which provides some new strategies for clinical genetic therapy of tumors.

  16. Killed whole-HIV vaccine; employing a well established strategy for antiviral vaccines.

    PubMed

    Kang, C Yong; Gao, Yong

    2017-09-12

    The development of an efficient prophylactic HIV vaccine has been one of the major challenges in infectious disease research during the last three decades. Here, we present a mini review on strategies employed for the development of HIV vaccines with an emphasis on a well-established vaccine technology, the killed whole-virus vaccine approach. Recently, we reported an evaluation of the safety and the immunogenicity of a genetically modified and killed whole-HIV-1 vaccine designated as SAV001 [1]. HIV-1 Clade B NL4-3 was genetically modified by deleting the nef and vpu genes and substituting the coding sequence of the Env signal peptide with that of honeybee melittin to produce an avirulent and replication efficient HIV-1. This genetically modified virus (gmHIV-1 NL4-3 ) was propagated in a human T cell line followed by virus purification and inactivation by aldrithiol-2 and γ-irradiation. We found that SAV001 was well tolerated with no serious adverse events. HIV-1 NL4-3 -specific polymerase chain reaction showed no evidence of vaccine virus replication in participants receiving SAV001 and in human T cells infected in vitro. Furthermore, SAV001 with an adjuvant significantly increased the antibody response to HIV-1 structural proteins. Moreover, antibodies in the plasma from these vaccinations neutralized tier I and tier II of HIV-1 B, A, and D subtypes. These results indicated that the killed whole-HIV vaccine is safe and may trigger appropriate immune responses to prevent HIV infection. Utilization of this killed whole-HIV vaccine strategy may pave the way to develop an effective HIV vaccine.

  17. Genetically Engineered, Live Attenuated Vaccines Protect Nonhuman Primates Against Aerosol Challenge with a Virulent IE Strain of Venezuelan Equine Encephalitis Virus

    DTIC Science & Technology

    2005-01-21

    integrated moving average ( ARIMA ) model [15,19]. Fore- casted values for the postexposure time periods were based on the training model extrapolated...Smith JF. Genetically engineered, live attenuated vaccines or Venezuelan equine encephalitis: testing in animal models . Vaccine 2003;21(25–26):3854–62...encephalitis: testing in animal models . Vaccine 2003;21(25-26):3854-62] and IE strains of VEE viruses. 15. SUBJECT TERMS Venezuelan equine

  18. Intrajugular Vein Delivery of AAV9-RNAi Prevents Neuropathological Changes and Weight Loss in Huntington's Disease Mice

    PubMed Central

    Dufour, Brett D; Smith, Catherine A; Clark, Randall L; Walker, Timothy R; McBride, Jodi L

    2014-01-01

    Huntington's disease (HD) is a fatal neurological disorder caused by a CAG repeat expansion in the HTT gene, which encodes a mutant huntingtin protein (mHTT). The mutation confers a toxic gain of function on huntingtin, leading to widespread neurodegeneration and inclusion formation in many brain regions. Although the hallmark symptom of HD is hyperkinesia stemming from striatal degeneration, several other brain regions are affected which cause psychiatric, cognitive, and metabolic symptoms. Additionally, mHTT expression in peripheral tissue is associated with skeletal muscle atrophy, cardiac failure, weight loss, and diabetes. We, and others, have demonstrated a prevention of motor symptoms in HD mice following direct striatal injection of adeno-associated viral vector (AAV) serotype 1 encoding an RNA interference (RNAi) construct targeting mutant HTT mRNA (mHTT). Here, we expand these efforts and demonstrate that an intrajugular vein injection of AAV serotype 9 (AAV9) expressing a mutant HTT-specific RNAi construct significantly reduced mHTT expression in multiple brain regions and peripheral tissues affected in HD. Correspondingly, this approach prevented atrophy and inclusion formation in key brain regions as well as the severe weight loss germane to HD transgenic mice. These results demonstrate that systemic delivery of AAV9-RNAi may provide more widespread clinical benefit for patients suffering from HD. PMID:24390280

  19. Convection-enhanced delivery of AAV2 in white matter--a novel method for gene delivery to cerebral cortex.

    PubMed

    Barua, N U; Woolley, M; Bienemann, A S; Johnson, D; Wyatt, M J; Irving, C; Lewis, O; Castrique, E; Gill, S S

    2013-10-30

    Convection-enhanced delivery (CED) is currently under investigation for delivering therapeutic agents to subcortical targets in the brain. Direct delivery of therapies to the cerebral cortex, however, remains a significant challenge. We describe a novel method of targeting adeno-associated viral vector (AAV) mediated gene therapies to specific cerebral cortical regions by performing high volume, high flow rate infusions into underlying white matter in a large animal (porcine) model. Infusion volumes of up to 700 μl at flow rates as high as 10 μl/min were successfully performed in white matter without adverse neurological sequelae. Co-infusion of AAV2/5-GFP with 0.2% Gadolinium in artificial CSF confirmed transgene expression in the deep layers of cerebral cortex overlying the infused areas of white matter. AAV-mediated gene therapies have been previously targeted to the cerebral cortex by performing intrathalamic CED and exploiting axonal transport. The novel method described in this study facilitates delivery of gene therapies to specific regions of the cerebral cortex without targeting deep brain structures. AAV-mediated gene therapies can be targeted to specific cortical regions by performing CED into underlying white matter. This technique could be applied to the treatment of neurological disorders characterised by cerebral cortical degeneration. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. DNA-launched live-attenuated vaccines for biodefense applications

    PubMed Central

    Pushko, Peter; Lukashevich, Igor S.; Weaver, Scott C.; Tretyakova, Irina

    2016-01-01

    Summary A novel vaccine platform uses DNA immunization to launch live-attenuated virus vaccines in vivo. This technology has been applied for vaccine development against positive-strand RNA viruses with global public health impact including alphaviruses and flaviviruses. The DNA-launched vaccine represents the recombinant plasmid that encodes the full-length genomic RNA of live-attenuated virus downstream from a eukaryotic promoter. When administered in vivo, the genomic RNA of live-attenuated virus is transcribed. The RNA initiates limited replication of a genetically defined, live-attenuated vaccine virus in the tissues of the vaccine recipient, thereby inducing a protective immune response. This platform combines the strengths of reverse genetics, DNA immunization and the advantages of live-attenuated vaccines, resulting in a reduced chance of genetic reversions, increased safety, and improved immunization. With this vaccine technology, the field of DNA vaccines is expanded from those that express subunit antigens to include a novel type of DNA vaccines that launch live-attenuated viruses. PMID:27055100

  1. Sieve analysis in HIV-1 vaccine efficacy trials.

    PubMed

    Edlefsen, Paul T; Gilbert, Peter B; Rolland, Morgane

    2013-09-01

    The genetic characterization of HIV-1 breakthrough infections in vaccine and placebo recipients offers new ways to assess vaccine efficacy trials. Statistical and sequence analysis methods provide opportunities to mine the mechanisms behind the effect of an HIV vaccine. The release of results from two HIV-1 vaccine efficacy trials, Step/HVTN-502 (HIV Vaccine Trials Network-502) and RV144, led to numerous studies in the last 5 years, including efforts to sequence HIV-1 breakthrough infections and compare viral characteristics between the vaccine and placebo groups. Novel genetic and statistical analysis methods uncovered features that distinguished founder viruses isolated from vaccinees from those isolated from placebo recipients, and identified HIV-1 genetic targets of vaccine-induced immune responses. Studies of HIV-1 breakthrough infections in vaccine efficacy trials can provide an independent confirmation to correlates of risk studies, as they take advantage of vaccine/placebo comparisons, whereas correlates of risk analyses are limited to vaccine recipients. Through the identification of viral determinants impacted by vaccine-mediated host immune responses, sieve analyses can shed light on potential mechanisms of vaccine protection.

  2. AAV-mediated in vivo functional selection of tissue-protective factors against ischaemia

    PubMed Central

    Ruozi, Giulia; Bortolotti, Francesca; Falcione, Antonella; Dal Ferro, Matteo; Ukovich, Laura; Macedo, Antero; Zentilin, Lorena; Filigheddu, Nicoletta; Cappellari, Gianluca Gortan; Baldini, Giovanna; Zweyer, Marina; Barazzoni, Rocco; Graziani, Andrea; Zacchigna, Serena; Giacca, Mauro

    2015-01-01

    Functional screening of expression libraries in vivo would offer the possibility of identifying novel biotherapeutics without a priori knowledge of their biochemical function. Here we describe a procedure for the functional selection of tissue-protective factors based on the in vivo delivery of arrayed cDNA libraries from the mouse secretome using adeno-associated virus (AAV) vectors. Application of this technique, which we call FunSel, in the context of acute ischaemia, revealed that the peptide ghrelin protects skeletal muscle and heart from ischaemic damage. When delivered to the heart using an AAV9 vector, ghrelin markedly reduces infarct size and preserves cardiac function over time. This protective activity associates with the capacity of ghrelin to sustain autophagy and remove dysfunctional mitochondria after myocardial infarction. Our findings describe an innovative tool to identify biological therapeutics and reveal a novel role of ghrelin as an inducer of myoprotective autophagy. PMID:26066847

  3. A recombinant vesicular stomatitis virus-based Lassa fever vaccine protects guinea pigs and macaques against challenge with geographically and genetically distinct Lassa viruses.

    PubMed

    Safronetz, David; Mire, Chad; Rosenke, Kyle; Feldmann, Friederike; Haddock, Elaine; Geisbert, Thomas; Feldmann, Heinz

    2015-04-01

    Lassa virus (LASV) is endemic in several West African countries and is the etiological agent of Lassa fever. Despite the high annual incidence and significant morbidity and mortality rates, currently there are no approved vaccines to prevent infection or disease in humans. Genetically, LASV demonstrates a high degree of diversity that correlates with geographic distribution. The genetic heterogeneity observed between geographically distinct viruses raises concerns over the potential efficacy of a "universal" LASV vaccine. To date, several experimental LASV vaccines have been developed; however, few have been evaluated against challenge with various genetically unique Lassa virus isolates in relevant animal models. Here we demonstrate that a single, prophylactic immunization with a recombinant vesicular stomatitis virus (VSV) expressing the glycoproteins of LASV strain Josiah from Sierra Leone protects strain 13 guinea pigs from infection / disease following challenge with LASV isolates originating from Liberia, Mali and Nigeria. Similarly, the VSV-based LASV vaccine yields complete protection against a lethal challenge with the Liberian LASV isolate in the gold-standard macaque model of Lassa fever. Our results demonstrate the VSV-based LASV vaccine is capable of preventing morbidity and mortality associated with non-homologous LASV challenge in two animal models of Lassa fever. Additionally, this work highlights the need for the further development of disease models for geographical distinct LASV strains, particularly those from Nigeria, in order to comprehensively evaluate potential vaccines and therapies against this prominent agent of viral hemorrhagic fever.

  4. Genetic diversity of transmission-blocking vaccine candidate Pvs48/45 in Plasmodium vivax populations in China.

    PubMed

    Feng, Hui; Gupta, Bhavna; Wang, Meilian; Zheng, Wenqi; Zheng, Li; Zhu, Xiaotong; Yang, Yimei; Fang, Qiang; Luo, Enjie; Fan, Qi; Tsuboi, Takafumi; Cao, Yaming; Cui, Liwang

    2015-12-01

    The male gamete fertilization factor P48/45 in malaria parasites is a prime transmission-blocking vaccine (TBV) candidate. Efforts to develop antimalarial vaccines are often thwarted by genetic diversity of the target antigens. Here we evaluated the genetic diversity of Pvs48/45 gene in global Plasmodium vivax populations. We determined 200 Pvs48/45 sequences collected from temperate and subtropical parasite populations in China. Population genetic and evolutionary analyses were performed to determine the levels of genetic diversity, potential signature of selection, and population differentiation. Analysis of the Pvs48/45 sequences from 200 P. vivax parasites collected in a temperate and a tropical region revealed a low level of genetic diversity (π = 0.0012) with 14 single nucleotide polymorphisms, of which 11 were nonsynonymous. Analysis of 344 Pvs48/45 sequences from nine worldwide P. vivax populations detected a total of 38 haplotypes, of which 13 haplotypes were present only once. Multiple tests for selection confirmed a signature of positive selection on Pvs48/45 with selection skewed to the second cysteine domain. Haplotype network analysis and Wright's fixation index showed large geographical differentiation with the presence of continent-or region-specific mutations in this gene. Pvs48/45 displays low levels of genetic diversity with the presence of region-specific mutations. Some of the mutations may be potential epitope targets based on their positions in the predicted structure, highlighting the need for future evaluation of these mutations in designing Pvs48/45-based TBV.

  5. AAV-mediated targeting of gene expression to the peri-infarct region in rat cortical stroke model.

    PubMed

    Mätlik, Kert; Abo-Ramadan, Usama; Harvey, Brandon K; Arumäe, Urmas; Airavaara, Mikko

    2014-10-30

    For stroke patients the recovery of cognitive and behavioral functions is often incomplete. Functional recovery is thought to be mediated largely by connectivity rearrangements in the peri-infarct region. A method for manipulating gene expression in this region would be useful for identifying new recovery-enhancing treatments. We have characterized a way of targeting adeno-associated virus (AAV) vectors to the peri-infarct region of cortical ischemic lesion in rats 2days after middle cerebral artery occlusion (MCAo). We used magnetic resonance imaging (MRI) to show that the altered properties of post-ischemic brain tissue facilitate the spreading of intrastriatally injected nanoparticles toward the infarct. We show that subcortical injection of green fluorescent protein-encoding dsAAV7-GFP resulted in transduction of cells in and around the white matter tract underlying the lesion, and in the cortex proximal to the lesion. A similar result was achieved with dsAAV7 vector encoding the cerebral dopamine neurotrophic factor (CDNF), a protein with therapeutic potential. Viral vector-mediated intracerebral gene delivery has been used before in rodent models of ischemic injury. However, the method of targeting gene expression to the peri-infarct region, after the initial phase of ischemic cell death, has not been described before. We demonstrate a straightforward and robust way to target AAV vector-mediated over-expression of genes to the peri-infarct region in a rat stroke model. This method will be useful for studying the action of specific proteins in peri-infarct region during the recovery process. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Rhodococcus equi (Prescottella equi) vaccines; the future of vaccine development.

    PubMed

    Giles, C; Vanniasinkam, T; Ndi, S; Barton, M D

    2015-09-01

    For decades researchers have been targeting prevention of Rhodococcus equi (Rhodococcus hoagui/Prescottella equi) by vaccination and the horse breeding industry has supported the ongoing efforts by researchers to develop a safe and cost effective vaccine to prevent disease in foals. Traditional vaccines including live, killed and attenuated (physical and chemical) vaccines have proved to be ineffective and more modern molecular-based vaccines including the DNA plasmid, genetically attenuated and subunit vaccines have provided inadequate protection of foals. Newer, bacterial vector vaccines have recently shown promise for R. equi in the mouse model. This article describes the findings of key research in R. equi vaccine development and looks at alternative methods that may potentially be utilised. © 2014 EVJ Ltd.

  7. Single residue AAV capsid mutation improves transduction of photoreceptors in the Abca4-/- mouse and bipolar cells in the rd1 mouse and human retina ex vivo.

    PubMed

    De Silva, Samantha R; Charbel Issa, Peter; Singh, Mandeep S; Lipinski, Daniel M; Barnea-Cramer, Alona O; Walker, Nathan J; Barnard, Alun R; Hankins, Mark W; MacLaren, Robert E

    2016-11-01

    Gene therapy using adeno-associated viral (AAV) vectors for the treatment of retinal degenerations has shown safety and efficacy in clinical trials. However, very high levels of vector expression may be necessary for the treatment of conditions such as Stargardt disease where a dual vector approach is potentially needed, or in optogenetic strategies for end-stage degeneration in order to achieve maximal light sensitivity. In this study, we assessed two vectors with single capsid mutations, rAAV2/2(Y444F) and rAAV2/8(Y733F) in their ability to transduce retina in the Abca4 -/- and rd1 mouse models of retinal degeneration. We noted significantly increased photoreceptor transduction using rAAV2/8(Y733F) in the Abca4 -/- mouse, in contrast to previous work where vectors tested in this model have shown low levels of photoreceptor transduction. Bipolar cell transduction was achieved following subretinal delivery of both vectors in the rd1 mouse, and via intravitreal delivery of rAAV2/2(Y444F). The successful use of rAAV2/8(Y733F) to target bipolar cells was further validated on human tissue using an ex vivo culture system of retinal explants. Capsid mutant AAV vectors transduce human retinal cells and may be particularly suited to treat retinal degenerations in which high levels of transgene expression are required.

  8. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.

    PubMed

    Manno, Catherine S; Pierce, Glenn F; Arruda, Valder R; Glader, Bertil; Ragni, Margaret; Rasko, John J; Rasko, John; Ozelo, Margareth C; Hoots, Keith; Blatt, Philip; Konkle, Barbara; Dake, Michael; Kaye, Robin; Razavi, Mahmood; Zajko, Albert; Zehnder, James; Rustagi, Pradip K; Nakai, Hiroyuki; Chew, Amy; Leonard, Debra; Wright, J Fraser; Lessard, Ruth R; Sommer, Jürg M; Tigges, Michael; Sabatino, Denise; Luk, Alvin; Jiang, Haiyan; Mingozzi, Federico; Couto, Linda; Ertl, Hildegund C; High, Katherine A; Kay, Mark A

    2006-03-01

    We have previously shown that a single portal vein infusion of a recombinant adeno-associated viral vector (rAAV) expressing canine Factor IX (F.IX) resulted in long-term expression of therapeutic levels of F.IX in dogs with severe hemophilia B. We carried out a phase 1/2 dose-escalation clinical study to extend this approach to humans with severe hemophilia B. rAAV-2 vector expressing human F.IX was infused through the hepatic artery into seven subjects. The data show that: (i) vector infusion at doses up to 2 x 10(12) vg/kg was not associated with acute or long-lasting toxicity; (ii) therapeutic levels of F.IX were achieved at the highest dose tested; (iii) duration of expression at therapeutic levels was limited to a period of approximately 8 weeks; (iv) a gradual decline in F.IX was accompanied by a transient asymptomatic elevation of liver transaminases that resolved without treatment. Further studies suggested that destruction of transduced hepatocytes by cell-mediated immunity targeting antigens of the AAV capsid caused both the decline in F.IX and the transient transaminitis. We conclude that rAAV-2 vectors can transduce human hepatocytes in vivo to result in therapeutically relevant levels of F.IX, but that future studies in humans may require immunomodulation to achieve long-term expression.

  9. Vaccination and allergy.

    PubMed

    Rottem, Menachem; Shoenfeld, Yehuda

    2004-06-01

    Vaccines have had a major effect on controlling the spread of infectious diseases, but use of certain vaccines was linked to potential allergic and autoimmune side effects in healthy and often in certain high-risk populations. In this review the authors summarize the current knowledge of such risks. Immediate systemic allergic reactions after vaccination with commonly used vaccines are extremely rare. Use of certain vaccines was linked to potential allergic side effects in healthy and often in certain high-risk populations. The authors review the data on the risk associated with important vaccines including influenza, smallpox, pneumococcus, Japanese encephalitis, Bacille Calmette-Guerin, pertussis, and measles, mumps, and rubella. Two main components were identified as a source for allergic reactions in vaccines: gelatin and egg protein. There is growing interest in the potential interactions between infant vaccination and risk for development of atopic disease. In addition, there is concern that genetic risk for atopy influences capacity to respond to vaccination during infancy. There is no evidence that vaccines such as Bacille Calmette-Guerin; pertussis; influenza; measles, mumps, and rubella; or smallpox have an effect on the risk of the development of atopy later in life. Immunotherapy provides an efficacious and safe method for the treatment of allergic conditions by immunomodulation of the immune system. The possibility of vaccination triggering or unmasking autoimmunity in genetically susceptible individuals cannot be ruled out, but for the general population the risk-to-benefit ratio is overwhelmingly in favor of vaccinations. Childhood vaccination remains an essential part of child health programs and should not be withheld, even from children predisposed to allergy. Vaccinations are safe, but special attention should be taken in high-risk individuals with anaphylactic reactions to foods, and in patients with autoimmune diseases.

  10. Sieve analysis in HIV-1 vaccine efficacy trials

    PubMed Central

    Edlefsen, Paul T.; Gilbert, Peter B.; Rolland, Morgane

    2013-01-01

    Purpose of review The genetic characterization of HIV-1 breakthrough infections in vaccine and placebo recipients offers new ways to assess vaccine efficacy trials. Statistical and sequence analysis methods provide opportunities to mine the mechanisms behind the effect of an HIV vaccine. Recent findings The release of results from two HIV-1 vaccine efficacy trials, Step/HVTN-502 and RV144, led to numerous studies in the last five years, including efforts to sequence HIV-1 breakthrough infections and compare viral characteristics between the vaccine and placebo groups. Novel genetic and statistical analysis methods uncovered features that distinguished founder viruses isolated from vaccinees from those isolated from placebo recipients, and identified HIV-1 genetic targets of vaccine-induced immune responses. Summary Studies of HIV-1 breakthrough infections in vaccine efficacy trials can provide an independent confirmation to correlates of risk studies, as they take advantage of vaccine/placebo comparisons while correlates of risk analyses are limited to vaccine recipients. Through the identification of viral determinants impacted by vaccine-mediated host immune responses, sieve analyses can shed light on potential mechanisms of vaccine protection. PMID:23719202

  11. A Recombinant Vesicular Stomatitis Virus-Based Lassa Fever Vaccine Protects Guinea Pigs and Macaques against Challenge with Geographically and Genetically Distinct Lassa Viruses

    PubMed Central

    Safronetz, David; Mire, Chad; Rosenke, Kyle; Feldmann, Friederike; Haddock, Elaine; Geisbert, Thomas; Feldmann, Heinz

    2015-01-01

    Background Lassa virus (LASV) is endemic in several West African countries and is the etiological agent of Lassa fever. Despite the high annual incidence and significant morbidity and mortality rates, currently there are no approved vaccines to prevent infection or disease in humans. Genetically, LASV demonstrates a high degree of diversity that correlates with geographic distribution. The genetic heterogeneity observed between geographically distinct viruses raises concerns over the potential efficacy of a “universal” LASV vaccine. To date, several experimental LASV vaccines have been developed; however, few have been evaluated against challenge with various genetically unique Lassa virus isolates in relevant animal models. Methodologies/principle findings Here we demonstrate that a single, prophylactic immunization with a recombinant vesicular stomatitis virus (VSV) expressing the glycoproteins of LASV strain Josiah from Sierra Leone protects strain 13 guinea pigs from infection / disease following challenge with LASV isolates originating from Liberia, Mali and Nigeria. Similarly, the VSV-based LASV vaccine yields complete protection against a lethal challenge with the Liberian LASV isolate in the gold-standard macaque model of Lassa fever. Conclusions/significance Our results demonstrate the VSV-based LASV vaccine is capable of preventing morbidity and mortality associated with non-homologous LASV challenge in two animal models of Lassa fever. Additionally, this work highlights the need for the further development of disease models for geographical distinct LASV strains, particularly those from Nigeria, in order to comprehensively evaluate potential vaccines and therapies against this prominent agent of viral hemorrhagic fever. PMID:25884628

  12. Tyrosine-mutant AAV8 delivery of human MERTK provides long-term retinal preservation in RCS rats.

    PubMed

    Deng, Wen-Tao; Dinculescu, Astra; Li, Qiuhong; Boye, Sanford L; Li, Jie; Gorbatyuk, Marina S; Pang, Jijing; Chiodo, Vince A; Matthes, Michael T; Yasumura, Douglas; Liu, Li; Alkuraya, Fowzan S; Zhang, Kang; Vollrath, Douglas; LaVail, Matthew M; Hauswirth, William W

    2012-04-06

    The absence of Mertk in RCS rats results in defective RPE phagocytosis, accumulation of outer segment (OS) debris in the subretinal space, and subsequent death of photoreceptors. Previous research utilizing Mertk gene replacement therapy in RCS rats provided proof of concept for treatment of this form of recessive retinitis pigmentosa (RP); however, the beneficial effects on retinal function were transient. In the present study, we evaluated whether delivery of a MERTK transgene using a tyrosine-mutant AAV8 capsid could lead to more robust and longer-term therapeutic outcomes than previously reported. An AAV8 Y733F vector expressing a human MERTK cDNA driven by a RPE-selective promoter was administrated subretinally at postnatal day 2. Functional and morphological analyses were performed at 4 months and 8 months post-treatment. Retinal vasculature and Müller cell activation were analyzed by quantifying acellular capillaries and glial fibrillary acidic protein immunostaining, respectively. Electroretinographic responses from treated eyes were more than one-third of wild-type levels and OS were well preserved in the injection area even at 8 months. Rescue of RPE phagocytosis, prevention of retinal vasculature degeneration, and inhibition of Müller cell activation were demonstrated in the treated eyes for at least 8 months. This research describes a longer and much more robust functional and morphological rescue than previous studies. We also demonstrate for the first time that an AAV8 mutant capsid serotype vector has a substantial therapeutic potential for RPE-specific gene delivery. These results suggest that tyrosine-mutant AAV8 vectors hold promise for the treatment of individuals with MERTK-associated RP.

  13. Tyrosine-Mutant AAV8 Delivery of Human MERTK Provides Long-Term Retinal Preservation in RCS Rats

    PubMed Central

    Deng, Wen-Tao; Dinculescu, Astra; Li, Qiuhong; Boye, Sanford L.; Li, Jie; Gorbatyuk, Marina S.; Pang, Jijing; Chiodo, Vince A.; Matthes, Michael T.; Yasumura, Douglas; Liu, Li; Alkuraya, Fowzan S.; Zhang, Kang; Vollrath, Douglas; LaVail, Matthew M.; Hauswirth, William W.

    2012-01-01

    Purpose. The absence of Mertk in RCS rats results in defective RPE phagocytosis, accumulation of outer segment (OS) debris in the subretinal space, and subsequent death of photoreceptors. Previous research utilizing Mertk gene replacement therapy in RCS rats provided proof of concept for treatment of this form of recessive retinitis pigmentosa (RP); however, the beneficial effects on retinal function were transient. In the present study, we evaluated whether delivery of a MERTK transgene using a tyrosine-mutant AAV8 capsid could lead to more robust and longer-term therapeutic outcomes than previously reported. Methods. An AAV8 Y733F vector expressing a human MERTK cDNA driven by a RPE-selective promoter was administrated subretinally at postnatal day 2. Functional and morphological analyses were performed at 4 months and 8 months post-treatment. Retinal vasculature and Müller cell activation were analyzed by quantifying acellular capillaries and glial fibrillary acidic protein immunostaining, respectively. Results. Electroretinographic responses from treated eyes were more than one-third of wild-type levels and OS were well preserved in the injection area even at 8 months. Rescue of RPE phagocytosis, prevention of retinal vasculature degeneration, and inhibition of Müller cell activation were demonstrated in the treated eyes for at least 8 months. Conclusions. This research describes a longer and much more robust functional and morphological rescue than previous studies. We also demonstrate for the first time that an AAV8 mutant capsid serotype vector has a substantial therapeutic potential for RPE-specific gene delivery. These results suggest that tyrosine-mutant AAV8 vectors hold promise for the treatment of individuals with MERTK-associated RP. PMID:22408006

  14. Systemic delivery of shRNA by AAV9 provides highly efficient knockdown of ubiquitously expressed GFP in mouse heart, but not liver.

    PubMed

    Piras, Bryan A; O'Connor, Daniel M; French, Brent A

    2013-01-01

    AAV9 is a powerful gene delivery vehicle capable of providing long-term gene expression in a variety of cell types, particularly cardiomyocytes. The use of AAV-delivery for RNA interference is an intense area of research, but a comprehensive analysis of knockdown in cardiac and liver tissues after systemic delivery of AAV9 has yet to be reported. We sought to address this question by using AAV9 to deliver a short-hairpin RNA targeting the enhanced green fluorescent protein (GFP) in transgenic mice that constitutively overexpress GFP in all tissues. The expression cassette was initially tested in vitro and we demonstrated a 61% reduction in mRNA and a 90% reduction in GFP protein in dual-transfected 293 cells. Next, the expression cassette was packaged as single-stranded genomes in AAV9 capsids to test cardiac GFP knockdown with several doses ranging from 1.8×10(10) to 1.8×10(11) viral genomes per mouse and a dose-dependent response was obtained. We then analyzed GFP expression in both heart and liver after delivery of 4.4×10(11) viral genomes per mouse. We found that while cardiac knockdown was highly efficient, with a 77% reduction in GFP mRNA and a 71% reduction in protein versus control-treated mice, there was no change in liver expression. This was despite a 4.5-fold greater number of viral genomes in the liver than in the heart. This study demonstrates that single-stranded AAV9 vectors expressing shRNA can be used to achieve highly efficient cardiac-selective knockdown of GFP expression that is sustained for at least 7 weeks after the systemic injection of 8 day old mice, with no change in liver expression and no evidence of liver damage despite high viral genome presence in the liver.

  15. Long-term safety and efficacy of AAV gene therapy in the canine model of glycogen storage disease type Ia.

    PubMed

    Lee, Young Mok; Conlon, Thomas J; Specht, Andrew; Coleman, Kirsten E; Brown, Laurie M; Estrella, Ana M; Dambska, Monika; Dahlberg, Kathryn R; Weinstein, David A

    2018-05-25

    Viral mediated gene therapy has progressed after overcoming early failures, and gene therapy has now been approved for several conditions in Europe and the USA. Glycogen storage disease (GSD) type Ia, caused by a deficiency of glucose-6-phosphatase-α, has been viewed as an outstanding candidate for gene therapy. This follow-up report describes the long-term outcome for the naturally occurring GSD-Ia dogs treated with rAAV-GPE-hG6PC-mediated gene therapy. A total of seven dogs were treated with rAAV-GPE-hG6PC-mediated gene therapy. The first four dogs were treated at birth, and three dogs were treated between 2 and 6 months of age to assess the efficacy and safety in animals with mature livers. Blood and urine samples, radiographic studies, histological evaluation, and biodistribution were assessed. Gene therapy improved survival in the GSD-Ia dogs. With treatment, the biochemical studies normalized for the duration of the study (up to 7 years). None of the rAAV-GPE-hG6PC-treated dogs had focal hepatic lesions or renal abnormalities. Dogs treated at birth required a second dose of rAAV after 2-4 months; gene therapy after hepatic maturation resulted in improved efficacy after a single dose. rAAV-GPE-hG6PC treatment in GSD-Ia dogs was found to be safe and efficacious. GSD-Ia is an attractive target for human gene therapy since it is a monogenic disorder with limited tissue involvement. Blood glucose and lactate monitoring can be used to assess effectiveness and as a biomarker of success. GSD-Ia can also serve as a model for other hepatic monogenic disorders.

  16. Cardiac AAV9-S100A1 gene therapy rescues postischemic heart failure in a preclinical large animal model

    PubMed Central

    Pleger, Sven T.; Shan, Changguang; Ksienzyk, Jan; Bekeredjian, Raffi; Boekstegers, Peter; Hinkel, Rabea; Schinkel, Stefanie; Leuchs, Barbara; Ludwig, Jochen; Qiu, Gang; Weber, Christophe; Kleinschmidt, Jürgen A.; Raake, Philip; Koch, Walter J.; Katus, Hugo A.; Müller, Oliver J.; Most, Patrick

    2014-01-01

    As a prerequisite to clinical application, we determined the long-term therapeutic effectiveness and safety of adeno-associated viral (AAV) S100A1 gene therapy in a preclinical, large animal model of heart failure. S100A1, a positive inotropic regulator of myocardial contractility, becomes depleted in failing cardiomyocytes in humans and various animal models, and myocardial-targeted S100A1 gene transfer rescues cardiac contractile function by restoring sarcoplasmic reticulum calcium Ca2+ handling in acutely and chronically failing hearts in small animal models. We induced heart failure in domestic pigs by balloon-occlusion of the left circumflex coronary artery, resulting in myocardial infarction. After 2 weeks, when the pigs displayed significant left ventricular contractile dysfunction, we administered through retrograde coronary venous delivery, AAV9-S100A1 to the left ventricular non-infarcted myocardium. AAV9-luciferase and saline treatment served as control. At 14 weeks, both control groups showed significantly decreased myocardial S100A1 protein expression along with progressive deterioration of cardiac performance and left ventricular remodeling. AAV9-S100A1 treatment prevented and reversed this phenotype by restoring cardiac S100A1 protein levels. S100A1 treatment normalized cardiomyocyte Ca2+ cycling, sarcoplasmic reticulum calcium handling and energy homeostasis. Transgene expression was restricted to cardiac tissue and extra-cardiac organ function was uncompromised indicating a favorable safety profile. This translational study shows the pre-clinical feasibility, long-term therapeutic effectiveness and a favorable safety profile of cardiac AAV9-S100A1 gene therapy in a preclinical model of heart failure. Our study presents a strong rational for a clinical trial of S100A1 gene therapy for human heart failure that could potentially complement current strategies to treat end-stage heart failure. PMID:21775667

  17. Vaccination for Disease

    NASA Astrophysics Data System (ADS)

    Oehen, Stephan; Hengartner, Hans; Zinkernagel, Rolf M.

    1991-01-01

    Recombinant virus vaccines that express a limited number of epitopes are currently being developed to prevent disease by changing the relative balance between viral spread and the immune response. Some circumstances, however, were found in infections with a noncytopathic virus in which vaccination caused disease; sensitive parameters included the genetic background of the host, the time or dose of infection, and the constituents of the vaccine. Thus, immunopathologic damage by T cells may be an unwanted consequence of vaccination with the new types of peptide or recombinant vaccines that are being investigated for the human immunodeficiency viruses and other pathogens.

  18. Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease

    PubMed Central

    Trapani, Ivana; Toriello, Elisabetta; de Simone, Sonia; Colella, Pasqualina; Iodice, Carolina; Polishchuk, Elena V.; Sommella, Andrea; Colecchi, Linda; Rossi, Settimio; Simonelli, Francesca; Giunti, Massimo; Bacci, Maria L.; Polishchuk, Roman S.; Auricchio, Alberto

    2015-01-01

    Stargardt disease (STGD1) due to mutations in the large ABCA4 gene is the most common inherited macular degeneration in humans. We have shown that dual adeno-associated viral (AAV) vectors effectively transfer ABCA4 to the retina of Abca4−/− mice. However, they express both lower levels of transgene compared with a single AAV and truncated proteins. To increase productive dual AAV concatemerization, which would overcome these limitations, we have explored the use of either various regions of homology or heterologous inverted terminal repeats (ITR). In addition, we tested the ability of various degradation signals to decrease the expression of truncated proteins. We found the highest levels of transgene expression using regions of homology based on either alkaline phosphatase or the F1 phage (AK). The use of heterologous ITR does not decrease the levels of truncated proteins relative to full-length ABCA4 and impairs AAV vector production. Conversely, the inclusion of the CL1 degradation signal results in the selective degradation of truncated proteins from the 5′-half without affecting full-length protein production. Therefore, we developed dual AAV hybrid ABCA4 vectors including homologous ITR2, the photoreceptor-specific G protein-coupled receptor kinase 1 promoter, the AK region of homology and the CL1 degradation signal. We show that upon subretinal administration these vectors are both safe in pigs and effective in Abca4−/− mice. Our data support the use of improved dual AAV vectors for gene therapy of STGD1. PMID:26420842

  19. Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease.

    PubMed

    Trapani, Ivana; Toriello, Elisabetta; de Simone, Sonia; Colella, Pasqualina; Iodice, Carolina; Polishchuk, Elena V; Sommella, Andrea; Colecchi, Linda; Rossi, Settimio; Simonelli, Francesca; Giunti, Massimo; Bacci, Maria L; Polishchuk, Roman S; Auricchio, Alberto

    2015-12-01

    Stargardt disease (STGD1) due to mutations in the large ABCA4 gene is the most common inherited macular degeneration in humans. We have shown that dual adeno-associated viral (AAV) vectors effectively transfer ABCA4 to the retina of Abca4-/- mice. However, they express both lower levels of transgene compared with a single AAV and truncated proteins. To increase productive dual AAV concatemerization, which would overcome these limitations, we have explored the use of either various regions of homology or heterologous inverted terminal repeats (ITR). In addition, we tested the ability of various degradation signals to decrease the expression of truncated proteins. We found the highest levels of transgene expression using regions of homology based on either alkaline phosphatase or the F1 phage (AK). The use of heterologous ITR does not decrease the levels of truncated proteins relative to full-length ABCA4 and impairs AAV vector production. Conversely, the inclusion of the CL1 degradation signal results in the selective degradation of truncated proteins from the 5'-half without affecting full-length protein production. Therefore, we developed dual AAV hybrid ABCA4 vectors including homologous ITR2, the photoreceptor-specific G protein-coupled receptor kinase 1 promoter, the AK region of homology and the CL1 degradation signal. We show that upon subretinal administration these vectors are both safe in pigs and effective in Abca4-/- mice. Our data support the use of improved dual AAV vectors for gene therapy of STGD1. © The Author 2015. Published by Oxford University Press.

  20. Long-term correction of very long-chain acyl-coA dehydrogenase deficiency in mice using AAV9 gene therapy.

    PubMed

    Keeler, Allison M; Conlon, Thomas; Walter, Glenn; Zeng, Huadong; Shaffer, Scott A; Dungtao, Fu; Erger, Kirsten; Cossette, Travis; Tang, Qiushi; Mueller, Christian; Flotte, Terence R

    2012-06-01

    Very long-chain acyl-coA dehydrogenase (VLCAD) is the rate-limiting step in mitochondrial fatty acid oxidation. VLCAD-deficient mice and patients clinical symptoms stem from not only an energy deficiency but also long-chain metabolite accumulations. VLCAD-deficient mice were treated systemically with 1 × 10(12) vector genomes of recombinant adeno-associated virus 9 (rAAV9)-VLCAD. Biochemical correction was observed in vector-treated mice beginning 2 weeks postinjection, as characterized by a significant drop in long-chain fatty acyl accumulates in whole blood after an overnight fast. Changes persisted through the termination point around 20 weeks postinjection. Magnetic resonance spectroscopy (MRS) and tandem mass spectrometry (MS/MS) revealed normalization of intramuscular lipids in treated animals. Correction was not observed in liver tissue extracts, but cardiac muscle extracts showed significant reduction of long-chain metabolites. Disease-specific phenotypes were characterized, including thermoregulation and maintenance of euglycemia after a fasting cold challenge. Internal body temperatures of untreated VLCAD(-/-) mice dropped below 20 °C and the mice became lethargic, requiring euthanasia. In contrast, all rAAV9-treated VLCAD(-/-) mice and the wild-type controls maintained body temperatures. rAAV9-treated VLCAD(-/-) mice maintained euglycemia, whereas untreated VLCAD(-/-) mice suffered hypoglycemia following a fasting cold challenge. These promising results suggest rAAV9 gene therapy as a potential treatment for VLCAD deficiency in humans.

  1. Safety and tolerability of MRI-guided infusion of AAV2-hAADC into the mid-brain of nonhuman primate

    PubMed Central

    Sebastian, Waldy San; Kells, Adrian P; Bringas, John; Samaranch, Lluis; Hadaczek, Piotr; Ciesielska, Agnieszka; Macayan, Michael J; Pivirotto, Phillip J; Forsayeth, John; Osborne, Sheryl; Wright, J Fraser; Green, Foad; Heller, Gregory; Bankiewicz, Krystof S

    2014-01-01

    Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare, autosomal-recessive neurological disorder caused by mutations in the DDC gene that leads to an inability to synthesize catecholamines and serotonin. As a result, patients suffer compromised development, particularly in motor function. A recent gene replacement clinical trial explored putaminal delivery of recombinant adeno-associated virus serotype 2 vector encoding human AADC (AAV2-hAADC) in AADC-deficient children. Unfortunately, patients presented only modest amelioration of motor symptoms, which authors acknowledged could be due to insufficient transduction of putamen. We hypothesize that, with the development of a highly accurate MRI-guided cannula placement technology, a more effective approach might be to target the affected mid-brain neurons directly. Transduction of AADC-deficient dopaminergic neurons in the substantia nigra and ventral tegmental area with locally infused AAV2-hAADC would be expected to lead to restoration of normal dopamine levels in affected children. The objective of this study was to assess the long-term safety and tolerability of bilateral AAV2-hAADC MRI-guided pressurized infusion into the mid-brain of nonhuman primates. Animals received either vehicle, low or high AAV2-hAADC vector dose and were euthanized 1, 3, or 9 months after surgery. Our data indicate that effective mid-brain transduction was achieved without untoward effects. PMID:25541617

  2. Vaccines and vaccination strategies against human cutaneous leishmaniasis.

    PubMed

    Okwor, Ifeoma; Uzonna, Jude

    2009-05-01

    One might think that the development of a vaccine against cutaneous leishmaniasis would be relatively straightforward because the type of immune response required for protection is known and natural immunity occurs following recovery from primary infection. However, there is as yet no effective vaccine against the disease in humans. Although vaccination in murine studies has yielded promising results, these vaccines have failed miserably when tested in primates or humans. The reasons behind these failures are unknown and remain a major hurdle for vaccine design and development against cutaneous leishmaniasis. In contrast, recovery from natural, deliberate or experimental infections results in development of long-lasting immunity to re-infection. This so called infection-induced resistance is the strongest anti-Leishmania immunity known. Here, we briefly review the different approaches to vaccination against cutaneous leishmaniasis and argue that vaccines composed of genetically modified (attenuated) parasites, which induce immunity akin to infection-induced resistance, may provide best protection against cutaneous leishmaniasis in humans.

  3. Novel mucosal vaccines generated by genetic conjugation of heterologous proteins to pneumolysin (PLY) from Streptococcus pneumoniae.

    PubMed

    Douce, Gill; Ross, Kirsty; Cowan, Graeme; Ma, Jiangtao; Mitchell, Tim J

    2010-04-19

    Induction of immunity at mucosal surfaces is thought to be an essential feature in the protection of the host against the many pathogens that gain access through these surfaces. Here we describe how strong local and systemic immune responses can be generated when proteins are genetically conjugated to pneumolysin (PLY) from Streptococcus pneumoniae. Using green fluorescent protein (eGFP) and PsaA from S. pneumoniae, we have shown that genetic fusion (eGFPPLY and PsaAPLY) is essential to ensure high levels of antigen specific IgG and IgA in the serum and at mucosal surfaces. This form of vaccination is highly effective with antigen specific antibodies detected after a single dose of nanogram quantities of the conjugated proteins. In addition, generation of a non-toxic variant (eGFPDelta6PLY) indicated that while the toxic activity of PLY was not essential for adjuvanticity, it contributed to the magnitude of the response generated. Whilst vaccination with the PsaAPLY fusion proteins did not protect the animals from challenge, these studies confirm the utility of pneumolysin to act as a novel mucosal adjuvant to substantially increase the local and systemic humoral response to genetically fused protein antigens. Copyright 2010 Elsevier Ltd. All rights reserved.

  4. Systemic injection of AAV9 carrying a periostin promoter targets gene expression to a myofibroblast-like lineage in mouse hearts after reperfused myocardial infarction.

    PubMed

    Piras, B A; Tian, Y; Xu, Y; Thomas, N A; O'Connor, D M; French, B A

    2016-05-01

    Adeno-associated virus (AAV) has been used to direct gene transfer to a variety of tissues, including heart, liver, skeletal muscle, brain, kidney and lung, but it has not previously been shown to effectively target fibroblasts in vivo, including cardiac fibroblasts. We constructed expression cassettes using a modified periostin promoter to drive gene expression in a cardiac myofibroblast-like lineage, with only occasional spillover into cardiomyocyte-like cells. We compared AAV serotypes 6 and 9 and found robust gene expression when the vectors were delivered by systemic injection after myocardial infarction (MI), with little expression in healthy, non-infarcted mice. AAV9 provided expression in a greater number of cells than AAV6, with reporter gene expression visible in the cardiac infarct and border zones from 5 to 62 days post MI, as assessed by luciferase and Cre-activated green fluorescent protein expression. Although common myofibroblast markers were expressed in low abundance, most of the targeted cells expressed myosin IIb, an embryonic form of smooth muscle myosin heavy chain that has previously been associated with myofibroblasts after reperfused MI. This study is the first to demonstrate AAV-mediated expression in a potentially novel myofibroblast-like lineage in mouse hearts post MI and may open new avenues of gene therapy to treat patients surviving MI.

  5. New genetic associations detected in a host response study to hepatitis B vaccine.

    PubMed

    Davila, S; Froeling, F E M; Tan, A; Bonnard, C; Boland, G J; Snippe, H; Hibberd, M L; Seielstad, M

    2010-04-01

    The immune response to hepatitis B vaccination differs greatly among individuals, with 5-10% of healthy people failing to produce protective levels of antibodies. Several factors have been implicated in determining this response, chiefly individual genetic variation and age. Aiming to identify genes involved in the response to hepatitis B vaccination, a two-stage investigation of 6091 single-nucleotide polymorphisms (SNPs) in 914 immune genes was performed in an Indonesian cohort of 981 individuals showing normal levels of anti-HBs versus 665 individuals displaying undetectable levels of anti-HBs 18 months after initial dose of the vaccine. Of 275 SNPs identified in the first stage (476 normal/372 nonresponders) with P<0.05, significant associations were replicated for 25 polymorphisms in 15 genes (503 normal/295 nonresponders). We validated previous findings (HLA-DRA, rs5000563, P-value combined=5.57 x 10(-10); OR (95%CI)=0.61 (0.52-0.71)). In addition, we detected a new association outside of the human leukocyte antigen loci region that passed correction for multiple testing. This SNP is in the 3' downstream region of FOXP1, a transcription factor involved in B-cell development (P-value combined=9.2 x 10(-6); OR (95%CI)=1.38 (1.2-1.6)).These findings might help to understand the biological reasons behind vaccine failure and other aspects of variation in the immune responses of healthy individuals.

  6. Characterization of intravitreally delivered capsid mutant AAV2-Cre vector to induce tissue-specific mutations in murine retinal ganglion cells.

    PubMed

    Langouet-Astrie, Christophe J; Yang, Zhiyong; Polisetti, Sraavya M; Welsbie, Derek S; Hauswirth, William W; Zack, Donald J; Merbs, Shannath L; Enke, Raymond A

    2016-10-01

    Targeted expression of Cre recombinase in murine retinal ganglion cells (RGCs) by viral vector is an effective strategy for creating tissue-specific gene knockouts for investigation of genetic contribution to RGC degeneration associated with optic neuropathies. Here we characterize dosage, efficacy and toxicity for sufficient intravitreal delivery of a capsid mutant Adeno-associated virus 2 (AAV2) vector encoding Cre recombinase. Wild type and Rosa26 (R26) LacZ mice were intravitreally injected with capsid mutant AAV2 viral vectors. Murine eyes were harvested at intervals ranging from 2 weeks to 15 weeks post-injection and were assayed for viral transduction, transgene expression and RGC survival. 10(9) vector genomes (vg) were sufficient for effective in vivo targeting of murine ganglion cell layer (GCL) retinal neurons. Transgene expression was observed as early as 2 weeks post-injection of viral vectors and persisted to 11 weeks. Early expression of Cre had no significant effect on RGC survival, while significant RGC loss was detected beginning 5 weeks post-injection. Early expression of viral Cre recombinase was robust, well-tolerated and predominantly found in GCL neurons suggesting this strategy can be effective in short-term RGC-specific mutation studies in experimental glaucoma models such as optic nerve crush and transection experiments. RGC degeneration with Cre expression for more than 4 weeks suggests that Cre toxicity is a limiting factor for targeted mutation strategies in RGCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Genetic variation in potential Giardia vaccine candidates cyst wall protein 2 and α1-giardin.

    PubMed

    Radunovic, Matej; Klotz, Christian; Saghaug, Christina Skår; Brattbakk, Hans-Richard; Aebischer, Toni; Langeland, Nina; Hanevik, Kurt

    2017-08-01

    Giardia is a prevalent intestinal parasitic infection. The trophozoite structural protein a1-giardin (a1-g) and the cyst protein cyst wall protein 2 (CWP2) have shown promise as Giardia vaccine antigen candidates in murine models. The present study assesses the genetic diversity of a1-g and CWP2 between and within assemblages A and B in human clinical isolates. a1-g and CWP2 sequences were acquired from 15 Norwegian isolates by PCR amplification and 20 sequences from German cultured isolates by whole genome sequencing. Sequences were aligned to reference genomes from assemblage A2 and B to identify genetic variance. Genetic diversity was found between assemblage A and B reference sequences for both a1-g (90.8% nucleotide identity) and CWP2 (82.5% nucleotide identity). However, for a1-g, this translated into only 3 amino acid (aa) substitutions, while for CWP2 there were 41 aa substitutions, and also one aa deletion. Genetic diversity within assemblage B was larger; nucleotide identity 92.0% for a1-g and 94.3% for CWP2, than within assemblage A (nucleotide identity 99.0% for a1-g and 99.7% for CWP2). For CWP2, the diversity on both nucleotide and protein level was higher in the C-terminal end. Predicted antigenic epitopes were not affected for a1-g, but partially for CWP2. Despite genetic diversity in a1-g, we found aa sequence, characteristics, and antigenicity to be well preserved. CWP2 showed more aa variance and potential antigenic differences. Several CWP2 antigens might be necessary in a future Giardia vaccine to provide cross protection against both Giardia assemblages infecting humans.

  8. Cloned cDNA of A/swine/Iowa/15/1930 internal genes as a candidate backbone for reverse genetics vaccine against influenza A viruses

    PubMed Central

    Lekcharoensuk, Porntippa; Wiriyarat, Witthawat; Petcharat, Nuntawan; Lekcharoensuk, Chalermpol; Auewarakul, Prasert; Richt, Juergen A

    2012-01-01

    Reverse genetics viruses for influenza vaccine production usually utilize the internal genes of the egg-adapted A/Puerto Rico/8/34 (PR8) strain. This egg-adapted strain provides high production yield in embryonated eggs but does not necessarily give the best yield in mammalian cell culture. In order to generate a reverse genetics viral backbone that is well-adapted to high growth in mammalian cell culture, a swine influenza isolate (A/swine/Iowa/15/30 (H1N1) (rg1930) that was shown to give high yield in Madin-Darby Canine Kidney (MDCK) cells was used as the internal gene donor for reverse genetics plasmids. In this report, the internal genes from rg1930 were used for construction of reverse genetics viruses carrying a cleavage site-modified hemagglutinin (HA) gene and neuraminidase (NA) gene from a highly pathogenic H5N1 virus. The resulting virus (rg1930H5N1) was low pathogenic in vivo. Inactivated rg1930H5N1 vaccine completely protected chickens from morbidity and mortality after challenge with highly pathogenic H5N1. Protective immunity was obtained when chickens were immunized with an inactivated vaccine consisting of at least 29 HA units of the rg1930H5N1 virus. In comparison to the PR8-based reverse genetics viruses carrying the same HA and NA genes from an H5N1 virus, rg1930 based viruses yielded higher viral titers in MDCK and Vero cells. In addition, the reverse genetics derived H3N2 and H5N2 viruses with the rg1930 backbone replicated in MDCK cells better than the cognate viruses with the rgPR8 backbone. It is concluded that this newly established reverse genetics backbone system could serve as a candidate for a master donor strain for development of inactivated influenza vaccines in cell-based systems. PMID:22230579

  9. Cloned cDNA of A/swine/Iowa/15/1930 internal genes as a candidate backbone for reverse genetics vaccine against influenza A viruses.

    PubMed

    Lekcharoensuk, Porntippa; Wiriyarat, Witthawat; Petcharat, Nantawan; Lekcharoensuk, Chalermpol; Auewarakul, Prasert; Richt, Juergen A

    2012-02-14

    Reverse genetics viruses for influenza vaccine production usually utilize the internal genes of the egg-adapted A/Puerto Rico/8/34 (PR8) strain. This egg-adapted strain provides high production yield in embryonated eggs but does not necessarily give the best yield in mammalian cell culture. In order to generate a reverse genetics viral backbone that is well-adapted to high growth in mammalian cell culture, a swine influenza isolate A/swine/Iowa/15/30 (H1N1) (rg1930) that was shown to give high yield in Madin-Darby canine kidney (MDCK) cells was used as the internal gene donor for reverse genetics plasmids. In this report, the internal genes from rg1930 were used for construction of reverse genetics viruses carrying a cleavage site-modified hemagglutinin (HA) gene and neuraminidase (NA) gene from a highly pathogenic H5N1 virus. The resulting virus (rg1930H5N1) was low pathogenic in vivo. Inactivated rg1930H5N1 vaccine completely protected chickens from morbidity and mortality after challenge with highly pathogenic H5N1. Protective immunity was obtained when chickens were immunized with an inactivated vaccine consisting of at least 2(9) HA units of the rg1930H5N1 virus. In comparison to the PR8-based reverse genetics viruses carrying the same HA and NA genes from an H5N1 virus, rg1930 based viruses yielded higher viral titers in MDCK and Vero cells. In addition, the reverse genetics derived H3N2 and H5N2 viruses with the rg1930 backbone replicated in MDCK cells better than the cognate viruses with the rgPR8 backbone. It is concluded that this newly established reverse genetics backbone system could serve as a candidate for a master donor strain for development of inactivated influenza vaccines in cell-based systems. Copyright © 2011 Elsevier Ltd. All rights reserved.

  10. Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer.

    PubMed

    Buchlis, George; Podsakoff, Gregory M; Radu, Antonetta; Hawk, Sarah M; Flake, Alan W; Mingozzi, Federico; High, Katherine A

    2012-03-29

    In previous work we transferred a human factor IX-encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer.

  11. Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer

    PubMed Central

    Buchlis, George; Podsakoff, Gregory M.; Radu, Antonetta; Hawk, Sarah M.; Flake, Alan W.; Mingozzi, Federico

    2012-01-01

    In previous work we transferred a human factor IX–encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer. PMID:22271447

  12. Rescue of Pompe disease in mice by AAV-mediated liver delivery of secretable acid α-glucosidase

    PubMed Central

    Puzzo, Francesco; Colella, Pasqualina; Biferi, Maria G.; Bali, Deeksha; Paulk, Nicole K.; Vidal, Patrice; Collaud, Fanny; Simon-Sola, Marcelo; Charles, Severine; Hardet, Romain; Leborgne, Christian; Meliani, Amine; Cohen-Tannoudji, Mathilde; Astord, Stephanie; Gjata, Bernard; Sellier, Pauline; van Wittenberghe, Laetitia; Vignaud, Alban; Boisgerault, Florence; Barkats, Martine; Laforet, Pascal; Kay, Mark A.; Koeberl, Dwight D.; Ronzitti, Giuseppe; Mingozzi, Federico

    2018-01-01

    Glycogen storage disease type II or Pompe disease is a severe neuromuscular disorder caused by mutations in the lysosomal enzyme, acid α-glucosidase (GAA), which result in pathological accumulation of glycogen throughout the body. Enzyme replacement therapy is available for Pompe disease; however, it has limited efficacy, has high immunogenicity, and fails to correct pathological glycogen accumulation in nervous tissue and skeletal muscle. Using bioinformatics analysis and protein engineering, we developed transgenes encoding GAA that could be expressed and secreted by hepatocytes. Then, we used adeno-associated virus (AAV) vectors optimized for hepatic expression to deliver the GAA transgenes to Gaa knockout (Gaa−/−) mice, a model of Pompe disease. Therapeutic gene transfer to the liver rescued glycogen accumulation in muscle and the central nervous system, and ameliorated cardiac hypertrophy as well as muscle and respiratory dysfunction in the Gaa−/− mice; mouse survival was also increased. Secretable GAA showed improved therapeutic efficacy and lower immunogenicity compared to nonengineered GAA. Scale-up to nonhuman primates, and modeling of GAA expression in primary human hepatocytes using hepatotropic AAV vectors, demonstrated the therapeutic potential of AAV vector–mediated liver expression of secretable GAA for treating pathological glycogen accumulation in multiple tissues in Pompe disease. PMID:29187643

  13. Neonatal Systemic AAV Induces Tolerance to CNS Gene Therapy in MPS I Dogs and Nonhuman Primates

    PubMed Central

    Hinderer, Christian; Bell, Peter; Louboutin, Jean-Pierre; Zhu, Yanqing; Yu, Hongwei; Lin, Gloria; Choa, Ruth; Gurda, Brittney L; Bagel, Jessica; O'Donnell, Patricia; Sikora, Tracey; Ruane, Therese; Wang, Ping; Tarantal, Alice F; Casal, Margret L; Haskins, Mark E; Wilson, James M

    2015-01-01

    The potential host immune response to a nonself protein poses a fundamental challenge for gene therapies targeting recessive diseases. We demonstrate in both dogs and nonhuman primates that liver-directed gene transfer using an adeno-associated virus (AAV) vector in neonates induces a persistent state of immunological tolerance to the transgene product, substantially improving the efficacy of subsequent vector administration targeting the central nervous system (CNS). We applied this approach to a canine model of mucopolysaccharidosis type I (MPS I), a progressive neuropathic lysosomal storage disease caused by deficient activity of the enzyme α-l-iduronidase (IDUA). MPS I dogs treated systemically in the first week of life with a vector expressing canine IDUA did not develop antibodies against the enzyme and exhibited robust expression in the CNS upon intrathecal AAV delivery at 1 month of age, resulting in complete correction of brain storage lesions. Newborn rhesus monkeys treated systemically with AAV vector expressing human IDUA developed tolerance to the transgene, resulting in high cerebrospinal fluid (CSF) IDUA expression and no antibody induction after subsequent CNS gene therapy. These findings suggest that inducing tolerance to the transgene product during a critical period in immunological development can improve the efficacy and safety of gene therapy. PMID:26022732

  14. Neonatal Systemic AAV Induces Tolerance to CNS Gene Therapy in MPS I Dogs and Nonhuman Primates.

    PubMed

    Hinderer, Christian; Bell, Peter; Louboutin, Jean-Pierre; Zhu, Yanqing; Yu, Hongwei; Lin, Gloria; Choa, Ruth; Gurda, Brittney L; Bagel, Jessica; O'Donnell, Patricia; Sikora, Tracey; Ruane, Therese; Wang, Ping; Tarantal, Alice F; Casal, Margret L; Haskins, Mark E; Wilson, James M

    2015-08-01

    The potential host immune response to a nonself protein poses a fundamental challenge for gene therapies targeting recessive diseases. We demonstrate in both dogs and nonhuman primates that liver-directed gene transfer using an adeno-associated virus (AAV) vector in neonates induces a persistent state of immunological tolerance to the transgene product, substantially improving the efficacy of subsequent vector administration targeting the central nervous system (CNS). We applied this approach to a canine model of mucopolysaccharidosis type I (MPS I), a progressive neuropathic lysosomal storage disease caused by deficient activity of the enzyme α-l-iduronidase (IDUA). MPS I dogs treated systemically in the first week of life with a vector expressing canine IDUA did not develop antibodies against the enzyme and exhibited robust expression in the CNS upon intrathecal AAV delivery at 1 month of age, resulting in complete correction of brain storage lesions. Newborn rhesus monkeys treated systemically with AAV vector expressing human IDUA developed tolerance to the transgene, resulting in high cerebrospinal fluid (CSF) IDUA expression and no antibody induction after subsequent CNS gene therapy. These findings suggest that inducing tolerance to the transgene product during a critical period in immunological development can improve the efficacy and safety of gene therapy.

  15. AAV2 production with optimized N/P ratio and PEI-mediated transfection results in low toxicity and high titer for in vitro and in vivo applications.

    PubMed

    Huang, Xinping; Hartley, Antja-Voy; Yin, Yishi; Herskowitz, Jeremy H; Lah, James J; Ressler, Kerry J

    2013-11-01

    The adeno-associated virus (AAV) is one of the most useful viral vectors for gene delivery for both in vivo and in vitro applications. A variety of methods have been established to produce and characterize recombinant AAV (rAAV) vectors; however most methods are quite cumbersome and obtaining consistently high titer can be problematic. This protocol describes a triple-plasmid co-transfection approach with 25 kDa linear polyethylenimine (PEI) in 293 T cells for the production of AAV serotype 2. Seventy-two hours post-transfection, supernatant and cells were harvested and purified by a discontinuous iodixanol density gradient ultracentrifugation, then dialyzed and concentrated with an Amicon 15 100,000 MWCO concentration unit. To optimize the protocol for AAV2 production using PEI, various N/P ratios and DNA amounts were compared. We found that an N/P ratio of 40 coupled with 1.05 μg DNA per ml of media (21 μg DNA/15 cm dish) was found to produce the highest yields for viral replication and assembly measured multiple ways. The infectious units, as determined by serial dilution, were between 1×10(8) and 2×10(9) IU/ml. The genomic titer of the viral stock was determined by qPCR and ranged from 2×10(12) to 6×10(13) VG/ml. These viral vectors showed high expression both in vivo within the brain and in vitro in cell culture. The use of linear 25 kDa polyethylenamine PEI as a transfection reagent is a simple, more cost-effective, and stable means of high-throughput production of high-titer AAV serotype 2. The use of PEI also eliminates the need to change cell medium post-transfection, lowering cost and workload, while producing high-titer, efficacious AAV2 vectors for routine gene transfer. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Newcastle disease virus vectored vaccines as bivalent or antigen delivery vaccines

    PubMed Central

    2017-01-01

    Recent advances in reverse genetics techniques make it possible to manipulate the genome of RNA viruses such as Newcastle disease virus (NDV). Several NDV vaccine strains have been used as vaccine vectors in poultry, mammals, and humans to express antigens of different pathogens. The safety, immunogenicity, and protective efficacy of these NDV-vectored vaccines have been evaluated in pre-clinical and clinical studies. The vaccines are safe in mammals, humans, and poultry. Bivalent NDV-vectored vaccines against pathogens of economic importance to the poultry industry have been developed. These bivalent vaccines confer solid protective immunity against NDV and other foreign antigens. In most cases, NDV-vectored vaccines induce strong local and systemic immune responses against the target foreign antigen. This review summarizes the development of NDV-vectored vaccines and their potential use as a base for designing other effective vaccines for veterinary and human use. PMID:28775971

  17. Lack of Humoral Immune Response to the Tetracycline (Tet) Activator in Rats Injected Intracranially with Tet-off rAAV Vectors

    PubMed Central

    Han, Ye; Chang, Qin A.; Virag, Tamas; West, Neva C.; George, David; Castro, Maria G.; Bohn, Martha C.

    2010-01-01

    The ability to safely control transgene expression from viral vectors is a long-term goal in the gene therapy field. We have previously reported tight regulation of GFP expression in rat brain using a self-regulating tet-off rAAV vector. The immune responses against tet regulatory elements observed by other groups in nonhuman primates after intramuscular injection of tet-on encoding vectors raise concerns about the clinical value of tet-regulated vectors. However, previous studies have not examined immune responses following injection of AAV vectors into brain. Therefore, rat striatum was injected with tet-off rAAV harboring a therapeutic gene for Parkinson's disease, either hAADC or hGDNF. The expression of each gene was tightly controlled by the tet-off regulatory system. Using an ELISA developed with purified GST-tTA protein, no detectable immunogenicity against tTA was observed in sera of rats that received an intrastriatal injection of either vector. In contrast, sera from rats intradermally injected with an adenovirus containing either tTA or rtTA, as positive controls, had readily detectable antibodies. These observations suggest that tet-off rAAV vectors do not elicit an immune response when injected into rat brain and that these may offer safer vectors for Parkinson's disease than vectors with constitutive expression. PMID:20164859

  18. A genetically engineered live attenuated vaccine of Coccidioides posadasii protects BALB/c mice against coccidioidomycosis.

    PubMed

    Xue, Jianmin; Chen, Xia; Selby, Dale; Hung, Chiung-Yu; Yu, Jieh-Juen; Cole, Garry T

    2009-08-01

    Coccidioidomycosis (also known as San Joaquin Valley fever) is an occupational disease. Workers exposed to outdoor dust which contains spores of the soil-inhabiting fungus have a significantly increased risk of respiratory infection. In addition, people with compromised T-cell immunity, the elderly, and certain racial groups, particularly African-Americans and Filipinos, who live in regions of endemicity in the southwestern United States have an elevated incidence of symptomatic infection caused by inhalation of spores of Coccidioides posadasii or Coccidioides immitis. Recurring epidemics and escalation of medical costs have helped to motivate production of a vaccine against valley fever. The major focus has been the development of a defined, T-cell-reactive, recombinant protein vaccine. However, none of the products described to date have provided full protection to coccidioidal disease-susceptible BALB/c mice. Here we describe the first genetically engineered, live, attenuated vaccine that protects both BALB/c and C57BL/6 mice against coccidioidomycosis. Two chitinase genes (CTS2 and CTS3) were disrupted to yield the attenuated strain, which was unable to endosporulate and was no longer infectious. Vaccinated survivors mounted an immune response characterized by production of both T-helper-1- and T-helper-2-type cytokines. Histology revealed well-formed granulomas and markedly diminished inflammation. Significantly fewer organisms were observed in the lungs of survivors than in those of nonvaccinated mice. Additional investigations are required to further define the nature of the live, attenuated vaccine-induced immunity against Coccidioides infection.

  19. A genetically inactivated two-component acellular pertussis vaccine, alone or combined with tetanus and reduced-dose diphtheria vaccines, in adolescents: a phase 2/3, randomised controlled non-inferiority trial.

    PubMed

    Sricharoenchai, Sirintip; Sirivichayakul, Chukiat; Chokephaibulkit, Kulkanya; Pitisuttithum, Punnee; Dhitavat, Jittima; Pitisuthitham, Arom; Phongsamart, Wanatpreeya; Boonnak, Kobporn; Lapphra, Keswadee; Sabmee, Yupa; Wittawatmongkol, Orasri; Chinwangso, Pailinrut; Poredi, Indrajeet Kumar; Petre, Jean; Thai, Pham Hong; Viviani, Simonetta

    2018-01-01

    Increasing evidence shows that protection induced by acellular pertussis vaccines is short-lived, requiring repeated booster vaccination to control pertussis disease. We aimed to assess the safety and immunogenicity of a recombinant acellular pertussis vaccine containing genetically inactivated pertussis toxin and filamentous haemagglutinin, as either a monovalent vaccine (aP [PTgen/FHA] ) or in combination with tetanus and reduced-dose diphtheria vaccines (TdaP [PTgen/FHA] ), versus a licensed tetanus and reduced-dose diphtheria and acellular pertussis combination vaccine (Tdap). We did this phase 2/3, randomised controlled non-inferiority trial at two sites in Bangkok, Thailand. Healthy adolescents (aged 12-17 years) were randomly assigned (1:1:1), via a computer-generated randomisation list with block sizes of three, to receive one dose (0·5 mL) of aP (PTgen/FHA) , TdaP (PTgen/FHA) , or Tdap (comparator). Clinical research staff responsible for participant randomisation, vaccine preparation and administration, and accountability were aware of group allocation. However, allocation was concealed from all other site study staff, data management personnel, statisticians, laboratory staff, and study participants. The primary outcome was non-inferior immunogenicity of TdaP (PTgen/FHA) to Tdap based on seroconversion rates (a four-fold increase or more) for pertussis toxin and filamentous haemagglutinin IgG antibodies 28 days after vaccination, with a predefined 10% margin of equivalence. We did analysis by per protocol. This study is registered with the Thai Clinical Trial Registry, number TCTR20150703002. Between July 6 and Aug 20, 2015, we allocated 450 participants to receive one dose of TdaP (PTgen/FHA) (n=150), aP (PTgen/FHA) (n=150), or comparator Tdap (n=150). 28 days after vaccination, seroconversion rates for anti-pertussis toxin IgG were 96·6% (95% CI 93·8-99·5; n=144) in the TdaP (PTgen/FHA) group and 55·0% (47·1-63·0; n=82) in the comparator Tdap

  20. [Construction of rAAV2-GPIIb/IIIa vector and test of its expression and function in vitro].

    PubMed

    Wang, Kai; Peng, Jian-Qiang; Chen, Fang-Ping; Wu, Xiao-Bin

    2006-04-01

    This study was aimed to explore the possibility of rAAV2 vector-mediating gene therapy for Glanzmann' s thrombasthenia. The rAAV2-GPIIb/IIIa vector was constructed. The GPIIb/IIIa gene expression in mammal cell were examined by different methods, such as: detection of mRNA expression in BHK-21 cells after 24 hours of infection (MOI = 1 x 10(5) v.g/cell) was performed by RT-PCR; the relation between MOI and quantity of GPII6/IIIa gene expression was detected by FACS after 48 hours of infection; GPIIb/IIIa protein expression in BHK-21 cells after 48 hours of infection (MOI = 10(5) v x g/cell) was assayed by Western blot, GPIIb/IIIa protein expression on cell surface was detected by immunofluorescence, and the biological function of expressing product was determined by PAC-1 conjunct experiments. The results showed that GPIIb/IIIa gene expression in mRNA level could be detected in BHK-21 cells after 24 hours of infection at MOI = 1 x 10(5) v x g/cell and the GPIIb/IIIa gene expression in protein level could be detected in BHK-21 cells after 48 hours of infection at MOI = 1 x 10(5) v x g/cell. In certain range, quantity of GPIIb/IIIa gene expression increased with MOI, but overdose of MOI decreased quantity of GPIIb/IIIa gene expression. Activated product of GPIIb/IIIa gene expression could combined with PAC-I, and possesed normal biological function. In conclusion, rAAV2 vactor can effectively mediate GPIIb and GPIIIa gene expressing in mammal cells, and the products of these genes exhibit biological function. This result may provide a basis for application of rAAV2 vector in Glanzmann's thrombasthenia gene therapy in furture.

  1. Gene Therapy for Osteoarthritis: Pharmacokinetics of Intra-articular scAAV.IL-1Ra Delivery in an Equine Model.

    PubMed

    Watson Levings, Rachael; Broome, Ted A; Smith, Andrew D; Rice, Brett L; Gibbs, Eric P; Myara, D Alex; Hyddmark, E Viktoria; Nasri, Elham; Zarezadeh, Ali; Levings, Padraic P; Lu, Yuan; Dacanay, E Anthony; Foremny, Gregory B; Evans, Christopher H; Morton, Alison J; Winter, Mathew; Dark, Michael J; Nickerson, David M; Colahan, Patrick T; Ghivizzani, Steven Craig

    2018-06-05

    142: Toward the treatment of osteoarthritis (OA), we have been investigating self-complementary adeno-associated virus (scAAV) for intra-articular delivery of gene products with therapeutic potential. As OA frequently affects weight-bearing joints, we performed pharmacokinetic studies in the equine forelimb to identify parameters of scAAV gene delivery relevant to clinical translation. Using the coding sequence for interleukin-1 receptor antagonist (IL-1Ra) as a secreted therapeutic reporter we first generated an scAAV vector containing an optimized cDNA for equine IL-1Ra. In dosing studies in vivo we identified a putative ceiling dose of 5 x 1012 viral genomes (vg) which elevated the steady-state eqIL-1Ra in synovial fluids >50-fold for over 6 months. No adverse effects of treatment were seen, and eqIL-1Ra in serum and urine remained at background. Using 5 x 1012 vg and GFP as a cytologic marker, we compared the local and systemic distribution of vector and transduced cells in healthy joints and those with late stage, naturally-occurring OA. Strikingly, a substantial increase in transgenic expression was associated with the articular pathologies characteristic of OA, including synovitis, osteophyte formation and damaged cartilage. Nonetheless, in both the healthy and OA environments the vector and transgene expression were effectively contained within the injected joint. 143: We are investigating self-complementary adeno-associated virus (scAAV) as a vector for intra-articular gene-delivery of IL-1Ra, and its therapeutic capacity in the treatment of osteoarthritis (OA). To model gene-transfer on a scale proportional to the human knee, a frequent site of OA incidence, we focused our studies on the joints of the equine forelimb. Using AAV2.5 capsid and equine IL-1Ra as a homologous transgene, we previously identified a functional ceiling dose of ~5 x 1012 viral genomes, which elevated the steady state levels of eqIL-1Ra in synovial fluids by more than 40-fold over

  2. 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.

  3. 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

  4. Vaccines Against Malaria

    PubMed Central

    Ouattara, Amed; Laurens, Matthew B.

    2015-01-01

    Despite global efforts to control malaria, the illness remains a significant public health threat. Currently, there is no licensed vaccine against malaria, but an efficacious vaccine would represent an important public health tool for successful malaria elimination. Malaria vaccine development continues to be hindered by a poor understanding of antimalarial immunity, a lack of an immune correlate of protection, and the genetic diversity of malaria parasites. Current vaccine development efforts largely target Plasmodium falciparum parasites in the pre-erythrocytic and erythrocytic stages, with some research on transmission-blocking vaccines against asexual stages and vaccines against pregnancy-associated malaria. The leading pre-erythrocytic vaccine candidate is RTS,S, and early results of ongoing Phase 3 testing show overall efficacy of 46% against clinical malaria. The next steps for malaria vaccine development will focus on the design of a product that is efficacious against the highly diverse strains of malaria and the identification of a correlate of protection against disease. PMID:25452593

  5. Gene therapy mediated seizure suppression in Genetic Generalised Epilepsy: Neuropeptide Y overexpression in a rat model.

    PubMed

    Powell, Kim L; Fitzgerald, Xavier; Shallue, Claire; Jovanovska, Valentina; Klugmann, Matthias; Von Jonquieres, Georg; O'Brien, Terence J; Morris, Margaret J

    2018-05-01

    Neuropeptide Y (NPY) is an important 36 amino acid peptide that is abundantly expressed in the mammalian CNS and is known to be an endogenous modulator of seizure activity, including in rat models of Genetic Generalised Epilepsy (GGE) with absence seizures. Studies have shown that viral-mediated "gene therapy" with overexpression of NPY in the hippocampus can suppress seizures in acquired epilepsy animal models. This study investigated whether NPY gene delivery to the thalamus or somatosensory cortex, using recombinant adeno-associated viral vector (rAAV), could produce sustained seizure suppression in the GAERS model of GGE with absence seizures. Three cohorts of GAERS were injected bilaterally into the thalamus (short term n = 14 and long term n = 8) or the somatosensory cortex (n = 26) with rAAV-NPY or rAAV-empty. EEG recordings were acquired weekly post-treatment and seizure expression was quantified. Anxiety levels were tested using elevated plus maze and open field test. NPY and NPY receptor mRNA and protein expression were evaluated using quantitative PCR, immunohistochemistry and immunofluorescence. Viral overexpression of human NPY in the thalamus and somatosensory cortex in GAERS significantly reduced the time spent in seizure activity and number of seizures, whereas seizure duration was only reduced after thalamic NPY overexpression. Human and rat NPY and rat Y2 receptor mRNA expression was significantly increased in the somatosensory cortex. NPY overexpression in the thalamus was observed in rAAV-NPY treated rats compared to controls in the long term cohort. No effect was observed on anxiety behaviour. We conclude that virally-mediated human NPY overexpression in the thalamus or somatosensory cortex produces sustained anti-epileptic effects in GAERS. NPY gene therapy may represent a novel approach for the treatment of patients with genetic generalised epilepsies. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. [Immune response to live influenza vaccine].

    PubMed

    Naĭkhin, A N; Rekstin, A R; Barantseva, I B; Donina, S A; Desheva, Iu A; Grigor'eva, E P; Kiseleva, I V; Rudenko, L G

    2002-01-01

    Priority data on the induction, by using a Russian live cold-adapted reassortant influenza vaccine (LIV), of the cellular and humoral immunity with regard for attenuation and genetic reassortment of vaccine stains as well as with regard for the age of vaccinated persons and the production of Th1 (IFNY, IL-2) and Th2 (IL-4) cytokine markers in vitro are presented. It was demonstrated in vivo that a pathogenic virus of the A group by far more actively induced the lymphocyte apoptosis as compared with attenuated genetically reassorted stains. Unlike the influenza pathogenic virus, the genetically attenuated and reassorted strain did not produce any negative effects on the induction of cellular immunity. A comparative study of the LIV immunogenic properties in vaccinated persons showed an advantage of LIV over inactivated influenza vaccine (IIV) in stimulating the cellular and local immunity in the elderly. Unlike IIV, LIV induced an active and balanced immune response developing due to Th1 and Th2 activation. LIV was found to stimulate well enough the production of IFN and IL-2 in both young and old persons.

  7. Variability in Humoral Immunity to Measles Vaccine: New Developments

    PubMed Central

    Haralambieva, Iana H.; Kennedy, Richard B.; Ovsyannikova, Inna G.; Whitaker, Jennifer A.; Poland, Gregory A.

    2015-01-01

    Despite the existence of an effective measles vaccine, resurgence in measles cases in the United States and across Europe has occurred, including in individuals vaccinated with two doses of the vaccine. Host genetic factors result in inter-individual variation in measles vaccine-induced antibodies, and play a role in vaccine failure. Studies have identified HLA and non-HLA genetic influences that individually or jointly contribute to the observed variability in the humoral response to vaccination among healthy individuals. In this exciting era, new high-dimensional approaches and techniques including vaccinomics, systems biology, GWAS, epitope prediction and sophisticated bioinformatics/statistical algorithms, provide powerful tools to investigate immune response mechanisms to the measles vaccine. These might predict, on an individual basis, outcomes of acquired immunity post measles vaccination. PMID:26602762

  8. Flying vaccinator; a transgenic mosquito delivers a Leishmania vaccine via blood feeding.

    PubMed

    Yamamoto, D S; Nagumo, H; Yoshida, S

    2010-06-01

    'Flying vaccinator' is the concept of using genetically engineered hematophagous insects to deliver vaccines. Here we show the generation of a transgenic anopheline mosquito that expresses the Leishmania vaccine candidate, SP15, fused to monomeric red fluorescent protein (mDsRed) in its salivary glands. Importantly, mice bitten repeatedly by the transgenic mosquitoes raised anti-SP15 antibodies, indicating delivery of SP15 via blood feeding with its immunogenicity intact. Thus, this technology makes possible the generation of transgenic mosquitoes that match the original concept of a 'flying vaccinator'. However, medical safety issues and concerns about informed consent mitigate the use of the 'flying vaccinator' as a method to deliver vaccines. We propose that this expression system could be applied to elucidate saliva-malaria sporozoite interactions.

  9. Vaccines: the fourth century.

    PubMed

    Plotkin, Stanley A

    2009-12-01

    Vaccine development, which began with Edward Jenner's observations in the late 18th century, has entered its 4th century. From its beginnings, with the use of whole organisms that had been weakened or inactivated, to the modern-day use of genetic engineering, it has taken advantage of the tools discovered in other branches of microbiology. Numerous successful vaccines are in use, but the list of diseases for which vaccines do not exist is long. However, the multiplicity of strategies now available, discussed in this article, portends even more successful development of vaccines.

  10. Report of the 2014 Cent Gardes HIV Vaccine Conference-Part 2: Cell-mediated immunity, mucosal protection, and clinical trials: Fondation Mérieux Conference Center, Veyrier du Lac, France, 5-7 October, 2014.

    PubMed

    Girard, Marc P; Picot, Valentina; Longuet, Christophe; Nabel, Gary J

    2015-08-07

    The 2014 Cent Gardes Conference took place on October 5-7, 2014, at the Fondation Mérieux Conference Center, on the shores of the Annecy Lake and aimed to review the progress and promise of HIV vaccines. The elicitation of broadly neutralizing antibodies (bNAbs), their use in passive immunization, as well as their genetic delivery (vector immunoprophylaxis) by a recombinant Adenovirus-associated virus (AAV) vector were reviewed in a preceding article [1]. Approaches to the elicitation of long-lasting T cell or mucosal immunity were also discussed and are now reviewed here. The possibility of eliciting mucosal IgAs was discussed, since it was demonstrated that transcytosis-blocking IgAs can protect monkeys against repeated vaginal challenge with a pathogenic chimeric simian and human immunodeficiency virus (SHIV). The possibility of purging the HIV reservoirs from HIV-infected persons and developing a cure of the disease was also discussed. Copyright © 2015. Published by Elsevier Ltd.. All rights reserved.

  11. [Genetic characterisation of Powassan virus (POWV) isolated from Haemophysalis longicornis ticks in Primorye and two strains of Tick-borne encephalitis virus (TBEV) (Flaviviridae, Flavivirus): Alma-Arasan virus (AAV) isolated from Ixodes persulcatus ticks in Kazakhstan and Malyshevo virus isolated from Aedes vexans nipponii mosquitoes in Khabarovsk kray].

    PubMed

    L'vov, D K; Al'khovskiĭ, S V; Shchelkanov, M Iu; Deriabin, P G; Gitel'man, A K; Botikov, A G; Aristova, V A

    2014-01-01

    The complete genomes of the three tick-borne flaviviruses (genus Flavivirus, fam. Bunyaviridae) were sequenced: Povassan virus (POWV, strain LEIV-3070Prm, isolated from Haemophysalis logicornis in Primorsky Krai, Russia in 1977), Alma-Arasan virus (AAV, strain LEIV-1380Kaz, isolated from Ixodes persulcatus ticks in Kazakhstan in 1977) and Malyshevo virus (isolated from a pool of Aedes vexans nipponii mosquitoes, in the Khabarovsk Krai, Russia in 1978). It is shown that AAV and Malyshevo virus are the strains of Tick-borne encephalitis virus (TBEV) and belong to Sibirian and Far-Eastern genotypes, respectively (GenBank ID: AAV KJ744033; strain Malyshevo KJ744034). Phylogenetically AAV is closest related (94,6% nt and 98,3% aa identity) to TBEV strains, isolated in Sibiria (Vasilchenko, Aino, Chita-653, Irkutsk-12). Malyshevo virus is closest related (96,4% nt and 98,3% nt identity) to strains of TBEV, isolated in Far Eastern part of Russia (1230, Spassk-72, Primorye-89). POWV LEIV-3070Prm has 99.7% identity with the prototype strain POWV LB, isolated in Canada and 99.5% of isolates with Far-Eastern strains of POWV (Spassk-9 and Nadezdinsk-1991).

  12. Worldwide genetic variability of the Duffy binding protein: insights into Plasmodium vivax vaccine development.

    PubMed

    Nóbrega de Sousa, Taís; Carvalho, Luzia Helena; Alves de Brito, Cristiana Ferreira

    2011-01-01

    The dependence of Plasmodium vivax on invasion mediated by Duffy binding protein (DBP) makes this protein a prime candidate for development of a vaccine. However, the development of a DBP-based vaccine might be hampered by the high variability of the protein ligand (DBP(II)), known to bias the immune response toward a specific DBP variant. Here, the hypothesis being investigated is that the analysis of the worldwide DBP(II) sequences will allow us to determine the minimum number of haplotypes (MNH) to be included in a DBP-based vaccine of broad coverage. For that, all DBP(II) sequences available were compiled and MNH was based on the most frequent nonsynonymous single nucleotide polymorphisms, the majority mapped on B and T cell epitopes. A preliminary analysis of DBP(II) genetic diversity from eight malaria-endemic countries estimated that a number between two to six DBP haplotypes (17 in total) would target at least 50% of parasite population circulating in each endemic region. Aiming to avoid region-specific haplotypes, we next analyzed the MNH that broadly cover worldwide parasite population. The results demonstrated that seven haplotypes would be required to cover around 60% of DBP(II) sequences available. Trying to validate these selected haplotypes per country, we found that five out of the eight countries will be covered by the MNH (67% of parasite populations, range 48-84%). In addition, to identify related subgroups of DBP(II) sequences we used a Bayesian clustering algorithm. The algorithm grouped all DBP(II) sequences in six populations that were independent of geographic origin, with ancestral populations present in different proportions in each country. In conclusion, in this first attempt to undertake a global analysis about DBP(II) variability, the results suggest that the development of DBP-based vaccine should consider multi-haplotype strategies; otherwise a putative P. vivax vaccine may not target some parasite populations.

  13. Elucidation of the full genetic information of Japanese rubella vaccines and the genetic changes associated with in vitro and in vivo vaccine virus phenotypes.

    PubMed

    Otsuki, Noriyuki; Abo, Hitoshi; Kubota, Toru; Mori, Yoshio; Umino, Yukiko; Okamoto, Kiyoko; Takeda, Makoto; Komase, Katsuhiro

    2011-02-24

    Rubella is a mild disease characterized by low-grade fever, and a morbilliform rash, but causes congenital defects in neonates born from mothers who suffered from rubella during the pregnancy. After many passages of wild-type rubella virus strains in various types of cultured cells, five live attenuated rubella vaccines were developed in Japan. An inability to elicit anti-rubella virus antibodies in experimentally infected animals was used as an in vivo marker phenotype of Japanese rubella vaccines. All Japanese rubella vaccine viruses exhibit a temperature-sensitive (ts) phenotype, and replicate very poorly at a high temperature. We determined the entire genome sequences of three Japanese rubella vaccines (Matsuba, TCRB19, and Matsuura), thereby completing the sequencing of all five Japanese rubella vaccines. In addition, the entire genome sequences of three vaccine progenitors were determined. Comparative nucleotide sequence analyses revealed mutations that were introduced into the genomes of the TO-336 and Matsuura vaccines during their production by laboratory passaging. Analyses involving cellular expression of viral P150 nonstructural protein-derived peptides revealed that the N1159S mutation conferred the ts phenotype on the TO-336 vaccine, and that reduced thermal stability of the P150 protease domain was a cause of the ts phenotype of some rubella vaccine viruses. The ts phenotype of vaccine viruses was not necessarily correlated with their inability to elicit humoral immune responses in animals. Therefore, the molecular mechanisms underlying the inability of these vaccines to elicit humoral responses in animals are more complicated than the previously considered mechanism involving the ts phenotype as the major cause. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. AAV5-Factor VIII Gene Transfer in Severe Hemophilia A.

    PubMed

    Rangarajan, Savita; Walsh, Liron; Lester, Will; Perry, David; Madan, Bella; Laffan, Michael; Yu, Hua; Vettermann, Christian; Pierce, Glenn F; Wong, Wing Y; Pasi, K John

    2017-12-28

    Patients with hemophilia A rely on exogenous factor VIII to prevent bleeding in joints, soft tissue, and the central nervous system. Although successful gene transfer has been reported in patients with hemophilia B, the large size of the factor VIII coding region has precluded improved outcomes with gene therapy in patients with hemophilia A. We infused a single intravenous dose of a codon-optimized adeno-associated virus serotype 5 (AAV5) vector encoding a B-domain-deleted human factor VIII (AAV5-hFVIII-SQ) in nine men with severe hemophilia A. Participants were enrolled sequentially into one of three dose cohorts (low dose [one participant], intermediate dose [one participant], and high dose [seven participants]) and were followed through 52 weeks. Factor VIII activity levels remained at 3 IU or less per deciliter in the recipients of the low or intermediate dose. In the high-dose cohort, the factor VIII activity level was more than 5 IU per deciliter between weeks 2 and 9 after gene transfer in all seven participants, and the level in six participants increased to a normal value (>50 IU per deciliter) that was maintained at 1 year after receipt of the dose. In the high-dose cohort, the median annualized bleeding rate among participants who had previously received prophylactic therapy decreased from 16 events before the study to 1 event after gene transfer, and factor VIII use for participant-reported bleeding ceased in all the participants in this cohort by week 22. The primary adverse event was an elevation in the serum alanine aminotransferase level to 1.5 times the upper limit of the normal range or less. Progression of preexisting chronic arthropathy in one participant was the only serious adverse event. No neutralizing antibodies to factor VIII were detected. The infusion of AAV5-hFVIII-SQ was associated with the sustained normalization of factor VIII activity level over a period of 1 year in six of seven participants who received a high dose, with

  15. Military vaccines in today's environment.

    PubMed

    Schmaljohn, Connie S; Smith, Leonard A; Friedlander, Arthur M

    2012-08-01

    The US military has a long and highly distinguished record of developing effective vaccines against pathogens that threaten the armed forces. Many of these vaccines have also been of significant benefit to civilian populations around the world. The current requirements for force protection include vaccines against endemic disease threats as well as against biological warfare or bioterrorism agents, to include novel or genetically engineered threats. The cost of vaccine development and the modern regulatory requirements for licensing vaccines have strained the ability of the program to maintain this broad mission. Without innovative vaccine technologies, streamlined regulatory strategies, and coordinating efforts for use in civilian populations where appropriate, the military vaccine development program is in jeopardy.

  16. Biomarkers of safety and immune protection for genetically modified live attenuated leishmania vaccines against visceral leishmaniasis - discovery and implications.

    PubMed

    Gannavaram, Sreenivas; Dey, Ranadhir; Avishek, Kumar; Selvapandiyan, Angamuthu; Salotra, Poonam; Nakhasi, Hira L

    2014-01-01

    Despite intense efforts there is no safe and efficacious vaccine against visceral leishmaniasis, which is fatal and endemic in many tropical countries. A major shortcoming in the vaccine development against blood-borne parasitic agents such as Leishmania is the inadequate predictive power of the early immune responses mounted in the host against the experimental vaccines. Often immune correlates derived from in-bred animal models do not yield immune markers of protection that can be readily extrapolated to humans. The limited efficacy of vaccines based on DNA, subunit, heat killed parasites has led to the realization that acquisition of durable immunity against the protozoan parasites requires a controlled infection with a live attenuated organism. Recent success of irradiated malaria parasites as a vaccine candidate further strengthens this approach to vaccination. We developed several gene deletion mutants in Leishmania donovani as potential live attenuated vaccines and reported extensively on the immunogenicity of LdCentrin1 deleted mutant in mice, hamsters, and dogs. Additional limited studies using genetically modified live attenuated Leishmania parasites as vaccine candidates have been reported. However, for the live attenuated parasite vaccines, the primary barrier against widespread use remains the absence of clear biomarkers associated with protection and safety. Recent studies in evaluation of vaccines, e.g., influenza and yellow fever vaccines, using systems biology tools demonstrated the power of such strategies in understanding the immunological mechanisms that underpin a protective phenotype. Applying similar tools in isolated human tissues such as PBMCs from healthy individuals infected with live attenuated parasites such as LdCen(-/-) in vitro followed by human microarray hybridization experiments will enable us to understand how early vaccine-induced gene expression profiles and the associated immune responses are coordinately regulated in normal

  17. [Genetic recombination in vaccine poliovirus: comparative study in strains excreted in course of vaccination by oral poliovirus vaccine and circulating strains].

    PubMed

    Haddad-Boubaker, S; Ould-Mohamed-Abdallah, M V; Ben-Yahia, A; Triki, H

    2010-12-01

    Recombination is one of the major mechanisms of evolution in poliovirus. In this work, recombination was assessed in children during vaccination with OPV and among circulating vaccine strains isolated in Tunisia during the last 15 years in order to identify a possible role of recombination in the response to the vaccine or the acquisition of an increased transmissibility. This study included 250 poliovirus isolates: 137 vaccine isolates, excreted by children during primary vaccination with OPV and 113 isolates obtained from acute flaccid paralytic (AFP) cases and healthy contacts. Recombination was first assessed using a double PCR-RFLP, and sequencing. Nineteen per cent of recombinant strains were identified: 20% of strains excreted by vaccinees among 18% of circulating strains. The proportion of recombinant in isolates of serotype1 was very low in the two groups while the proportions of recombinants in serotypes 2 and 3 were different. In vaccinees, the frequency of recombinants in serotype3 decreased during the course of vaccination: 54% after the first dose, 32% after the second and 14% after the third dose. These results suggest that recombination enhances the ability of serotype3 vaccine strains to induce an immune response. Apart from recent vaccination, it may contribute to a more effective transmissibility of vaccine strains among human population. Copyright © 2009 Elsevier Masson SAS. All rights reserved.

  18. Muscle function recovery in golden retriever muscular dystrophy after AAV1-U7 exon skipping.

    PubMed

    Vulin, Adeline; Barthélémy, Inès; Goyenvalle, Aurélie; Thibaud, Jean-Laurent; Beley, Cyriaque; Griffith, Graziella; Benchaouir, Rachid; le Hir, Maëva; Unterfinger, Yves; Lorain, Stéphanie; Dreyfus, Patrick; Voit, Thomas; Carlier, Pierre; Blot, Stéphane; Garcia, Luis

    2012-11-01

    Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder resulting from lesions of the gene encoding dystrophin. These usually consist of large genomic deletions, the extents of which are not correlated with the severity of the phenotype. Out-of-frame deletions give rise to dystrophin deficiency and severe DMD phenotypes, while internal deletions that produce in-frame mRNAs encoding truncated proteins can lead to a milder myopathy known as Becker muscular dystrophy (BMD). Widespread restoration of dystrophin expression via adeno-associated virus (AAV)-mediated exon skipping has been successfully demonstrated in the mdx mouse model and in cardiac muscle after percutaneous transendocardial delivery in the golden retriever muscular dystrophy dog (GRMD) model. Here, a set of optimized U7snRNAs carrying antisense sequences designed to rescue dystrophin were delivered into GRMD skeletal muscles by AAV1 gene transfer using intramuscular injection or forelimb perfusion. We show sustained correction of the dystrophic phenotype in extended muscle areas and partial recovery of muscle strength. Muscle architecture was improved and fibers displayed the hallmarks of mature and functional units. A 5-year follow-up ruled out immune rejection drawbacks but showed a progressive decline in the number of corrected muscle fibers, likely due to the persistence of a mild dystrophic process such as occurs in BMD phenotypes. Although AAV-mediated exon skipping was shown safe and efficient to rescue a truncated dystrophin, it appears that recurrent treatments would be required to maintain therapeutic benefit ahead of the progression of the disease.

  19. Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation

    PubMed Central

    Ramsingh, Arlene I.; Gray, Steven J.; Reilly, Andrew; Koday, Michael; Bratt, Debbie; Koday, Merika Treants; Murnane, Robert; Hu, Yuhui; Messer, Anne

    2018-01-01

    A critical issue in transgene delivery studies is immune reactivity to the transgene- encoded protein and its impact on sustained gene expression. Here, we test the hypothesis that immunomodulation by rapamycin can decrease immune reactivity after intrathecal AAV9 delivery of a transgene (GFP) in non-human primates, resulting in sustained GFP expression in the CNS. We show that rapamycin treatment clearly reduced the overall immunogenicity of the AAV9/GFP vector by lowering GFP- and AAV9-specific antibody responses, and decreasing T cell responses including cytokine and cytolytic effector responses. Spinal cord GFP protein expression was sustained for twelve weeks, with no toxicity. Immune correlates of robust transgene expression include negligible GFP-specific CD4 and CD8 T cell responses, absence of GFP-specific IFN-γ producing T cells, and absence of GFP-specific cytotoxic T cells, which support the hypothesis that decreased T cell reactivity results in sustained transgene expression. These data strongly support the use of modest doses of rapamycin to modulate immune responses for intrathecal gene therapies, and potentially a much wider range of viral vector-based therapeutics. PMID:29874260

  20. Intracellular generation of single-strand template increases the knock-in efficiency by combining CRISPR/Cas9 with AAV.

    PubMed

    Xiao, Qing; Min, Taishan; Ma, Shuangping; Hu, Lingna; Chen, Hongyan; Lu, Daru

    2018-04-18

    Targeted integration of transgenes facilitates functional genomic research and holds prospect for gene therapy. The established microhomology-mediated end-joining (MMEJ)-based strategy leads to the precise gene knock-in with easily constructed donor, yet the limited efficiency remains to be further improved. Here, we show that single-strand DNA (ssDNA) donor contributes to efficient increase of knock-in efficiency and establishes a method to achieve the intracellular linearization of long ssDNA donor. We identified that the CRISPR/Cas9 system is responsible for breaking double-strand DNA (dsDNA) of palindromic structure in inverted terminal repeats (ITRs) region of recombinant adeno-associated virus (AAV), leading to the inhibition of viral second-strand DNA synthesis. Combing Cas9 plasmids targeting genome and ITR with AAV donor delivery, the precise knock-in of gene cassette was achieved, with 13-14% of the donor insertion events being mediated by MMEJ in HEK 293T cells. This study describes a novel method to integrate large single-strand transgene cassettes into the genomes, increasing knock-in efficiency by 13.6-19.5-fold relative to conventional AAV-mediated method. It also provides a comprehensive solution to the challenges of complicated production and difficult delivery with large exogenous fragments.

  1. AAV9-based gene therapy partially ameliorates the clinical phenotype of a mouse model of Leigh syndrome

    PubMed Central

    Di Meo, I; Marchet, S; Lamperti, C; Zeviani, M; Viscomi, C

    2017-01-01

    Leigh syndrome (LS) is the most common infantile mitochondrial encephalopathy. No treatment is currently available for this condition. Mice lacking Ndufs4, encoding NADH: ubiquinone oxidoreductase iron-sulfur protein 4 (NDUFS4) recapitulates the main findings of complex I (cI)-related LS, including severe multisystemic cI deficiency and progressive neurodegeneration. In order to develop a gene therapy approach for LS, we used here an AAV2/9 vector carrying the human NDUFS4 coding sequence (hNDUFS4). We administered AAV2/9-hNDUFS4 by intravenous (IV) and/or intracerebroventricular (ICV) routes to either newborn or young Ndufs4−/− mice. We found that IV administration alone was only able to correct the cI deficiency in peripheral organs, whereas ICV administration partially corrected the deficiency in the brain. However, both treatments failed to improve the clinical phenotype or to prolong the lifespan of Ndufs4−/− mice. In contrast, combined IV and ICV treatments resulted, along with increased cI activity, in the amelioration of the rotarod performance and in a significant prolongation of the lifespan. Our results indicate that extraneurological organs have an important role in LS pathogenesis and provide an insight into current limitations of adeno-associated virus (AAV)-mediated gene therapy in multisystem disorders. These findings warrant future investigations to develop new vectors able to efficiently target multiple organs. PMID:28753212

  2. AAV9-based gene therapy partially ameliorates the clinical phenotype of a mouse model of Leigh syndrome.

    PubMed

    Di Meo, I; Marchet, S; Lamperti, C; Zeviani, M; Viscomi, C

    2017-10-01

    Leigh syndrome (LS) is the most common infantile mitochondrial encephalopathy. No treatment is currently available for this condition. Mice lacking Ndufs4, encoding NADH: ubiquinone oxidoreductase iron-sulfur protein 4 (NDUFS4) recapitulates the main findings of complex I (cI)-related LS, including severe multisystemic cI deficiency and progressive neurodegeneration. In order to develop a gene therapy approach for LS, we used here an AAV2/9 vector carrying the human NDUFS4 coding sequence (hNDUFS4). We administered AAV2/9-hNDUFS4 by intravenous (IV) and/or intracerebroventricular (ICV) routes to either newborn or young Ndufs4 -/- mice. We found that IV administration alone was only able to correct the cI deficiency in peripheral organs, whereas ICV administration partially corrected the deficiency in the brain. However, both treatments failed to improve the clinical phenotype or to prolong the lifespan of Ndufs4 -/- mice. In contrast, combined IV and ICV treatments resulted, along with increased cI activity, in the amelioration of the rotarod performance and in a significant prolongation of the lifespan. Our results indicate that extraneurological organs have an important role in LS pathogenesis and provide an insight into current limitations of adeno-associated virus (AAV)-mediated gene therapy in multisystem disorders. These findings warrant future investigations to develop new vectors able to efficiently target multiple organs.

  3. Targeted gene knock-in by homology-directed genome editing using Cas9 ribonucleoprotein and AAV donor delivery.

    PubMed

    Gaj, Thomas; Staahl, Brett T; Rodrigues, Gonçalo M C; Limsirichai, Prajit; Ekman, Freja K; Doudna, Jennifer A; Schaffer, David V

    2017-06-20

    Realizing the full potential of genome editing requires the development of efficient and broadly applicable methods for delivering programmable nucleases and donor templates for homology-directed repair (HDR). The RNA-guided Cas9 endonuclease can be introduced into cells as a purified protein in complex with a single guide RNA (sgRNA). Such ribonucleoproteins (RNPs) can facilitate the high-fidelity introduction of single-base substitutions via HDR following co-delivery with a single-stranded DNA oligonucleotide. However, combining RNPs with transgene-containing donor templates for targeted gene addition has proven challenging, which in turn has limited the capabilities of the RNP-mediated genome editing toolbox. Here, we demonstrate that combining RNP delivery with naturally recombinogenic adeno-associated virus (AAV) donor vectors enables site-specific gene insertion by homology-directed genome editing. Compared to conventional plasmid-based expression vectors and donor templates, we show that combining RNP and AAV donor delivery increases the efficiency of gene addition by up to 12-fold, enabling the creation of lineage reporters that can be used to track the conversion of striatal neurons from human fibroblasts in real time. These results thus illustrate the potential for unifying nuclease protein delivery with AAV donor vectors for homology-directed genome editing. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. 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.

  5. Japanese encephalitis vaccines: current vaccines and future prospects.

    PubMed

    Monath, T P

    2002-01-01

    Vaccination against JE ideally should be practiced in all areas of Asia where the virus is responsible for human disease. The WHO has placed a high priority on the development of a new vaccine for prevention of JE. Some countries in Asia (Japan, South Korea, North Korea, Taiwan, Vietnam, Thailand, and the PRC) manufacture JE vaccines and practice childhood immunization, while other countries suffering endemic or epidemic disease (India, Nepal, Laos, Cambodia, Bangladesh, Myanmar, Malaysia, Indonesia and the Philippines) have no JE vaccine manufacturing or policy for use. With the exception of the PRC, all countries practicing JE vaccination use formalin inactivated mouse brain vaccines, which are relatively expensive and are associated with rare but clinically significant allergic and neurological adverse events. New inactivated JE vaccines manufactured in Vero cells are in advanced preclinical or early clinical development in Japan, South Korea, Taiwan, and the PRC. An empirically derived, live attenuated vaccine (SA14-14-2) is widely used in the PRC. Trials in the PRC have shown SA14-14-2 to be safe and effective when administered in a two-dose regimen, but regulatory concerns over manufacturing and control have restricted international distribution. The genetic basis of attenuation of SA14-14-2 has been partially defined. A new live attenuated vaccine (ChimeriVax-JE) that uses a reliable flavivirus vaccine--yellow fever 17D--as a live vector for the envelope genes of SA14-14-2 virus is in early clinical trials and appears to be well tolerated and immunogenic after a single dose. Vaccinia and avipox vectored vaccines have also been tested clinically, but are no longer being pursued due to restricted effectiveness mediated by anti-vector immunity. Other approaches to JE vaccines--including naked DNA, oral vaccination, and recombinant subunit vaccines--have been reviewed.

  6. Rabies virus vaccines: is there a need for a pan-lyssavirus vaccine?

    PubMed

    Evans, Jennifer S; Horton, Daniel L; Easton, Andrew J; Fooks, Anthony R; Banyard, Ashley C

    2012-12-14

    All members of the lyssavirus genus are capable of causing disease that invariably results in death following the development of clinical symptoms. The recent detection of several novel lyssavirus species across the globe, in different animal species, has demonstrated that the lyssavirus genus contains a greater degree of genetic and antigenic variation than previously suspected. The divergence of species within the genus has led to a differentiation of lyssavirus isolates based on both antigenic and genetic data into two, and potentially a third phylogroup. Critically, from both a human and animal health perspective, current rabies vaccines appear able to protect against lyssaviruses classified within phylogroup I. However no protection is afforded against phylogroup II viruses or other more divergent viruses. Here we review current knowledge regarding the diversity and antigenicity of the lyssavirus glycoprotein. We review the degree of cross protection afforded by rabies vaccines, the genetic and antigenic divergence of the lyssaviruses and potential mechanisms for the development of novel lyssavirus vaccines for use in areas where divergent lyssaviruses are known to circulate, as well as for use by those at occupational risk from these pathogens. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  7. Protective efficacy of a high-growth reassortant swine H3N2 inactivated vaccine constructed by reverse genetic manipulation

    PubMed Central

    Wen, Feng; Ma, Ji-Hong; Yang, Fu-Ru; Huang, Meng; Zhou, Yan-Jun; Li, Ze-Jun

    2014-01-01

    Novel reassortant H3N2 swine influenza viruses (SwIV) with the matrix gene from the 2009 H1N1 pandemic virus have been isolated in many countries as well as during outbreaks in multiple states in the United States, indicating that H3N2 SwIV might be a potential threat to public health. Since southern China is the world's largest producer of pigs, efficient vaccines should be developed to prevent pigs from acquiring H3N2 subtype SwIV infections, and thus limit the possibility of SwIV infection at agricultural fairs. In this study, a high-growth reassortant virus (GD/PR8) was generated by plasmid-based reverse genetics and tested as a candidate inactivated vaccine. The protective efficacy of this vaccine was evaluated in mice by challenging them with another H3N2 SwIV isolate [A/Swine/Heilongjiang/1/05 (H3N2) (HLJ/05)]. Prime and booster inoculation with GD/PR8 vaccine yielded high-titer serum hemagglutination inhibiting antibodies and IgG antibodies. Complete protection of mice against H3N2 SwIV was observed, with significantly reduced lung lesion and viral loads in vaccine-inoculated mice relative to mock-vaccinated controls. These results suggest that the GD/PR8 vaccine may serve as a promising candidate for rapid intervention of H3N2 SwIV outbreaks in China. PMID:24675833

  8. Optimized AAV rh.10 Vectors That Partially Evade Neutralizing Antibodies during Hepatic Gene Transfer

    PubMed Central

    Selot, Ruchita; Arumugam, Sathyathithan; Mary, Bertin; Cheemadan, Sabna; Jayandharan, Giridhara R.

    2017-01-01

    Of the 12 common serotypes used for gene delivery applications, Adeno-associated virus (AAV)rh.10 serotype has shown sustained hepatic transduction and has the lowest seropositivity in humans. We have evaluated if further modifications to AAVrh.10 at its phosphodegron like regions or predicted immunogenic epitopes could improve its hepatic gene transfer and immune evasion potential. Mutant AAVrh.10 vectors were generated by site directed mutagenesis of the predicted targets. These mutant vectors were first tested for their transduction efficiency in HeLa and HEK293T cells. The optimal vector was further evaluated for their cellular uptake, entry, and intracellular trafficking by quantitative PCR and time-lapse confocal microscopy. To evaluate their potential during hepatic gene therapy, C57BL/6 mice were administered with wild-type or optimal mutant AAVrh.10 and the luciferase transgene expression was documented by serial bioluminescence imaging at 14, 30, 45, and 72 days post-gene transfer. Their hepatic transduction was further verified by a quantitative PCR analysis of AAV copy number in the liver tissue. The optimal AAVrh.10 vector was further evaluated for their immune escape potential, in animals pre-immunized with human intravenous immunoglobulin. Our results demonstrate that a modified AAVrh.10 S671A vector had enhanced cellular entry (3.6 fold), migrate rapidly to the perinuclear region (1 vs. >2 h for wild type vectors) in vitro, which further translates to modest increase in hepatic gene transfer efficiency in vivo. More importantly, the mutant AAVrh.10 vector was able to partially evade neutralizing antibodies (~27–64 fold) in pre-immunized animals. The development of an AAV vector system that can escape the circulating neutralizing antibodies in the host will substantially widen the scope of gene therapy applications in humans. PMID:28769791

  9. Development of a chimeric Zika vaccine using a licensed live-attenuated flavivirus vaccine as backbone.

    PubMed

    Li, Xiao-Feng; Dong, Hao-Long; Wang, Hong-Jiang; Huang, Xing-Yao; Qiu, Ye-Feng; Ji, Xue; Ye, Qing; Li, Chunfeng; Liu, Yang; Deng, Yong-Qiang; Jiang, Tao; Cheng, Gong; Zhang, Fu-Chun; Davidson, Andrew D; Song, Ya-Jun; Shi, Pei-Yong; Qin, Cheng-Feng

    2018-02-14

    The global spread of Zika virus (ZIKV) and its unexpected association with congenital defects necessitates the rapid development of a safe and effective vaccine. Here we report the development and characterization of a recombinant chimeric ZIKV vaccine candidate (termed ChinZIKV) that expresses the prM-E proteins of ZIKV using the licensed Japanese encephalitis live-attenuated vaccine SA14-14-2 as the genetic backbone. ChinZIKV retains its replication activity and genetic stability in vitro, while exhibiting an attenuation phenotype in multiple animal models. Remarkably, immunization of mice and rhesus macaques with a single dose of ChinZIKV elicits robust and long-lasting immune responses, and confers complete protection against ZIKV challenge. Significantly, female mice immunized with ChinZIKV are protected against placental and fetal damage upon ZIKV challenge during pregnancy. Overall, our study provides an alternative vaccine platform in response to the ZIKV emergency, and the safety, immunogenicity, and protection profiles of ChinZIKV warrant further clinical development.

  10. Global genetic diversity of the Plasmodium vivax transmission-blocking vaccine candidate Pvs48/45.

    PubMed

    Vallejo, Andres F; Martinez, Nora L; Tobon, Alejandra; Alger, Jackeline; Lacerda, Marcus V; Kajava, Andrey V; Arévalo-Herrera, Myriam; Herrera, Sócrates

    2016-04-12

    Plasmodium vivax 48/45 protein is expressed on the surface of gametocytes/gametes and plays a key role in gamete fusion during fertilization. This protein was recently expressed in Escherichia coli host as a recombinant product that was highly immunogenic in mice and monkeys and induced antibodies with high transmission-blocking activity, suggesting its potential as a P. vivax transmission-blocking vaccine candidate. To determine sequence polymorphism of natural parasite isolates and its potential influence on the protein structure, all pvs48/45 sequences reported in databases from around the world as well as those from low-transmission settings of Latin America were compared. Plasmodium vivax parasite isolates from malaria-endemic regions of Colombia, Brazil and Honduras (n = 60) were used to sequence the Pvs48/45 gene, and compared to those previously reported to GenBank and PlasmoDB (n = 222). Pvs48/45 gene haplotypes were analysed to determine the functional significance of genetic variation in protein structure and vaccine potential. Nine non-synonymous substitutions (E35K, Y196H, H211N, K250N, D335Y, E353Q, A376T, K390T, K418R) and three synonymous substitutions (I73, T149, C156) that define seven different haplotypes were found among the 282 isolates from nine countries when compared with the Sal I reference sequence. Nucleotide diversity (π) was 0.00173 for worldwide samples (range 0.00033-0.00216), resulting in relatively high diversity in Myanmar and Colombia, and low diversity in Mexico, Peru and South Korea. The two most frequent substitutions (E353Q: 41.9 %, K250N: 39.5 %) were predicted to be located in antigenic regions without affecting putative B cell epitopes or the tertiary protein structure. There is limited sequence polymorphism in pvs48/45 with noted geographical clustering among Asian and American isolates. The low genetic diversity of the protein does not influence the predicted antigenicity or protein structure and, therefore

  11. Biomarkers of Safety and Immune Protection for Genetically Modified Live Attenuated Leishmania Vaccines Against Visceral Leishmaniasis – Discovery and Implications

    PubMed Central

    Gannavaram, Sreenivas; Dey, Ranadhir; Avishek, Kumar; Selvapandiyan, Angamuthu; Salotra, Poonam; Nakhasi, Hira L.

    2014-01-01

    Despite intense efforts there is no safe and efficacious vaccine against visceral leishmaniasis, which is fatal and endemic in many tropical countries. A major shortcoming in the vaccine development against blood-borne parasitic agents such as Leishmania is the inadequate predictive power of the early immune responses mounted in the host against the experimental vaccines. Often immune correlates derived from in-bred animal models do not yield immune markers of protection that can be readily extrapolated to humans. The limited efficacy of vaccines based on DNA, subunit, heat killed parasites has led to the realization that acquisition of durable immunity against the protozoan parasites requires a controlled infection with a live attenuated organism. Recent success of irradiated malaria parasites as a vaccine candidate further strengthens this approach to vaccination. We developed several gene deletion mutants in Leishmania donovani as potential live attenuated vaccines and reported extensively on the immunogenicity of LdCentrin1 deleted mutant in mice, hamsters, and dogs. Additional limited studies using genetically modified live attenuated Leishmania parasites as vaccine candidates have been reported. However, for the live attenuated parasite vaccines, the primary barrier against widespread use remains the absence of clear biomarkers associated with protection and safety. Recent studies in evaluation of vaccines, e.g., influenza and yellow fever vaccines, using systems biology tools demonstrated the power of such strategies in understanding the immunological mechanisms that underpin a protective phenotype. Applying similar tools in isolated human tissues such as PBMCs from healthy individuals infected with live attenuated parasites such as LdCen−/− in vitro followed by human microarray hybridization experiments will enable us to understand how early vaccine-induced gene expression profiles and the associated immune responses are coordinately regulated in normal

  12. AAV-PHP.B-Mediated Global-Scale Expression in the Mouse Nervous System Enables GBA1 Gene Therapy for Wide Protection from Synucleinopathy.

    PubMed

    Morabito, Giuseppe; Giannelli, Serena G; Ordazzo, Gabriele; Bido, Simone; Castoldi, Valerio; Indrigo, Marzia; Cabassi, Tommaso; Cattaneo, Stefano; Luoni, Mirko; Cancellieri, Cinzia; Sessa, Alessandro; Bacigaluppi, Marco; Taverna, Stefano; Leocani, Letizia; Lanciego, José L; Broccoli, Vania

    2017-12-06

    The lack of technology for direct global-scale targeting of the adult mouse nervous system has hindered research on brain processing and dysfunctions. Currently, gene transfer is normally achieved by intraparenchymal viral injections, but these injections target a restricted brain area. Herein, we demonstrated that intravenous delivery of adeno-associated virus (AAV)-PHP.B viral particles permeated and diffused throughout the neural parenchyma, targeting both the central and the peripheral nervous system in a global pattern. We then established multiple procedures of viral transduction to control gene expression or inactivate gene function exclusively in the adult nervous system and assessed the underlying behavioral effects. Building on these results, we established an effective gene therapy strategy to counteract the widespread accumulation of α-synuclein deposits throughout the forebrain in a mouse model of synucleinopathy. Transduction of A53T-SCNA transgenic mice with AAV-PHP.B-GBA1 restored physiological levels of the enzyme, reduced α-synuclein pathology, and produced significant behavioral recovery. Finally, we provided evidence that AAV-PHP.B brain penetration does not lead to evident dysfunctions in blood-brain barrier integrity or permeability. Altogether, the AAV-PHP.B viral platform enables non-invasive, widespread, and long-lasting global neural expression of therapeutic genes, such as GBA1, providing an invaluable approach to treat neurodegenerative diseases with diffuse brain pathology such as synucleinopathies. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  13. Vaccines against malaria.

    PubMed

    Ouattara, Amed; Laurens, Matthew B

    2015-03-15

    Despite global efforts to control malaria, the illness remains a significant public health threat. Currently, there is no licensed vaccine against malaria, but an efficacious vaccine would represent an important public health tool for successful malaria elimination. Malaria vaccine development continues to be hindered by a poor understanding of antimalarial immunity, a lack of an immune correlate of protection, and the genetic diversity of malaria parasites. Current vaccine development efforts largely target Plasmodium falciparum parasites in the pre-erythrocytic and erythrocytic stages, with some research on transmission-blocking vaccines against asexual stages and vaccines against pregnancy-associated malaria. The leading pre-erythrocytic vaccine candidate is RTS,S, and early results of ongoing Phase 3 testing show overall efficacy of 46% against clinical malaria. The next steps for malaria vaccine development will focus on the design of a product that is efficacious against the highly diverse strains of malaria and the identification of a correlate of protection against disease. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Rhabdoviruses as vaccine platforms for infectious disease and cancer.

    PubMed

    Zemp, Franz; Rajwani, Jahanara; Mahoney, Douglas J

    2018-05-21

    The family Rhabdoviridae (RV) comprises a large, genetically diverse collection of single-stranded, negative sense RNA viruses from the order Mononegavirales. Several RV members are being developed as live-attenuated vaccine vectors for the prevention or treatment of infectious disease and cancer. These include the prototype recombinant Vesicular Stomatitis Virus (rVSV) and the more recently developed recombinant Maraba Virus, both species within the genus Vesiculoviridae. A relatively strong safety profile in humans, robust immunogenicity and genetic malleability are key features that make the RV family attractive vaccine platforms. Currently, the rVSV vector is in preclinical development for vaccination against numerous high-priority infectious diseases, with clinical evaluation underway for HIV/AIDS and Ebola virus disease. Indeed, the success of the rVSV-ZEBOV vaccine during the 2014-15 Ebola virus outbreak in West Africa highlights the therapeutic potential of rVSV as a vaccine vector for acute, life-threatening viral illnesses. The rVSV and rMaraba platforms are also being tested as 'oncolytic' cancer vaccines in a series of phase 1-2 clinical trials, after being proven effective at eliciting immune-mediated tumour regression in preclinical mouse models. In this review, we discuss the biological and genetic features that make RVs attractive vaccine platforms and the development and ongoing testing of rVSV and rMaraba strains as vaccine vectors for infectious disease and cancer.

  15. 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

  16. In-depth genome analyses of viruses from vaccine-derived rabies cases and corresponding live-attenuated oral rabies vaccines.

    PubMed

    Pfaff, Florian; Müller, Thomas; Freuling, Conrad M; Fehlner-Gardiner, Christine; Nadin-Davis, Susan; Robardet, Emmanuelle; Cliquet, Florence; Vuta, Vlad; Hostnik, Peter; Mettenleiter, Thomas C; Beer, Martin; Höper, Dirk

    2018-02-10

    Live-attenuated rabies virus strains such as those derived from the field isolate Street Alabama Dufferin (SAD) have been used extensively and very effectively as oral rabies vaccines for the control of fox rabies in both Europe and Canada. Although these vaccines are safe, some cases of vaccine-derived rabies have been detected during rabies surveillance accompanying these campaigns. In recent analysis it was shown that some commercial SAD vaccines consist of diverse viral populations, rather than clonal genotypes. For cases of vaccine-derived rabies, only consensus sequence data have been available to date and information concerning their population diversity was thus lacking. In our study, we used high-throughput sequencing to analyze 11 cases of vaccine-derived rabies, and compared their viral population diversity to the related oral rabies vaccines using pairwise Manhattan distances. This extensive deep sequencing analysis of vaccine-derived rabies cases observed during oral vaccination programs provided deeper insights into the effect of accidental in vivo replication of genetically diverse vaccine strains in the central nervous system of target and non-target species under field conditions. The viral population in vaccine-derived cases appeared to be clonal in contrast to their parental vaccines. The change from a state of high population diversity present in the vaccine batches to a clonal genotype in the affected animal may indicate the presence of a strong bottleneck during infection. In conclusion, it is very likely that these few cases are the consequence of host factors and not the result of the selection of a more virulent genotype. Furthermore, this type of vaccine-derived rabies leads to the selection of clonal genotypes and the selected variants were genetically very similar to potent SAD vaccines that have undergone a history of in vitro selection. Copyright © 2018. Published by Elsevier Ltd.

  17. Rheumatoid arthritis and Swine influenza vaccine: a case report.

    PubMed

    Basra, Gurjot; Jajoria, Praveen; Gonzalez, Emilio

    2012-01-01

    Rheumatoid arthritis (RA) is the most common chronic inflammatory joint disease. Multiple scientific articles have documented that vaccinations for influenza, MMR, and HBV, to name a few, could be triggers of RA in genetically predisposed individuals. However, there is limited data regarding the association of swine flu vaccine (H1N1) and RA. We report the case of a Mexican American female who developed RA right after vaccination with H1N1 vaccine. Genetically, RA has consistently been associated with an epitope in the third hypervariable region of the HLA-DR β chains, known as the "shared epitope", which is found primarily in DR4 and DR1 regions. The presence of HLA-DRB1 alleles is associated with susceptibility to RA in Mexican Americans. Hence, certain individuals with the presence of the "shared epitope" may develop RA following specific vaccinations. To our knowledge, this is the first reported case of RA following vaccination with the swine flu vaccine.

  18. The safety of influenza vaccines in children: An Institute for Vaccine Safety white paper.

    PubMed

    Halsey, Neal A; Talaat, Kawsar R; Greenbaum, Adena; Mensah, Eric; Dudley, Matthew Z; Proveaux, Tina; Salmon, Daniel A

    2015-12-30

    Most influenza vaccines are generally safe, but influenza vaccines can cause rare serious adverse events. Some adverse events, such as fever and febrile seizures, are more common in children than adults. There can be differences in the safety of vaccines in different populations due to underlying differences in genetic predisposition to the adverse event. Live attenuated vaccines have not been studied adequately in children under 2 years of age to determine the risks of adverse events; more studies are needed to address this and several other priority safety issues with all influenza vaccines in children. All vaccines intended for use in children require safety testing in the target age group, especially in young children. Safety of one influenza vaccine in children should not be extrapolated to assumed safety of all influenza vaccines in children. The low rates of adverse events from influenza vaccines should not be a deterrent to the use of influenza vaccines because of the overwhelming evidence of the burden of disease due to influenza in children. Copyright © 2016. Published by Elsevier Ltd.

  19. Viruses - from pathogens to vaccine carriers.

    PubMed

    Small, Juliana C; Ertl, Hildegund C J

    2011-10-01

    Vaccination is mankind's greatest public health success story. By now vaccines to many of the viruses that once caused fatal childhood diseases are routinely used throughout the world. Traditional methods of vaccine development through inactivation or attenuation of viruses have failed for some of the most deadly human pathogens, necessitating new approaches. Genetic modification of viruses not only allows for their attenuation but also for incorporation of sequences from other viruses, turning one pathogen into a vaccine carrier for another. Recombinant viruses have pros and cons as vaccine carriers, as discussed below using vectors based on adenovirus, herpesvirus, flavivirus, and rhabdovirus as examples.

  20. Virus vaccines: principles and prospects.

    PubMed Central

    Melnick, J. L.

    1989-01-01

    The present status of vaccination for controlling viral diseases is reviewed, and the needs and directions for future investigations are discussed. A survey of viral vaccines now in use has shown that knowledge about the viral agents and about the hosts' responses to infection was essential for their development. The steps needed to demonstrate the efficacy and safety of a viral vaccine are summarized; the final requirement for a successful vaccine is that it be administered in proper dosage and potency to the target populations. After general remarks on the proper use of current vaccines there follows an overview of various developments in creating new vaccines, along with the predicted time-frames for their coming into general use. Topics considered include vaccines to be administered locally at the portal of entry, subunit vaccines, viruses attenuated by genetic manipulation, use of viral vectors, vaccines developed by means of recombinant DNA, synthetic peptides, and anti-idiotype vaccines, as well as new vaccines being developed by more conventional methods. PMID:2663217

  1. Recent tissue engineering-based advances for effective rAAV-mediated gene transfer in the musculoskeletal system.

    PubMed

    Rey-Rico, Ana; Cucchiarini, Magali

    2016-04-01

    Musculoskeletal tissues are diverse and significantly different in their ability to repair upon injury. Current treatments often fail to reproduce the natural functions of the native tissue, leading to an imperfect healing. Gene therapy might improve the repair of tissues by providing a temporarily and spatially defined expression of the therapeutic gene(s) at the site of the injury. Several gene transfer vehicles have been developed to modify various human cells and tissues from musculoskeletal system among which the non-pathogenic, effective, and relatively safe recombinant adeno-associated viral (rAAV) vectors that have emerged as the preferred gene delivery system to treat human disorders. Adapting tissue engineering platforms to gene transfer approaches mediated by rAAV vectors is an attractive tool to circumvent both the limitations of the current therapeutic options to promote an effective healing of the tissue and the natural obstacles from these clinically adapted vectors to achieve an efficient and durable gene expression of the therapeutic sequences within the lesions.

  2. scAAV-Mediated IL-1Ra gene delivery for the Treatment of Osteoarthritis: Test of Efficacy in an Equine Model.

    PubMed

    Watson Levings, Rachael; Smith, Andrew D; Broome, Ted A; Rice, Brett L; Gibbs, Eric P; Myara, D Alex; Hyddmark, Viktoria; Nasri, Elham; Zarezadeh, Ali; Levings, Padraic P; Lu, Yuan; Dacanay, E Anthony; Foremny, Gregory B; Evans, Christopher H; Morton, Alison J; Winter, Mathew; Dark, Michael J; Nickerson, David M; Colahan, Patrick T; Ghivizzani, Steven Craig

    2018-06-05

    We are investigating self-complementary adeno-associated virus (scAAV) as a vector for intra-articular gene-delivery of IL-1Ra, and its therapeutic capacity in the treatment of osteoarthritis (OA). To model gene-transfer on a scale proportional to the human knee, a frequent site of OA incidence, we focused our studies on the joints of the equine forelimb. Using AAV2.5 capsid and equine IL-1Ra as a homologous transgene, we previously identified a functional ceiling dose of ~5 x 1012 viral genomes, which elevated the steady state levels of eqIL-1Ra in synovial fluids by more than 40-fold over endogenous production for at least 6 months. Here, using an osteochondral fragmentation model of early OA, we examined the functional capacity of scAAV.IL-1Ra gene-delivery in equine joints over a period of 12 weeks. In the disease model, transgenic eqIL-1Ra expression was several-fold higher than seen previously in healthy joints, and correlated directly with the severity of joint pathology at the time of treatment. Despite wide variation in expression, the steady-state eqIL-1Ra in synovial fluids exceeded that of IL-1 by > 400-fold in all animals, and a consistent treatment effect was observed. This included a 30-40% reduction in lameness and ~25% improvement in total joint pathology by both MRI and arthroscopic assessments, which included reduced joint effusion and synovitis, and improved repair of the osteochondral lesion. No vector-related increase in eqIL-1Ra levels in blood or urine was noted. Cumulatively our studies in the equine model indicate scAAV.IL-1Ra administration is reasonably safe and capable of sustained therapeutic IL-1Ra production intra-articularly in joints of human scale. This profile supports consideration for human testing in OA.

  3. Current status of flavivirus vaccines.

    PubMed

    Barrett, A D

    2001-12-01

    Although there are approximately 68 flaviviruses recognized, vaccines have been developed to control very few human flavivirus diseases. Licensed live attenuated vaccines have been developed for yellow fever (strain 17D) and Japanese encephalitis (strain SA14-14-2) viruses, and inactivated vaccines have been developed for Japanese encephalitis and tick-borne encephalitis viruses. The yellow fever live attenuated 17D vaccine is one of the most efficacious and safe vaccines developed to date and has been used to immunize more than 300 million people. A number of experimental vaccines are being developed, most notably for dengue. Candidate tetravalent live attenuated dengue vaccines are undergoing clinical trials. Other vaccines are being developed using reverse genetics, DNA vaccines, and recombinant immunogens. In addition, the yellow fever 17D vaccine has been used as a backbone to generate chimeric viruses containing the premembrane and envelope protein genes from other flaviviruses. The "Chimerivax" platform has been used to construct chimeric Japanese encephalitis and dengue viruses that are in different phases of development. Similar strategies are being used by other laboratories.

  4. Virus vector-mediated genetic modification of brain tumor stromal cells after intravenous delivery.

    PubMed

    Volak, Adrienn; LeRoy, Stanley G; Natasan, Jeya Shree; Park, David J; Cheah, Pike See; Maus, Andreas; Fitzpatrick, Zachary; Hudry, Eloise; Pinkham, Kelsey; Gandhi, Sheetal; Hyman, Bradley T; Mu, Dakai; GuhaSarkar, Dwijit; Stemmer-Rachamimov, Anat O; Sena-Esteves, Miguel; Badr, Christian E; Maguire, Casey A

    2018-05-16

    The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV genome, limiting duration of transgene expression. Intravenous (IV) injection gives widespread distribution of AAV in normal brain, however poor transgene expression in tumor, and high expression in non-target cells which may lead to ineffective therapy and high toxicity, respectively. Delivery of transgenes encoding secreted, anti-tumor proteins to tumor stromal cells may provide a more stable and localized reservoir of therapy as they are more differentiated than fast-dividing tumor cells. Reactive astrocytes and tumor-associated macrophage/microglia (TAMs) are stromal cells that comprise a large portion of the tumor mass and are associated with tumorigenesis. In mouse models of GBM, we used IV delivery of exosome-associated AAV vectors driving green fluorescent protein expression by specific promoters (NF-κB-responsive promoter and a truncated glial fibrillary acidic protein promoter), to obtain targeted transduction of TAMs and reactive astrocytes, respectively, while avoiding transgene expression in the periphery. We used our approach to express the potent, yet toxic anti-tumor cytokine, interferon beta, in tumor stroma of a mouse model of GBM, and achieved a modest, yet significant enhancement in survival compared to controls. Noninvasive genetic modification of tumor microenvironment represents a promising approach for therapy against cancers. Additionally, the vectors described here may facilitate basic research in the study of tumor stromal cells in situ.

  5. MyD88/CD40 Genetic Adjuvant Function in Cutaneous Atypical Antigen-Presenting Cells Contributes to DNA Vaccine Immunogenicity

    PubMed Central

    Slawin, Kevin M.; Levitt, Jonathan M.; Spencer, David M.

    2016-01-01

    Therapeutic DNA-based vaccines aim to prime an adaptive host immune response against tumor-associated antigens, eliminating cancer cells primarily through CD8+ cytotoxic T cell-mediated destruction. To be optimally effective, immunological adjuvants are required for the activation of tumor-specific CD8+ T cells responses by DNA vaccination. Here, we describe enhanced anti-tumor efficacy of an in vivo electroporation-delivered DNA vaccine by inclusion of a genetically encoded chimeric MyD88/CD40 (MC) adjuvant, which integrates both innate and adaptive immune signaling pathways. When incorporated into a DNA vaccine, signaling by the MC adjuvant increased antigen-specific CD8+ T cells and promoted elimination of pre-established tumors. Interestingly, MC-enhanced vaccine efficacy did not require direct-expression of either antigen or adjuvant by local antigen-presenting cells, but rather our data supports a key role for MC function in “atypical” antigen-presenting cells of skin. In particular, MC adjuvant-modified keratinocytes increased inflammatory cytokine secretion, upregulated surface MHC class I, and were able to increase in vitro and in vivo priming of antigen-specific CD8+ T cells. Furthermore, in the absence of critical CD8α+/CD103+ cross-priming dendritic cells, MC was still able to promote immune priming in vivo, albeit at a reduced level. Altogether, our data support a mechanism by which MC signaling activates an inflammatory phenotype in atypical antigen-presenting cells within the cutaneous vaccination site, leading to an enhanced CD8+ T cell response against DNA vaccine-encoded antigens, through both CD8α+/CD103+ dendritic cell-dependent and independent pathways. PMID:27741278

  6. Epilepsy and vaccinations: Italian guidelines.

    PubMed

    Pruna, Dario; Balestri, Paolo; Zamponi, Nelia; Grosso, Salvatore; Gobbi, Giuseppe; Romeo, Antonino; Franzoni, Emilio; Osti, Maria; Capovilla, Giuseppe; Longhi, Riccardo; Verrotti, Alberto

    2013-10-01

    Reports of childhood epilepsies in temporal association with vaccination have had a great impact on the acceptance of vaccination programs by health care providers, but little is known about this possible temporal association and about the types of seizures following vaccinations. For these reasons the Italian League Against Epilepsy (LICE), in collaboration with other Italian scientific societies, has decided to generate Guidelines on Vaccinations and Epilepsy. The aim of Guidelines on Vaccinations and Epilepsy is to present recent unequivocal evidence from published reports on the possible relationship between vaccines and epilepsy in order to provide information about contraindications and risks of vaccinations in patients with epilepsy. The following main issues have been addressed: (1) whether contraindications to vaccinations exist in patients with febrile convulsions, epilepsy, and/or epileptic encephalopathies; and (2) whether any vaccinations can cause febrile seizures, epilepsy, and/or epileptic encephalopathies. Diphtheria-tetanus-pertussis (DTP) vaccination and measles, mumps, and rubella vaccination (MMR) increase significantly the risk of febrile seizures. Recent observations and data about the relationships between vaccination and epileptic encephalopathy show that some cases of apparent vaccine-induced encephalopathy could in fact be caused by an inherent genetic defect with no causal relationship with vaccination. Wiley Periodicals, Inc. © 2013 International League Against Epilepsy.

  7. Status of vaccine research and development of vaccines for HIV-1.

    PubMed

    Safrit, Jeffrey T; Fast, Patricia E; Gieber, Lisa; Kuipers, Hester; Dean, Hansi J; Koff, Wayne C

    2016-06-03

    Human immunodeficiency virus (HIV) is the cause of one of the most lethal pandemics in human history, although in recent years access to highly effective anti-retroviral therapy has provided new hope worldwide. Transmission of HIV by sexual contact, childbirth and injection drug use has been reduced, but 2 million are newly infected each year, and much of the transmission is from people who do not know their status. In addition to known methods, a preventive vaccine is needed to end the pandemic. The extraordinary mutability and genetic diversity of HIV is an enormous challenge, but vaccines are being designed for broad coverage. Computer-aided design of mosaic immunogens, incorporating many epitopes from the entire genome or from conserved regions aim to induce CD8+ T cells to kill virus-infected cells or inhibit virus replication, while trimeric envelope proteins or synthetic mimics aim to induce broadly reactive neutralizing antibodies similar to those cloned from some infected patients. Induction of more potent and durable responses may require new adjuvants or replicating chimeric vectors chimeras that bear HIV genes. Passive or genetic delivery of broadly neutralizing antibodies may provide broad protection and/or lead to insights for vaccine designers. Proof-of-concept trials in non-human primates and in one human efficacy trial have provided scientific clues for a vaccine that could provide broad and durable protection against HIV. The use of vaccines to destroy HIV reservoirs as part of therapy or cure is now also being explored. Copyright © 2016 World Health Organization. Published by Elsevier Ltd.. All rights reserved.

  8. Ab-initio conformational epitope structure prediction using genetic algorithm and SVM for vaccine design.

    PubMed

    Moghram, Basem Ameen; Nabil, Emad; Badr, Amr

    2018-01-01

    T-cell epitope structure identification is a significant challenging immunoinformatic problem within epitope-based vaccine design. Epitopes or antigenic peptides are a set of amino acids that bind with the Major Histocompatibility Complex (MHC) molecules. The aim of this process is presented by Antigen Presenting Cells to be inspected by T-cells. MHC-molecule-binding epitopes are responsible for triggering the immune response to antigens. The epitope's three-dimensional (3D) molecular structure (i.e., tertiary structure) reflects its proper function. Therefore, the identification of MHC class-II epitopes structure is a significant step towards epitope-based vaccine design and understanding of the immune system. In this paper, we propose a new technique using a Genetic Algorithm for Predicting the Epitope Structure (GAPES), to predict the structure of MHC class-II epitopes based on their sequence. The proposed Elitist-based genetic algorithm for predicting the epitope's tertiary structure is based on Ab-Initio Empirical Conformational Energy Program for Peptides (ECEPP) Force Field Model. The developed secondary structure prediction technique relies on Ramachandran Plot. We used two alignment algorithms: the ROSS alignment and TM-Score alignment. We applied four different alignment approaches to calculate the similarity scores of the dataset under test. We utilized the support vector machine (SVM) classifier as an evaluation of the prediction performance. The prediction accuracy and the Area Under Receiver Operating Characteristic (ROC) Curve (AUC) were calculated as measures of performance. The calculations are performed on twelve similarity-reduced datasets of the Immune Epitope Data Base (IEDB) and a large dataset of peptide-binding affinities to HLA-DRB1*0101. The results showed that GAPES was reliable and very accurate. We achieved an average prediction accuracy of 93.50% and an average AUC of 0.974 in the IEDB dataset. Also, we achieved an accuracy of 95

  9. Heritability of vaccine-induced measles neutralizing antibody titers.

    PubMed

    Schaid, Daniel J; Haralambieva, Iana H; Larrabee, Beth R; Ovsyannikova, Inna G; Kennedy, Richard B; Poland, Gregory A

    2017-03-07

    Understanding how genetics influences inter-individual variation of antibody titers in response to measles vaccination is vital to understanding possible sources of vaccine failure as well as improved vaccine development. Although it is recognized that both the human leukocyte antigen (HLA) genes and the immunoglobulin allotype genes play significant roles in immune response, there is significant variation in antibody titers that is not explained by these genes. To obtain a more complete estimate of the role of the entire genome, we used a large panel of single nucleotide polymorphisms to estimate the heritability of antibody response to measles vaccine. Based on 935 subjects with European ancestry, we estimated the heritability to be 49% (standard error 0.17). We also estimated the heritability attributable to each chromosome, and found a large range in chromosome-specific heritabilities. Notably, chromosome 1 had the largest estimate (28%), while chromosome 6, which harbors HLA, had an estimated heritability of 13%. Compared with a prior study of twins in the same community, which resulted in a heritability estimate of 88.5%, our study suggests there are either many rare genetic variants, or many common genetic variants of small effect sizes that contribute to variations of antibody titers in response to measles vaccine. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. A survey of ex vivo/in vitro transduction efficiency of mammalian primary cells and cell lines with Nine natural adeno-associated virus (AAV1-9) and one engineered adeno-associated virus serotype.

    PubMed

    Ellis, Brian L; Hirsch, Matthew L; Barker, Jenny C; Connelly, Jon P; Steininger, Robert J; Porteus, Matthew H

    2013-03-06

    The ability to deliver a gene of interest into a specific cell type is an essential aspect of biomedical research. Viruses can be a useful tool for this delivery, particularly in difficult to transfect cell types. Adeno-associated virus (AAV) is a useful gene transfer vector because of its ability to mediate efficient gene transduction in numerous dividing and quiescent cell types, without inducing any known pathogenicity. There are now a number of natural for that designed AAV serotypes that each has a differential ability to infect a variety of cell types. Although transduction studies have been completed, the bulk of the studies have been done in vivo, and there has never been a comprehensive study of transduction ex vivo/in vitro. Each cell type was infected with each serotype at a multiplicity of infection of 100,000 viral genomes/cell and transduction was analyzed by flow cytometry + . We found that AAV1 and AAV6 have the greatest ability to transduce a wide range of cell types, however, for particular cell types, there are specific serotypes that provide optimal transduction. In this work, we describe the transduction efficiency of ten different AAV serotypes in thirty-four different mammalian cell lines and primary cell types. Although these results may not be universal due to numerous factors such as, culture conditions and/ or cell growth rates and cell heterogeneity, these results provide an important and unique resource for investigators who use AAV as an ex vivo gene delivery vector or who work with cells that are difficult to transfect.

  11. Primary vaccine failure to routine vaccines: Why and what to do?

    PubMed

    Wiedermann, Ursula; Garner-Spitzer, Erika; Wagner, Angelika

    2016-01-01

    There are 2 major factors responsible for vaccine failures, the first is vaccine-related such as failures in vaccine attenuation, vaccination regimes or administration. The other is host-related, of which host genetics, immune status, age, health or nutritional status can be associated with primary or secondary vaccine failures. The first describes the inability to respond to primary vaccination, the latter is characterized by a loss of protection after initial effectiveness. Our studies concentrate on the evaluation of immunological characteristics responsible for primary vaccine failures in different (risk) populations for which the underlying mechanisms are currently unknown. Here we summarise current knowledge and findings from our studies. About 2-10% of healthy individuals fail to mount antibody levels to routine vaccines. Comparing the immune responses to different vaccines in non-responder and high-responder vaccinees revealed that hypo-responsiveness is antigen/vaccine-specific at the humoral but not at the cellular level. We found that T-regulatory as well as B-regulatory cells and the production of IL-10 are involved in non/hypo-responsiveness. Non-responsiveness increases with age and in particular vaccination to a novel vaccine in persons > 65 years is associated with a high low/non-responder rate, indicating that vaccine schedules and doses (at least for primary vaccination) should be adapted according to age. In light of the growing number of allergic but also obese people, our current studies concentrate on these risk groups to reveal whether different vaccination approaches are necessary for optimal protection compared to healthy individuals. These studies are in line with the significant paradigm shift taking place in many fields of medical research and care, and will extend the concept of personalised medicine into the field of vaccinology.

  12. Prospects for new viral vaccines.

    PubMed

    Marmion, B P

    1980-08-11

    Animal virology has made outstanding contributions to preventive medicine by the development of vaccines for the control of infectious disease in man and animals. Cost-benefit analysis indicates substantial savings in health care costs from the control of diseases such as smallpox, poliomyelitis, yellow fever and measels. Areas for further development include vaccines for influenza (living, attenuated virus), the herpes group (varicella: cytomegalovirus), respiratory syncytial virus, rotavirus and hepatitis A, B, and non A/non B. The general options for vaccine formulation are discussed with particular emphasis on approaches with the use of viral genetics to 'tailor make' vaccine viruses with defined growth potential in laboratory systems, low pathogenicity, and defined antigens. Current progress with the development of an inactivated hepatitis B vaccine is reviewed as a case study in vaccine development. The impact of recent experiments in cloning hepatitis B virus DNA in E. coli on the production of a purified viral polypeptide vaccine is assessed.

  13. The future of human DNA vaccines

    PubMed Central

    Li, Lei; Saade, Fadi; Petrovsky, Nikolai

    2012-01-01

    DNA vaccines have evolved greatly over the last 20 years since their invention, but have yet to become a competitive alternative to conventional protein or carbohydrate based human vaccines. Whilst safety concerns were an initial barrier, the Achilles heel of DNA vaccines remains their poor immunogenicity when compared to protein vaccines. A wide variety of strategies have been developed to optimize DNA vaccine immunogenicity, including codon optimization, genetic adjuvants, electroporation and sophisticated prime-boost regimens, with each of these methods having its advantages and limitations. Whilst each of these methods has contributed to incremental improvements in DNA vaccine efficacy, more is still needed if human DNA vaccines are to succeed commercially. This review foresees a final breakthrough in human DNA vaccines will come from application of the latest cutting-edge technologies, including “epigenetics” and “omics” approaches, alongside traditional techniques to improve immunogenicity such as adjuvants and electroporation, thereby overcoming the current limitations of DNA vaccines in humans PMID:22981627

  14. The future of human DNA vaccines.

    PubMed

    Li, Lei; Saade, Fadi; Petrovsky, Nikolai

    2012-12-31

    DNA vaccines have evolved greatly over the last 20 years since their invention, but have yet to become a competitive alternative to conventional protein or carbohydrate based human vaccines. Whilst safety concerns were an initial barrier, the Achilles heel of DNA vaccines remains their poor immunogenicity when compared to protein vaccines. A wide variety of strategies have been developed to optimize DNA vaccine immunogenicity, including codon optimization, genetic adjuvants, electroporation and sophisticated prime-boost regimens, with each of these methods having its advantages and limitations. Whilst each of these methods has contributed to incremental improvements in DNA vaccine efficacy, more is still needed if human DNA vaccines are to succeed commercially. This review foresees a final breakthrough in human DNA vaccines will come from application of the latest cutting-edge technologies, including "epigenetics" and "omics" approaches, alongside traditional techniques to improve immunogenicity such as adjuvants and electroporation, thereby overcoming the current limitations of DNA vaccines in humans. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Measles Vaccine.

    PubMed

    Griffin, Diane E

    2018-03-01

    Measles remains an important cause of child morbidity and mortality worldwide despite the availability of a safe and efficacious vaccine. The current measles virus (MeV) vaccine was developed empirically by attenuation of wild-type (WT) MeV by in vitro passage in human and chicken cells and licensed in 1963. Additional passages led to further attenuation and the successful vaccine strains in widespread use today. Attenuation is associated with decreased replication in lymphoid tissue, but the molecular basis for this restriction has not been identified. The immune response is age dependent, inhibited by maternal antibody (Ab) and involves induction of both Ab and T cell responses that resemble the responses to WT MeV infection, but are lower in magnitude. Protective immunity is correlated with levels of neutralizing Ab, but the actual immunologic determinants of protection are not known. Because measles is highly transmissible, control requires high levels of population immunity. Delivery of the two doses of vaccine needed to achieve >90% immunity is accomplished by routine immunization of infants at 9-15 months of age followed by a second dose delivered before school entry or by periodic mass vaccination campaigns. Because delivery by injection creates hurdles to sustained high coverage, there are efforts to deliver MeV vaccine by inhalation. In addition, the safety record for the vaccine combined with advances in reverse genetics for negative strand viruses has expanded proposed uses for recombinant versions of measles vaccine as vectors for immunization against other infections and as oncolytic agents for a variety of tumors.

  16. Injection of AAV2-BMP2 and AAV2-TIMP1 into the nucleus pulposus slows the course of intervertebral disc degeneration in an in vivo rabbit model.

    PubMed

    Leckie, Steven K; Bechara, Bernard P; Hartman, Robert A; Sowa, Gwendolyn A; Woods, Barrett I; Coelho, Joao P; Witt, William T; Dong, Qing D; Bowman, Brent W; Bell, Kevin M; Vo, Nam V; Wang, Bing; Kang, James D

    2012-01-01

    Intervertebral disc degeneration (IDD) is a common cause of back pain. Patients who fail conservative management may face the morbidity of surgery. Alternative treatment modalities could have a significant impact on disease progression and patients' quality of life. To determine if the injection of a virus vector carrying a therapeutic gene directly into the nucleus pulposus improves the course of IDD. Prospective randomized controlled animal study. Thirty-four skeletally mature New Zealand white rabbits were used. In the treatment group, L2-L3, L3-L4, and L4-L5 discs were punctured in accordance with a previously validated rabbit annulotomy model for IDD and then subsequently treated with adeno-associated virus serotype 2 (AAV2) vector carrying genes for either bone morphogenetic protein 2 (BMP2) or tissue inhibitor of metalloproteinase 1 (TIMP1). A nonoperative control group, nonpunctured sham surgical group, and punctured control group were also evaluated. Serial magnetic resonance imaging (MRI) studies at 0, 6, and 12 weeks were obtained, and a validated MRI analysis program was used to quantify degeneration. The rabbits were sacrificed at 12 weeks, and L4-L5 discs were analyzed histologically. Viscoelastic properties of the L3-L4 discs were analyzed using uniaxial load-normalized displacement testing. Creep curves were mathematically modeled according to a previously validated two-phase exponential model. Serum samples obtained at 0, 6, and 12 weeks were assayed for biochemical evidence of degeneration. The punctured group demonstrated MRI and histologic evidence of degeneration as expected. The treatment groups demonstrated less MRI and histologic evidence of degeneration than the punctured group. The serum biochemical marker C-telopeptide of collagen type II increased rapidly in the punctured group, but the treated groups returned to control values by 12 weeks. The treatment groups demonstrated several viscoelastic properties that were distinct from control

  17. AAV delivery of GRP78/BiP promotes adaptation of human RPE cell to ER stress.

    PubMed

    Ghaderi, Shima; Ahmadian, Shahin; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Samiei, Shahram; Kheitan, Samira; Pirmardan, Ehsan R

    2018-02-01

    Adeno associated virus (AAV)-mediated gene delivery of GRP78 (78 kDa glucose-regulated protein) attenuates the condition of endoplasmic reticulum (ER) stress and prevents apoptotic loss of photoreceptors in Retinitis pigmentosa (RP) rats. In the current study we overexpressed Grp78 with the help of AAV-2 in primary human retinal pigmented epithelium (hRPE) cell cultures and examined its effect on cell response to ER stress. The purpose of this work was studying potential stimulating effect of GRP78 on adaptation/pro-survival of hRPE cells under ER stress, as an in vitro model for RPE degeneration. To investigate the effect of Grp78 overexpression on unfolded protein response (UPR) markers under ER stress, hRPE primary cultures were transduced by recombinant virus rAAV/Grp78, and treated with ER stressor drug, tunicamycin. Expression changes of four UPR markers including GRP78, PERK, ATF6α, and GADD153/CHOP, were assessed by real-time PCR and western blotting. We found that GRP78 has a great contribution in modulation of UPR markers to favor adaptive response in ER-stressed hRPE cells. In fact, GRP78 overexpression affected adaptation and apoptotic phases of early UPR, through enhancement of two master regulators/ER stress sensors (PERK and ATF6α) and down-regulation of a key pro-apoptotic cascade activator (GADD153/CHOP). Together these findings demonstrate the promoting effect of GRP78 on adaptation/pro-survival of hRPE cells under ER stress. This protein with anti-apoptotic actions in the early UPR and important role in cell fate regulation, can be recruited as a useful candidate for future investigations of RPE degenerative diseases. © 2017 Wiley Periodicals, Inc.

  18. In Situ Gene Therapy via AAV-CRISPR-Cas9-Mediated Targeted Gene Regulation.

    PubMed

    Moreno, Ana M; Fu, Xin; Zhu, Jie; Katrekar, Dhruva; Shih, Yu-Ru V; Marlett, John; Cabotaje, Jessica; Tat, Jasmine; Naughton, John; Lisowski, Leszek; Varghese, Shyni; Zhang, Kang; Mali, Prashant

    2018-04-25

    Development of efficacious in vivo delivery platforms for CRISPR-Cas9-based epigenome engineering will be critical to enable the ability to target human diseases without permanent modification of the genome. Toward this, we utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted in vivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from in vivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for in vivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner. Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  19. The AAV-mediated and RNA-guided CRISPR/Cas9 system for gene therapy of DMD and BMD.

    PubMed

    Wang, Jing-Zhang; Wu, Peng; Shi, Zhi-Min; Xu, Yan-Li; Liu, Zhi-Jun

    2017-08-01

    Mutations in the dystrophin gene (Dmd) result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), which afflict many newborn boys. In 2016, Brain and Development published several interesting articles on DMD treatment with antisense oligonucleotide, kinase inhibitor, and prednisolone. Even more strikingly, three articles in the issue 6271 of Science in 2016 provide new insights into gene therapy of DMD and BMD via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). In brief, adeno-associated virus (AAV) vectors transport guided RNAs (gRNAs) and Cas9 into mdx mouse model, gRNAs recognize the mutated Dmd exon 23 (having a stop codon), and Cas9 cut the mutated exon 23 off the Dmd gene. These manipulations restored expression of truncated but partially functional dystrophin, improved skeletal and cardiac muscle function, and increased survival of mdx mice significantly. This review concisely summarized the related advancements and discussed their primary implications in the future gene therapy of DMD, including AAV-vector selection, gRNA designing, Cas9 optimization, dystrophin-restoration efficiency, administration routes, and systemic and long-term therapeutic efficacy. Future orientations, including off-target effects, safety concerns, immune responses, precision medicine, and Dmd-editing in the brain (potentially blocked by the blood-brain barrier) were also elucidated briefly. Collectively, the AAV-mediated and RNA-guided CRISPR/Cas9 system has major superiorities compared with traditional gene therapy, and might contribute to the treatment of DMD and BMD substantially in the near future. Copyright © 2017 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.

  20. Replication of Rubella Vaccine Population Genetic Studies: Validation of HLA Genotype and Humoral Response Associations

    PubMed Central

    Ovsyannikova, Inna G.; Jacobson, Robert M.; Vierkant, Robert A.; O’Byrne, Megan M.; Poland, Gregory A.

    2009-01-01

    Purported genetic associations found in population studies require validation for confirmation. We previously reported rubella vaccine-induced immune responses and HLA associations in 346 adolescents, age 12–18 years (1st cohort), following two doses of a rubella-containing vaccine. We sought to replicate the associations discovered in that work by verifying these associations in a new cohort of 396 subjects, age 11–19 years (2nd cohort), all having had two doses of a rubella-containing vaccine. We found that B*2705 (median 1st cohort 20.9 IU/ml, p=0.028; 2nd cohort 20.5 IU/ml, p=0.001) and DPA1*0201 (median 1st cohort 32.5 IU/ml, p=0.048; 2nd cohort 25.8 IU/ml, p=0.025) alleles were consistently associated with lower rubella-induced antibodies. Further, DPB1*0401 (median 1st cohort 43.5 IU/ml, p=0.021; 2nd cohort 36.2 IU/ml, p=0.002) alleles were associated with higher antibody levels in both populations. The association of DRB1*04-DQB1*03-DPB1*03 (mean 1st cohort 25.2 IU/ml, p=0.011; 2nd cohort 21.4 IU/ml, p=0.032) and DRB1*15/16-DQB1*06-DPB1*03 (1st cohort 16.3 IU/ml, p=0.043; 2nd cohort 19.1 IU/ml, p=0.023) haplotypes with lower rubella-specific antibodies were observed in both studies. This study provides confirmatory evidence for an association between specific class I and II HLA markers and haplotypes with rubella vaccine-induced humoral responses and lends further weight to their influence on rubella immune responses. PMID:19761839

  1. Live bacterial vaccines--a review and identification of potential hazards.

    PubMed

    Detmer, Ann; Glenting, Jacob

    2006-06-23

    The use of live bacteria to induce an immune response to itself or to a carried vaccine component is an attractive vaccine strategy. Advantages of live bacterial vaccines include their mimicry of a natural infection, intrinsic adjuvant properties and their possibility to be administered orally. Derivatives of pathogenic and non-pathogenic food related bacteria are currently being evaluated as live vaccines. However, pathogenic bacteria demands for attenuation to weaken its virulence. The use of bacteria as vaccine delivery vehicles implies construction of recombinant strains that contain the gene cassette encoding the antigen. With the increased knowledge of mucosal immunity and the availability of genetic tools for heterologous gene expression the concept of live vaccine vehicles gains renewed interest. However, administration of live bacterial vaccines poses some risks. In addition, vaccination using recombinant bacteria results in the release of live recombinant organisms into nature. This places these vaccines in the debate on application of genetically modified organisms. In this review we give an overview of live bacterial vaccines on the market and describe the development of new live vaccines with a focus on attenuated bacteria and food-related lactic acid bacteria. Furthermore, we outline the safety concerns and identify the hazards associated with live bacterial vaccines and try to give some suggestions of what to consider during their development.

  2. Nicotine vaccines: Will smokers take a shot at quitting?

    PubMed Central

    Lerman, Caryn; Cappella, Joseph N.

    2010-01-01

    Introduction: A vaccine against nicotine may soon be available to smokers who want to quit. The vaccine stimulates the production of antibodies that bind to nicotine, thereby impeding nicotine from crossing the blood-brain barrier and exerting psychoactive effects. The primary purpose of this study was to evaluate intentions to try a nicotine vaccine if one were to become available among a nationally representative sample of smokers. The secondary purpose was to assess whether information about genetic susceptibility to nicotine addiction had an effect on smokers’ interest in receiving the vaccine. Methods: Four hundred and twenty-seven adults were randomized to read one of two versions of a short description about the vaccine. One version framed addiction as genetically influenced, while the other framed it as environmentally influenced. Smokers were then asked about their intentions to use a nicotine vaccine if one were to become available in the future. Results: Across both groups, 53% indicated that they would be likely or very likely to try the vaccine. Using multivariate linear regression, the strongest predictors of vaccination intention were having a favorable attitude toward a nicotine vaccine (β = .41) and having a favorable attitude toward vaccination in general (β = .22). There were no significant effects of the framing conditions on intention to receive the vaccine. Discussion: Intentions to try a nicotine vaccine as a cessation method are relatively high among smokers. If the vaccine becomes available, specific groups of smokers may be more interested than others; education and recruitment efforts could be targeted appropriately. PMID:20185516

  3. Brucellosis vaccines for livestock.

    PubMed

    Goodwin, Zakia I; Pascual, David W

    2016-11-15

    Brucellosis is a livestock disease responsible for fetal loss due to abortions. Worldwide, this disease has profound economic and social impact by reducing the ability of livestock producers to provide an adequate supply of disease-free meat and dairy products. In addition to its presence in domesticated animals, brucellosis is harbored in a number of wildlife species creating new disease reservoirs, which adds to the difficulty of eradicating this disease. Broad and consistent use of the available vaccines would contribute in reducing the incidence of brucellosis. Unfortunately, this practice is not common. In addition, the current brucellosis vaccines cannot provide sterilizing immunity, and in certain circumstances, vaccinated livestock are not protected against co-mingling Brucella-infected wildlife. Given that these vaccines are inadequate for conferring complete protection for some vaccinated livestock, alternatives are being sought, and these include genetic modifications of current vaccines or their reformulations. Alternatively, many groups have sought to develop new vaccines. Subunit vaccines, delivered as a combination of soluble vaccine plus adjuvant or the heterologous expression of Brucella epitopes by different vaccine vectors are currently being tested. New live attenuated Brucella vaccines are also being developed and tested in their natural hosts. Yet, what is rarely considered is the route of vaccination which could improve vaccine efficacy. Since Brucella infections are mostly transmitted mucosally, mucosal delivery of a vaccine has the potential of eliciting a more robust protective immune response for improved efficacy. Hence, this review will examine these questions and provide the status of new vaccines for livestock brucellosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Immunogenetics of Seasonal Influenza Vaccine Response*

    PubMed Central

    Poland, Gregory A.; Ovsyannikova, Inna G.; Jacobson, Robert M.

    2008-01-01

    Seasonal influenza causes significant morbidity, mortality, and economic costs. Vaccines against influenza, though both safe and effective, are imperfect. Notably, these vaccines result in significant immune response variability across the population. The mechanism for this variability, in part, appears to lie in the polymorphisms of key immune response genes. Despite the importance of this variability, little in the way of genetic polymorphisms and its association with vaccine immune response to viral vaccines has been performed. Herein, we review and synthesize what is known about the immune response pathway and influenza viral immunity and then present original data from our laboratory on the immunogenetic relationships between HLA, cytokine and cytokine receptor gene polymorphisms and the variations in humoral immune response to inactivated seasonal influenza vaccine. Finally, we propose that a better understanding of vaccine immunogenetics offers insight towards the development of better influenza vaccines. PMID:19230157

  5. Optimization model of vaccination strategy for dengue transmission

    NASA Astrophysics Data System (ADS)

    Widayani, H.; Kallista, M.; Nuraini, N.; Sari, M. Y.

    2014-02-01

    Dengue fever is emerging tropical and subtropical disease caused by dengue virus infection. The vaccination should be done as a prevention of epidemic in population. The host-vector model are modified with consider a vaccination factor to prevent the occurrence of epidemic dengue in a population. An optimal vaccination strategy using non-linear objective function was proposed. The genetic algorithm programming techniques are combined with fourth-order Runge-Kutta method to construct the optimal vaccination. In this paper, the appropriate vaccination strategy by using the optimal minimum cost function which can reduce the number of epidemic was analyzed. The numerical simulation for some specific cases of vaccination strategy is shown.

  6. Vaccinia Virus Vaccines: Past, Present and Future

    PubMed Central

    Jacobs, Bertram L.; Langland, Jeffrey O.; Kibler, Karen V.; Denzler, Karen L.; White, Stacy D.; Holechek, Susan A.; Wong, Shukmei; Huynh, Trung; Baskin, Carole R.

    2009-01-01

    Vaccinia virus (VACV) has been used more extensively for human immunization than any other vaccine. For almost two centuries, VACV was employed to provide cross-protection against variola virus, the causative agent of smallpox, until the disease was eradicated in the late 1970s. Since that time, continued research on VACV has produced a number of modified vaccines with improved safety profiles. Attenuation has been achieved through several strategies, including sequential passage in an alternative host, deletion of specific genes or genetic engineering of viral genes encoding immunomodulatory proteins. Some highly attenuated third- and fourth-generation VACV vaccines are now being considered for stockpiling against a possible re-introduction of smallpox through bioterrorism. Researchers have also taken advantage of the ability of the VACV genome to accommodate additional genetic material to produce novel vaccines against a wide variety of infectious agents, including a recombinant VACV encoding the rabies virus glycoprotein that is administered orally to wild animals. This review provides an in-depth examination of these successive generations of VACV vaccines, focusing on how the understanding of poxviral replication and viral gene function permits the deliberate modification of VACV immunogenicity and virulence. PMID:19563829

  7. Genomic Variation between Genetic lines of White Leghorns Differed in Resistance to Marek’s Disease

    USDA-ARS?s Scientific Manuscript database

    Genetic resistance to avian tumor virus-induced tumorigenesis and vaccine protective efficacy preventing such tumorigenicity are determined by multiple factors including host genetics, viral virulence, dose of challenge viruses, type of vaccine, vaccine dosage, and interval between vaccination and v...

  8. Isolation and genetic characterization of a novel 2.2.1.2a H5N1 virus from a vaccinated meat-turkeys flock in Egypt.

    PubMed

    Salaheldin, Ahmed H; Veits, Jutta; Abd El-Hamid, Hatem S; Harder, Timm C; Devrishov, Davud; Mettenleiter, Thomas C; Hafez, Hafez M; Abdelwhab, Elsayed M

    2017-03-09

    Vaccination of poultry to control highly pathogenic avian influenza virus (HPAIV) H5N1 is used in several countries. HPAIV H5N1 of clade 2.2.1 which is endemic in Egypt has diversified into two genetic clades. Clade 2.2.1.1 represents antigenic drift variants in vaccinated commercial poultry while clade 2.2.1.2 variants are detected in humans and backyard poultry. Little is known about H5N1 infection in vaccinated turkeys under field conditions. Here, we describe an HPAI H5N1 outbreak in a vaccinated meat-turkey flock in Egypt. Birds were vaccinated with inactivated H5N2 and H5N1 vaccines at 8 and 34 days of age, respectively. At 72 nd day of age (38 days post last vaccination), turkeys exhibited mild respiratory signs, cyanosis of snood and severe congestion of the internal organs. Survivors had a reduction in feed consumption and body gain. A mortality of ~29% cumulated within 10 days after the onset of clinical signs. Laboratory diagnosis using RT-qPCRs revealed presence of H5N1 but was negative for H7 and H9 subtypes. A substantial antigenic drift against different serum samples from clade 2.2.1.1 and clade 2.3.4.4 was observed. Based on full genome sequence analysis the virus belonged to clade 2.2.1.2 but clustered with recent H5N1 viruses from 2015 in poultry in Israel, Gaza and Egypt in a novel subclade designated here 2.2.1.2a which is distinct from 2014/2015 2.2.1.2 viruses. These viruses possess 2.2.1.2 clade-specific genetic signatures and also mutations in the HA similar to those in clade 2.2.1.1 that enabled evasion from humoral immune response. Taken together, this manuscript describes a recent HPAI H5N1 outbreak in vaccinated meat-turkeys in Egypt after infection with a virus representing novel distinct 2.2.1.2a subclade. Infection with HPAIV H5N1 in commercial turkeys resulted in significant morbidity and mortality despite of vaccination using H5 vaccines. The isolated virus showed antigenic drift and clustered in a novel cluster designated here

  9. Swine influenza virus vaccines: to change or not to change-that's the question.

    PubMed

    Van Reeth, Kristien; Ma, Wenjun

    2013-01-01

    Commercial vaccines currently available against swine influenza virus (SIV) are inactivated, adjuvanted, whole virus vaccines, based on H1N1 and/or H3N2 and/or H1N2 SIVs. In keeping with the antigenic and genetic differences between SIVs circulating in Europe and the US, the vaccines for each region are produced locally and contain different strains. Even within a continent, there is no standardization of vaccine strains, and the antigen mass and adjuvants can also differ between different commercial products. Recombinant protein vaccines against SIV, vector, and DNA vaccines, and vaccines attenuated by reverse genetics have been tested in experimental studies, but they have not yet reached the market. In this review, we aim to present a critical analysis of the performance of commercial inactivated and novel generation SIV vaccines in experimental vaccination challenge studies in pigs. We pay special attention to the differences between commercial SIV vaccines and vaccination attitudes in Europe and in North America, to the issue of vaccine strain selection and changes, and to the potential advantages of novel generation vaccines over the traditional killed SIV vaccines.

  10. Bacterial Artificial Chromosome Clones of Viruses Comprising the Towne Cytomegalovirus Vaccine

    PubMed Central

    Cui, Xiaohong; Adler, Stuart P.; Davison, Andrew J.; Smith, Larry; Habib, EL-Sayed E.; McVoy, Michael A.

    2012-01-01

    Bacterial artificial chromosome (BAC) clones have proven invaluable for genetic manipulation of herpesvirus genomes. BAC cloning can also be useful for capturing representative genomes that comprise a viral stock or mixture. The Towne live attenuated cytomegalovirus vaccine was developed in the 1970s by serial passage in cultured fibroblasts. Although its safety, immunogenicity, and efficacy have been evaluated in nearly a thousand human subjects, the vaccine itself has been little studied. Instead, genetic composition and in vitro growth properties have been inferred from studies of laboratory stocks that may not always accurately represent the viruses that comprise the vaccine. Here we describe the use of BAC cloning to define the genotypic and phenotypic properties of viruses from the Towne vaccine. Given the extensive safety history of the Towne vaccine, these BACs provide a logical starting point for the development of next-generation rationally engineered cytomegalovirus vaccines. PMID:22187535

  11. Bacterial artificial chromosome clones of viruses comprising the towne cytomegalovirus vaccine.

    PubMed

    Cui, Xiaohong; Adler, Stuart P; Davison, Andrew J; Smith, Larry; Habib, El-Sayed E; McVoy, Michael A

    2012-01-01

    Bacterial artificial chromosome (BAC) clones have proven invaluable for genetic manipulation of herpesvirus genomes. BAC cloning can also be useful for capturing representative genomes that comprise a viral stock or mixture. The Towne live attenuated cytomegalovirus vaccine was developed in the 1970s by serial passage in cultured fibroblasts. Although its safety, immunogenicity, and efficacy have been evaluated in nearly a thousand human subjects, the vaccine itself has been little studied. Instead, genetic composition and in vitro growth properties have been inferred from studies of laboratory stocks that may not always accurately represent the viruses that comprise the vaccine. Here we describe the use of BAC cloning to define the genotypic and phenotypic properties of viruses from the Towne vaccine. Given the extensive safety history of the Towne vaccine, these BACs provide a logical starting point for the development of next-generation rationally engineered cytomegalovirus vaccines.

  12. Adverse Reactions to Vaccination: From Anaphylaxis to Autoimmunity.

    PubMed

    Gershwin, Laurel J

    2018-03-01

    Vaccines are important for providing protection from infectious diseases. Vaccination initiates a process that stimulates development of a robust and long-lived immune response to the disease agents in the vaccine. Side effects are sometimes associated with vaccination. These vary from development of acute hypersensitivity responses to vaccine components to local tissue reactions that are annoying but not significantly detrimental to the patient. The pathogenesis of these responses and the consequent clinical outcomes are discussed. Overstimulation of the immune response and the potential relationship to autoimmunity is evaluated in relation to genetic predisposition. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Identification of Brucella melitensis Rev.1 vaccine-strain genetic markers: Towards understanding the molecular mechanism behind virulence attenuation.

    PubMed

    Issa, Mohammad Nouh; Ashhab, Yaqoub

    2016-09-22

    Brucella melitensis Rev.1 is an avirulent strain that is widely used as a live vaccine to control brucellosis in small ruminants. Although an assembled draft version of Rev.1 genome has been available since 2009, this genome has not been investigated to characterize this important vaccine. In the present work, we used the draft genome of Rev.1 to perform a thorough genomic comparison and sequence analysis to identify and characterize the panel of its unique genetic markers. The draft genome of Rev.1 was compared with genome sequences of 36 different Brucella melitensis strains from the Brucella project of the Broad Institute of MIT and Harvard. The comparative analyses revealed 32 genetic alterations (30 SNPs, 1 single-bp insertion and 1 single-bp deletion) that are exclusively present in the Rev.1 genome. In silico analyses showed that 9 out of the 17 non-synonymous mutations are deleterious. Three ABC transporters are among the disrupted genes that can be linked to virulence attenuation. Out of the 32 mutations, 11 Rev.1 specific markers were selected to test their potential to discriminate Rev.1 using a bi-directional allele-specific PCR assay. Six markers were able to distinguish between Rev.1 and a set of control strains. We succeeded in identifying a panel of 32 genome-specific markers of the B. melitensis Rev.1 vaccine strain. Extensive in silico analysis showed that a considerable number of these mutations could severely affect the function of the associated genes. In addition, some of the discovered markers were able to discriminate Rev.1 strain from a group of control strains using practical PCR tests that can be applied in resource-limited settings. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Genetic variability of porcine circovirus 2 (PCV2) field isolates from vaccinated and non-vaccinated pig herds in Germany.

    PubMed

    Reiner, Gerald; Hofmeister, Regina; Willems, Hermann

    2015-10-22

    Porcine circovirus 2 (PCV2) is responsible for a wide range of associated diseases (PCVD) affecting swine production worldwide. Highly efficient commercial vaccines induce protective immunity, but PCV2 is still circulating in vaccinated farms. Thus, and because of the viruś high mutation rate, recent findings provide concerns about PCV2 strains capable to escape vaccination. Based on 2156 samples from individual pigs of 315 herds from Germany we describe a high effectivity of vaccination between 2008 and the third quarter of 2011. In this period, virus load dropped continuously and at the end of this period it hardly reached the limit of quantification. Thereafter, virus loads re-increased, although most of the herds were still vaccinated. Sixty-two randomly selected samples from vaccinated (n=28) and non-vaccinated (n=26) herds between 2008 and 2012 were completely sequenced. As compared to the PCV2b reference sequence 259 polymorphisms were detected. Polymorhisms were analysed for associations to vaccination status, genotype (PCV2a/PCV2b), and virus load. PCV2a sequences were significantly repelled by PCV2b. One SNP at position 1182 (g.1182G>T), involved in capsid epitope formation, was significantly associated with the PCV2 genotype (2a/2b). Moreover, this SNP was affected by vaccination, with effects on allele frequencies and viral load, independent from the PCV2 genotype (2a/2b). We conclude that there is indeed evidence for a selectional impact of vaccination on the PCV2 sequence, especially on nucleotides involved in epitope formation. Such variation might be responsible for the observed re-increase of PCV2-loads in samples from the end of 2011 in Germany. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. DNA and RNA-based vaccines: principles, progress and prospects

    PubMed Central

    Leitner, Wolfgang W.; Ying, Han; Restifo, Nicholas P.

    2007-01-01

    DNA vaccines were introduced less than a decade ago but have already been applied to a wide range of infectious and malignant diseases. Here we review the current understanding of the mechanisms underlying the activities of these new vaccines. We focus on recent strategies designed to enhance their function including the use of immunostimulatory (CpG) sequences, dendritic cells (DC), co-stimulatory molecules and cytokine- and chemokine-adjuvants. Although genetic vaccines have been significantly improved, they may not be sufficiently immunogenic for the therapeutic vaccination of patients with infectious diseases or cancer in clinical trials. One promising approach aimed at dramatically increasing the immunogenicity of genetic vaccines involves making them ‘self-replicating’. This can be accomplished by using a gene encoding RNA replicase, a polyprotein derived from alphaviruses, such as Sindbis virus. Replicase-containing RNA vectors are significantly more immunogenic than conventional plasmids, immunizing mice at doses as low as 0.1 μg of nucleic acid injected once intramuscularly. Cells transfected with ‘self-replicating’ vectors briefly produce large amounts of antigen before undergoing apoptotic death. This death is a likely result of requisite double-stranded (ds) RNA intermediates, which also have been shown to super-activate DC. Thus, the enhanced immunogenicity of ‘self-replicating’ genetic vaccines may be a result of the production of pro-inflammatory dsRNA, which mimics an RNA-virus infection of host cells. PMID:10580187

  16. Cas9/sgRNA selective targeting of the P23H Rhodopsin mutant allele for treating retinitis pigmentosa by intravitreal AAV9.PHP.B-based delivery.

    PubMed

    Giannelli, Serena G; Luoni, Mirko; Castoldi, Valerio; Massimino, Luca; Cabassi, Tommaso; Angeloni, Debora; Demontis, Gian Carlo; Leocani, Letizia; Andreazzoli, Massimiliano; Broccoli, Vania

    2018-03-01

    P23H is the most common mutation in the RHODOPSIN (RHO) gene leading to a dominant form of retinitis pigmentosa (RP), a rod photoreceptor degeneration that invariably causes vision loss. Specific disruption of the disease P23H RHO mutant while preserving the wild-type (WT) functional allele would be an invaluable therapy for this disease. However, various technologies tested in the past failed to achieve effective changes and consequently therapeutic benefits. We validated a CRISPR/Cas9 strategy to specifically inactivate the P23H RHO mutant, while preserving the WT allele in vitro. We, then, translated this approach in vivo by delivering the CRISPR/Cas9 components in murine Rho+/P23H mutant retinae. Targeted retinae presented a high rate of cleavage in the P23H but not WT Rho allele. This gene manipulation was sufficient to slow photoreceptor degeneration and improve retinal functions. To improve the translational potential of our approach, we tested intravitreal delivery of this system by means of adeno-associated viruses (AAVs). To this purpose, the employment of the AAV9-PHP.B resulted the most effective in disrupting the P23H Rho mutant. Finally, this approach was translated successfully in human cells engineered with the homozygous P23H RHO gene mutation. Overall, this is a significant proof-of-concept that gene allele specific targeting by CRISPR/Cas9 technology is specific and efficient and represents an unprecedented tool for treating RP and more broadly dominant genetic human disorders affecting the eye, as well as other tissues.

  17. Personalized vaccines: the emerging field of vaccinomics

    PubMed Central

    Poland, Gregory A; Ovsyannikova, Inna G; Jacobson, Robert M

    2010-01-01

    The next ‘golden age’ in vaccinology will be ushered in by the new science of vaccinomics. In turn, this will inform and allow the development of personalized vaccines, based on our increasing understanding of immune response phenotype: genotype information. Rapid advances in developing such data are already occurring for hepatitis B, influenza, measles, mumps, rubella, anthrax and smallpox vaccines. In addition, newly available data suggest that some vaccine-related adverse events may also be genetically determined and, therefore, predictable. This paper reviews the basis and logic of personalized vaccines, and describes recent advances in the field. PMID:18847302

  18. Comparative analysis of pentavalent rotavirus vaccine strains and G8 rotaviruses identified during vaccine trial in Africa.

    PubMed

    Heylen, Elisabeth; Zeller, Mark; Ciarlet, Max; Lawrence, Jody; Steele, Duncan; Van Ranst, Marc; Matthijnssens, Jelle

    2015-10-06

    RotaTeqTM is a pentavalent rotavirus vaccine based on a bovine rotavirus genetic backbone in vitro reassorted with human outer capsid genes. During clinical trials of RotaTeqTM in Sub-Saharan Africa, the vaccine efficacy over a 2-year follow-up was lower against the genotypes contained in the vaccine than against the heterotypic G8P[6] and G8P[1] rotavirus strains of which the former is highly prevalent in Africa. Complete genome analyses of 43 complete rotavirus genomes collected during phase III clinical trials of RotaTeqTM in Sub-Saharan Africa, were conducted to gain insight into the high level of cross-protection afforded by RotaTeqTM against these G8 strains. Phylogenetic analysis revealed the presence of a high number of bovine rotavirus gene segments in these human G8 strains. In addition, we performed an in depth analysis on the individual amino acid level which showed that G8 rotaviruses were more similar to the RotaTeqTM vaccine than non-G8 strains. Because RotaTeqTM possesses a bovine genetic backbone, the high vaccine efficacy against G8 strains might be partially explained by the fact that all these strains contain a complete or partial bovine-like backbone. Altogether, this study supports the hypothesis that gene segments other than VP7 and VP4 play a role in vaccine-induced immunity.

  19. A Translational Pathway Toward a Clinical Trial Using the Second-Generation AAV Micro Dystrophin Vector

    DTIC Science & Technology

    2017-09-01

    future experimental therapeutic studies in the canine model such as CRISPR -mediated gene editing, stem cell therapy, dystrophin-independent disease...There is no scientific/budget overlap with the current proposal.) CRISPR /Cas9-based gene editing for the correction of Duchenne muscular dystrophy...lab will perform in vivo gene delivery and functional outcome measurements in mice treated by AAV- CRISPR gene repair vectors and if needed will also

  20. Bringing DNA vaccines closer to commercial use.

    PubMed

    Carvalho, Joana A; Prazeres, Duarte M F; Monteiro, Gabriel A

    2009-10-01

    Progress in the application of DNA vaccines as an immunization protocol is evident from the increasing number of such vaccines under evaluation in clinical trials and by the recent approval of several DNA vaccine products for veterinary applications. DNA vaccine technology offers important therapeutic and commercial advantages compared with conventional approaches, including the opportunity to target pathogens characterized by significant genetic diversity using a safe immunization platform, and the ability to use a simple, rapid and well-characterized production method. However, further optimization of DNA vaccine technology through the use of improved constructs, delivery systems and immunization protocols is necessary to clinically achieve the promising results that have been demonstrated in preclinical models.

  1. Immunogenicity of next-generation HPV vaccines in non-human primates: Measles-vectored HPV vaccine versus Pichia pastoris recombinant protein vaccine.

    PubMed

    Gupta, Gaurav; Giannino, Viviana; Rishi, Narayan; Glueck, Reinhard

    2016-09-07

    Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide. HPVs are oncogenic small double-stranded DNA viruses that are the primary causal agent of cervical cancer and other types of cancers, including in the anus, oropharynx, vagina, vulva, and penis. Prophylactic vaccination against HPV is an attractive strategy for preventing cervical cancer and some other types of cancers. However, there are few safe and effective vaccines against HPV infections. Current first-generation commercial HPV vaccines are expensive to produce and deliver. The goal of this study was to develop an alternate potent HPV recombinant L1-based vaccines by producing HPV virus-like particles into a vaccine that is currently used worldwide. Live attenuated measles virus (MV) vaccines have a well-established safety and efficacy record, and recombinant MV (rMV) produced by reverse genetics may be useful for generating candidate HPV vaccines to meet the needs of the developing world. We studied in non-human primate rMV-vectored HPV vaccine in parallel with a classical alum adjuvant recombinant HPV16L1 and 18L1 protein vaccine produced in Pichia pastoris. A combined prime-boost approach using both vaccines was evaluated, as well as immune interference due to pre-existing immunity against the MV. The humoral immune response induced by the MV, Pichia-expressed vaccine, and their combination as priming and boosting approaches was found to elicit HPV16L1 and 18L1 specific total IgG and neutralizing antibody titres. Pre-existing antibodies against measles did not prevent the immune response against HPV16L1 and 18L1. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Benefits of gene transduction of granulocyte macrophage colony-stimulating factor in cancer vaccine using genetically modified dendritic cells.

    PubMed

    Ojima, Toshiyasu; Iwahashi, Makoto; Nakamura, Masaki; Matsuda, Kenji; Nakamori, Mikihito; Ueda, Kentaro; Naka, Teiji; Katsuda, Masahiro; Miyazawa, Motoki; Yamaue, Hiroki

    2007-10-01

    Granulocyte macrophage colony-stimulating factor (GM-CSF) is a key cytokine for the generation and stimulation of dendritic cells (DCs), and it may also play a pivotal role in promoting the survival of DCs. In this study, the feasibility of creating a cancer vaccine using DCs adenovirally transduced with the carcinoembryonic antigen (CEA) gene and the GM-CSF gene was examined. In addition, the effect of the co-transduction of GM-CSF gene on the lifespan of these genetically modified DCs was determined. A cytotoxic assay using peripheral blood mononuclear cell (PBMC)-derived cytotoxic T lymphocytes (CTLs) was performed in a 4-h 51Cr release assay. The apoptosis of DCs was examined by TdT-mediated dUTP-FITC nick end labeling (TUNEL) assay. CEA-specific CTLs were generated from PBMCs stimulated with genetically modified DCs expressing CEA. The cytotoxicity of these CTLs was augmented by co-transduction of DCs with the GM-CSF gene. Co-transduction of the GM-CSF gene into DCs inhibited apoptosis of these DCs themselves via up-regulation of Bcl-x(L) expression, leading to the extension of the lifespan of these DCs. Furthermore, the transduction of the GM-CSF gene into DCs also suppressed the incidence of apoptosis of DCs induced by transforming growth factor-beta1 (TGFbeta-1). Immunotherapy using these genetically modified DCs may therefore be useful with several advantages as follows: i) adenoviral toxicity to DCs can be reduced; ii) the lifespan of vaccinated DCs can be prolonged; and iii) GM-CSF may protect DCs from apoptosis induced by tumor-derived TGFbeta-1 in the regional lymph nodes.

  3. When gene medication is also genetic modification--regulating DNA treatment.

    PubMed

    Foss, Grethe S; Rogne, Sissel

    2007-07-26

    The molecular methods used in DNA vaccination and gene therapy resemble in many ways the methods applied in genetic modification of organisms. In some regulatory regimes, this creates an overlap between 'gene medication' and genetic modification. In Norway, an animal injected with plasmid DNA, in the form of DNA vaccine or gene therapy, currently is viewed as being genetically modified for as long as the added DNA is present in the animal. However, regulating a DNA-vaccinated animal as genetically modified creates both regulatory and practical challenges. It is also counter-intuitive to many biologists. Since immune responses can be elicited also to alter traits, the borderline between vaccination and the modification of properties is no longer distinct. In this paper, we discuss the background for the Norwegian interpretation and ways in which the regulatory challenge can be handled.

  4. Plastid Molecular Pharming I. Production of Oral Vaccines via Plastid Transformation.

    PubMed

    Berecz, Bernadett; Zelenyánszki, Helga; Pólya, Sára; Tamás-Nyitrai, Cecília; Oszvald, Mária

    2017-01-01

    Vaccines produced in plants have opened up new opportunities in vaccination. Among the various categories of vaccines, the recombinant vaccine is generally regarded as the most economical and safest type because it cannot cause disease and does not require large-scale cultivation of pathogens. Due to the low cost of their cultivation, plants may represent viable alternative platforms for producing subunit vaccines. Genetic engineering of plastids is the innovation of the last three decades and has numerous benefits when compared to nuclear transformation. Due to the high level of expression, oral vaccines produced in transplastomic plants do not have to be purified as they can be consumed raw, which, therefore, reduces the cost of preparation, transportation and handling of the vaccines. Oral vaccination also excludes the risk of other infections or contaminations, while compartmentation of the plant cell provides an excellent encapsulation to the antigen within the plastid. Herein we review the main biotechnological and immunological aspects of the progress achieved in the field of plastid derived edible vaccines during the last decade. As there is a public debate against genetically modified crops, the advantages and limitations of oral vaccines are also discussed. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Intracranial AAV-sTRAIL combined with lanatoside C prolongs survival in an orthotopic xenograft mouse model of invasive glioblastoma.

    PubMed

    Crommentuijn, Matheus H W; Maguire, Casey A; Niers, Johanna M; Vandertop, W Peter; Badr, Christian E; Würdinger, Thomas; Tannous, Bakhos A

    2016-04-01

    Glioblastoma (GBM) is the most common malignant brain tumor in adults. We designed an adeno-associated virus (AAV) vector for intracranial delivery of secreted, soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) to GBM tumors in mice and combined it with the TRAIL-sensitizing cardiac glycoside, lanatoside C (lan C). We applied this combined therapy to two different GBM models using human U87 glioma cells and primary patient-derived GBM neural spheres in culture and in orthotopic GBM xenograft models in mice. In U87 cells, conditioned medium from AAV2-sTRAIL expressing cells combined with lan C induced 80% cell death. Similarly, lan C sensitized primary GBM spheres to sTRAIL causing over 90% cell death. In mice bearing intracranial U87 tumors treated with AAVrh.8-sTRAIL, administration of lan C caused a decrease in tumor-associated Fluc signal, while tumor size increased within days of stopping the treatment. Another round of lan C treatment re-sensitized GBM tumor to sTRAIL-induced cell death. AAVrh.8-sTRAIL treatment alone and combined with lanatoside C resulted in a significant decrease in tumor growth and longer survival of mice bearing orthotopic invasive GBM brain tumors. In summary, AAV-sTRAIL combined with lanatoside C induced cell death in U87 glioma cells and patient-derived GBM neural spheres in culture and in vivo leading to an increased in overall mice survival. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  6. Molecular analysis of varicella vaccines and varicella-zoster virus from vaccine-related skin lesions.

    PubMed

    Thiele, Sonja; Borschewski, Aljona; Küchler, Judit; Bieberbach, Marc; Voigt, Sebastian; Ehlers, Bernhard

    2011-07-01

    To prevent complications that might follow an infection with varicella-zoster virus (VZV), the live attenuated Oka strain (V-Oka) is administered to children in many developed countries. Three vaccine brands (Varivax from Sanofi Pasteur MSD; Varilrix and Priorix-Tetra, both from Glaxo-Smith-Kline) are licensed in Germany and have been associated with both different degrees of vaccine effectiveness and adverse effects. To identify genetic variants in the vaccines that might contribute to rash-associated syndromes, single nucleotide polymorphism (SNP) profiles of variants from the three vaccines and rash-associated vaccine-type VZV from German vaccinees were quantitatively compared by PCR-based pyrosequencing (PSQ). The Varivax vaccine contained an estimated 3-fold higher diversity of VZV variants, with 20% more wild-type (wt) SNPs than Varilrix and Priorix-Tetra. These minor VZV variants in the vaccines were identified by analyzing cloned full-length open reading frame (ORF) orf62 sequences by chain termination sequencing and PSQ. Some of these sequences amplified from vaccine VZV were very similar or identical to those of the rash-associated vaccine-type VZV from vaccinees and were almost exclusively detected in Varivax. Therefore, minorities of rash-associated VZV variants are present in varicella vaccine formulations, and it can be concluded that the analysis of a core set of four SNPs is required as a minimum for a firm diagnostic differentiation of vaccine-type VZV from wt VZV.

  7. Genetic and antigenic relationship of foot-and-mouth disease virus serotype O isolates with the vaccine strain O1/BFS.

    PubMed

    Xu, Wanhong; Zhang, Zhidong; Nfon, Charles; Yang, Ming

    2018-05-15

    Foot-and-mouth disease serotype O viruses (FMDV/O) are responsible for the most outbreaks in FMD endemic countries. O1/BFS is one of the recommended FMD/O vaccine strains by World Reference Laboratory for FMD. In the current study, FMDV/O1 BFS vaccine strain and serotype O field isolates (45) were analyzed phylogenetically and antigenically to gain more insight into the genetic and antigenic characteristics of the vaccine strain and field isolates. O1/BFS showed similarity with 89% of the field isolates using a virus neutralization test (VNT). The P1 region encoding the FMDV capsid was sequenced and analysed for 46 strains of FMDV/O. Phylogenetic analysis showed these viruses originated from five continents and covered eight of 11 reported topotypes. Five isolates that demonstrated low antigenic similarities with O1/BFS were analyzed for their antigenic variation at the known neutralizing antigenic sites. Three of the five isolates demonstrated unique amino acid substitutions at various antigenic sites. No unique amino acid substitutions were observed for the other two unmatched isolates. Positively selected residues were identified on the surface of the FMD virus capsid supporting that it is important to continuously monitor field isolates for their antigenic and phenotypic changes. In conclusion, the vaccine strain O1/BFS is likely to confer protection against 89% of the 45 FMDV/O isolates based on VNT. Thus O1/BFS vaccine strain is still suitable for use in global FMD serotype O outbreak control. Combining data from phylogenetic, molecular and antigenic analysis can provide improvements in the process of vaccine selection. Crown Copyright © 2018. Published by Elsevier Ltd. All rights reserved.

  8. Update on Vaccine-Derived Polioviruses - Worldwide, January 2015-May 2016.

    PubMed

    Jorba, Jaume; Diop, Ousmane M; Iber, Jane; Sutter, Roland W; Wassilak, Steven G; Burns, Cara C

    2016-08-05

    In 1988, the World Health Assembly resolved to eradicate poliomyelitis worldwide (1). One of the main tools used in polio eradication efforts has been the live, attenuated, oral poliovirus vaccine (OPV) (2), an inexpensive vaccine easily administered by trained volunteers. OPV might require several doses to induce immunity, but provides long-term protection against paralytic disease. Through effective use of OPV, the Global Polio Eradication Initiative (GPEI) has brought wild polioviruses to the threshold of eradication (1). However, OPV use, particularly in areas with low routine vaccination coverage, is associated with the emergence of genetically divergent vaccine-derived polioviruses (VDPVs) whose genetic drift from the parental OPV strains indicates prolonged replication or circulation (3). VDPVs can emerge among immunologically normal vaccine recipients and their contacts as well as among persons with primary immunodeficiencies (PIDs). Immunodeficiency-associated VDPVs (iVDPVs) can replicate for years in some persons with PIDs. In addition, circulating vaccine-derived polioviruses (cVDPVs) (3) can emerge in areas with low OPV coverage and can cause outbreaks of paralytic polio. This report updates previous summaries regarding VDPVs (4).

  9. Hepatitis B vaccine in celiac disease: yesterday, today and tomorrow.

    PubMed

    Vitaliti, Giovanna; Praticò, Andrea Domenico; Cimino, Carla; Di Dio, Giovanna; Lionetti, Elena; La Rosa, Mario; Leonardi, Salvatore

    2013-02-14

    Some studies showed that in celiac patients the immunological response to vaccination is similar to that one found in general population except for vaccine against hepatitis B virus (HBV). The non-responsiveness to HBV vaccine has also been described in healthy people, nevertheless the number of non-responders has been demonstrated to be higher in celiac disease (CD) patients than in healthy controls. Several hypothesis explaining this higher rate of unresponsiveness to HBV vaccine in CD patients have been described, such as the genetic hypothesis, according with CD patients carrying the disease-specific haplotype HLA-B8, DR3, and DQ2, show a lower response to HBV vaccine both in clinical expressed CD patients and in healthy people carrying the same haplotype. On the other hand, it has been demonstrated that the gluten intake during the vaccination seems to influence the response to the same vaccine. Moreover, it has been demonstrated a possible genetic predisposition to hepatitis B vaccine non-responsiveness likely due to the presence of specific human leukocyte antigen haplotypes and specific single nucleotide polymorphism in genes of cytokine/cytokine receptors and toll like receptors, but the pathogenic mechanism responsible for this low responsiveness still remains unclear. The aim of this review is to focus on the possible pathogenic causes of unresponsiveness to HBV vaccine in CD patients and to propose an alternative vaccination schedule in order to improve the responsiveness to HBV vaccine in this at-risk patients.

  10. Reemergence of recombinant vaccine-derived poliovirus outbreak in Madagascar.

    PubMed

    Rakoto-Andrianarivelo, Mala; Gumede, Nicksy; Jegouic, Sophie; Balanant, Jean; Andriamamonjy, Seta N; Rabemanantsoa, Sendraharimanana; Birmingham, Maureen; Randriamanalina, Bakolalao; Nkolomoni, Léon; Venter, Marietjie; Schoub, Barry D; Delpeyroux, Francis; Reynes, Jean-Marc

    2008-05-15

    After the 2001-2002 poliomyelitis outbreak due to recombinant vaccine-derived polioviruses (VDPVs) in the Toliara province of Madagascar, another outbreak reoccurred in the same province in 2005. We conducted epidemiological and virological investigations for each polio case patient and for their contacts. From May to August 2005, a total of 5 cases of acute flaccid paralysis were reported among unvaccinated or partially vaccinated children 2-3 years old. Type-3 or type-2 VDPV was isolated from case patients and from healthy contacts. These strains were classified into 4 recombinant lineages that showed complex mosaic genomic structures originating from different vaccine strain serotypes and probably from human enterovirus C (HEV-C) species. Genetic relatedness could be observed among these 4 lineages. Vaccination coverage of the population was very low (<50%). The broad distribution of VDPVs in the province and their close genetic relationship indicate intense and rapid cocirculation and coevolution of the vaccine strains and of their related HEV-C strains. The occurrence of an outbreak due to VDPV 3 years after a previous outbreak indicates that a short period with low vaccination coverage is enough to create favorable conditions for the emergence of VDPV in this setting.

  11. Evaluation of AAV-mediated Gene Therapy for Central Nervous System Disease in Canine Mucopolysaccharidosis VII

    PubMed Central

    Gurda, Brittney L; De Guilhem De Lataillade, Adrien; Bell, Peter; Zhu, Yanqing; Yu, Hongwei; Wang, Ping; Bagel, Jessica; Vite, Charles H; Sikora, Tracey; Hinderer, Christian; Calcedo, Roberto; Yox, Alexander D; Steet, Richard A; Ruane, Therese; O'Donnell, Patricia; Gao, Guangping; Wilson, James M; Casal, Margret; Ponder, Katherine P; Haskins, Mark E

    2016-01-01

    Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease arising from mutations in β-d-glucuronidase (GUSB), which results in glycosaminoglycan (GAG) accumulation and a variety of clinical manifestations including neurological disease. Herein, MPS VII dogs were injected intravenously (i.v.) and/or intrathecally (i.t.) via the cisterna magna with AAV9 or AAVrh10 vectors carrying the canine GUSB cDNA. Although i.v. injection alone at 3 days of age resulted in normal cerebrospinal fluid (CSF) GUSB activity, brain tissue homogenates had only ~1 to 6% normal GUSB activity and continued to have elevated GAG storage. In contrast, i.t. injection at 3 weeks of age resulted in CSF GUSB activity 44-fold normal while brain tissue homogenates had >100% normal GUSB activity and reduced GAGs compared with untreated dogs. Markers for secondary storage and inflammation were eliminated in i.t.-treated dogs and reduced in i.v.-treated dogs compared with untreated dogs. Given that i.t.-treated dogs expressed higher levels of GUSB in the CNS tissues compared to those treated i.v., we conclude that i.t. injection of AAV9 or AAVrh10 vectors is more effective than i.v. injection alone in the large animal model of MPS VII. PMID:26447927

  12. Systemic AAV8-Mediated Gene Therapy Drives Whole-Body Correction of Myotubular Myopathy in Dogs.

    PubMed

    Mack, David L; Poulard, Karine; Goddard, Melissa A; Latournerie, Virginie; Snyder, Jessica M; Grange, Robert W; Elverman, Matthew R; Denard, Jérôme; Veron, Philippe; Buscara, Laurine; Le Bec, Christine; Hogrel, Jean-Yves; Brezovec, Annie G; Meng, Hui; Yang, Lin; Liu, Fujun; O'Callaghan, Michael; Gopal, Nikhil; Kelly, Valerie E; Smith, Barbara K; Strande, Jennifer L; Mavilio, Fulvio; Beggs, Alan H; Mingozzi, Federico; Lawlor, Michael W; Buj-Bello, Ana; Childers, Martin K

    2017-04-05

    X-linked myotubular myopathy (XLMTM) results from MTM1 gene mutations and myotubularin deficiency. Most XLMTM patients develop severe muscle weakness leading to respiratory failure and death, typically within 2 years of age. Our objective was to evaluate the efficacy and safety of systemic gene therapy in the p.N155K canine model of XLMTM by performing a dose escalation study. A recombinant adeno-associated virus serotype 8 (rAAV8) vector expressing canine myotubularin (cMTM1) under the muscle-specific desmin promoter (rAAV8-cMTM1) was administered by simple peripheral venous infusion in XLMTM dogs at 10 weeks of age, when signs of the disease are already present. A comprehensive analysis of survival, limb strength, gait, respiratory function, neurological assessment, histology, vector biodistribution, transgene expression, and immune response was performed over a 9-month study period. Results indicate that systemic gene therapy was well tolerated, prolonged lifespan, and corrected the skeletal musculature throughout the body in a dose-dependent manner, defining an efficacious dose in this large-animal model of the disease. These results support the development of gene therapy clinical trials for XLMTM. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

  13. Evaluation of AAV-mediated Gene Therapy for Central Nervous System Disease in Canine Mucopolysaccharidosis VII.

    PubMed

    Gurda, Brittney L; De Guilhem De Lataillade, Adrien; Bell, Peter; Zhu, Yanqing; Yu, Hongwei; Wang, Ping; Bagel, Jessica; Vite, Charles H; Sikora, Tracey; Hinderer, Christian; Calcedo, Roberto; Yox, Alexander D; Steet, Richard A; Ruane, Therese; O'Donnell, Patricia; Gao, Guangping; Wilson, James M; Casal, Margret; Ponder, Katherine P; Haskins, Mark E

    2016-02-01

    Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease arising from mutations in β-d-glucuronidase (GUSB), which results in glycosaminoglycan (GAG) accumulation and a variety of clinical manifestations including neurological disease. Herein, MPS VII dogs were injected intravenously (i.v.) and/or intrathecally (i.t.) via the cisterna magna with AAV9 or AAVrh10 vectors carrying the canine GUSB cDNA. Although i.v. injection alone at 3 days of age resulted in normal cerebrospinal fluid (CSF) GUSB activity, brain tissue homogenates had only ~1 to 6% normal GUSB activity and continued to have elevated GAG storage. In contrast, i.t. injection at 3 weeks of age resulted in CSF GUSB activity 44-fold normal while brain tissue homogenates had >100% normal GUSB activity and reduced GAGs compared with untreated dogs. Markers for secondary storage and inflammation were eliminated in i.t.-treated dogs and reduced in i.v.-treated dogs compared with untreated dogs. Given that i.t.-treated dogs expressed higher levels of GUSB in the CNS tissues compared to those treated i.v., we conclude that i.t. injection of AAV9 or AAVrh10 vectors is more effective than i.v. injection alone in the large animal model of MPS VII.

  14. Genetic stability of Rift Valley fever virus MP-12 vaccine during serial passages in culture cells.

    PubMed

    Lokugamage, Nandadeva; Ikegami, Tetsuro

    2017-01-01

    Rift Valley fever (RVF) is a mosquito-borne zoonotic disease endemic to Africa which affects both ruminants and humans. RVF causes serious damage to the livestock industry and is also a threat to public health. The Rift Valley fever virus has a segmented negative-stranded RNA genome consisting of Large (L)-, Medium (M)-, and Small (S)-segments. The live-attenuated MP-12 vaccine is immunogenic in livestock and humans, and is conditionally licensed for veterinary use in the U.S. The MP-12 strain encodes 23 mutations (nine amino acid substitutions) and is attenuated through a combination of mutations in the L-, M-, and S-segments. Among them, the M-U795C, M-A3564G, and L-G3104A mutations contribute to viral attenuation through the L- and M-segments. The M-U795C, M-A3564G, L-U533C, and L-G3750A mutations are also independently responsible for temperature-sensitive (ts) phenotype. We hypothesized that a serial passage of the MP-12 vaccine in culture cells causes reversions of the MP-12 genome. The MP-12 vaccine and recombinant rMP12-ΔNSs16/198 were serially passaged 25 times. Droplet digital PCR analysis revealed that the reversion occurred at L-G3750A during passages of MP-12 in Vero or MRC-5 cells. The reversion also occurred at M-A3564G and L-U533C of rMP12-ΔNSs16/198 in Vero cells. Reversion mutations were not found in MP-12 or the variant, rMP12-TOSNSs, in the brains of mice with encephalitis. This study characterized genetic stability of the MP-12 vaccine and the potential risk of reversion mutation at the L-G3750A ts mutation after excessive viral passages in culture cells.

  15. Update on autism spectrum disorder: vaccines, genomes, and social skills training.

    PubMed

    McGuinness, Teena M

    2015-04-01

    Despite making significant progress in understanding autism spectrum disorder (ASD) and its genetic underpinnings, controversy remains regarding ASD and its historical, erroneous association with vaccines. This controversy includes the latest anti-vaccine movement that caused a recurrence of the almost vanquished measles and mumps diseases. The history of ASD, complexities of research involving ASD genetics, and benefits of social skills training are explored. Copyright 2015, SLACK Incorporated.

  16. Designing malaria vaccines to circumvent antigen variability.

    PubMed

    Ouattara, Amed; Barry, Alyssa E; Dutta, Sheetij; Remarque, Edmond J; Beeson, James G; Plowe, Christopher V

    2015-12-22

    Prospects for malaria eradication will be greatly enhanced by an effective vaccine, but parasite genetic diversity poses a major impediment to malaria vaccine efficacy. In recent pre-clinical and field trials, vaccines based on polymorphic Plasmodium falciparum antigens have shown efficacy only against homologous strains, raising the specter of allele-specific immunity such as that which plagues vaccines against influenza and HIV. The most advanced malaria vaccine, RTS,S, targets relatively conserved epitopes on the P. falciparum circumsporozoite protein. After more than 40 years of development and testing, RTS,S, has shown significant but modest efficacy against clinical malaria in phase 2 and 3 trials. Ongoing phase 2 studies of an irradiated sporozoite vaccine will ascertain whether the full protection against homologous experimental malaria challenge conferred by high doses of a whole organism vaccine can provide protection against diverse strains in the field. Here we review and evaluate approaches being taken to design broadly cross-protective malaria vaccines. Copyright © 2015. Published by Elsevier Ltd.

  17. Designing malaria vaccines to circumvent antigen variability✩

    PubMed Central

    Ouattara, Amed; Barry, Alyssa E.; Dutta, Sheetij; Remarque, Edmond J.; Beeson, James G.; Plowe, Christopher V.

    2016-01-01

    Prospects for malaria eradication will be greatly enhanced by an effective vaccine, but parasite genetic diversity poses a major impediment to malaria vaccine efficacy. In recent pre-clinical and field trials, vaccines based on polymorphic Plasmodium falciparum antigens have shown efficacy only against homologous strains, raising the specter of allele-specific immunity such as that which plagues vaccines against influenza and HIV. The most advanced malaria vaccine, RTS,S, targets relatively conserved epitopes on the P. falciparum circumsporozoite protein. After more than 40 years of development and testing, RTS,S, has shown significant but modest efficacy against clinical malaria in phase 2 and 3 trials. Ongoing phase 2 studies of an irradiated sporozoite vaccine will ascertain whether the full protection against homologous experimental malaria challenge conferred by high doses of a whole organism vaccine can provide protection against diverse strains in the field. Here we review and evaluate approaches being taken to design broadly cross-protective malaria vaccines. PMID:26475447

  18. Induction of immunity to antigens expressed by recombinant adeno-associated virus depends on the route of administration.

    PubMed

    Brockstedt, D G; Podsakoff, G M; Fong, L; Kurtzman, G; Mueller-Ruchholtz, W; Engleman, E G

    1999-07-01

    Recombinant adeno-associated virus (rAAV) is a replication-defective parvovirus which is being explored as a vector for gene therapy because of its broad host range, excellent safety profile, and durable transgene expression in infected hosts. rAAV has also been reported by several groups to induce little or no immune response to its encoded transgene products. In this study we examined the immunogenicity of rAAV by studying the immune response of C57BL/6 mice to a single dose of rAAV-encoding ovalbumin (AAV-Ova) administered by a variety of routes. Mice injected with AAV-Ova intraperitoneally (ip), intravenously, or subcutaneously developed potent ovalbumin-specific cytotoxic T lymphocytes (CTL) as well as anti-ovalbumin antibodies and antibodies to AAV. In contrast, mice injected with AAV-Ova intramuscularly developed a humoral response to the virus and the transgene but minimal ovalbumin-specific CTLs. The induced CTL response after ip administration of AAV-Ova protected mice against a subsequent tumor challenge with an ovalbumin-transfected B16 melanoma cell line. Studies of the mechanism by which AAV-Ova induces CTL confirmed that the virus delivers the transgene product into the classical MHC class I pathway of antigen processing. Mice that previously had been exposed to rAAV vectors failed to develop ovalbumin-specific CTL following administration of AAV-Ova. Analysis of these mice revealed the presence of circulating anti-AAV antibodies that blocked rAAV transduction in vitro and inhibited CTL induction in vivo. These results suggest a possible role for rAAV in the immunotherapy of malignancies and viral infections, although induced antibody responses to AAV may limit its ability to be administered for repeated vaccinations. Copyright 1999 Academic Press.

  19. Germline viral "fossils" guide in silico reconstruction of a mid-Cenozoic era marsupial adeno-associated virus.

    PubMed

    Smith, Richard H; Hallwirth, Claus V; Westerman, Michael; Hetherington, Nicola A; Tseng, Yu-Shan; Cecchini, Sylvain; Virag, Tamas; Ziegler, Mona-Larissa; Rogozin, Igor B; Koonin, Eugene V; Agbandje-McKenna, Mavis; Kotin, Robert M; Alexander, Ian E

    2016-07-05

    Germline endogenous viral elements (EVEs) genetically preserve viral nucleotide sequences useful to the study of viral evolution, gene mutation, and the phylogenetic relationships among host organisms. Here, we describe a lineage-specific, adeno-associated virus (AAV)-derived endogenous viral element (mAAV-EVE1) found within the germline of numerous closely related marsupial species. Molecular screening of a marsupial DNA panel indicated that mAAV-EVE1 occurs specifically within the marsupial suborder Macropodiformes (present-day kangaroos, wallabies, and related macropodoids), to the exclusion of other Diprotodontian lineages. Orthologous mAAV-EVE1 locus sequences from sixteen macropodoid species, representing a speciation history spanning an estimated 30 million years, facilitated compilation of an inferred ancestral sequence that recapitulates the genome of an ancient marsupial AAV that circulated among Australian metatherian fauna sometime during the late Eocene to early Oligocene. In silico gene reconstruction and molecular modelling indicate remarkable conservation of viral structure over a geologic timescale. Characterisation of AAV-EVE loci among disparate species affords insight into AAV evolution and, in the case of macropodoid species, may offer an additional genetic basis for assignment of phylogenetic relationships among the Macropodoidea. From an applied perspective, the identified AAV "fossils" provide novel capsid sequences for use in translational research and clinical applications.

  20. Vaccine-derived polioviruses.

    PubMed

    Agol, Vadim I

    2006-06-01

    The Sabin oral poliovaccine (OPV) is extremely efficacious and safe, despite its inherent genetic instability. While reversion to nearly wild-type phenotype regularly occurs soon after the onset of OPV reproduction in the gastro-intestinal tract of vaccine recipients or their contacts, this is usually not a big problem, provided the vaccine is used either for mass vaccination or in populations with a relatively high level of anti-polio immunity. However, if these conditions are not met, the vaccine viruses are likely to be converted into highly transmissible agents with a nearly wild-type level of neurovirulence. Moreover, OPV viruses may persist and evolve even in adequately immunized populations. The current strategy for the "endgame" of poliovirus eradication envisions cessation of OPV usage shortly after the last isolation of a wild poliovirus. If implemented, this strategy would result in rapid growth of non-immune human populations at the time when OPV derivatives would very likely be persisting. Therefore, the planned cessation of OPV vaccination is associated with a very high, and in the author's opinion, unacceptable risk of polio outbreaks caused by OPV derivatives. The only currently available tool to curb such outbreaks is OPV, which should have been used at a global scale. Safe discontinuation of OPV vaccination will be possible only after an efficient new vaccine or an anti-poliovirus drug is available. To achieve this goal, stimulation of poliovirus research and elimination of organizational and financial obstacles preventing it are needed.

  1. [Rubella virus genetic determinant of attenuation].

    PubMed

    Dmitriev, G V; Borisova, T K; Faizuloev, E B; Desiatskova, R G; Zverev, V V

    2014-01-01

    Vaccination is the most effective and available way to prevent Rubella. Presently, 9 vaccine strains were registered. Nevertheless, the molecular mechanisms of the attenuation were poorly elucidated for the rubella virus. However, the study of these mechanisms identifying genotypic and phenotypic markers of attenuation, which together with sequence analysis could be used for the genetic stability control of vaccine strains, is still of current interest. Common trends of genetic changes in the process of adaptation to cold were found due to comparison of nucleic acid and amino acid sequences of the Russian strain C-77 with corresponding positions of the known rubella virus strains and its wild type progenitors, if available.

  2. Multigenic Control of Measles Vaccine Immunity Mediated by Polymorphisms in Measles Receptor, Innate Pathway, and Cytokine Genes

    PubMed Central

    Kennedy, Richard B.; Ovsyannikova, Inna G.; Haralambieva, Iana H.; O’Byrne, Megan; Jacobson, Robert M.; Pankratz, V. Shane; Poland, Gregory A.

    2012-01-01

    Measles infection and vaccine response are complex biological processes that involve both viral and host genetic factors. We have previously investigated the influence of genetic polymorphisms on vaccine immune response, including measles vaccines, and have shown that polymorphisms in HLA, cytokine, cytokine receptor, and innate immune response genes are associated with variation in vaccine response but do not account for all of the inter-individual variance seen in vaccinated populations. In the current study we report the findings of a multigenic analysis of measles vaccine immunity, indicating a role for the measles virus receptor CD46, innate pattern-recognition receptors (DDX58, TLR2, 4, 5,7 and 8) and intracellular signaling intermediates (MAP3K7, NFKBIA), and key antiviral molecules (VISA, OAS2, MX1, PKR) as well as cytokines (IFNA1, IL4, IL6, IL8, IL12B) and cytokine receptor genes (IL2RB, IL6R, IL8RA) in the genetic control of both humoral and cellular immune responses. This multivariate approach provided additional insights into the genetic control of measles vaccine responses over and above the information gained by our previous univariate SNP association analyses. PMID:22265947

  3. Rift Valley fever MP-12 vaccine Phase 2 clinical trial: Safety, immunogenicity, and genetic characterization of virus isolates.

    PubMed

    Pittman, Phillip R; Norris, Sarah L; Brown, Elizabeth S; Ranadive, Manmohan V; Schibly, Barbara A; Bettinger, George E; Lokugamage, Nandadeva; Korman, Lawrence; Morrill, John C; Peters, Clarence J

    2016-01-20

    An outbreak or deliberate release of Rift Valley fever (RVF) virus could have serious public health and socioeconomic consequences. A safe RVF vaccine capable of eliciting long-lasting immunity after a single injection is urgently needed. The live attenuated RVF MP-12 vaccine candidate has shown promise in Phase 1 clinical trials; no evidence of reversion to virulence has been identified in numerous animal studies. The objective of this Phase 2 clinical trial was to (a) further examine the safety and immunogenicity of RVF MP-12 in RVF virus-naïve humans and (b) characterize isolates of RVF MP-12 virus recovered from the blood of vaccinated subjects to evaluate the genetic stability of MP-12 attenuation. We found that RVF MP-12 was well tolerated, causing mostly mild reactions that resolved without sequelae. Of 19 subjects, 18 (95%) and 19 (100%) achieved, respectively, 80% and 50% plaque reduction neutralization titers (PRNT80 and PRNT50)≥1:20 by postvaccination day 28. All 18 PRNT80 responders maintained PRNT80 and PRNT50≥1:40 until at least postvaccination month 12. Viremia was undetectable in the plasma of any subject by direct plaque assay techniques. However, 5 of 19 vaccinees were positive for MP-12 isolates in plasma by blind passage of plasma on Vero cells. Vaccine virus was also recovered from buffy coat material from one of those vaccinees and from one additional vaccinee. Through RNA sequencing of MP-12 isolates, we found no reversions of amino acids to those of the parent virulent virus (strain ZH548). Five years after a single dose of RVF MP-12 vaccine, 8 of 9 vaccinees (89%) maintained a PRNT80≥1:20. These findings support the continued development of RVF MP-12 as a countermeasure against RVF virus in humans. Published by Elsevier Ltd.

  4. The impact of new technologies on vaccines.

    PubMed

    Talwar, G P; Diwan, M; Razvi, F; Malhotra, R

    1999-01-01

    Vast changes are taking place in vaccinology consequent to the introduction of new technologies. Amongst the vaccines included in the Expanded Programme of Immunization (EPI), the pertussis vaccine has been replaced by acellular purified fractions devoid of side-effects. Non-pathogenic but immunogenic mutants of tetanus and diptheria toxins are likely to replace the toxoids. An effective vaccine against hepatitis B prepared by recombinant technology is in large-scale use. Conjugated vaccines against Haemophilus influenzae b, S. pneumococcus and meningococcus are now available, as also vaccines against mumps, rubella and measles. Combination vaccines have been devised to limit the number of injections. Vaccine delivery systems have been developed to deliver multiple doses of the vaccine at a single contact point. A genetically-engineered oral vaccine for typhoid imparts better and longer duration of immunity. Oral vaccines for cholera and other enteric infections are under clinical trials. The nose as a route for immunization is showing promise for mucosal immunity and for anti-inflammatory experimental vaccines against multiple sclerosis and insulin-dependent diabetes mellitus. The range of vaccines has expanded to include pathogens resident in the body such as Helicobacter pylori (duodenal ulcer), S. mutans (dental caries), and human papilloma virus (carcinoma of the cervix). An important progress is the recognition that DNA alone can constitute the vaccines, inducing both humoral and cell-mediated immune responses. A large number of DNA vaccines have been made and shown interesting results in experimental animals. Live recombinant vaccines against rabies and rinderpest have proven to be highly effective for controlling these infections in the field, and those for AIDS are under clinical trial. Potent adjuvants have added to the efficacy of the vaccines. New technologies have emerged to 'humanize' mouse monoclonals by genetic engineering and express these

  5. A Translational Pathway Toward a Clinical Trial Using the Second-Generation AAV Micro-Dystrophin Vector

    DTIC Science & Technology

    2016-09-01

    minidystrophin gene (a gift from Dr Jeffrey Chamberlain at the University of Washington, Seattle, WA) and the bovine growth hormone polyadenylation...full-length micro-dystrophin protein. Dys-2 is a short peptide in the wild-type full-length dystrophin. It can be recognized by the Dys-2...muscle. In one approach, a muscle homing peptide is inserted on the surface of the capsid to facilitate the entry of AAV into muscle cells. In the

  6. Traditional and New Influenza Vaccines

    PubMed Central

    Wong, Sook-San

    2013-01-01

    SUMMARY The challenges in successful vaccination against influenza using conventional approaches lie in their variable efficacy in different age populations, the antigenic variability of the circulating virus, and the production and manufacturing limitations to ensure safe, timely, and adequate supply of vaccine. The conventional influenza vaccine platform is based on stimulating immunity against the major neutralizing antibody target, hemagglutinin (HA), by virus attenuation or inactivation. Improvements to this conventional system have focused primarily on improving production and immunogenicity. Cell culture, reverse genetics, and baculovirus expression technology allow for safe and scalable production, while adjuvants, dose variation, and alternate routes of delivery aim to improve vaccine immunogenicity. Fundamentally different approaches that are currently under development hope to signal new generations of influenza vaccines. Such approaches target nonvariable regions of antigenic proteins, with the idea of stimulating cross-protective antibodies and thus creating a “universal” influenza vaccine. While such approaches have obvious benefits, there are many hurdles yet to clear. Here, we discuss the process and challenges of the current influenza vaccine platform as well as new approaches that are being investigated based on the same antigenic target and newer technologies based on different antigenic targets. PMID:23824369

  7. Evolution and Vaccination of Influenza Virus.

    PubMed

    Lam, Ham Ching; Bi, Xuan; Sreevatsan, Srinand; Boley, Daniel

    2017-08-01

    In this study, we present an application paradigm in which an unsupervised machine learning approach is applied to the high-dimensional influenza genetic sequences to investigate whether vaccine is a driving force to the evolution of influenza virus. We first used a visualization approach to visualize the evolutionary paths of vaccine-controlled and non-vaccine-controlled influenza viruses in a low-dimensional space. We then quantified the evolutionary differences between their evolutionary trajectories through the use of within- and between-scatter matrices computation to provide the statistical confidence to support the visualization results. We used the influenza surface Hemagglutinin (HA) gene for this study as the HA gene is the major target of the immune system. The visualization is achieved without using any clustering methods or prior information about the influenza sequences. Our results clearly showed that the evolutionary trajectories between vaccine-controlled and non-vaccine-controlled influenza viruses are different and vaccine as an evolution driving force cannot be completely eliminated.

  8. Evolution of equine influenza virus in vaccinated horses.

    PubMed

    Murcia, Pablo R; Baillie, Gregory J; Stack, J Conrad; Jervis, Carley; Elton, Debra; Mumford, Jennifer A; Daly, Janet; Kellam, Paul; Grenfell, Bryan T; Holmes, Edward C; Wood, James L N

    2013-04-01

    Influenza A viruses are characterized by their ability to evade host immunity, even in vaccinated individuals. To determine how prior immunity shapes viral diversity in vivo, we studied the intra- and interhost evolution of equine influenza virus in vaccinated horses. Although the level and structure of genetic diversity were similar to those in naïve horses, intrahost bottlenecks may be more stringent in vaccinated animals, and mutations shared among horses often fall close to putative antigenic sites.

  9. [Development of new vaccines].

    PubMed

    González-Romo, Fernando; Picazo, Juan J

    2015-10-01

    Recent and important advances in the fields of immunology, genomics, functional genomics, immunogenetics, immunogenomics, bioinformatics, microbiology, genetic engineering, systems biology, synthetic biochemistry, proteomics, metabolomics and nanotechnology, among others, have led to new approaches in the development of vaccines. The better identification of ideal epitopes, the strengthening of the immune response due to new adjuvants, and the search of new routes of vaccine administration, are good examples of advances that are already a reality and that will favour the development of more vaccines, their use in indicated population groups, or its production at a lower cost. There are currently more than 130 vaccines are under development against the more wished (malaria or HIV), difficult to get (CMV or RSV), severe re-emerging (Dengue or Ebola), increasing importance (Chagas disease or Leishmania), and nosocomial emerging (Clostridium difficile or Staphylococcus aureus) infectious diseases. Copyright © 2015. Published by Elsevier España, S.L.U.

  10. A Recombinant Measles Vaccine with Enhanced Resistance to Passive Immunity.

    PubMed

    Julik, Emily; Reyes-Del Valle, Jorge

    2017-09-21

    Current measles vaccines suffer from poor effectiveness in young infants due primarily to the inhibitory effect of residual maternal immunity on vaccine responses. The development of a measles vaccine that resists such passive immunity would strongly contribute to the stalled effort toward measles eradication. In this concise communication, we show that a measles virus (MV) with enhanced hemagglutinin (H) expression and incorporation, termed MVvac2-H2, retained its enhanced immunogenicity, previously established in older mice, when administered to very young, genetically modified, MV-susceptible mice in the presence of passive anti-measles immunity. This immunity level mimics the sub-neutralizing immunity prevalent in infants too young to be vaccinated. Additionally, toward a more physiological small animal model of maternal anti-measles immunity interference, we document vertical transfer of passive anti-MV immunity in genetically-modified, MV susceptible mice and show in this physiological model a better MVvac2-H2 immunogenic profile than that of the parental vaccine strain. In sum, these data support the notion that enhancing MV hemagglutinin incorporation can circumvent in vivo neutralization. This strategy merits additional exploration as an alternative pediatric measles vaccine.

  11. A Recombinant Measles Vaccine with Enhanced Resistance to Passive Immunity

    PubMed Central

    Julik, Emily; Reyes-del Valle, Jorge

    2017-01-01

    Current measles vaccines suffer from poor effectiveness in young infants due primarily to the inhibitory effect of residual maternal immunity on vaccine responses. The development of a measles vaccine that resists such passive immunity would strongly contribute to the stalled effort toward measles eradication. In this concise communication, we show that a measles virus (MV) with enhanced hemagglutinin (H) expression and incorporation, termed MVvac2-H2, retained its enhanced immunogenicity, previously established in older mice, when administered to very young, genetically modified, MV-susceptible mice in the presence of passive anti-measles immunity. This immunity level mimics the sub-neutralizing immunity prevalent in infants too young to be vaccinated. Additionally, toward a more physiological small animal model of maternal anti-measles immunity interference, we document vertical transfer of passive anti-MV immunity in genetically-modified, MV susceptible mice and show in this physiological model a better MVvac2-H2 immunogenic profile than that of the parental vaccine strain. In sum, these data support the notion that enhancing MV hemagglutinin incorporation can circumvent in vivo neutralization. This strategy merits additional exploration as an alternative pediatric measles vaccine. PMID:28934110

  12. New developments and concepts related to biomarker application to vaccines

    PubMed Central

    Ahmed, S. Sohail; Black, Steve; Ulmer, Jeffrey

    2012-01-01

    Summary This minireview will provide a perspective on new developments and concepts related to biomarker applications for vaccines. In the context of preventive vaccines, biomarkers have the potential to predict adverse events in select subjects due to differences in genetic make‐up/underlying medical conditions or to predict effectiveness (good versus poor response). When expanding them to therapeutic vaccines, their utility in identification of patients most likely to respond favourably (or avoid potentially negative effects of treatment) becomes self‐explanatory. Despite the progress made so far on dissection of various pathways of biological significance in humans, there is still plenty to unravel about the mysteries related to the quantitative and qualitative aspects of the human host response. This review will provide a focused overview of new concepts and developments in the field of vaccine biomarkers including (i) vaccine‐dependent signatures predicting subject response and safety, (ii) predicting therapeutic vaccine efficacy in chronic diseases, (iii) exploring the genetic make‐up of the host that may modulate subject‐specific adverse events or affect the quality of immune responses, and (iv) the topic of volunteer stratification as a result of biomarker screening (e.g. for therapeutic vaccines but also potentially for preventive vaccines) or as a reflection of an effort to compare select groups (e.g. vaccinated subjects versus patients recovering from infection) to enable the discovery of clinically relevant biomarkers for preventive vaccines. PMID:21895991

  13. Immunogenicity of Novel Mumps Vaccine Candidates Generated by Genetic Modification

    PubMed Central

    Xu, Pei; Chen, Zhenhai; Phan, Shannon; Pickar, Adrian

    2014-01-01

    Mumps is a highly contagious human disease, characterized by lateral or bilateral nonsuppurative swelling of the parotid glands and neurological complications that can result in aseptic meningitis or encephalitis. A mumps vaccination program implemented since the 1960s reduced mumps incidence by more than 99% and kept the mumps case numbers as low as hundreds of cases per year in the United States before 2006. However, a large mumps outbreak occurred in vaccinated populations in 2006 and again in 2009 in the United States, raising concerns about the efficacy of the vaccination program. Previously, we have shown that clinical isolate-based recombinant mumps viruses lacking expression of either the V protein (rMuVΔV) or the SH protein (rMuVΔSH) are attenuated in a neurovirulence test using newborn rat brains (P. Xu et al., Virology 417:126–136, 2011, http://dx.doi.org/10.1016/j.virol.2011.05.003; P. Xu et al., J. Virol. 86:1768–1776, 2012, http://dx.doi.org/10.1128/JVI.06019-11) and may be good candidates for vaccine development. In this study, we examined immunity induced by rMuVΔSH and rMuVΔV in mice. Furthermore, we generated recombinant mumps viruses lacking expression of both the V protein and the SH protein (rMuVΔSHΔV). Analysis of rMuVΔSHΔV indicated that it was stable in tissue culture cell lines. Importantly, rMuVΔSHΔV was immunogenic in mice, indicating that it is a promising candidate for mumps vaccine development. PMID:24352450

  14. Immunogenicity of novel mumps vaccine candidates generated by genetic modification.

    PubMed

    Xu, Pei; Chen, Zhenhai; Phan, Shannon; Pickar, Adrian; He, Biao

    2014-03-01

    Mumps is a highly contagious human disease, characterized by lateral or bilateral nonsuppurative swelling of the parotid glands and neurological complications that can result in aseptic meningitis or encephalitis. A mumps vaccination program implemented since the 1960s reduced mumps incidence by more than 99% and kept the mumps case numbers as low as hundreds of cases per year in the United States before 2006. However, a large mumps outbreak occurred in vaccinated populations in 2006 and again in 2009 in the United States, raising concerns about the efficacy of the vaccination program. Previously, we have shown that clinical isolate-based recombinant mumps viruses lacking expression of either the V protein (rMuVΔV) or the SH protein (rMuVΔSH) are attenuated in a neurovirulence test using newborn rat brains (P. Xu et al., Virology 417:126-136, 2011, http://dx.doi.org/10.1016/j.virol.2011.05.003; P. Xu et al., J. Virol. 86:1768-1776, 2012, http://dx.doi.org/10.1128/JVI.06019-11) and may be good candidates for vaccine development. In this study, we examined immunity induced by rMuVΔSH and rMuVΔV in mice. Furthermore, we generated recombinant mumps viruses lacking expression of both the V protein and the SH protein (rMuVΔSHΔV). Analysis of rMuVΔSHΔV indicated that it was stable in tissue culture cell lines. Importantly, rMuVΔSHΔV was immunogenic in mice, indicating that it is a promising candidate for mumps vaccine development.

  15. Accelerating Next Generation Vaccine Development for Global Disease Prevention

    PubMed Central

    Koff, Wayne C; Burton, Dennis R.; R.Johnson, Philip; Walker, Bruce D.; King, Charles R.; Nabel, Gary J.; Ahmed, Rafi; Bhan, Maharaj Kishan; Plotkin, Stanley A.

    2014-01-01

    Summary Vaccines are among the greatest successes in the history of public health. However, past strategies for vaccine development are unlikely to succeed in the future against major global diseases such as AIDS, TB, and malaria. For such diseases, the correlates of protection are poorly defined and the pathogens evade immune detection and/or exhibit extensive genetic variability. Recent advances have heralded in a new era of vaccine discovery. However, translation of these advances into vaccines remains impeded by lack of understanding of key vaccinology principles in humans. We review these advances towards vaccine discovery and suggest that for accelerating successful vaccine development, new human immunology-based clinical research initiatives be implemented with the goal of elucidating and more effectively inducing vaccine-induced protective immune responses. PMID:23723240

  16. Safety and Efficacy of a Genetic Vaccine Targeting Telomerase Plus Chemotherapy for the Therapy of Canine B-Cell Lymphoma

    PubMed Central

    Gavazza, Alessandra; Lubas, George; Fridman, Arthur; Peruzzi, Daniela; Impellizeri, Joseph A.; Luberto, Laura; Marra, Emanuele; Roscilli, Giuseppe; Ciliberto, Gennaro

    2013-01-01

    Abstract Client-owned pet dogs represent exceptional translational models for advancement of cancer research because they reflect the complex heterogeneity observed in human cancer. We have recently shown that a genetic vaccine targeting dog telomerase reverse transcriptase (dTERT) and based on adenovirus DNA electro-gene-transfer (Ad/DNA-EGT) technology can induce strong cell-mediated immune responses against this tumor antigen and increase overall survival of dogs affected by B-cell lymphosarcoma (LSA) in comparison with historical controls when combined with a cyclophosphamide, vincristine, and prednisone (COP) chemotherapy regimen. Here, we have conducted a double-arm clinical trial with an extended number of LSA patients, measured the antigen-specific immune response, and evaluated potential toxic effects of the immunotherapy along with a follow-up of patients survival for 3.5 years. The immune response was measured by enzyme-linked immunospot assay. The expression of dTERT was quantified by quantitative polymerase chain reaction. Changes in hematological parameters, local/systemic toxicity or organic dysfunction and fever were monitored over time during the treatment. dTERT-specific cell-mediated immune responses were induced in almost all treated animals. No adverse effects were observed in any dog patient that underwent treatment. The overall survival time of vaccine/COP-treated dogs was significantly increased over the COP-only cohort (>76.1 vs. 29.3 weeks, respectively, p<0.0001). There was a significant association between dTERT expression levels in LSA cells and overall survival among vaccinated patients. In conclusion, Ad/DNA-EGT-based cancer vaccine against dTERT in combination with COP chemotherapy is safe and significantly prolongs the survival of LSA canine patients. These data confirm the therapeutic efficacy of dTERT vaccine and support the evaluation of this approach for other cancer types as well as the translation of this approach to human

  17. A novel approach to generating morbillivirus vaccines: negatively marking the rinderpest vaccine.

    PubMed

    Buczkowski, Hubert; Parida, Satya; Bailey, Dalan; Barrett, Thomas; Banyard, Ashley C

    2012-03-02

    The eradication of rinderpest virus (RPV) from the globe was possible through the availability of a safe and effective live attenuated vaccine and a suitable companion diagnostic test. However, the inability to serologically 'Differentiate between naturally Infected and Vaccinated Animals' (DIVA) meant that both the time taken to complete the eradication programme and the economic burden on countries involved was significantly greater than if a vaccine and companion diagnostic test that fulfilled the DIVA concept had been available. During the RPV eradication campaign serosurveillance for RPV was primarily based on a competitive ELISA using a RPV specific (C1) monoclonal antibody (mAb) directed against the viral haemagglutinin (H) protein but this test was not able to meet DIVA requirements. To provide proof of concept for the generation of novel morbillivirus DIVA vaccines we have identified, by phage display, and mutated residues critical for C1 mAb binding and assessed the functionality of mutants in an in vitro fusion assay. Finally we have incorporated mutated epitopes into a full length clone and rescued recombinant RPV using reverse genetics techniques. Here we describe a novel mechanism of marking morbillivirus vaccines, using RPV as a proof of concept, and discuss the applicability of this method to the development of marked vaccines for peste des petits ruminants virus (PPRV). Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  18. [Current events in vaccination].

    PubMed

    Aubert, M; Aumaître, H; Beytout, J; Bloch, K; Bouhour, D; Callamand, P; Chave, C; Cheymol, J; Combadière, B; Dahlab, A; Denis, F; De Pontual, L; Dodet, B; Dommergues, M-A; Dufour, V; Gagneur, A; Gaillat, J; Gaudelus, J; Gavazzi, G; Gillet, Y; Gras-le-Guen, C; Haas, H; Hanslik, T; Hau-Rainsard, I; Larnaudie, S; Launay, O; Lorrot, M; Loulergue, P; Malvy, D; Marchand, S; Picherot, G; Pinquier, D; Pulcini, C; Rabaud, C; Regnier, F; Reinert, P; Sana, C; Savagner, C; Soubeyrand, B; Stephan, J-L; Strady, C

    2011-11-01

    The annual meeting of the Infectious Disease Society of America (IDSA) ; which brought together nearly 5000 participants from over 80 countries in Vancouver, Canada, October 21 to 24, 2010 ; provided a review of the influenza (H1N1) 2009 pandemic, evaluated vaccination programmes and presented new vaccines under development. With 12,500 deaths in the United States in 2009-2010, the influenza (H1N1) 2009 pandemic was actually less deadly than the seasonal flu. But it essentially hit the young, and the toll calculated in years of life lost is high. The monovalent vaccines, whether live attenuated or inactivated with or without adjuvants, were well tolerated in toddlers, children, adults and pregnant women. In order to protect infants against pertussis, family members are urged to get their booster shots. The introduction of the 13-valent Pneumococcal conjugated vaccine in the beginning of 2010 may solve - but for how long ? - the problem of serotype replacement, responsible for the re-increasing incidence of invasive Pneumococcal infections observed in countries that had introduced the 7-valent vaccine. The efficacy of a rotavirus vaccine has been confirmed, with a reduction in hospitalization in the United States and a reduction in gastroenteritis-related deaths in Mexico. In the United States, vaccination of pre-adolescents against human papillomavirus (HPV) has not resulted in any specific undesirable effects. Routine vaccination against chicken pox, recommended since 1995, has not had an impact on the evolution of the incidence of shingles. Vaccination against shingles, recommended in the United States for subjects 60 years and over, shows an effectiveness of 55 %, according to a cohort study (Kaiser Permanente, Southern California). Although some propose the development of personalized vaccines according to individual genetic characteristics, the priority remains with increasing vaccine coverage, not only in infants but also in adults and the elderly. Vaccine

  19. [Current events in vaccination].

    PubMed

    Aubert, M; Aumaître, H; Beytout, J; Bloch, K; Bouhour, D; Callamand, P; Chave, C; Cheymol, J; Combadière, B; Dahlab, A; Denis, F; De Pontual, L; Dodet, B; Dommergues, M A; Dufour, V; Gagneur, A; Gaillat, J; Gaudelus, J; Gavazzi, G; Gillet, Y; Gras-le-Guen, C; Haas, H; Hanslik, T; Hau-Rainsard, I; Larnaudie, S; Launay, O; Lorrot, M; Loulergue, P; Malvy, D; Marchand, S; Picherot, G; Pinquier, D; Pulcini, C; Rabaud, C; Regnier, F; Reinert, P; Sana, C; Savagner, C; Soubeyrand, B; Stephan, J L; Strady, C

    2011-05-01

    The annual meeting of the Infectious Disease Society of America (IDSA); which brought together nearly 5000 participants from over 80 countries in Vancouver, Canada, October 21 to 24, 2010; provided a review of the influenza (H1N1) 2009 pandemic, evaluated vaccination programmes and presented new vaccines under development. With 12,500 deaths in the United States in 2009-2010, the influenza (H1N1) 2009 pandemic was actually less deadly than the seasonal flu. But it essentially hit the young, and the toll calculated in years of life lost is high. The monovalent vaccines, whether live attenuated or inactivated with or without adjuvants, were well tolerated in toddlers, children, adults and pregnant women. In order to protect infants against pertussis, family members are urged to get their booster shots. The introduction of the 13-valent Pneumococcal conjugated vaccine in the beginning of 2010 may solve--but for how long?--the problem of serotype replacement, responsible for the re-increasing incidence of invasive Pneumococcal infections observed in countries that had introduced the 7-valent vaccine. The efficacy of a rotavirus vaccine has been confirmed, with a reduction in hospitalization in the United States and a reduction in gastroenteritis-related deaths in Mexico. In the United States, vaccination of pre-adolescents against human papillomavirus (HPV) has not resulted in any specific undesirable effects. Routine vaccination against chicken pox, recommended since 1995, has not had an impact on the evolution of the incidence of shingles. Vaccination against shingles, recommended in the United States for subjects 60 years and over, shows an effectiveness of 55%, according to a cohort study (Kaiser Permanente, Southern California). Although some propose the development of personalized vaccines according to individual genetic characteristics, the priority remains with increasing vaccine coverage, not only in infants but also in adults and the elderly. Vaccine

  20. [Approaches and problems in vaccine development against leishmaniasis].

    PubMed

    Allahverdiyev, Adil; Bağirova, Melahat; Cakir Koç, Rabia; Oztel, Olga Nehir; Elçıçek, Serhat; Ateş, Sezen Canım; Karaca, Tuğçe Deniz

    2010-01-01

    Leishmaniasis is a major public health problem of the world and Turkey. Recently there has been increasing interest in vaccine studies among strategies for control of leishmaniasis. Recently the increase of interest in vaccine studies among leishmaniasis control strategies makes the subject more up to date. So the aim of this review is to present information about recent vaccine studies, problems and new strategies for vaccine development studies. There are 3 generations of vaccine against leishmaniasis. First-generation vaccines are killed or live attenuated parasites; second-generation vaccines are recombinant or native antigens and live genetically modified parasites (knock out and suicidal cassettes), third generation vaccines are DNA vaccines. Also vector salivary proteins, dendritic cells and non-pathogenic L. tarentolae have been used as vaccine candidates. However there is still no effective vaccine against leishmaniasis. Since polymer conjugates considerably increase immunogenicity, polymer based vaccine studies have gained importance in recent years. However, there has not been such a study for an antileishmanial vaccine yet. LPG, surface antigen of Leishmania promastigotes, and polymer conjugates may be promising in antileishmanial vaccine studies so we are carrying out a TUBITAK Project on this subject which has been given the number, 1085170SBAG-4007.

  1. Use of adenoviral vectors as veterinary vaccines.

    PubMed

    Ferreira, T B; Alves, P M; Aunins, J G; Carrondo, M J T

    2005-10-01

    Vaccines are the most effective and inexpensive prophylactic tool in veterinary medicine. Ideally, vaccines should induce a lifelong protective immunity against the target pathogen while not causing clinical or pathological signs of diseases in the vaccinated animals. However, such ideal vaccines are rare in the veterinary field. Many vaccines are either of limited effectiveness or have harmful side effects. In addition, there are still severe diseases with no effective vaccines. A very important criterion for an ideal vaccine in veterinary medicine is low cost; this is especially important in developing countries and even more so for poultry vaccination, where vaccines must sell for a few cents a dose. Traditional approaches include inactivated vaccines, attenuated live vaccines and subunit vaccines. Recently, genetic engineering has been applied to design new, improved vaccines. Adenovirus vectors are highly efficient for gene transfer in a broad spectrum of cell types and species. Moreover, adenoviruses often induce humoral, mucosal and cellular immune responses to antigens encoded by the inserted foreign genes. Thus, adenoviruses have become a vector of choice for delivery and expression of foreign proteins for vaccination. Consequently, the market requirements for adenovirus vaccines are increasing, creating a need for production methodologies of concentrated vectors with warranted purity and efficacy. This review summarizes recent developments and approaches of adenovirus production and purification as the application of these vectors, including successes and failures in clinical applications to date.

  2. Improving the Immunogenicity of the Mycobacterium bovis BCG Vaccine by Non-Genetic Bacterial Surface Decoration Using the Avidin-Biotin System.

    PubMed

    Liao, Ting-Yu Angela; Lau, Alice; Joseph, Sunil; Hytönen, Vesa; Hmama, Zakaria

    2015-01-01

    Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

  3. Recirculating cardiac delivery of AAV2/1SERCA2a improves myocardial function in an experimental model of heart failure in large animals.

    PubMed

    Byrne, M J; Power, J M; Preovolos, A; Mariani, J A; Hajjar, R J; Kaye, D M

    2008-12-01

    Abnormal excitation-contraction coupling is a key pathophysiologic component of heart failure (HF), and at a molecular level reduced expression of the sarcoplasmic reticulum (SR) Ca(2+) ATPase (SERCA2a) is a major contributor. Previous studies in small animals have suggested that restoration of SERCA function is beneficial in HF. Despite this promise, the means by which this information might be translated into potential clinical application remains uncertain. Using a recently established cardiac-directed recirculating method of gene delivery, we administered adeno-associated virus 2 (AAV2)/1SERCA2a to sheep with pacing-induced HF. We explored the effects of differing doses of AAV2/1SERCA2a (low 1 x 10(10) d.r.p.; medium 1 x 10(12) d.r.p. and high 1 x 10(13) d.r.p.) in conjunction with an intra-coronary delivery group (2.5 x 10(13) d.r.p.). At the end of the study, haemodynamic, echocardiographic, histopathologic and molecular biologic assessments were performed. Cardiac recirculation delivery of AAV2/1SERCA2a elicited a dose-dependent improvement in cardiac performance determined by left ventricular pressure analysis, (+d P/d t(max); low dose -220+/-70, P>0.05; medium dose 125+/-53, P<0.05; high dose 287+/-104, P<0.05) and echocardiographically (fractional shortening: low dose -3+/-2, P>0.05; medium dose 1+/-2, P>0.05; high dose 6.5+/-3.9, P<0.05). In addition to favourable haemodynamic effects, brain natriuretic peptide expression was reduced consistent with reversal of the HF molecular phenotype. In contrast, direct intra-coronary infusion did not elicit any effect on ventricular function. As such, AAV2/1SERCA2a elicits favourable functional and molecular actions when delivered in a mechanically targeted manner in an experimental model of HF. These observations lay a platform for potential clinical translation.

  4. Disease correction by AAV-mediated gene therapy in a new mouse model of mucopolysaccharidosis type IIID.

    PubMed

    Roca, Carles; Motas, Sandra; Marcó, Sara; Ribera, Albert; Sánchez, Víctor; Sánchez, Xavier; Bertolin, Joan; León, Xavier; Pérez, Jennifer; Garcia, Miguel; Villacampa, Pilar; Ruberte, Jesús; Pujol, Anna; Haurigot, Virginia; Bosch, Fatima

    2017-04-15

    Gene therapy is a promising therapeutic alternative for Lysosomal Storage Disorders (LSD), as it is not necessary to correct the genetic defect in all cells of an organ to achieve therapeutically significant levels of enzyme in body fluids, from which non-transduced cells can uptake the protein correcting their enzymatic deficiency. Animal models are instrumental in the development of new treatments for LSD. Here we report the generation of the first mouse model of the LSD Muccopolysaccharidosis Type IIID (MPSIIID), also known as Sanfilippo syndrome type D. This autosomic recessive, heparan sulphate storage disease is caused by deficiency in N-acetylglucosamine 6-sulfatase (GNS). Mice deficient in GNS showed lysosomal storage pathology and loss of lysosomal homeostasis in the CNS and peripheral tissues, chronic widespread neuroinflammation, reduced locomotor and exploratory activity and shortened lifespan, a phenotype that closely resembled human MPSIIID. Moreover, treatment of the GNS-deficient animals with GNS-encoding adeno-associated viral (AAV) vectors of serotype 9 delivered to the cerebrospinal fluid completely corrected pathological storage, improved lysosomal functionality in the CNS and somatic tissues, resolved neuroinflammation, restored normal behaviour and extended lifespan of treated mice. Hence, this work represents the first step towards the development of a treatment for MPSIIID. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  5. Genome-Wide Associations of CD46 and IFI44L Genetic Variants with Neutralizing Antibody Response to Measles Vaccine

    PubMed Central

    Haralambieva, Iana H.; Ovsyannikova, Inna G.; Kennedy, Richard B.; Larrabee, Beth R.; Zimmermann, Michael T.; Grill, Diane E.; Schaid, Daniel J.; Poland, Gregory A.

    2017-01-01

    Background Population-based studies have revealed 2 to 10% measles vaccine failure rate even after two vaccine doses. While the mechanisms behind this remain unknown, we hypothesized that host genetic factors are likely to be involved. Methods We performed a genome-wide association study of measles specific neutralizing antibody and IFNγ ELISPOT response in a combined sample of 2,872 subjects. Results We identified two distinct chromosome 1 regions (previously associated with MMR-related febrile seizures), associated with vaccine-induced measles neutralizing antibody titers. The 1q32 region contained 20 significant SNPs in/around the measles virus receptor-encoding CD46 gene, including the intronic rs2724384 (p-value = 2.64x10−09) and rs2724374 (p-value = 3.16x10−09) SNPs. The 1q31.1 region contained nine significant SNPs in/around IFI44L, including the intronic rs1333973 (p-value = 1.41x10−10) and the missense rs273259 (His73Arg, p-value = 2.87x10−10) SNPs. Analysis of differential exon usage with mRNA-Seq data and RT-PCR suggests the involvement of rs2724374 minor G allele in the CD46 STP region exon B skipping, resulting in shorter CD46 isoforms. Conclusions Our study reveals common CD46 and IFI44L SNPs associated with measles-specific humoral immunity, and highlights the importance of alternative splicing/virus cellular receptor isoform usage as a mechanism explaining inter-individual variation in immune response after live measles vaccine. PMID:28289848

  6. Bovine adenovirus-3 as a vaccine delivery vehicle.

    PubMed

    Ayalew, Lisanework E; Kumar, Pankaj; Gaba, Amit; Makadiya, Niraj; Tikoo, Suresh K

    2015-01-15

    The use of vaccines is an effective and relatively inexpensive means of controlling infectious diseases, which cause heavy economic losses to the livestock industry through animal loss, decreased productivity, treatment expenses and decreased carcass quality. However, some vaccines produced by conventional means are imperfect in many respects including virulence, safety and efficacy. Moreover, there are no vaccines for some animal diseases. Although genetic engineering has provided new ways of producing effective vaccines, the cost of production for veterinary use is a critical criterion for selecting the method of production and delivery of vaccines. The cost effective production and intrinsic ability to enter cells has made adenovirus vectors a highly efficient tool for delivery of vaccine antigens. Moreover, adenoviruses induce both humoral and cellular immune responses to expressed vaccine antigens. Since nonhuman adenoviruses are species specific, the development of animal specific adenoviruses as vaccine delivery vectors is being evaluated. This review summarizes the work related to the development of bovine adenovirus-3 as a vaccine delivery vehicle in animals, particularly cattle. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. Vaccination of small Asian mongoose (Herpestes javanicus) against rabies.

    PubMed

    Blanton, Jesse D; Meadows, Anastasia; Murphy, Staci M; Manangan, Jamie; Hanlon, Cathleen A; Faber, Marie-Luise; Dietzschold, Bernhard; Rupprecht, Charles E

    2006-07-01

    Oral vaccination of free-ranging wildlife is a promising technique in rabies control. The small Asian mongoose (Herpestes javanicus) is an important reservoir of rabies on several Caribbean islands, but no vaccines have been evaluated for this species. Captive mongooses were used to test the safety and efficacy of the commercially licensed vaccinia-rabies glycoprotein (V-RG) recombinant vaccine and a newly developed genetically engineered oral rabies virus vaccine (SPBNGA-S). In one study using V-RG, no vaccinated animals developed detectable rabies virus-neutralizing antibodies, and all but one died after experimental challenge with rabies virus. In contrast, all animals given SPBNGA-S demonstrated seroconversion within 7 to 14 days after vaccination and survived rabies virus challenge. On the basis of these preliminary results indicating the greater efficacy of SPBNGA-S vs. V-RG vaccine, additional investigations will be necessary to determine the optimal dose and duration of vaccination, as well as incorporation of the SPBNGA-S vaccine into edible bait.

  8. BVDV vaccination in North America: risks versus benefits.

    PubMed

    Griebel, Philip J

    2015-06-01

    The control and prevention of bovine viral diarrhea virus (BVDV) infections has provided substantial challenges. Viral genetic variation, persistent infections, and viral tropism for immune cells have complicated disease control strategies. Vaccination has, however, provided an effective tool to prevent acute systemic infections and increase reproductive efficiency through fetal protection. There has been substantial controversy about the safety and efficacy of BVDV vaccines, especially when comparing killed versus modified-live viral (MLV) vaccines. Furthermore, numerous vaccination protocols have been proposed to protect the fetus and ensure maternal antibody transfer to the calf. These issues have been further complicated by reports of immune suppression during natural infections and following vaccination. While killed BVDV vaccines provide the greatest safety, their limited immunogenicity makes multiple vaccinations necessary. In contrast, MLV BVDV vaccines induce a broader range of immune responses with a longer duration of immunity, but require strategic vaccination to minimize potential risks. Vaccination strategies for breeding females and young calves, in the face of maternal antibody, are discussed. With intranasal vaccination of young calves it is possible to avoid maternal antibody interference and induce immune memory that persists for 6-8 months. Thus, with an integrated vaccination protocol for both breeding cows and calves it is possible to maximize disease protection while minimizing vaccine risks.

  9. In silico identification of genetically attenuated vaccine candidate genes for Plasmodium liver stage.

    PubMed

    Kumar, Hirdesh; Frischknecht, Friedrich; Mair, Gunnar R; Gomes, James

    2015-12-01

    Genetically attenuated parasites (GAPs) that lack genes essential for the liver stage of the malaria parasite, and therefore cause developmental arrest, have been developed as live vaccines in rodent malaria models and recently been tested in humans. The genes targeted for deletion were often identified by trial and error. Here we present a systematic gene - protein and transcript - expression analyses of several Plasmodium species with the aim to identify candidate genes for the generation of novel GAPs. With a lack of liver stage expression data for human malaria parasites, we used data available for liver stage development of Plasmodium yoelii, a rodent malaria model, to identify proteins expressed in the liver stage but absent from blood stage parasites. An orthology-based search was then employed to identify orthologous proteins in the human malaria parasite Plasmodium falciparum resulting in a total of 310 genes expressed in the liver stage but lacking evidence of protein expression in blood stage parasites. Among these 310 possible GAP candidates, we further studied Plasmodium liver stage proteins by phyletic distribution and functional domain analyses and shortlisted twenty GAP-candidates; these are: fabB/F, fabI, arp, 3 genes encoding subunits of the PDH complex, dnaJ, urm1, rS5, ancp, mcp, arh, gk, lisp2, valS, palm, and four conserved Plasmodium proteins of unknown function. Parasites lacking one or several of these genes might yield new attenuated malaria parasites for experimental vaccination studies. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Vaccines to prevent severe acute respiratory syndrome coronavirus-induced disease

    PubMed Central

    Enjuanes, Luis; DeDiego, Marta L.; Álvarez, Enrique; Deming, Damon; Sheahan, Tim; Baric, Ralph

    2009-01-01

    An important effort has been performed after the emergence of severe acute respiratory syndrome (SARS) epidemic in 2003 to diagnose and prevent virus spreading. Several types of vaccines have been developed including inactivated viruses, subunit vaccines, virus-like particles (VLPs), DNA vaccines, heterologous expression systems, and vaccines derived from SARS-CoV genome by reverse genetics. This review describes several aspects essential to develop SARS-CoV vaccines, such as the correlates of protection, virus serotypes, vaccination side effects, and bio-safeguards that can be engineered into recombinant vaccine approaches based on the SARS-CoV genome. The production of effective and safe vaccines to prevent SARS has led to the development of promising vaccine candidates, in contrast to the design of vaccines for other coronaviruses, that in general has been less successful. After preclinical trials in animal models, efficacy and safety evaluation of the most promising vaccine candidates described has to be performed in humans. PMID:17416434

  11. Seropositivity to non-vaccine incorporated genotypes induced by the bivalent and quadrivalent HPV vaccines: A systematic review and meta-analysis.

    PubMed

    Bissett, Sara L; Godi, Anna; Jit, Mark; Beddows, Simon

    2017-07-13

    Human papillomavirus vaccines have demonstrated remarkable efficacy against persistent infection and disease associated with vaccine-incorporated genotypes and a degree of efficacy against some genetically related, non-vaccine-incorporated genotypes. The vaccines differ in the extent of cross-protection against these non-vaccine genotypes. Data supporting the role for neutralizing antibodies as a correlate or surrogate of cross-protection are lacking, as is a robust assessment of the seroconversion rates against these non-vaccine genotypes. We performed a systematic review and meta-analysis of available data on vaccine-induced neutralizing antibody seropositivity to non-vaccine incorporated HPV genotypes. Of 304 articles screened, 9 were included in the analysis representing ca. 700 individuals. The pooled estimate for seropositivity against HPV31 for the bivalent vaccine (86%; 95%CI 78-91%) was higher than that for the quadrivalent vaccine (61%; 39-79%; p=0.011). The pooled estimate for seropositivity against HPV45 for the bivalent vaccine (50%; 37-64%) was also higher than that for the quadrivalent vaccine (16%; 6-36%; p=0.007). Seropositivity against HPV33, HPV52 and HPV58 were similar between the vaccines. Mean seropositivity rates across non-vaccine genotypes were positively associated with the corresponding vaccine efficacy data reported from vaccine trials. These data improve our understanding of vaccine-induced functional antibody specificity against non-vaccine incorporated genotypes and may help to parameterize vaccine-impact models and improve patient management in a post-vaccine setting. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

  12. Rift Valley Fever vaccines: An overview of the safety and efficacy of the live-attenuated MP-12 vaccine candidate

    PubMed Central

    Ikegami, Tetsuro

    2017-01-01

    Introduction Rift Valley fever (RVF) is a mosquito-borne zoonotic viral disease endemic to Africa and the Arabian Peninsula. High rates of abortion among infected ruminants and hemorrhagic fever in infected humans are major public health concerns. Commercially available veterinary RVF vaccines are important for preventing the spread of the Rift Valley fever virus (RVFV) in endemic countries; however, RVFV outbreaks continue to occur frequently in endemic countries in the 21st century. In the U.S., the live-attenuated MP-12 vaccine has been developed for both animal and human vaccination. This vaccine strain is well attenuated, and a single dose induces neutralizing antibodies in both ruminants and humans. Areas covered This review describes scientific evidences of MP-12 vaccine efficacy and safety, as well as MP-12 variants recently developed by reverse genetics, in comparison with other RVF vaccines. Expert commentary The containment of active RVF outbreaks and long-term protection from RVF exposure to infected mosquitoes are important goals for RVF vaccination. MP-12 vaccine will allow immediate vaccination of susceptible animals in case of an unexpected RVF outbreak in the U.S., whereas MP-12 vaccine may be also useful for the RVF control in endemic regions. PMID:28425834

  13. Rift Valley fever vaccines: an overview of the safety and efficacy of the live-attenuated MP-12 vaccine candidate.

    PubMed

    Ikegami, Tetsuro

    2017-06-01

    Rift Valley fever (RVF) is a mosquito-borne zoonotic viral disease endemic to Africa and the Arabian Peninsula. High rates of abortion among infected ruminants and hemorrhagic fever in infected humans are major public health concerns. Commercially available veterinary RVF vaccines are important for preventing the spread of the Rift Valley fever virus (RVFV) in endemic countries; however, RVFV outbreaks continue to occur frequently in endemic countries in the 21st century. In the U.S., the live-attenuated MP-12 vaccine has been developed for both animal and human vaccination. This vaccine strain is well attenuated, and a single dose induces neutralizing antibodies in both ruminants and humans. Areas covered: This review describes scientific evidences of MP-12 vaccine efficacy and safety, as well as MP-12 variants recently developed by reverse genetics, in comparison with other RVF vaccines. Expert commentary: The containment of active RVF outbreaks and long-term protection from RVF exposure to infected mosquitoes are important goals for RVF vaccination. MP-12 vaccine will allow immediate vaccination of susceptible animals in case of an unexpected RVF outbreak in the U.S., whereas MP-12 vaccine may be also useful for the RVF control in endemic regions.

  14. Pneumococcal vaccine failure: can it be a primary immunodeficiency?

    PubMed

    Moinho, Rita; Brett, Ana; Ferreira, Gisela; Lemos, Sónia

    2014-06-12

    Vaccine failure is a rare condition and the need to investigate a primary immunodeficiency is controversial. We present the case of a 4-year-old boy, with complete antipneumococcal vaccination, who had necrotising pneumonia with pleural effusion and severe pancytopaenia with need for transfusion. A vaccine-serotype Streptococcus pneumoniae was isolated in the blood culture. On follow-up, detailed medical history, laboratory and genetic investigation led to the diagnosis of X linked dyskeratosis congenita. Dyskeratosis congenita is an inherited disorder that causes shortening or dysfunction of telomeres, affecting mainly rapidly dividing cells (particularly in the skin and haematopoietic system). It leads to bone marrow failure, combined immunodeficiency and predisposition to cancer. The confirmation of this diagnosis allows genetic counselling and medical monitoring of these patients, in order to detect early complications such as bone marrow aplasia or malignancies. 2014 BMJ Publishing Group Ltd.

  15. Exosomes derived from tumor cells genetically modified to express Mycobacterium tuberculosis antigen: a novel vaccine for cancer therapy.

    PubMed

    Koyama, Yoshiyuki; Ito, Tomoko; Hasegawa, Aya; Eriguchi, Masazumi; Inaba, Toshio; Ushigusa, Takahiro; Sugiura, Kikuya

    2016-11-01

    To examine the potential of exosomes derived from the tumor cells, which had been genetically modified to express a Mycobacterium tuberculosis antigen, as a cancer vaccine aimed at overcoming the weak immunogenicity of tumor antigens. We transfected B16 melanoma cells with a plasmid encoding the M. tuberculosis antigen, early secretory antigenic target-6 (ESAT-6). The secreted exosomes bearing both tumor-associated antigens and the pathogenic antigen (or their epitopes) were collected. When the exosomes were injected into foot pads of mice, they significantly (p < 0.05) evoked cellular immunity against both ESAT-6, and B16 tumor cells. Intra-tumoral injection of the exosomes significantly suppressed (p < 0.001) tumor growth in syngeneic B16 tumor-bearing mice, while the exosomes derived from the non-transfected B16 cells showed no effect on tumor growth, although both exosomes should have similar tumor antigens. Exosomes bearing both tumor antigens and the M. tuberculosis antigen (or their epitopes) have a high potential as a candidate for cancer vaccine to overcome the immune escape by tumor cells.

  16. Utilization of feline ELISPOT for mapping vaccine epitopes.

    PubMed

    Abbott, Jeffrey R; Pu, Ruiyu; Coleman, James K; Yamamoto, Janet K

    2012-01-01

    A commercial feline immunodeficiency virus (FIV) vaccine consisting of inactivated dual-subtype viruses was released in the USA in 2002 and released subsequently over the next 6 years in Canada, Australia, New Zealand, and Japan. Based on the genetic, morphologic, and biochemical similarities between FIV and human immunodeficiency virus-1 (HIV-1), FIV infection of domestic cats is being used as a small animal model of HIV/AIDS vaccine. Studies on prototype and commercial FIV vaccines provide new insights to the types of immunity and the vaccine epitopes required for an effective human HIV-1 vaccine. ELISPOT assays to detect cytokines, chemokines, and cytolytic mediators are widely used to measure the magnitude and the types of cellular immunity produced by vaccination. Moreover, such approach has identified regions on both HIV-1 and FIV proteins that induce robust antiviral cellular immunity in infected hosts. Using the same strategy, cats immunized with prototype and commercial FIV vaccines are being analyzed by feline interferon-γ and IL-2 ELISPOT systems to identify the vaccine epitope repertoire for prophylaxis.

  17. Biosafety evaluation of recombinant live oral bacterial vaccines in the context of European regulation.

    PubMed

    Favre, Didier; Viret, Jean-François

    2006-05-01

    Live bacterial vaccines represent a highly valid preventive strategy in the fight against infectious disease. However, the road from research to market is peppered with hurdles, one of which is the requirement for high biosafety characteristics, which the candidate vaccine has to display. In Europe, the European Agency for the evaluation of medicinal products (EMEA) is the relevant authority regulating the licensure of genetically engineered vaccines. For this purpose, the agency may rely on several directives and guidelines defined in the past 15 years. As for live vaccines containing genetically modified organisms (GMOs) susceptible to be released into the environment, Directive 2001/18/EC determines the framework and principles of an environmental risk assessment (ERA) process, the results of which constitute an important section of the vaccine registration package submitted to registration authorities. In this article, we address the implications of current European regulations for the approval of live oral bacterial vaccines with emphasis on the assessment of potential risks associated with environmental release. Biosafety aspects of already registered and some promising live bacterial vaccine strains will be briefly discussed.

  18. Current Status of Veterinary Vaccines

    PubMed Central

    Meeusen, Els N. T.; Walker, John; Peters, Andrew; Pastoret, Paul-Pierre; Jungersen, Gregers

    2007-01-01

    The major goals of veterinary vaccines are to improve the health and welfare of companion animals, increase production of livestock in a cost-effective manner, and prevent animal-to-human transmission from both domestic animals and wildlife. These diverse aims have led to different approaches to the development of veterinary vaccines from crude but effective whole-pathogen preparations to molecularly defined subunit vaccines, genetically engineered organisms or chimeras, vectored antigen formulations, and naked DNA injections. The final successful outcome of vaccine research and development is the generation of a product that will be available in the marketplace or that will be used in the field to achieve desired outcomes. As detailed in this review, successful veterinary vaccines have been produced against viral, bacterial, protozoal, and multicellular pathogens, which in many ways have led the field in the application and adaptation of novel technologies. These veterinary vaccines have had, and continue to have, a major impact not only on animal health and production but also on human health through increasing safe food supplies and preventing animal-to-human transmission of infectious diseases. The continued interaction between animals and human researchers and health professionals will be of major importance for adapting new technologies, providing animal models of disease, and confronting new and emerging infectious diseases. PMID:17630337

  19. Reverse genetics of Mononegavirales: How they work, new vaccines, and new cancer therapeutics

    PubMed Central

    Pfaller, Christian K.; Cattaneo, Roberto; Schnell, Matthias J.

    2015-01-01

    The order Mononegavirales includes five families: Bornaviridae, Filoviridae, Nyamaviridae, Paramyxoviridae, and Rhabdoviridae. The genome of these viruses is one molecule of negative-sense single strand RNA coding for five to ten genes in a conserved order. The RNA is not infectious until packaged by the nucleocapsid protein and transcribed by the polymerase and co-factors. Reverse genetics approaches have answered fundamental questions about the biology of Mononegavirales. The lack of icosahedral symmetry and modular organization in the genome of these viruses has facilitated engineering of viruses expressing fluorescent proteins, and these fluorescent proteins have provided important insights about the molecular and cellular basis of tissue tropism and pathogenesis. Studies have assessed the relevance for virulence of different receptors and the interactions with cellular proteins governing the innate immune responses. Research has also analyzed the mechanisms of attenuation. Based on these findings, ongoing clinical trials are exploring new live attenuated vaccines and the use of viruses re-engineered as cancer therapeutics. PMID:25702088

  20. Vaccine platforms to control Lassa fever.

    PubMed

    Lukashevich, Igor S; Pushko, Peter

    2016-09-01

    Lassa virus (LASV), the most prominent human pathogen of the Arenaviridae, is transmitted to humans from infected rodents and can cause Lassa Fever (LF). The sizeable disease burden in West Africa, numerous imported LF cases worldwide, and the possibility that LASV can be used as an agent of biological warfare make a strong case for vaccine development. There are no licensed LASV vaccines and the antiviral treatment is limited to an off-label use of ribavirin that is only partially effective. LASV vaccine development is hampered by high cost of biocontainment requirement, the absence of appropriate small animal models, genetic diversity of LASV species, and by high HIV-1 prevalence in LASV endemic areas. Over the past 15 years several vaccine platforms have been developed. Natural history of LASV and pathogenesis of the disease provide strong justification for replication-competent (RC) vaccine as one of the most feasible approaches to control LF. Development of LASV vaccine candidates based on reassortant, recombinant, and alphavirus replicon technologies is covered in this review. Expert commentary: Two lead RC vaccine candidates, reassortant ML29 and recombinant VSV/LASV, have been successfully tested in non-human primates and have been recommended by international vaccine experts for rapid clinical development. Both platforms have powerful molecular tools to further secure safety, improve immunogenicity, and cross-protection. These platforms are well positioned to design multivalent vaccines to protect against all LASV strains citculatrd in West Africa. The regulatory pathway of Candid #1, the first live-attenuated arenaviral vaccine against Argentine hemorrhagic, will be a reasonable guideline for LASV vaccine efficacy trials.

  1. Application of Mutated miR-206 Target Sites Enables Skeletal Muscle-specific Silencing of Transgene Expression of Cardiotropic AAV9 Vectors

    PubMed Central

    Geisler, Anja; Schön, Christian; Größl, Tobias; Pinkert, Sandra; Stein, Elisabeth A; Kurreck, Jens; Vetter, Roland; Fechner, Henry

    2013-01-01

    Insertion of completely complementary microRNA (miR) target sites (miRTS) into a transgene has been shown to be a valuable approach to specifically repress transgene expression in non-targeted tissues. miR-122TS have been successfully used to silence transgene expression in the liver following systemic application of cardiotropic adeno-associated virus (AAV) 9 vectors. For miR-206–mediated skeletal muscle-specific silencing of miR-206TS–bearing AAV9 vectors, however, we found this approach failed due to the expression of another member (miR-1) of the same miR family in heart tissue, the intended target. We introduced single-nucleotide substitutions into the miR-206TS and searched for those which prevented miR-1–mediated cardiac repression. Several mutated miR-206TS (m206TS), in particular m206TS-3G, were resistant to miR-1, but remained fully sensitive to miR-206. All these variants had mismatches in the seed region of the miR/m206TS duplex in common. Furthermore, we found that some m206TS, containing mismatches within the seed region or within the 3′ portion of the miR-206, even enhanced the miR-206– mediated transgene repression. In vivo expression of m206TS-3G– and miR-122TS–containing transgene of systemically applied AAV9 vectors was strongly repressed in both skeletal muscle and the liver but remained high in the heart. Thus, site-directed mutagenesis of miRTS provides a new strategy to differentiate transgene de-targeting of related miRs. PMID:23439498

  2. A Novel Method Combining Vitreous Aspiration and Intravitreal AAV2/8 Injection Results in Retina-Wide Transduction in Adult Mice.

    PubMed

    Da Costa, Romain; Röger, Carsten; Segelken, Jasmin; Barben, Maya; Grimm, Christian; Neidhardt, John

    2016-10-01

    Gene therapies to treat eye disorders have been extensively studied in the past 20 years. Frequently, adeno-associated viruses were applied to the subretinal or intravitreal space of the eye to transduce retinal cells with nucleotide sequences of therapeutic potential. In this study we describe a novel intravitreal injection procedure that leads to a reproducible adeno-associated virus (AAV)2/8-mediated transduction of more than 70% of the retina. Prior to a single intravitreal injection of a enhanced green fluorescent protien (GFP)-expressing viral suspension, we performed an aspiration of vitreous tissue from wild-type C57Bl/6J mice. One and one-half microliters of AAV2/8 suspension was injected. Funduscopy, optical coherence tomography (OCT), laser scanning microscopy of retinal flat mounts, cryosections of eye cups, and ERG recordings verified the efficacy and safety of the method. The combination of vitreous aspiration and intravitreal injection resulted in an almost complete transduction of the retina in approximately 60% of the eyes and showed transduced cells in all retinal layers. Photoreceptors and RPE cells were predominantly transduced. Eyes presented with well-preserved retinal morphology. Electroretinographic recordings suggested that the new combination of techniques did not cause significant alterations of the retinal physiology. We show a novel application technique of AAV2/8 to the vitreous of mice that leads to widespread transduction of the retina. The results of this study have implications for virus-based gene therapies and basic science; for example, they might provide an approach to apply gene replacement strategies or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in vivo. It may further help to develop similar techniques for larger animal models or humans.

  3. Varicella-zoster virus vaccine, successes and difficulties.

    PubMed

    Sarkadi, Julia

    2013-12-01

    Despite intensive efforts in recent decades to develop preventive or therapeutic vaccines against diseases caused by herpes simplex virus (HSV), or varicella-zoster virus (VZV), members of the Alpha herpes virinae subfamily of human herpes viruses,a safe and efficient vaccine has been approved for commercial development only against VZV. The VZV vaccine contains a live attenuated strain, OKA. It consists of amixture of at least 13 subpopulations of viruses, all with deletions, insertions or mutations in the genome; the most common mutations are observed in the open reading frame 62 (ORF62). Experience over more than 30 years in Japan, the USA and other countries where VZV vaccination is provided has demonstrated that the vaccine is safe and the effectiveness of two doses compared to unvaccinated children is 98-99%. When administered in a higher dose to stimulate the declining cell-mediated immunity, the same vaccine has been shown to reduce the incidence and severity of herpes zoster in immunocompetent individuals older than 60 years. Vaccination of immuno-compromised subjects with this VZV vaccine is problematic and various strategies need to be explored. Differences in the pathomechanisms of infection, latency and immune evasion of VZV and HSV, together with host genetic factors, may explain the availability of the successful VZV vaccine and the failures of the past HSV vaccine candidates.

  4. Reverse genetics of Newcastle disease virus

    USDA-ARS?s Scientific Manuscript database

    Reverse genetics allows the generation of recombinant viruses or vectors used in functional studies, vaccine development, and gene therapy. This technique allows genetic manipulation and cloning of viral genomes, mutation through site-directed mutagenesis, and gene insertion or deletion, among othe...

  5. Synthetic biology devices and circuits for RNA-based 'smart vaccines': a propositional review.

    PubMed

    Andries, Oliwia; Kitada, Tasuku; Bodner, Katie; Sanders, Niek N; Weiss, Ron

    2015-02-01

    Nucleic acid vaccines have been gaining attention as an alternative to the standard attenuated pathogen or protein based vaccine. However, an unrealized advantage of using such DNA or RNA based vaccination modalities is the ability to program within these nucleic acids regulatory devices that would provide an immunologist with the power to control the production of antigens and adjuvants in a desirable manner by administering small molecule drugs as chemical triggers. Advances in synthetic biology have resulted in the creation of highly predictable and modular genetic parts and devices that can be composed into synthetic gene circuits with complex behaviors. With the recent advent of modified RNA gene delivery methods and developments in the RNA replicon platform, we foresee a future in which mammalian synthetic biologists will create genetic circuits encoded exclusively on RNA. Here, we review the current repertoire of devices used in RNA synthetic biology and propose how programmable 'smart vaccines' will revolutionize the field of RNA vaccination.

  6. Emergence of Vaccine-Derived Polioviruses during Ebola Virus Disease Outbreak, Guinea, 2014-2015.

    PubMed

    Fernandez-Garcia, Maria Dolores; Majumdar, Manasi; Kebe, Ousmane; Fall, Aichatou D; Kone, Moussa; Kande, Mouctar; Dabo, Moustapha; Sylla, Mohamed Salif; Sompare, Djenou; Howard, Wayne; Faye, Ousmane; Martin, Javier; Ndiaye, Kader

    2018-01-01

    During the 2014-2015 outbreak of Ebola virus disease in Guinea, 13 type 2 circulating vaccine-derived polioviruses (cVDPVs) were isolated from 6 polio patients and 7 healthy contacts. To clarify the genetic properties of cVDPVs and their emergence, we combined epidemiologic and virologic data for polio cases in Guinea. Deviation of public health resources to the Ebola outbreak disrupted polio vaccination programs and surveillance activities, which fueled the spread of neurovirulent VDPVs in an area of low vaccination coverage and immunity. Genetic properties of cVDPVs were consistent with their capacity to cause paralytic disease in humans and capacity for sustained person-to-person transmission. Circulation ceased when coverage of oral polio vaccine increased. A polio outbreak in the context of the Ebola virus disease outbreak highlights the need to consider risks for polio emergence and spread during complex emergencies and urges awareness of the challenges in polio surveillance, vaccination, and diagnosis.

  7. Vaccination against typhoid fever: present status.

    PubMed Central

    Ivanoff, B.; Levine, M. M.; Lambert, P. H.

    1994-01-01

    Typhoid fever remains an underestimated important health problem in many developing countries, causing more than 600,000 deaths annually in the world. Because of the reactogenicity of the parenteral, killed whole-cell vaccine, research has been oriented towards vaccination orally using live organisms and purified antigen. Live vaccine Ty21a, given by the oral route, has been extensively tested in several studies in developing countries. Its liquid formulation was the most effective, providing more than 60% protection after 7 years of follow-up. A Vi polysaccharide vaccine has been elaborated and provided more than 65% protection; after 3 years of follow-up the Vi antibody level was still at a high level. These two vaccines are therefore candidates for use in public health control programmes. Before such use, however, they need further evaluation for safety and protective efficacy when administered to the EPI-targeted age groups. The question of whether typhoid fever vaccines interfere with the response to simultaneously administered measles vaccine must also be studied. New live vaccines, given by the oral route in one dose, have been constructed through genetic engineering. The first results are promising, but they must be improved before use in a large-scale study. These strains could be used as live vector to deliver foreign antigens to the intestinal mucosa. PMID:7867143

  8. Correlative Gene Expression to Protective Seroconversion in Rift Valley Fever Vaccinates.

    PubMed

    Laughlin, Richard C; Drake, Kenneth L; Morrill, John C; Adams, L Garry

    2016-01-01

    Rift Valley fever Virus (RVFV), a negative-stranded RNA virus, is the etiological agent of the vector-borne zoonotic disease, Rift Valley fever (RVF). In both humans and livestock, protective immunity can be achieved through vaccination. Earlier and more recent vaccine trials in cattle and sheep demonstrated a strong neutralizing antibody and total IgG response induced by the RVF vaccine, authentic recombinant MP-12 (arMP-12). From previous work, protective immunity in sheep and cattle vaccinates normally occurs from 7 to 21 days after inoculation with arMP-12. While the serology and protective response induced by arMP-12 has been studied, little attention has been paid to the underlying molecular and genetic events occurring prior to the serologic immune response. To address this, we isolated RNA from whole blood of vaccinated calves over a time course of 21 days before and after vaccination with arMP-12. The time course RNAs were sequenced by RNASeq and bioinformatically analyzed. Our results revealed time-dependent activation or repression of numerous gene ontologies and pathways related to the vaccine induced immune response and its regulation. Additional bioinformatic analyses identified a correlative relationship between specific host immune response genes and protective immunity prior to the detection of protective serum neutralizing antibody responses. These results contribute an important proof of concept for identifying molecular and genetic components underlying the immune response to RVF vaccination and protection prior to serologic detection.

  9. Intravitreous injection of AAV2-sFLT01 in patients with advanced neovascular age-related macular degeneration: a phase 1, open-label trial.

    PubMed

    Heier, Jeffrey S; Kherani, Saleema; Desai, Shilpa; Dugel, Pravin; Kaushal, Shalesh; Cheng, Seng H; Delacono, Cheryl; Purvis, Annie; Richards, Susan; Le-Halpere, Annaig; Connelly, John; Wadsworth, Samuel C; Varona, Rafael; Buggage, Ronald; Scaria, Abraham; Campochiaro, Peter A

    2017-07-01

    Long-term intraocular injections of vascular endothelial growth factor (VEGF)-neutralising proteins can preserve central vision in many patients with neovascular age-related macular degeneration. We tested the safety and tolerability of a single intravitreous injection of an AAV2 vector expressing the VEGF-neutralising protein sFLT01 in patients with advanced neovascular age-related macular degeneration. This was a phase 1, open-label, dose-escalating study done at four outpatient retina clinics in the USA. Patients were assigned to each cohort in order of enrolment, with the first three patients being assigned to and completing the first cohort before filling positions in the following treatment groups. Patients aged 50 years or older with neovascular age-related macular degeneration and a baseline best-corrected visual acuity score of 20/100 or less in the study eye were enrolled in four dose-ranging cohorts (cohort 1, 2 × 10 8 vector genomes (vg); cohort 2, 2 × 10 9 vg; cohort 3, 6 × 10 9 vg; and cohort 4, 2 × 10 10 vg, n=3 per cohort) and one maximum tolerated dose cohort (cohort 5, 2 × 10 10 vg, n=7) and followed up for 52 weeks. The primary objective of the study was to assess the safety and tolerability of a single intravitreous injection of AAV2-sFLT01, through the measurement of eye-related adverse events. This trial is registered with ClinicalTrials.gov, number NCT01024998. 19 patients with advanced neovascular age-related macular degeneration were enrolled in the study between May 18, 2010, and July 14, 2014. All patients completed the 52-week trial period. Two patients in cohort 4 (2 × 10 10 vg) experienced adverse events that were possibly study-drug related: pyrexia and intraocular inflammation that resolved with a topical steroid. Five of ten patients who received 2 × 10 10 vg had aqueous humour concentrations of sFLT01 that peaked at 32·7-112·0 ng/mL (mean 73·7 ng/mL, SD 30·5) by week 26 with a slight decrease to

  10. rAAV-compatible MiniPromoters for restricted expression in the brain and eye.

    PubMed

    de Leeuw, Charles N; Korecki, Andrea J; Berry, Garrett E; Hickmott, Jack W; Lam, Siu Ling; Lengyell, Tess C; Bonaguro, Russell J; Borretta, Lisa J; Chopra, Vikramjit; Chou, Alice Y; D'Souza, Cletus A; Kaspieva, Olga; Laprise, Stéphanie; McInerny, Simone C; Portales-Casamar, Elodie; Swanson-Newman, Magdalena I; Wong, Kaelan; Yang, George S; Zhou, Michelle; Jones, Steven J M; Holt, Robert A; Asokan, Aravind; Goldowitz, Daniel; Wasserman, Wyeth W; Simpson, Elizabeth M

    2016-05-10

    Small promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters-however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo. For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were "cut down" to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP. The data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia. Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.

  11. Rotavirus Diversity and Evolution in the Post-Vaccine World

    PubMed Central

    Patton, John T.

    2013-01-01

    Rotaviruses (RVs) are a large genetically diverse population of segmented double-stranded (ds) RNA viruses that are important causes of gastroenteritis in many animal species. The human RVs are responsible for the deaths of nearly 450,000 infants and young children each year, most occurring in developing countries. Recent large-scale sequencing efforts have revealed that the genomes of human RVs typically consist of phylogenetically linked constellations of eleven dsRNA segments. The presence of such preferred constellations indicate that the human RV genes have co-evolved to produce protein sets that work optimally together to support virus replication. Two of the viral genes encode virion outer capsid proteins (VP7 and VP4) whose antigenic properties define the G/P type of the virus. From year-to-year and place-to-place, the G/P type of human RVs associated with disease can fluctuate dramatically, phenomena that can be associated with the presence and behavior of genetically distinct RV clades. The recent introduction of two live attenuated RV vaccines (RotaReq™ and Rotarix™) into the childhood vaccination programs of various countries has been highly effective in reducing the incidence of RV diarrheal disease. Whether the widespread use of these vaccines will introduce selective pressures on human RVs, triggering genetic and antigenic changes that undermine the effectiveness of vaccinations programs, is uncertain and will require continued surveillance of human RVs. PMID:22284787

  12. Predicting post-vaccination autoimmunity: who might be at risk?

    PubMed

    Soriano, Alessandra; Nesher, Gideon; Shoenfeld, Yehuda

    2015-02-01

    Vaccinations have been used as an essential tool in the fight against infectious diseases, and succeeded in improving public health. However, adverse effects, including autoimmune conditions may occur following vaccinations (autoimmune/inflammatory syndrome induced by adjuvants--ASIA syndrome). It has been postulated that autoimmunity could be triggered or enhanced by the vaccine immunogen contents, as well as by adjuvants, which are used to increase the immune reaction to the immunogen. Fortunately, vaccination-related ASIA is uncommon. Yet, by defining individuals at risk we may further limit the number of individuals developing post-vaccination ASIA. In this perspective we defined four groups of individuals who might be susceptible to develop vaccination-induced ASIA: patients with prior post-vaccination autoimmune phenomena, patients with a medical history of autoimmunity, patients with a history of allergic reactions, and individuals who are prone to develop autoimmunity (having a family history of autoimmune diseases; asymptomatic carriers of autoantibodies; carrying certain genetic profiles, etc.). Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Current status and future prospects of yellow fever vaccines.

    PubMed

    Beck, Andrew S; Barrett, Alan D T

    2015-01-01

    Yellow fever 17D vaccine is one of the oldest live-attenuated vaccines in current use that is recognized historically for its immunogenic and safe properties. These unique properties of 17D are presently exploited in rationally designed recombinant vaccines targeting not only flaviviral antigens but also other pathogens of public health concern. Several candidate vaccines based on 17D have advanced to human trials, and a chimeric recombinant Japanese encephalitis vaccine utilizing the 17D backbone has been licensed. The mechanism(s) of attenuation for 17D are poorly understood; however, recent insights from large in silico studies have indicated particular host genetic determinants contributing to the immune response to the vaccine, which presumably influences the considerable durability of protection, now in many cases considered to be lifelong. The very rare occurrence of severe adverse events for 17D is discussed, including a recent fatal case of vaccine-associated viscerotropic disease.

  14. Current Status and Future Prospects of Yellow Fever Vaccines

    PubMed Central

    Beck, Andrew S.; Barrett, Alan D.T.

    2017-01-01

    Summary Yellow fever 17D vaccine is one of the oldest live-attenuated vaccines in current use that is recognized for historically immunogenic and safe properties. These unique properties of 17D are presently exploited in rationally designed recombinant vaccines targeting not only flaviviral antigens but also other pathogens of public health concern. Several candidate vaccines based on 17D have advanced to human trials, and a chimeric recombinant Japanese encephalitis vaccine utilizing the 17D backbone has been licensed. The mechanism(s) of attenuation for 17D are poorly understood; however, recent insights from large in silico studies have indicated particular host genetic determinants contributing to the immune response to the vaccine, which presumably influences the considerable durability of protection, now in many cases considered to be life-long. The very rare occurrence of severe adverse events for 17D is discussed, including a recent fatal case of vaccine-associated viscerotropic disease. PMID:26366673

  15. Update on bacterial meningitis: epidemiology, trials and genetic association studies.

    PubMed

    Kasanmoentalib, E Soemirien; Brouwer, Matthijs C; van de Beek, Diederik

    2013-06-01

    Bacterial meningitis is a life-threatening disease that continues to inflict a heavy toll. We reviewed recent advances in vaccination, randomized studies on treatment, and genetic association studies in bacterial meningitis. The incidence of bacterial meningitis has decreased after implementation of vaccines, and further implementation of existing conjugate vaccines particularly in low-income countries is expected to reduce the global disease burden. Several randomized studies have been performed recently in this field. Clinical studies showed that short duration (5 days) of antibiotic treatment is as effective as longer duration treatment in low-income countries, and that dexamethasone decreases death and neurological sequelae in high-income countries. Ongoing trials will further define the role of paracetamol, glycerol and hypothermia in bacterial meningitis. Genetic association studies identified pathophysiological mechanisms that could be counteracted in experimental meningitis, providing promising leads for future treatments. Conjugate vaccines have reduced the burden of bacterial meningitis in high-income countries, but implementation of available vaccines in low-income countries is necessary to reduce disease burden worldwide. Adjunctive dexamethasone therapy has beneficial effects in patients with bacterial meningitis but only in high-income countries. Genetic association studies may reveal targets for new treatment strategies.

  16. Middle East respiratory syndrome: obstacles and prospects for vaccine development

    PubMed Central

    Papaneri, Amy B.; Johnson, Reed F.; Wada, Jiro; Bollinger, Laura; Jahrling, Peter B.; Kuhn, Jens H.

    2016-01-01

    Summary The recent emergence of Middle East respiratory syndrome (MERS) highlights the need to engineer new methods for expediting vaccine development against emerging diseases. However, several obstacles prevent pursuit of a licensable MERS vaccine. First, the lack of a suitable animal model for MERS complicates in vivo testing of candidate vaccines. Second, due to the low number of MERS cases, pharmaceutical companies have little incentive to pursue MERS vaccine production as the costs of clinical trials are high. In addition, the timeline from bench research to approved vaccine use is 10 years or longer. Using novel methods and cost-saving strategies, genetically engineered vaccines can be produced quickly and cost-effectively. Along with progress in MERS animal model development, these obstacles can be circumvented or at least mitigated. PMID:25864502

  17. Influenza A virus vaccines for swine.

    PubMed

    Vincent, Amy L; Perez, Daniel R; Rajao, Daniela; Anderson, Tavis K; Abente, Eugenio J; Walia, Rasna R; Lewis, Nicola S

    2017-07-01

    Economic losses due to influenza A virus (IAV) infections are substantial and a global problem, ranking among the top three major health challenges in the swine industry. Currently, H1 and H3 subtypes circulate in pigs globally associated with different combinations of N1 and N2 subtypes; however, the origin, gene constellation, and antigenic makeup of IAV vary greatly on different continents. Vaccination is one means of mitigating the effects of IAV disease, and vaccines are most effective if the strains included closely match the currently circulating strains in pigs. Genetic analyses provide panoramic views of the virus landscape at the sequence level and, thus, can aid in the selection of well-matched swine IAV vaccine strains, but is not sufficient alone. Additionally, a major challenge in selecting appropriate swine IAV vaccine strains is the co-circulation of multiple lineages of viruses in the same region, requiring multivalent or broadly cross-reacting antigens. Due to this complex IAV ecology in swine, new vaccination strategies and vaccine platforms are needed. The hemagglutinin (HA) viral protein is the major target of neutralizing antibodies, which are widely considered to be correlated with protection. Virus variants that are not recognized by previously elicited antibodies can render traditional vaccines that primarily elicit humoral responses ineffective, and therefore result in the need for vaccine strain reformulation and re-vaccination. In the future, new vaccine platforms may be on the market that will provide alternative options to those currently available. Nonetheless, a collaborative approach is needed to improve IAV vaccine strain selection for use in swine. Published by Elsevier B.V.

  18. Different evolutionary trajectories of vaccine-controlled and non-controlled avian infectious bronchitis viruses in commercial poultry

    PubMed Central

    Lee, Dong-Hun

    2017-01-01

    To determine the genetic and epidemiological relationship of infectious bronchitis virus (IBV) isolates from commercial poultry to attenuated live IBV vaccines we conducted a phylogenetic network analysis on the full-length S1 sequence for Arkansas (Ark), Massachusetts (Mass) and Delmarva/1639 (DMV/1639) type viruses isolated in 2015 from clinical cases by 3 different diagnostic laboratories. Phylogenetic network analysis of Ark isolates showed two predominant groups linked by 2 mutations, consistent with subpopulations found in commercial vaccines for this IBV type. In addition, a number of satellite groups surrounding the two predominant populations were observed for the Ark type virus, which is likely due to mutations associated with the nature of this vaccine to persist in flocks. The phylogenetic network analysis of Mass-type viruses shows two groupings corresponding to different manufacturers vaccine sequences. No satellite groups were observed for Mass-type viruses, which is consistent with no persistence of this vaccine type in the field. At the time of collection, no vaccine was being used for the DMV/1639 type viruses and phylogenetic network analysis showed a dispersed network suggesting no clear change in genetic distribution. Selection pressure analysis showed that the DMV/1639 and Mass-type strains were evolving under negative selection, whereas the Ark type viruses had evolved under positive selection. This data supports the hypothesis that live attenuated vaccine usage does play a role in the genetic profile of similar IB viruses in the field and phylogenetic network analysis can be used to identify vaccine and vaccine origin isolates, which is important for our understanding of the role live vaccines play in the evolutionary trajectory of those viruses. PMID:28472110

  19. Vaccine breaks: Outbreaks of myxomatosis on Spanish commercial rabbit farms.

    PubMed

    Dalton, K P; Nicieza, I; de Llano, D; Gullón, J; Inza, M; Petralanda, M; Arroita, Z; Parra, F

    2015-08-05

    Despite the success of vaccination against myxoma virus, myxomatosis remains a problem on rabbit farms throughout Spain and Europe. In this study we set out to evaluate possible causes of myxoma virus (MYXV) vaccine failures addressing key issues with regard to pathogen, vaccine and vaccination strategies. This was done by genetically characterising MYXV field isolates from farm outbreaks, selecting a representative strain for which to assay its virulence and measuring the protective capability of a commercial vaccine against this strain. Finally, we compare methods (route) of vaccine administration under farm conditions and evaluate immune response in vaccinated rabbits. The data presented here show that the vaccine tested is capable of eliciting protection in rabbits that show high levels of seroconversion. However, the number of animals failing to seroconvert following subcutaneous vaccination may leave a large number of rabbits unprotected following vaccine administration. Successful vaccination requires the strict implication of workable, planned, on farm programs. Following this, analysis to confirm seroconversion rates may be advisable. Factors such as the wild rabbit reservoir, control of biting insects and good hygienic practices must be taken into consideration to prevent vaccine failures from occurring. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Novel Platforms for the Development of a Universal Influenza Vaccine

    PubMed Central

    Kumar, Arun; Meldgaard, Trine Sundebo; Bertholet, Sylvie

    2018-01-01

    Despite advancements in immunotherapeutic approaches, influenza continues to cause severe illness, particularly among immunocompromised individuals, young children, and elderly adults. Vaccination is the most effective way to reduce rates of morbidity and mortality caused by influenza viruses. Frequent genetic shift and drift among influenza-virus strains with the resultant disparity between circulating and vaccine virus strains limits the effectiveness of the available conventional influenza vaccines. One approach to overcome this limitation is to develop a universal influenza vaccine that could provide protection against all subtypes of influenza viruses. Moreover, the development of a novel or improved universal influenza vaccines may be greatly facilitated by new technologies including virus-like particles, T-cell-inducing peptides and recombinant proteins, synthetic viruses, broadly neutralizing antibodies, and nucleic acid-based vaccines. This review discusses recent scientific advances in the development of next-generation universal influenza vaccines. PMID:29628926

  1. Development of whole sporozoite malaria vaccines.

    PubMed

    Hollingdale, Michael R; Sedegah, Martha

    2017-01-01

    Despite recent advances, malaria remains a major health threat both to populations in endemic areas as well travelers, including military personnel, to these areas. Subunit vaccines have not yet achieved sufficient efficacy needed for use in any of these at risk populations. Areas covered: This review discusses the current status of various whole sporozoite vaccine approaches and is mainly focused on current clinical trials. Expert commentary: Nearly 100% efficacy was achieved by administering multiple bites of radiation-attenuated sporozoite (RAS) Plasmodium falciparum-infected mosquitoes; this is impractical for widespread use. Now, this high level efficacy has been reproduced using purified, metabolically active RAS (PfSPZ Sanaria® Vaccine), which is undergoing extensive clinical testing. Alternative whole sporozoite vaccines include immunization with fully infectious sporozoites under chloroquine prophylaxis (CPS) or as genetically-attenuated parasites (GAP). By also manufacturing purified infectious sporozoites, it is now possible to combine these with CPS and GAP, as well as perform challenge studies using controlled doses of sporozoites.

  2. Humoral Immune Response After Intravitreal But Not After Subretinal AAV8 in Primates and Patients.

    PubMed

    Reichel, Felix F; Peters, Tobias; Wilhelm, Barbara; Biel, Martin; Ueffing, Marius; Wissinger, Bernd; Bartz-Schmidt, Karl U; Klein, Reinhild; Michalakis, Stylianos; Fischer, M Dominik

    2018-04-01

    To study longitudinal changes of anti-drug antibody (ADA) titers to recombinant adeno-associated virus serotype 8 (rAAV8) capsid epitopes in nonhuman primates (NHP) and patients. Three groups of six NHP each received subretinal injections (high dose: 1 × 1012 vector genomes [vg], low dose: 1 × 1011 vg, or vehicle only). Four additional animals received intravitreal injections of the high dose (1 × 1012 vg). Three patients received 1 × 1010 vg as subretinal injections. ELISA quantified ADA levels at baseline and 1, 2, 3, 7, 28, and 90 days after surgery in NHP and at baseline and 1, 3, and 6 months after surgery in patients. Two out of 22 animals lacked ADA titers at baseline and developed low ADA titers toward the end of the study. Titers in the low-dose group stayed constant, while two of six animals from the high-dose group developed titers that rose beyond the range of the assay. All animals from the intravitreal control group showed a rise in ADA titer by day 7 that peaked at day 28. Preliminary data from the clinical trial (NCT02610582) show no humoral immune response in patients following subretinal delivery of 1 × 1010 vg. No significant induction of ADA occurred in NHP when mimicking the clinical scenario of subretinal delivery with a clinical-grade rAAV8 and concomitant immunosuppression. Likewise, clinical data showed no humoral immune response in patients. In contrast, intravitreal delivery was associated with a substantial humoral immune response. Subretinal delivery might be superior to an intravitreal application regarding immunologic aspects.

  3. Emergence of Vaccine-Derived Polioviruses during Ebola Virus Disease Outbreak, Guinea, 2014–2015

    PubMed Central

    Majumdar, Manasi; Kebe, Ousmane; Fall, Aichatou D.; Kone, Moussa; Kande, Mouctar; Dabo, Moustapha; Sylla, Mohamed Salif; Sompare, Djenou; Howard, Wayne; Faye, Ousmane; Martin, Javier; Ndiaye, Kader

    2018-01-01

    During the 2014–2015 outbreak of Ebola virus disease in Guinea, 13 type 2 circulating vaccine-derived polioviruses (cVDPVs) were isolated from 6 polio patients and 7 healthy contacts. To clarify the genetic properties of cVDPVs and their emergence, we combined epidemiologic and virologic data for polio cases in Guinea. Deviation of public health resources to the Ebola outbreak disrupted polio vaccination programs and surveillance activities, which fueled the spread of neurovirulent VDPVs in an area of low vaccination coverage and immunity. Genetic properties of cVDPVs were consistent with their capacity to cause paralytic disease in humans and capacity for sustained person-to-person transmission. Circulation ceased when coverage of oral polio vaccine increased. A polio outbreak in the context of the Ebola virus disease outbreak highlights the need to consider risks for polio emergence and spread during complex emergencies and urges awareness of the challenges in polio surveillance, vaccination, and diagnosis. PMID:29260690

  4. Cancer and Stroma-Targeted Immunotherapy with a Genetically Modified DC Vaccine

    DTIC Science & Technology

    2011-05-01

    targeting the tumor stroma in addition to breast cancer cells may produce the desired increase in antitumor activity of DC vaccines for breast cancer...vaccination inhibits 4T1-neu progression. We investigated whether DC-shA20-FAP- HER2 may induce more potent anti- stroma and anti-tumor immunity with the...the immunosuppressive tumor microenviroment resulting in potent antitumor activity. Zhu W, Zhou X, Rollins L , Rooney CM, Gottschalk S, Song XT

  5. The combined measles, mumps, and rubella vaccines and the total number of vaccines are not associated with development of autism spectrum disorder: the first case-control study in Asia.

    PubMed

    Uno, Yota; Uchiyama, Tokio; Kurosawa, Michiko; Aleksic, Branko; Ozaki, Norio

    2012-06-13

    The aim of this study was to investigate the relationship between autism spectrum disorder (ASD) and general vaccinations, including measles-mumps-rubella (MMR) vaccine, in Japanese subjects, a population with high genetic homogeneity. A case-control study was performed. Cases (n=189) were diagnosed with ASD, while controls (n=224) were volunteers from general schools, matched by sex and birth year to cases. Vaccination history and prenatal, perinatal, and neonatal factors from the Maternal and Child Health handbook, which was part of each subject's file, were examined. To determine the relationship between potential risk factors and ASD, crude odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated, and the differences in mean values of the quantitative variables between cases and controls were analyzed using an unpaired t-test. Moreover, MMR vaccination and the effect of the number of vaccine injections were investigated using a conditional multiple regression model. For MMR vaccination, the OR was 1.04 (95% CI, 0.65-1.68), and no significant differences were found for the other vaccines. For all of the prenatal, perinatal and neonatal factors, there were no significant differences between cases and controls. Furthermore, regarding the presence of ASD, MMR vaccination and the number of vaccine injections had ORs of 1.10 (95% CI, 0.64-1.90) and 1.10 (95% CI, 0.95-1.26), respectively, in the conditional multiple regression model; no significant differences were found. In this study, there were not any convincing evidences that MMR vaccination and increasing the number of vaccine injections were associated with an increased risk of ASD in a genetically homogeneous population. Therefore, these findings indicate that there is no basis for avoiding vaccination out of concern for ASD. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Engineering Foot-and-Mouth Disease Viruses with Improved Growth Properties for Vaccine Development

    PubMed Central

    Zheng, Haixue; Guo, Jianhong; Jin, Ye; Yang, Fan; He, Jijun; Lv, Lv; Zhang, Kesan; Wu, Qiong; Liu, Xiangtao; Cai, Xuepeng

    2013-01-01

    Background No licensed vaccine is currently available against serotype A foot-and-mouth disease (FMD) in China, despite the isolation of A/WH/CHA/09 in 2009, partly because this strain does not replicate well in baby hamster kidney (BHK) cells. Methodology/Principal Findings A novel plasmid-based reverse genetics system was used to construct a chimeric strain by replacing the P1 gene in the vaccine strain O/CHA/99 with that from the epidemic stain A/WH/CHA/09. The chimeric virus displayed growth kinetics similar to those of O/CHA/99 and was selected for use as a candidate vaccine strain after 12 passages in BHK cells. Cattle were vaccinated with the inactivated vaccine and humoral immune responses were induced in most of the animals on day 7. A challenge infection with A/WH/CHA/09 on day 28 indicated that the group given a 4-µg dose was fully protected and neither developed viremia nor seroconverted to a 3ABC antigen. Conclusions/Significance Our data demonstrate that the chimeric virus not only propagates well in BHK cells and has excellent antigenic matching against serotype A FMD, but is also a potential marker vaccine to distinguish infection from vaccination. These results suggest that reverse genetics technology is a useful tool for engineering vaccines for the prevention and control of FMD. PMID:23372840

  7. Evaluation of vaccines in the EU TB Vaccine Cluster using a guinea pig aerosol infection model of tuberculosis.

    PubMed

    Williams, Ann; Hatch, Graham J; Clark, Simon O; Gooch, Karen E; Hatch, Kim A; Hall, Graham A; Huygen, Kris; Ottenhoff, Tom H M; Franken, Kees L M C; Andersen, Peter; Doherty, T Mark; Kaufmann, Stefan H E; Grode, Leander; Seiler, Peter; Martin, Carlos; Gicquel, Brigitte; Cole, Stewart T; Brodin, Priscille; Pym, Alexander S; Dalemans, Wilfried; Cohen, Joe; Lobet, Yves; Goonetilleke, Nilu; McShane, Helen; Hill, Adrian; Parish, Tanya; Smith, Debbie; Stoker, Neil G; Lowrie, Douglas B; Källenius, Gunilla; Svenson, Stefan; Pawlowski, Andrzej; Blake, Karen; Marsh, Philip D

    2005-01-01

    The TB Vaccine Cluster project funded by the EU Fifth Framework programme aims to provide novel vaccines against tuberculosis that are suitable for evaluation in humans. This paper describes the studies of the protective efficacy of vaccines in a guinea pig aerosol-infection model of primary tuberculosis. The objective was to conduct comparative evaluations of vaccines that had previously demonstrated efficacy in other animal models. Groups of 6 guinea pigs were immunized with vaccines provided by the relevant EU Vaccine Cluster partners. Survival over 17 or 26 weeks was used as the principal measure of vaccine efficacy following aerosol challenge with H37Rv. Counts of mycobacteria in lungs and spleens, and histopathological changes in the lungs, were also used to provide evidence of protection. A total of 24 vaccines were evaluated in 4 experiments each of a different design. A heterologous prime-boost strategy of DNA and MVA, each expressing Ag85A and a fusion protein of ESAT-6 and Ag85B in adjuvant, protected the guinea pigs to the same extent as BCG. Genetically modified BCG vaccines and boosted BCG strategies also protected guinea pigs to the same extent as BCG but not statistically significantly better. A relatively high aerosol-challenge dose and evaluation over a protracted time post-challenge allowed superior protection over BCG to be demonstrated by BCG boosted with MVA and fowl pox vectors expressing Ag85A.

  8. Enhanced pneumonia and disease in pigs vaccinated with an inactivated human-like (δ-cluster) H1N2 vaccine and challenged with pandemic 2009 H1N1 influenza virus.

    PubMed

    Gauger, Phillip C; Vincent, Amy L; Loving, Crystal L; Lager, Kelly M; Janke, Bruce H; Kehrli, Marcus E; Roth, James A

    2011-03-24

    Influenza is an economically important respiratory disease affecting swine world-wide with potential zoonotic implications. Genetic reassortment and drift has resulted in genetically and antigenically distinct swine influenza viruses (SIVs). Consequently, prevention of SIV infection is challenging due to the increased rate of genetic change and a potential lack of cross-protection between vaccine strains and circulating novel isolates. This report describes a vaccine-heterologous challenge model in which pigs were administered an inactivated H1N2 vaccine with a human-like (δ-cluster) H1 six and three weeks before challenge with H1 homosubtypic, heterologous 2009 pandemic H1N1. At necropsy, macroscopic and microscopic pneumonia scores were significantly higher in the vaccinated and challenged (Vx/Ch) group compared to non-vaccinated and challenged (NVx/Ch) pigs. The Vx/Ch group also demonstrated enhanced clinical disease and a significantly elevated pro-inflammatory cytokine profile in bronchoalveolar lavage fluid compared to the NVx/Ch group. In contrast, viral shedding and replication were significantly higher in NVx/Ch pigs although all challenged pigs, including Vx/Ch pigs, were shedding virus in nasal secretions. Hemagglutination inhibition (HI) and serum neutralizing (SN) antibodies were detected to the priming antigen in the Vx/Ch pigs but no measurable cross-reacting HI or SN antibodies were detected to pandemic H1N1 (pH1N1). Overall, these results suggest that inactivated SIV vaccines may potentiate clinical signs, inflammation and pneumonia following challenge with divergent homosubtypic viruses that do not share cross-reacting HI or SN antibodies. Published by Elsevier Ltd.

  9. Clinical protocol. Gene therapy of Canavan disease: AAV-2 vector for neurosurgical delivery of aspartoacylase gene (ASPA) to the human brain.

    PubMed

    Janson, Christopher; McPhee, Scott; Bilaniuk, Larissa; Haselgrove, John; Testaiuti, Mark; Freese, Andrew; Wang, Dah-Jyuu; Shera, David; Hurh, Peter; Rupin, Joan; Saslow, Elizabeth; Goldfarb, Olga; Goldberg, Michael; Larijani, Ghassem; Sharrar, William; Liouterman, Larisa; Camp, Angelique; Kolodny, Edwin; Samulski, Jude; Leone, Paola

    2002-07-20

    This clinical protocol describes virus-based gene transfer for Canavan disease, a childhood leukodystrophy. Canavan disease, also known as Van Bogaert-Bertrand disease, is a monogeneic, autosomal recessive disease in which the gene coding for the enzyme aspartoacylase (ASPA) is defective. The lack of functional enzyme leads to an increase in the central nervous system of the substrate molecule, N-acetyl-aspartate (NAA), which impairs normal myelination and results in spongiform degeneration of the brain. No effective treatment currently exists; however, virus-based gene transfer has the potential to arrest or reverse the course of this otherwise fatal condition. This procedure involves neurosurgical administration of approximately 900 billion genomic particles (approximately 10 billion infectious particles) of recombinant adeno-associated virus (AAV) containing the aspartoacylase gene (ASPA) directly to affected regions of the brain in each of 21 patients with Canavan disease. Pre- and post-delivery assessments include a battery of noninvasive biochemical, radiological, and neurological tests. This gene transfer study represents the first clinical use of AAV in the human brain and the first instance of viral gene transfer for a neurodegenerative disease.

  10. Efficacy of commercial vaccines against newly emerging avian influenza H5N8 virus in Egypt.

    PubMed

    Kandeil, Ahmed; Sabir, Jamal S M; Abdelaal, Ahmed; Mattar, Ehab H; El-Taweel, Ahmed N; Sabir, Mumdooh J; Khalil, Ahmed Aly; Webby, Richard; Kayali, Ghazi; Ali, Mohamed A

    2018-06-26

    The newly emerging, highly pathogenic avian influenza (HPAI) H5N8 virus of clade 2.3.4.4 was recently detected in wild birds and domestic poultry in Egypt in the 2016/2017 winter season. Vaccination based on commercial H5 vaccines is used as an essential control strategy in Egyptian poultry. Here, we studied the efficacy of the eight most common commercial H5 poultry vaccines in the Egyptian market and compared them with an experimental vaccine based on the Egyptian LPAI H5N8 virus that was prepared by using reverse genetics. The experimental vaccine and Re-5 commercial vaccine were able to completely protect chickens and significantly reduce virus shedding. Our results indicate that most of the commercial poultry H5 vaccines used in the present study were ineffective because the seed viruses in these vaccines are genetically distinct from the H5N8 viruses currently circulating in Egypt. Although some of the commercial vaccines protected chickens from mortality, they failed to prevent chickens from shedding the virus. Accordingly, we recommend updating and reinforcing the H5N8 prevention and control strategies in Egypt. The vaccination strategy should be reconsidered based on currently circulating viruses.

  11. Clinical experience with respiratory syncytial virus vaccines.

    PubMed

    Piedra, Pedro A

    2003-02-01

    Respiratory syncytial virus (RSV) infection is at times associated with life-threatening lower respiratory tract illness in infancy. Severe infection during the first year of life may be an important risk factor or indicator for the development of asthma in early childhood. Severe infections primarily occur in healthy infants, and young infants and children with specific risk factors. However, RSV causes respiratory infections in all age groups. Indeed it is now recognized that RSV disease is responsible for significant morbidity and mortality in the geriatric population. RSV infection remains difficult to treat, and prevention is a worldwide goal. For this reason there has been an intensive effort to develop an effective and safe RSV vaccine. Initial infection with RSV affords limited protection to reinfection, yet repeated episodes decrease the risk for lower respiratory tract illness. In the 20 years from 1960 to 1980, trials of several candidate RSV vaccines failed to attain the desired safety and protection against natural infection. Some vaccine types either failed to elicit immunogenicity, as with the live subcutaneous vaccine, or resulted in exaggerated disease on natural exposure to the virus, as with the formalin-inactivated (FI) type. Currently vaccine candidates are being developed based on the molecular virology of RSV. Recent formulations of candidate RSV vaccines have focused on subunit vaccines [such as purified fusion protein (PFP)], subunit vaccines combined with nonspecific immune activating adjuvants, live attenuated vaccines (including cold passaged, temperature-sensitive or cpts mutants), genetically engineered live attenuated vaccines and polypeptide vaccines.

  12. Application of new vaccine technology to improve immunity and productivity: advantages and challenges

    USDA-ARS?s Scientific Manuscript database

    Vaccines play a critical role in the poultry industry’s efforts to protect animals against disease. However, providing safe, efficacious, and cost-effective vaccines remains a constant concern to the industry. Recent advances in avian immunology, genetics, molecular biology, and pathogenesis have ...

  13. Vaccines for viral diseases with dermatologic manifestations.

    PubMed

    Brentjens, Mathijs H; Yeung-Yue, Kimberly A; Lee, Patricia C; Tyring, Stephen K

    2003-04-01

    Vaccines against infectious diseases have been available since the 1800s, when an immunization strategy against smallpox developed by Jenner gained wide acceptance. Until recently, the only vaccination strategies available involved the use of protein-based, whole killed, and attenuated live virus vaccines. These strategies have led to the development of effective vaccines against a variety of diseases with primary or prominent cutaneous manifestations. Effective and safe vaccines now used worldwide include those directed against measles and rubella (now commonly used together with a mumps vaccine as the trivalent MMR), chickenpox, and hepatitis B. The eradication of naturally occurring smallpox remains one of the greatest successes in the history of modern medicine, but stockpiles of live smallpox exist in the United States and Russia. Renewed interest in the smallpox vaccine reflects concerns about a possible bioterrorist threat using this virus. Yellow fever is a hemorrhagic virus endemic to tropical areas of South America and Africa. An effective vaccine for this virus has existed since 1937, and it is used widely in endemic areas of South America, and to a lesser extent in Africa. This vaccine is recommended once every 10 years for people who are traveling to endemic areas. Advances in immunology have led to a greater understanding of immune system function in viral diseases. Progress in genetics and molecular biology has allowed researchers to design vaccines with novel mechanisms of action (eg, DNA, vector, and VLP vaccines). Vaccines have also been designed to specifically target particular viral components, allowing for stimulation of various arms of the immune system as desired. Ongoing research shows promise in prophylactic and therapeutic vaccination for viral infections with cutaneous manifestations. Further studies are necessary before vaccines for HSV, HPV, and HIV become commercially available.

  14. [Towards a new vaccine economy?].

    PubMed

    Poirot, P; Martin, J F

    1994-01-01

    When Jonas Salk announced in the mid-50s the availability of a new vaccine against poliomyelitis, the world had the impression that it was now controlling infectious diseases. In fact, the success of this vaccine has been considerable and although some innovations lead to the launch of vaccines against flu, measles, rubella or mumps, the world vaccine market remained remarkably stable till the mid-80s. However, since 1984 (launch of the hepatitis B vaccine) there have been very substantial changes and further change is expected in the next ten years in the world market. Today, big companies are making a concentrated supply: Pasteur Mérieux with its subsidiary Connaught, SmithKline Beecham who acquired the Belgian company RIT, and Merck & Co. who is joining its forces with Pasteur Mérieux. Medium sized and small companies remain and reflect the situation of the past, but must work hard to secure their long term existence eventhough the world demand is going to double before the year 2000. Very substantial technological innovations explain to a large extent the development of the supply: progress in molecular biology, and particularly genetic engineering, lead to recombinant vaccines of which hepatitis B is the best example with worldwide sales in the range of $600 million a year. Similarly, conjugation technologies have allowed the development of new vaccines against meningitis, particularly Haemophilus influenzae type b. More recently, an efficacious vaccine against hepatitis A has been launched and many new products will be marketed in the next years against herpes, Lyme disease, and agents of other meningitis, etc.(ABSTRACT TRUNCATED AT 250 WORDS)

  15. Rescue of a vaccine strain of peste des petits ruminants virus: In vivo evaluation and comparison with standard vaccine

    PubMed Central

    Muniraju, Murali; Mahapatra, Mana; Buczkowski, Hubert; Batten, Carrie; Banyard, Ashley C.; Parida, Satya

    2015-01-01

    Across the developing world peste des petits ruminants virus places a huge disease burden on agriculture, primarily affecting the production of small ruminant. The disease is most effectively controlled by vaccinating sheep and goats with live attenuated vaccines that provide lifelong immunity. However, the current vaccines and serological tests are unable to enable Differentiation between naturally Infected and Vaccinated Animals (DIVA). This factor precludes meaningful assessment of vaccine coverage and epidemiological surveillance based on serology, in turn reducing the efficiency of control programmes. The availability of a recombinant PPRV vaccine with a proven functionality is a prerequisite for the development of novel vaccines that may enable the development of DIVA tools for PPRV diagnostics. In this study, we have established an efficient reverse genetics system for PPRV Nigeria 75/1 vaccine strain and, further rescued a version of PPRV Nigeria 75/1 vaccine strain that expresses eGFP as a novel transcription cassette and a version of PPRV Nigeria 75/1 vaccine strain with mutations in the haemagglutinin (H) gene to enable DIVA through disruption of binding to H by the C77 monoclonal antibody used in the competitive (c) H-ELISA. All three rescued viruses showed similar growth characteristics in vitro in comparison to parent vaccine strain and, following in vivo assessment the H mutant provided full protection in goats. Although the C77 monoclonal antibody used in the cH-ELISA was unable to bind to the mutated form of H in vitro, the mutation was not sufficient to enable DIVA in vivo. PMID:25444790

  16. Updates on the use of vaccines in dermatological conditions.

    PubMed

    Hanna, Edith; Dany, Mohammed; Abbas, Ossama; Kreidieh, Firas; Kurban, Mazen

    2018-05-22

    Numerous vaccines are being actively developed for use in dermatologic diseases. Advances in the fields of immunotherapy, genetics and molecular medicine have allowed for the design of prophylactic and therapeutic vaccines with immense potential in managing infections and malignancies of the skin. This review addresses the different vaccines available for use in dermatological diseases and those under development for future potential use. The major limitation of our review is its complete reliance on published data. Our review is strictly limited to the availability of published research online through available databases. We do not cite any of the authors' previous publications nor have we conducted previous original research studies regarding vaccines in dermatology. Strength would have been added to our paper had we conducted original studies by our research team regarding the candidate vaccines delineated in the paper.

  17. Complete Protection against Pneumonic and Bubonic Plague after a Single Oral Vaccination.

    PubMed

    Derbise, Anne; Hanada, Yuri; Khalifé, Manal; Carniel, Elisabeth; Demeure, Christian E

    2015-01-01

    No efficient vaccine against plague is currently available. We previously showed that a genetically attenuated Yersinia pseudotuberculosis producing the Yersinia pestis F1 antigen was an efficient live oral vaccine against pneumonic plague. This candidate vaccine however failed to confer full protection against bubonic plague and did not produce F1 stably. The caf operon encoding F1 was inserted into the chromosome of a genetically attenuated Y. pseudotuberculosis, yielding the VTnF1 strain, which stably produced the F1 capsule. Given orally to mice, VTnF1 persisted two weeks in the mouse gut and induced a high humoral response targeting both F1 and other Y. pestis antigens. The strong cellular response elicited was directed mostly against targets other than F1, but also against F1. It involved cells with a Th1-Th17 effector profile, producing IFNγ, IL-17, and IL-10. A single oral dose (108 CFU) of VTnF1 conferred 100% protection against pneumonic plague using a high-dose challenge (3,300 LD50) caused by the fully virulent Y. pestis CO92. Moreover, vaccination protected 100% of mice from bubonic plague caused by a challenge with 100 LD50 Y. pestis and 93% against a high-dose infection (10,000 LD50). Protection involved fast-acting mechanisms controlling Y. pestis spread out of the injection site, and the protection provided was long-lasting, with 93% and 50% of mice surviving bubonic and pneumonic plague respectively, six months after vaccination. Vaccinated mice also survived bubonic and pneumonic plague caused by a high-dose of non-encapsulated (F1-) Y. pestis. VTnF1 is an easy-to-produce, genetically stable plague vaccine candidate, providing a highly efficient and long-lasting protection against both bubonic and pneumonic plague caused by wild type or un-encapsulated (F1-negative) Y. pestis. To our knowledge, VTnF1 is the only plague vaccine ever reported that could provide high and durable protection against the two forms of plague after a single oral

  18. Outer membrane vesicles as platform vaccine technology

    PubMed Central

    Stork, Michiel; van der Ley, Peter

    2015-01-01

    Abstract Outer membrane vesicles (OMVs) are released spontaneously during growth by many Gram‐negative bacteria. They present a range of surface antigens in a native conformation and have natural properties like immunogenicity, self‐adjuvation and uptake by immune cells which make them attractive for application as vaccines against pathogenic bacteria. In particular with Neisseria meningitidis, they have been investigated extensively and an OMV‐containing meningococcal vaccine has recently been approved by regulatory agencies. Genetic engineering of the OMV‐producing bacteria can be used to improve and expand their usefulness as vaccines. Recent work on meningitis B vaccines shows that OMVs can be modified, such as for lipopolysaccharide reactogenicity, to yield an OMV product that is safe and effective. The overexpression of crucial antigens or simultaneous expression of multiple antigenic variants as well as the expression of heterologous antigens enable expansion of their range of applications. In addition, modifications may increase the yield of OMV production and can be combined with specific production processes to obtain high amounts of well‐defined, stable and uniform OMV particle vaccine products. Further improvement can facilitate the development of OMVs as platform vaccine product for multiple applications. PMID:26912077

  19. Gene transfer as a strategy to achieve permanent cardioprotection II: rAAV-mediated gene therapy with heme oxygenase-1 limits infarct size 1 year later without adverse functional consequences.

    PubMed

    Li, Qianhong; Guo, Yiru; Ou, Qinghui; Wu, Wen-Jian; Chen, Ning; Zhu, Xiaoping; Tan, Wei; Yuan, Fangping; Dawn, Buddhadeb; Luo, Li; Hunt, Gregory N; Bolli, Roberto

    2011-11-01

    Extensive evidence indicates that heme oxygenase-1 (HO-1) exerts potent cytoprotective effects in response to stress. Previous studies have shown that gene therapy with HO-1 protects against myocardial ischemia/reperfusion injury for up to 8 weeks after gene transfer. However, the long-term effects of HO-1 gene therapy on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the HO-1 gene (rAAV/HO-1) that enables long-lasting transgene expression. Mice received injections in the anterior LV wall of rAAV/LacZ (LacZ group) or rAAV/HO-1 (HO-1 group); 1 year later, they were subjected to a 30-min coronary occlusion (O) and 4 h of reperfusion (R). Cardiac HO-1 gene expression was confirmed at 1 month and 1 year after gene transfer by immunoblotting and immunohistochemistry analyses. In the HO-1 group, infarct size (% of risk region) was dramatically reduced at 1 year after gene transfer (11.2 ± 2.1%, n = 12, vs. 44.7 ± 3.6%, n = 8, in the LacZ group; P < 0.05). The infarct-sparing effects of HO-1 gene therapy at 1 year were as powerful as those observed 24 h after ischemic PC (six 4-min O/4-min R cycles) (15.0 ± 1.7%, n = 10). There were no appreciable changes in LV fractional shortening, LV ejection fraction, or LV end-diastolic or end-systolic diameter at 1 year after HO-1 gene transfer as compared to the age-matched controls or with the LacZ group. Histology showed no inflammation in the myocardium 1 year after rAAV/HO-1-mediated gene transfer. These results demonstrate, for the first time, that rAAV-mediated HO-1 gene transfer confers long-term (1 year), possibly permanent, cardioprotection without adverse functional consequences, providing proof of principle for the concept of achieving prophylactic cardioprotection (i.e., "immunization against infarction").

  20. Vaccine-preventable infections in Systemic Lupus Erythematosus

    PubMed Central

    Murdaca, Giuseppe; Orsi, Andrea; Spanò, Francesca; Faccio, Valeria; Puppo, Francesco; Durando, Paolo; Icardi, Giancarlo; Ansaldi, Filippo

    2016-01-01

    Systemic Lupus Erythematosus (SLE) is characterized by abnormal autoantibody production and clearance. Infections are among the most important causes of morbidity and mortality in SLE patients; they have an increased frequency of severe bacterial and viral infections possibly due to inherited genetic and immunologic defects and to immunosuppressive therapies. In addition, infectious agents can switch on lupus disease expression and activity. Among the strategies to reduce the risk of infection, vaccination can be considered the most reliable option. Most vaccines are effective and safe in SLE patients, although in certain cases immunogenicity may be sub-optimal and vaccination can trigger a flare. Although these issues are currently unresolved, the risk benefit balance is in favor for vaccination to reduce the risk of infection in SLE patients. In the present review we discuss the preventive strategies currently recommended to reduce bacterial and viral infections in SLE. PMID:26750996

  1. Antigenic analysis of genetic variants of Canine distemper virus.

    PubMed

    Anis, Eman; Holford, Amy L; Galyon, Gina D; Wilkes, Rebecca P

    2018-06-01

    Canine distemper virus (CDV) is an RNA virus of the genus Morbillivirus within the family Paramyxoviridae. CDV produces multi-systemic disease in dogs and other terrestrial carnivores. With the development of modified live vaccines in the 1950s and 1960s, the disease, with a few exceptions, has been successfully controlled. However, recently the cases of CDV in vaccinated dogs have been increasing throughout the world, including the United States. There are many reasons that can lead to vaccine failure, including antigenic differences between the vaccine strains and the currently circulating wild-type strains. Currently, there are at least three genetically different CDV lineages circulating in the US. Therefore, in this study, we evaluated various wild-type CDV and vaccine isolates to determine if the genetic differences observed among various strains result in significant antigenic differences based on changes to the neutralizing epitopes. The results of a cross-neutralization assay revealed that there are antigenic differences among the tested CDV wild-type isolates as well as between the tested isolates and the vaccine strains currently used in the US. Therefore, these results suggest the need to develop an updated CDV vaccine. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Phylogenetic analysis of human influenza A/H3N2 viruses isolated in 2015 in Germany indicates significant genetic divergence from vaccine strains.

    PubMed

    Mostafa, Ahmed; Abdelwhab, El-Sayed M; Slanina, Heiko; Hussein, Mohamed A; Kuznetsova, Irina; Schüttler, Christian G; Ziebuhr, John; Pleschka, Stephan

    2016-06-01

    Infections by H3N2-type influenza A viruses (IAV) resulted in significant numbers of hospitalization in several countries in 2014-2015, causing disease also in vaccinated individuals and, in some cases, fatal outcomes. In this study, sequence analysis of H3N2 viruses isolated in Germany from 1998 to 2015, including eleven H3N2 isolates collected early in 2015, was performed. Compared to the vaccine strain A/Texas/50/2012 (H3N2), the 2015 strains from Germany showed up to 4.5 % sequence diversity in their HA1 protein, indicating substantial genetic drift. The data further suggest that two distinct phylogroups, 3C.2 and 3C.3, with 1.6-2.3 % and 0.3-2.4 % HA1 nucleotide and amino acid sequence diversity, respectively, co-circulated in Germany in the 2014/2015 season. Distinct glycosylation patterns and amino acid substitutions in the hemagglutinin and neuraminidase proteins were identified, possibly contributing to the unusually high number of H3N2 infections in this season and providing important information for developing vaccines that are effective against both genotypes.

  3. Reverse Genetics for Mammalian Orthoreovirus.

    PubMed

    Stuart, Johnasha D; Phillips, Matthew B; Boehme, Karl W

    2017-01-01

    Reverse genetics allows introduction of specific alterations into a viral genome. Studies performed with mutant viruses generated using reverse genetics approaches have contributed immeasurably to our understanding of viral replication and pathogenesis, and also have led to development of novel vaccines and virus-based vectors. Here, we describe the reverse genetics system that allows for production and recovery of mammalian orthoreovirus, a double-stranded (ds) RNA virus, from plasmids that encode the viral genome.

  4. [PERSPECTIVES OF DEVELOPMENT OF LIVE RECOMBINANT ANTHRAX VACCINES BASED ON OPPORTUNISTIC AND APATHOGENIC MICROORGANISMS].

    PubMed

    Popova, P Yu; Mikshis, N I

    2016-01-01

    Live genetic engineering anthrax vaccines on the platform of avirulent and probiotic micro-organisms are a safe and adequate alternative to preparations based on attenuated Bacillus anthracis strains. Mucosal application results in a direct contact of the vaccine preparations with mucous membranes in those organs arid tissues of the macro-organisms, that are exposed to the pathogen in the first place, resulting in a development of local and systemic immune response. Live recombinant anthrax vaccines could be used both separately as well as in a prime-boost immunization scheme. The review focuses on immunogenic and protective properties of experimental live genetic engineering prearations, created based on members of geni of Salmonella, Lactobacillus and adenoviruses.

  5. Associations between race, sex and immune response variations to rubella vaccination in two independent cohorts

    PubMed Central

    Haralambieva, Iana H.; Salk, Hannah M.; Lambert, Nathaniel D.; Ovsyannikova, Inna G.; Kennedy, Richard B.; Warner, Nathaniel D.; Pankratz, V.Shane; Poland, Gregory A.

    2014-01-01

    Introduction Immune response variations after vaccination are influenced by host genetic factors and demographic variables, such as race, ethnicity and sex. The latter have not been systematically studied in regard to live rubella vaccine, but are of interest for developing next generation vaccines for diverse populations, for predicting immune responses after vaccination, and for better understanding the variables that impact immune response. Methods We assessed associations between demographic variables, including race, ethnicity and sex, and rubella-specific neutralizing antibody levels and secreted cytokines (IFN! , IL-6) in two independent cohorts (1,994 subjects), using linear and linear mixed models approaches, and genetically defined racial and ethnic categorizations. Results Our replicated findings in two independent, large, racially diverse cohorts indicate that individuals of African descent have significantly higher rubella-specific neutralizing antibody levels compared to individuals of European descent and/or Hispanic ethnicity (p! 0.001). Conclusion Our study provides consistent evidence for racial/ethnic differences in humoral immune response following rubella vaccination. PMID:24530932

  6. Sex and Gender Differences in the Outcomes of Vaccination over the Life Course.

    PubMed

    Flanagan, Katie L; Fink, Ashley L; Plebanski, Magdalena; Klein, Sabra L

    2017-10-06

    Both sex (i.e., biological differences) and gender (i.e., social or cultural influences) impact vaccine acceptance, responses, and outcomes. Clinical data illustrate that among children, young adults, and aged individuals, males and females differ in vaccine-induced immune responses, adverse events, and protection. Although males are more likely to receive vaccines, following vaccination, females typically develop higher antibody responses and report more adverse effects of vaccination than do males. Human, nonhuman animal, and in vitro studies reveal numerous immunological, genetic, hormonal, and environmental factors that differ between males and females and contribute to sex- and gender-specific vaccine responses and outcomes. Herein, we address the impact of sex and gender variables that should be considered in preclinical and clinical studies of vaccines.

  7. Novel immunotherapy vaccine development.

    PubMed

    Jutel, Marek; Akdis, Cezmi A

    2014-12-01

    Allergen-specific immunotherapy is the only curative treatment for allergic diseases. In spite of the great progress in both vaccine development and the methods of allergen immunotherapy (AIT) in recent years, several key problems related to limited efficacy, side-effects, low patient adherence and the relatively high costs due to the long duration (3-5 years) remain to be solved. The current approaches aiming at optimization of AIT are reviewed, including both conceptual studies in experimental models and proof-of-concept - as well as large, multicenter clinical studies. The most promising approaches to improve efficacy and safety of vaccine-based AIT include bypassing IgE binding and targeting allergen-specific T cells using hypoallergenic recombinant allergen derivatives and immunogenic peptides, the use of new adjuvants and stimulators of the innate immune response, the fusion of allergens to immune modifiers and peptide carrier proteins and new routes of vaccine administration. The cloning of allergen proteins and genetic engineering enabled the production of vaccines that have well defined molecular, immunologic and biologic characteristics as well as modified molecular structure. These new compounds along with new immunization protocols can bring us closer to the ultimate goal of AIT, that is, complete cure of a large number of allergic patients.

  8. The use of transgenic parasites in malaria vaccine research.

    PubMed

    Othman, Ahmad Syibli; Marin-Mogollon, Catherin; Salman, Ahmed M; Franke-Fayard, Blandine M; Janse, Chris J; Khan, Shahid M

    2017-07-01

    Transgenic malaria parasites expressing foreign genes, for example fluorescent and luminescent proteins, are used extensively to interrogate parasite biology and host-parasite interactions associated with malaria pathology. Increasingly transgenic parasites are also exploited to advance malaria vaccine development. Areas covered: We review how transgenic malaria parasites are used, in vitro and in vivo, to determine protective efficacy of different antigens and vaccination strategies and to determine immunological correlates of protection. We describe how chimeric rodent parasites expressing P. falciparum or P. vivax antigens are being used to directly evaluate and rank order human malaria vaccines before their advancement to clinical testing. In addition, we describe how transgenic human and rodent parasites are used to develop and evaluate live (genetically) attenuated vaccines. Expert commentary: Transgenic rodent and human malaria parasites are being used to both identify vaccine candidate antigens and to evaluate both sub-unit and whole organism vaccines before they are advanced into clinical testing. Transgenic parasites combined with in vivo pre-clinical testing models (e.g. mice) are used to evaluate vaccine safety, potency and the durability of protection as well as to uncover critical protective immune responses and to refine vaccination strategies.

  9. Recombinant and epitope-based vaccines on the road to the market and implications for vaccine design and production.

    PubMed

    Oyarzún, Patricio; Kobe, Bostjan

    2016-03-03

    Novel vaccination approaches based on rational design of B- and T-cell epitopes - epitope-based vaccines - are making progress in the clinical trial pipeline. The epitope-focused recombinant protein-based malaria vaccine (termed RTS,S) is a next-generation approach that successfully reached phase-III trials, and will potentially become the first commercial vaccine against a human parasitic disease. Progress made on methods such as recombinant DNA technology, advanced cell-culture techniques, immunoinformatics and rational design of immunogens are driving the development of these novel concepts. Synthetic recombinant proteins comprising both B- and T-cell epitopes can be efficiently produced through modern biotechnology and bioprocessing methods, and can enable the induction of large repertoires of immune specificities. In particular, the inclusion of appropriate CD4+ T-cell epitopes is increasingly considered a key vaccine component to elicit robust immune responses, as suggested by results coming from HIV-1 clinical trials. In silico strategies for vaccine design are under active development to address genetic variation in pathogens and several broadly protective "universal" influenza and HIV-1 vaccines are currently at different stages of clinical trials. Other methods focus on improving population coverage in target populations by rationally considering specificity and prevalence of the HLA proteins, though a proof-of-concept in humans has not been demonstrated yet. Overall, we expect immunoinformatics and bioprocessing methods to become a central part of the next-generation epitope-based vaccine development and production process.

  10. A brief history of autism, the autism/vaccine hypothesis and a review of the genetic basis of autism spectrum disorders.

    PubMed

    Blake, Jerome; Hoyme, H Eugene; Crotwell, Patricia L

    2013-01-01

    Autism spectrum disorders (ASD) represent a common spectrum of developmental disabilities, sharing deficits in social interactions, communication and restricted interests or repetitive behaviors with difficult transitions. In this article, we review the history of the identification and classification of autism and the origin of the now widely-debunked autism/vaccine hypothesis. The differences between syndromal (complex) and non-syndromal (essential) autism are described and illustrated with case descriptions where appropriate. Finally, the evidence that autism is fundamentally a genetic disease is discussed, including family studies, the role of DNA copy number variation and known single gene mutations.

  11. Turning self-destructing Salmonella into a universal DNA vaccine delivery platform.

    PubMed

    Kong, Wei; Brovold, Matthew; Koeneman, Brian A; Clark-Curtiss, Josephine; Curtiss, Roy

    2012-11-20

    We previously developed a biological containment system using recombinant Salmonella Typhimurium strains that are attenuated yet capable of synthesizing protective antigens. The regulated delayed attenuation and programmed self-destructing features designed into these S. Typhimurium strains enable them to efficiently colonize host tissues and allow release of the bacterial cell contents after lysis. To turn such a recombinant attenuated Salmonella vaccine (RASV) strain into a universal DNA vaccine-delivery vehicle, our approach was to genetically modify RASV strains to display a hyperinvasive phenotype to maximize Salmonella host entry and host cell internalization, to enable Salmonella endosomal escape to release a DNA vaccine into the cytosol, and to decrease Salmonella-induced pyroptosis/apoptosis that allows the DNA vaccine time to traffic to the nucleus for efficient synthesis of encoded protective antigens. A DNA vaccine vector that encodes a domain that contributes to the arabinose-regulated lysis phenotype but has a eukaryotic promoter was constructed. The vector was then improved by insertion of multiple DNA nuclear-targeting sequences for efficient nuclear trafficking and gene expression, and by increasing nuclease resistance to protect the plasmid from host degradation. A DNA vaccine encoding influenza WSN virus HA antigen delivered by the RASV strain with the best genetic attributes induced complete protection to mice against a lethal influenza virus challenge. Adoption of these technological improvements will revolutionize means for effective delivery of DNA vaccines to stimulate mucosal, systemic, and cellular protective immunities, and lead to a paradigm shift in cost-effective control and prevention of a diversity of diseases.

  12. Turning self-destructing Salmonella into a universal DNA vaccine delivery platform

    PubMed Central

    Kong, Wei; Brovold, Matthew; Koeneman, Brian A.; Clark-Curtiss, Josephine; Curtiss, Roy

    2012-01-01

    We previously developed a biological containment system using recombinant Salmonella Typhimurium strains that are attenuated yet capable of synthesizing protective antigens. The regulated delayed attenuation and programmed self-destructing features designed into these S. Typhimurium strains enable them to efficiently colonize host tissues and allow release of the bacterial cell contents after lysis. To turn such a recombinant attenuated Salmonella vaccine (RASV) strain into a universal DNA vaccine-delivery vehicle, our approach was to genetically modify RASV strains to display a hyperinvasive phenotype to maximize Salmonella host entry and host cell internalization, to enable Salmonella endosomal escape to release a DNA vaccine into the cytosol, and to decrease Salmonella-induced pyroptosis/apoptosis that allows the DNA vaccine time to traffic to the nucleus for efficient synthesis of encoded protective antigens. A DNA vaccine vector that encodes a domain that contributes to the arabinose-regulated lysis phenotype but has a eukaryotic promoter was constructed. The vector was then improved by insertion of multiple DNA nuclear-targeting sequences for efficient nuclear trafficking and gene expression, and by increasing nuclease resistance to protect the plasmid from host degradation. A DNA vaccine encoding influenza WSN virus HA antigen delivered by the RASV strain with the best genetic attributes induced complete protection to mice against a lethal influenza virus challenge. Adoption of these technological improvements will revolutionize means for effective delivery of DNA vaccines to stimulate mucosal, systemic, and cellular protective immunities, and lead to a paradigm shift in cost-effective control and prevention of a diversity of diseases. PMID:23129620

  13. A New Whooping Cough Vaccine That May Prevent Colonization and Transmission.

    PubMed

    Brennan, Michael J

    2017-11-10

    This article is a Letter to the Editor. The major purpose of this Letter is to highlight the development of a new genetically altered whooping cough vaccine. Recently a baboon model has been used to show that this next generation pertussis vaccine can prevent colonization, as well as disease, and elicit antibodies against major pertussis antigens. Two phase I clinical trials have been performed, showing that this new vaccine is safe in humans, and a phase II trial will be performed in the US in 2018.

  14. rAAV Gene Therapy in a Canavan's Disease Mouse Model Reveals Immune Impairments and an Extended Pathology Beyond the Central Nervous System.

    PubMed

    Ahmed, Seemin Seher; Schattgen, Stefan A; Frakes, Ashley E; Sikoglu, Elif M; Su, Qin; Li, Jia; Hampton, Thomas G; Denninger, Andrew R; Kirschner, Daniel A; Kaspar, Brian; Matalon, Reuben; Gao, Guangping

    2016-06-01

    Aspartoacylase (AspA) gene mutations cause the pediatric lethal neurodegenerative Canavan disease (CD). There is emerging promise of successful gene therapy for CD using recombinant adeno-associated viruses (rAAVs). Here, we report an intracerebroventricularly delivered AspA gene therapy regime using three serotypes of rAAVs at a 20-fold reduced dose than previously described in AspA(-/-) mice, a bona-fide mouse model of CD. Interestingly, central nervous system (CNS)-restricted therapy prolonged survival over systemic therapy in CD mice but failed to sustain motor functions seen in systemically treated mice. Importantly, we reveal through histological and functional examination of untreated CD mice that AspA deficiency in peripheral tissues causes morphological and functional abnormalities in this heretofore CNS-defined disorder. We demonstrate for the first time that AspA deficiency, possibly through excessive N-acetyl aspartic acid accumulation, elicits both a peripheral and CNS immune response in CD mice. Our data establish a role for peripheral tissues in CD pathology and serve to aid the development of more efficacious and sustained gene therapy for this disease.

  15. 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

  16. Overview of recent DNA vaccine development for fish

    USGS Publications Warehouse

    Kurath, G.; ,

    2005-01-01

    Since the first description of DNA vaccines for fish in 1996, numerous studies of genetic immunisation against the rhabdovirus pathogens infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV) have established their potential as both highly efficacious biologicals and useful basic research tools. Single small doses of rhabdovirus DNA constructs provide extremely strong protection against severe viral challenge under a variety of conditions. DNA vaccines for several other important fish viruses, bacteria, and parasites are under investigation, but they have not yet shown high efficacy. Therefore, current research is focussed on mechanistic studies to understand the basis of protection, and on improvement of the nucleic acid vaccine applications against a wider range of fish pathogens.

  17. H5N1 vaccines in humans

    PubMed Central

    Baz, Mariana; Luke, Catherine J; Cheng, Xing; Jin, Hong; Subbarao, Kanta

    2013-01-01

    The spread of highly pathogenic avian H5N1 influenza viruses since 1997 and their virulence for poultry and humans has raised concerns about their potential to cause an influenza pandemic. Vaccines offer the most viable means to combat a pandemic threat. However, it will be a challenge to produce, distribute and implement a new vaccine if a pandemic spreads rapidly. Therefore, efforts are being undertaken to develop pandemic vaccines that use less antigen and induce cross-protective and long-lasting responses, that can be administered as soon as a pandemic is declared or possibly even before, in order to prime the population and allow for a rapid and protective antibody response. In the last few years, several vaccine manufacturers have developed candidate pandemic and pre-pandemic vaccines, based on reverse genetics and have improved the immunogenicity by formulating these vaccines with different adjuvants. Some of the important and consistent observations from clinical studies with H5N1 vaccines are as follows: two doses of inactivated vaccine are generally necessary to elicit the level of immunity required to meet licensure criteria, less antigen can be used if an oil-in-water adjuvant is included, in general antibody titers decline rapidly but can be boosted with additional doses of vaccine and if high titers of antibody are elicited, cross-reactivity against other clades is observed. Prime-boost strategies elicit a more robust immune response. In this review, we discuss data from clinical trials with a variety of H5N1 influenza vaccines. We also describe studies conducted in animal models to explore the possibility of reassortment between pandemic live attenuated vaccine candidates and seasonal influenza viruses, since this is an important consideration for the use of live vaccines in a pandemic setting. PMID:23726847

  18. Presence of mycobacterial L-forms in human blood: Challenge of BCG vaccination

    PubMed Central

    Markova, Nadya; Slavchev, Georgi; Michailova, Lilia

    2015-01-01

    Possible persistence of bacteria in human blood as cell wall deficient forms (L-forms) represents a top research priority for microbiologists. Application of live BCG vaccine and L-form transformation of vaccine strain may display a new intriguing aspect concerning the opportunity for occurrence of unpredictable colonization inside the human body by unusual microbial life forms. L-form cultures were isolated from 141 blood samples of people previously vaccinated with BCG, none with a history of exposure to tuberculosis. Innovative methodology to access the unusual L-form elements derived from human blood was developed. The methodology outlines the path of transformation of non- cultivable L-form element to cultivable bacteria and their adaptation for growth in vitro. All isolates showed typical L-forms growth features (“fried eggs” colonies and biofilm). Electron microscopy revealed morphology evidencing peculiar characteristics of bacterial L-form population (cell wall deficient polymorphic elements of variable shape and size). Regular detection of acid fast bacteria in smears of isolated blood L-form cultures, led us to start their identification by using specific Mycobactrium spp. genetic tests. Forty five of 97 genetically tested blood cultures provided specific positive signals for mycobacteria, confirmed by at least one of the 3 specific assays (16S rRNA PCR; IS6110 Real Time PCR and spoligotyping). In conclusion, the obtained genetic evidence suggests that these L-forms are of mycobacterial origin. As the investigated people had been vaccinated with BCG, we can assume that the identified mycobacterial L-forms may be produced by persisting live BCG vaccine. PMID:25874947

  19. Quantification of AAV particle titers by infrared fluorescence scanning of coomassie-stained sodium dodecyl sulfate-polyacrylamide gels.

    PubMed

    Kohlbrenner, Erik; Henckaerts, Els; Rapti, Kleopatra; Gordon, Ronald E; Linden, R Michael; Hajjar, Roger J; Weber, Thomas

    2012-06-01

    Adeno-associated virus (AAV)-based vectors have gained increasing attention as gene delivery vehicles in basic and preclinical studies as well as in human gene therapy trials. Especially for the latter two-for both safety and therapeutic efficacy reasons-a detailed characterization of all relevant parameters of the vector preparation is essential. Two important parameters that are routinely used to analyze recombinant AAV vectors are (1) the titer of viral particles containing a (recombinant) viral genome and (2) the purity of the vector preparation, most commonly assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining. An important, third parameter, the titer of total viral particles, that is, the combined titer of both genome-containing and empty viral capsids, is rarely determined. Here, we describe a simple and inexpensive method that allows the simultaneous assessment of both vector purity and the determination of the total viral particle titer. This method, which was validated by comparison with established methods to determine viral particle titers, is based on the fact that Coomassie Brilliant Blue, when bound to proteins, fluoresces in the infrared spectrum. Viral samples are separated by SDS-PAGE followed by Coomassie Brilliant Blue staining and gel analysis with an infrared laser-scanning device. In combination with a protein standard, our method allows the rapid and accurate determination of viral particle titers simultaneously with the assessment of vector purity.

  20. Poultry vaccination directed evolution of H9N2 low pathogenicity avian influenza viruses in Korea.

    PubMed

    Lee, Dong-Hun; Fusaro, Alice; Song, Chang-Seon; Suarez, David L; Swayne, David E

    2016-01-15

    Significant economic losses in the poultry industries have resulted from H9N2 low pathogenic avian influenza virus infections across North Africa, the Middle East and Asia. The present study investigated the evolutionary dynamics of H9N2 viruses circulating in Korea from 1996 to 2012. Our analysis of viral population dynamics revealed an increase in genetic diversity between the years 2003 and 2007, corresponding to the spread and diversification of H9N2 viruses into multiple genetic groups (named A and B), followed by a sudden decrease in 2007, which was associated with implementation of vaccination using a Clade A virus. Implementation of the H9N2 vaccination program in Korea has dramatically reduced the diversity of H9N2 virus, and only one sub-lineage of clade B has survived, expanded, and currently circulates in Korea. In addition, the antigenic drift of this new genetic group away from the current vaccine strain suggests the need to update the vaccine seed strain. Published by Elsevier Inc.

  1. [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.

  2. Excretion of Wild-Type and Vaccine-Derived Poliovirus in the Feces of Poliovirus Receptor-Transgenic Mice

    PubMed Central

    Boot, Hein J.; Kasteel, Daniella T. J.; Buisman, Anne-Marie; Kimman, Tjeerd G.

    2003-01-01

    The emergence of circulating vaccine-derived poliovirus (cVDPV) strains in suboptimally vaccinated populations is a serious threat to the global poliovirus eradication. The genetic determinants for the transmissibility phenotype of polioviruses, and in particularly of cVDPV strains, are currently unknown. Here we describe the fecal excretion of wild-type poliovirus, oral polio vaccine, and cVDPV (Hispaniola) strains after intraperitoneal injection in poliovirus receptor-transgenic mice. Both the pattern and the level of fecal excretion of the cVDPV strains resemble those of wild-type poliovirus type 1. In contrast, very little poliovirus was present in the feces after oral polio vaccine administration. This mouse model will be helpful in elucidating the genetic determinants for the high fecal-oral transmission phenotype of cVDPV strains. PMID:12743311

  3. Novel GMO-Based Vaccines against Tuberculosis: State of the Art and Biosafety Considerations.

    PubMed

    Leunda, Amaya; Baldo, Aline; Goossens, Martine; Huygen, Kris; Herman, Philippe; Romano, Marta

    2014-06-16

    Novel efficient vaccines are needed to control tuberculosis (TB), a major cause of morbidity and mortality worldwide. Several TB vaccine candidates are currently in clinical and preclinical development. They fall into two categories, the one of candidates designed as a replacement of the Bacille Calmette Guérin (BCG) to be administered to infants and the one of sub-unit vaccines designed as booster vaccines. The latter are designed as vaccines that will be administered to individuals already vaccinated with BCG (or in the future with a BCG replacement vaccine). In this review we provide up to date information on novel tuberculosis (TB) vaccines in development focusing on the risk assessment of candidates composed of genetically modified organisms (GMO) which are currently evaluated in clinical trials. Indeed, these vaccines administered to volunteers raise biosafety concerns with respect to human health and the environment that need to be assessed and managed.

  4. Engineering new mycobacterial vaccine design for HIV–TB pediatric vaccine vectored by lysine auxotroph of BCG

    PubMed Central

    Saubi, Narcís; Gea-Mallorquí, Ester; Ferrer, Pau; Hurtado, Carmen; Sánchez-Úbeda, Sara; Eto, Yoshiki; Gatell, Josep M; Hanke, Tomáš; Joseph, Joan

    2014-01-01

    In this study, we have engineered a new mycobacterial vaccine design by using an antibiotic-free plasmid selection system. We assembled a novel Escherichia coli (E. coli)–mycobacterial shuttle plasmid p2auxo.HIVA, expressing the HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism for plasmid selection and maintenance based on glycine complementation in E. coli and lysine complementation in mycobacteria. This plasmid was first transformed into glycine auxotroph of E. coli strain and subsequently transformed into lysine auxotroph of Mycobacterium bovis BCG strain to generate vaccine BCG.HIVA2auxo. We demonstrated that the episomal plasmid p2auxo.HIVA was stable in vivo over a 7-week period and genetically and phenotypically characterized the BCG.HIVA2auxo vaccine strain. The BCG.HIVA2auxo vaccine in combination with modified vaccinia virus Ankara (MVA). HIVA was safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. Polyfunctional HIV-1-specific CD8+ T cells, which produce interferon-γ and tumor necrosis factor-α and express the degranulation marker CD107a, were induced. Thus, we engineered a novel, safer, good laboratory practice–compatible BCG-vectored vaccine using prototype immunogen HIVA. This antibiotic-free plasmid selection system based on “double” auxotrophic complementation might be a new mycobacterial vaccine platform to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective response soon after birth. PMID:26015961

  5. Combating pertussis resurgence: One booster vaccination schedule does not fit all.

    PubMed

    Riolo, Maria A; Rohani, Pejman

    2015-02-03

    Pertussis has reemerged as a major public health concern in many countries where it was once considered well controlled. Although the mechanisms responsible for continued pertussis circulation and resurgence remain elusive and contentious, many countries have nevertheless recommended booster vaccinations, the timing and number of which vary widely. Here, using a stochastic, age-stratified transmission model, we searched for cost-effective booster vaccination strategies using a genetic algorithm. We did so assuming four hypothesized mechanisms underpinning contemporary pertussis epidemiology: (I) insufficient coverage, (II) frequent primary vaccine failure, (III) waning of vaccine-derived protection, and (IV) vaccine "leakiness." For scenarios I-IV, successful booster strategies were identified and varied considerably by mechanism. Especially notable is the inability of booster schedules to alleviate resurgence when vaccines are leaky. Critically, our findings argue that the ultimate effectiveness of vaccine booster schedules will likely depend on correctly pinpointing the causes of resurgence, with misdiagnosis of the problem epidemiologically ineffective and economically costly.

  6. High growth reassortant influenza vaccine viruses: new approaches to their control.

    PubMed

    Robertson, J S; Nicolson, C; Newman, R; Major, D; Dunleavy, U; Wood, J M

    1992-09-01

    When a new strain of an influenza virus is required to be incorporated into influenza vaccine, attempts are made to recombine such strains with laboratory adapted viruses, which will grow to high titre in order to improve the yield of the vaccine strain. It is important that such high growth reassortant vaccine strains are not contaminated with genes coding for the antigenic determinants of the high growth laboratory strain. We describe the characterization of two recent high growth reassortants and the application of the polymerase chain reaction to ensure their genetic identity and purity.

  7. Swimming against the current: genetic vaccination against Trypanosoma cruzi infection in mice.

    PubMed

    Rodrigues, Mauricio M; de Alencar, Bruna C; Claser, Carla; Tzelepis, Fanny; Silveira, Eduardo L; Haolla, Filipe A; Dominguez, Mariana R; Vasconcelos, José Ronnie

    2009-07-01

    Vaccines have had an unquestionable impact on public health during the last century. The most likely reason for the success of vaccines is the robust protective properties of specific antibodies. However, antibodies exert a strong selective pressure and many microorganisms, such as the obligatory intracellular parasite Trypanosoma cruzi, have been selected to survive in their presence. Although the host develops a strong immune response to T. cruzi, they do not clear the infection and instead progress to the chronic phase of the disease. Parasite persistence during the chronic phase of infection is now considered the main factor contributing to the chronic symptoms of the disease. Based on this finding, containment of parasite growth and survival may be one method to avoid the immunopathology of the chronic phase. In this context, vaccinologists have looked over the past 20 years for other immune effector mechanisms that could eliminate these antibody-resistant pathogens. We and others have tested the hypothesis that non-antibody-mediated cellular immune responses (CD4+ Th1 and CD8+ Tc1 cells) to specific parasite antigens/genes expressed by T. cruzi could indeed be used for the purpose of vaccination. This hypothesis was confirmed in different mouse models, indicating a possible path for vaccine development.

  8. Malaria vaccines: the case for a whole-organism approach.

    PubMed

    Pinzon-Charry, Alberto; Good, Michael F

    2008-04-01

    Malaria is a significant health problem causing morbidity and mortality worldwide. Vaccine development has been an imperative for decades. However, the intricacy of the parasite's lifecycle coupled with the lack of evidence for robust infection-induced immunity has made vaccine development exceptionally difficult. To review some of the key advances in the field and discuss potential ways forward for a whole-organism vaccine. The authors searched PubMed using the words 'malaria and vaccine'. We searched for manuscripts detailing antigen characterisation and vaccine strategies with emphasis on subunit versus whole-parasite approaches. Abstracts were selected and relevant articles are discussed. The searches were not restricted by language or date. The early cloning of malaria antigens has fuelled rapid development of subunit vaccines. However, the disappointing results of clinical trials have resulted in reappraisal of current strategies. Whole-parasite approaches have re-emerged as an alternative strategy. Immunization using radiation or genetically attenuated sporozoites has been shown to result in sterile immunity and immunization with blood-stage parasites curtailed by antimalarials has demonstrated delayed parasitemia in rodent models as well as in human malaria.

  9. Safety and durability of effect of contralateral-eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutations: a follow-on phase 1 trial.

    PubMed

    Bennett, Jean; Wellman, Jennifer; Marshall, Kathleen A; McCague, Sarah; Ashtari, Manzar; DiStefano-Pappas, Julie; Elci, Okan U; Chung, Daniel C; Sun, Junwei; Wright, J Fraser; Cross, Dominique R; Aravand, Puya; Cyckowski, Laura L; Bennicelli, Jeannette L; Mingozzi, Federico; Auricchio, Alberto; Pierce, Eric A; Ruggiero, Jason; Leroy, Bart P; Simonelli, Francesca; High, Katherine A; Maguire, Albert M

    2016-08-13

    Safety and efficacy have been shown in a phase 1 dose-escalation study involving a unilateral subretinal injection of a recombinant adeno-associated virus (AAV) vector containing the RPE65 gene (AAV2-hRPE65v2) in individuals with inherited retinal dystrophy caused by RPE65 mutations. This finding, along with the bilateral nature of the disease and intended use in treatment, prompted us to determine the safety of administration of AAV2-hRPE65v2 to the contralateral eye in patients enrolled in the phase 1 study. In this follow-on phase 1 trial, one dose of AAV2-hRPE65v2 (1.5 × 10(11) vector genomes) in a total volume of 300 μL was subretinally injected into the contralateral, previously uninjected, eyes of 11 children and adults (aged 11-46 years at second administration) with inherited retinal dystrophy caused by RPE65 mutations, 1.71-4.58 years after the initial subretinal injection. We assessed safety, immune response, retinal and visual function, functional vision, and activation of the visual cortex from baseline until 3 year follow-up, with observations ongoing. This study is registered with ClinicalTrials.gov, number NCT01208389. No adverse events related to the AAV were reported, and those related to the procedure were mostly mild (dellen formation in three patients and cataracts in two). One patient developed bacterial endophthalmitis and was excluded from analyses. We noted improvements in efficacy outcomes in most patients without significant immunogenicity. Compared with baseline, pooled analysis of ten participants showed improvements in mean mobility and full-field light sensitivity in the injected eye by day 30 that persisted to year 3 (mobility p=0.0003, white light full-field sensitivity p<0.0001), but no significant change was seen in the previously injected eyes over the same time period (mobility p=0.7398, white light full-field sensitivity p=0.6709). Changes in visual acuity from baseline to year 3 were not significant in pooled analysis in

  10. Immunoglobulin GM and KM genes and measles vaccine-induced humoral immunity.

    PubMed

    Ovsyannikova, Inna G; Larrabee, Beth R; Schaid, Daniel J; Poland, Gregory A

    2017-10-04

    Identifying genetic polymorphisms that explain variations in humoral immunity to live measles virus vaccine is of great interest. Immunoglobulin GM (heavy chain) and KM (light chain) allotypes are genetic markers known to be associated with susceptibility to several infectious diseases. We assessed associations between GM and KM genotypes and measles vaccine humoral immunity (neutralizing antibody titers) in a combined cohort (n=1796) of racially diverse healthy individuals (age 18-41years). We did not discover any significant associations between GM and/or KM genotypes and measles vaccine-induced neutralizing antibody titers. African-American subjects had higher neutralizing antibody titers than Caucasians (1260mIU/mL vs. 740mIU/mL, p=7.10×10 -13 ), and those titers remained statistically significant (p=1.68×10 -09 ) after adjusting for age at enrollment and time since last vaccination. There were no statistically significant sex-specific differences in measles-induced neutralizing antibody titers in our study (p=0.375). Our data indicate a surprising lack of evidence for an association between GM and KM genotypes and measles-specific neutralizing antibody titers, despite the importance of these immune response genes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. The kidneys and ANCA-associated vasculitis: from pathogenesis to diagnosis

    PubMed Central

    Rowaiye, Olumide Olatubosun; Kusztal, Mariusz; Klinger, Marian

    2015-01-01

    Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) are a group of pauci-immune small vessel vasculitides that often affect the kidneys manifesting as rapidly progressive glomerulonephritis. Although the exact pathogenesis of AAV is not fully known, evidence from in vitro, in vivo and clinical studies all point to the involvement of ANCA in the pathogenesis of AAV. In this review, we highlight the contributory roles played by various factors (e.g. genetics, environment, B and T-regulatory cells, toll-like receptors, etc.) in the pathogenesis of AAV. Furthermore, we discuss renal involvement in AAV in terms of clinical features and the various histopathological classification patterns, which are also known to be of prognostic importance. We also present information on useful imaging techniques for localizing kidney and other organ system involvement in AAV, and also on novel laboratory methods and assays useful for rapid and more specific determination of patients' ANCA status. Finally, we demonstrate evidence on novel serum biomarkers that have been shown to correlate with disease activity in AAV. PMID:26034600

  12. Vaccine adverse events in the new millennium: is there reason for concern?

    PubMed Central

    Ward, B. J.

    2000-01-01

    As more and more infectious agents become targets for immunization programmes, the spectrum of adverse events linked to vaccines has been widening. Although some of these links are tenuous, relatively little is known about the immunopathogenesis of even the best characterized vaccine-associated adverse events (VAAEs). The range of possible use of active immunization is rapidly expanding to include vaccines against infectious diseases that require cellular responses to provide protection (e.g. tuberculosis, herpes viral infections), therapeutic vaccines for chronic infections (e.g. human immunodeficiency virus (HIV) infection, viral hepatitis B and C), and vaccines against non-infectious conditions (e.g. cancer, autoimmune diseases). Less virulent pathogens (e.g. varicella, rotavirus in the developed world) are also beginning to be targeted, and vaccine use is being justified in terms of societal and parental "costs" rather than in straightforward morbidity and mortality costs. In the developed world the paediatric immunization schedule is becoming crowded, with pressure to administer increasing numbers of antigens simultaneously in ever simpler forms (e.g. subcomponent, peptide, and DNA vaccines). This trend, while attractive in many ways, brings hypothetical risks (e.g. genetic restriction, narrowed shield of protection, and loss of randomness), which will need to be evaluated and monitored. The available epidemiological and laboratory tools to address the issues outlined above are somewhat limited. As immunological and genetic tools improve in the years ahead, it is likely that we shall be able to explain the immunopathogenesis of many VAAEs and perhaps even anticipate and avoid some of them. However, this will only happen if the human and financial resources needed for monitoring and studying vaccine safety stay in step with the accelerating pace of vaccine development. Failure to make such a commitment would put all immunization programmes at risk. PMID

  13. [Eradication of poliomyelitis and emergence of pathogenic vaccine-derived polioviruses: from Madagascar to Cameroon].

    PubMed

    Delpeyroux, Francis; Colbère-Garapin, Florence; Razafindratsimandresy, Richter; Sadeuh-Mba, Serge; Joffret, Marie-Line; Rousset, Dominique; Blondel, Bruno

    2013-11-01

    The oral poliovaccine, a live vaccine made of attenuated poliovirus strains, is the main tool of the vaccination campaigns organised for eradicating poliomyelitis. these campaigns had led to the decline and, thereafter, to the disappearance of wild poliovirus strains of the three serotypes (1-3) in most parts of the world. However, when the poliovaccine coverage becomes too low, vaccine polioviruses can circulate in insufficiently immunized populations and become then pathogenic by mutations and genetic recombination with other enteroviruses of the same species, in particular some coxsackievirus A. These mutated and recombinant vaccine strains have been implicated in several epidemics of paralytic poliomyelitis. Two polio outbreaks associated with these pathogenic circulating vaccine-derived poliovirus (cVDPV) occurred in 2001-2002 and 2005 in the South of Madagascar where vaccine coverage was low. These cVDPV, of serotype 2 or 3, were isolated from paralyzed children and some of their healthy contacts. Other cVDPV were isolated in the same region from healthy children in 2011, indicating that these viruses were circulating again. Vaccination campaigns could stop the outbreaks in 2002 and 2005, and most probably prevent another one in 2011. Therefore, the genetic plasticity of poliovaccine strains that threatens the benefit of vaccination campaigns is the target of an accurate surveillance and an important theme of studies in the virology laboratories of the Institut Pasteur international network. © 2013 médecine/sciences – Inserm.

  14. Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines.

    PubMed

    Celma, Cristina C; Stewart, Meredith; Wernike, Kerstin; Eschbaumer, Michael; Gonzalez-Molleda, Lorenzo; Breard, Emmanuel; Schulz, Claudia; Hoffmann, Bernd; Haegeman, Andy; De Clercq, Kris; Zientara, Stephan; van Rijn, Piet A; Beer, Martin; Roy, Polly

    2017-01-01

    Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV. Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new approaches to produce

  15. Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines

    PubMed Central

    Celma, Cristina C.; Stewart, Meredith; Wernike, Kerstin; Eschbaumer, Michael; Gonzalez-Molleda, Lorenzo; Breard, Emmanuel; Schulz, Claudia; Hoffmann, Bernd; Haegeman, Andy; De Clercq, Kris; Zientara, Stephan; van Rijn, Piet A.; Beer, Martin

    2016-01-01

    ABSTRACT Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV. IMPORTANCE Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new

  16. Peptide nanofiber hydrogel adjuvanted live virus vaccine enhances cross-protective immunity to porcine reproductive and respiratory syndrome virus

    PubMed Central

    Li, Xiangdong; Galliher-Beckley, Amy; Huang, Hongzhou; Sun, Xiuzhi; Shi, Jishu

    2013-01-01

    Porcine reproductive and respiratory syndrome virus (PRRSV) is prevalent in swine farms worldwide and is a major source of economic loss and animal suffering. Rapid genetic variation of PRRSV makes it difficult for current vaccines to confer protection against newly emerging strains. We recently demonstrated that a novel peptide nanofiber hydrogel (H9e) could act as a potent adjuvant for killed H1N1 vaccines. Therefore, the objective of this study was to evaluate H9e as an adjuvant for PRRSV modified live virus (MLV) vaccines. Pigs were vaccinated with Ingelvac PRRSV MLV with or without H9e adjuvant before being challenged with the VR-2332 (parental vaccine strain) or MN184A (genetically diverse strain) PRRSV. Pigs vaccinated with MLV+H9e had higher levels of circulating vaccine virus. More importantly, pigs vaccinated with MLV+H9e had improved protection against challenge by both PRRSV strains, as demonstrated by reduced challenge-induced viremia compared with pigs vaccinated with MLV alone. Pigs vaccinated with MLV+H9e had lower frequency of T-regulatory cells and IL-10 production but higher frequency of Th/memory cells and IFN-γ secretion than that in pigs vaccinated with MLV alone. Taken together, our studies suggest that the peptide nanofiber hydrogel H9e, when combined with the PRRSV MLV vaccine, can enhance vaccine efficacy against two different PRRSV strains by modulating both host humoral and cellular immune responses. PMID:23933333

  17. Complete Protection against Pneumonic and Bubonic Plague after a Single Oral Vaccination

    PubMed Central

    Derbise, Anne; Hanada, Yuri; Khalifé, Manal; Carniel, Elisabeth; Demeure, Christian E.

    2015-01-01

    Background No efficient vaccine against plague is currently available. We previously showed that a genetically attenuated Yersinia pseudotuberculosis producing the Yersinia pestis F1 antigen was an efficient live oral vaccine against pneumonic plague. This candidate vaccine however failed to confer full protection against bubonic plague and did not produce F1 stably. Methodology/Principal Findings The caf operon encoding F1 was inserted into the chromosome of a genetically attenuated Y. pseudotuberculosis, yielding the VTnF1 strain, which stably produced the F1 capsule. Given orally to mice, VTnF1 persisted two weeks in the mouse gut and induced a high humoral response targeting both F1 and other Y. pestis antigens. The strong cellular response elicited was directed mostly against targets other than F1, but also against F1. It involved cells with a Th1—Th17 effector profile, producing IFNγ, IL-17, and IL-10. A single oral dose (108 CFU) of VTnF1 conferred 100% protection against pneumonic plague using a high-dose challenge (3,300 LD50) caused by the fully virulent Y. pestis CO92. Moreover, vaccination protected 100% of mice from bubonic plague caused by a challenge with 100 LD50 Y. pestis and 93% against a high-dose infection (10,000 LD50). Protection involved fast-acting mechanisms controlling Y. pestis spread out of the injection site, and the protection provided was long-lasting, with 93% and 50% of mice surviving bubonic and pneumonic plague respectively, six months after vaccination. Vaccinated mice also survived bubonic and pneumonic plague caused by a high-dose of non-encapsulated (F1-) Y. pestis. Significance VTnF1 is an easy-to-produce, genetically stable plague vaccine candidate, providing a highly efficient and long-lasting protection against both bubonic and pneumonic plague caused by wild type or un-encapsulated (F1-negative) Y. pestis. To our knowledge, VTnF1 is the only plague vaccine ever reported that could provide high and durable protection

  18. Lymph node fibrosis: a structural barrier to unleashing effective vaccine immunity.

    PubMed

    Julg, Boris; Alter, Galit

    2018-05-21

    There is marked variability in vaccine efficacy among global populations. In particular, individuals in low- to middle-income countries have been shown to be less responsive to vaccines than those from developed nations. Several factors, including endemic infections, nutrition, genetics, and gut microbiome composition, have been proposed to underlie discrepancies in vaccine response. In this issue of the JCI, Kityo et al. evaluated response to yellow fever virus vaccine, inflammation, and lymphatic tissue architecture and fibrosis in three cohorts: two from the U.S. and one from Uganda. Compared with the U.S. subjects, the Ugandan cohort exhibited enhanced cytokine responses, increased lymph node fibrosis, reduced CD4+ T cell levels, and reduced vaccine response. Together, these results provide a link among chronic inflammation, damaged lymphoid architecture, and poor vaccine outcome, and set the stage for future studies to identify strategies to overcome these barriers.

  19. Novel GMO-Based Vaccines against Tuberculosis: State of the Art and Biosafety Considerations

    PubMed Central

    Leunda, Amaya; Baldo, Aline; Goossens, Martine; Huygen, Kris; Herman, Philippe; Romano, Marta

    2014-01-01

    Novel efficient vaccines are needed to control tuberculosis (TB), a major cause of morbidity and mortality worldwide. Several TB vaccine candidates are currently in clinical and preclinical development. They fall into two categories, the one of candidates designed as a replacement of the Bacille Calmette Guérin (BCG) to be administered to infants and the one of sub-unit vaccines designed as booster vaccines. The latter are designed as vaccines that will be administered to individuals already vaccinated with BCG (or in the future with a BCG replacement vaccine). In this review we provide up to date information on novel tuberculosis (TB) vaccines in development focusing on the risk assessment of candidates composed of genetically modified organisms (GMO) which are currently evaluated in clinical trials. Indeed, these vaccines administered to volunteers raise biosafety concerns with respect to human health and the environment that need to be assessed and managed. PMID:26344627

  20. Identification of a cytoplasmic interaction partner of the large regulatory proteins Rep78/Rep68 of adeno-associated virus type 2 (AAV-2)

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

    Weger, Stefan; Hammer, Eva; Goetz, Anne

    2007-05-25

    Through yeast two-hybrid analysis and coimmunoprecipitation studies, we have identified a novel cellular AAV-2 Rep78/Rep68 interaction partner located predominantly in the cytoplasm. In public databases, it has been assigned as KCTD5, because of a region of high similarity to the cytoplasmic tetramerization domain of voltage-gated potassium channels. Whereas Rep/KCTD5 interaction relied on the region surrounding the Rep nuclear localization signal, nuclear accumulation of Rep was not required. Wildtype Rep78/Rep68 proteins induced the translocation of large portions of KCTD5 into the nucleus pointing to functional interactions both in the cytoplasm and the nucleus. In line with an anticipated functional interference inmore » the cytoplasm, KCTD5 overexpression completely abrogated Rep68-mediated posttranscriptional activation of a HIV-LTR driven luciferase reporter gene. Our study expands the panel of already identified nuclear Rep interaction partners to a cytoplasmic protein, which raises the awareness that important steps in the AAV life cycle may be regulated in this compartment.« less

  1. Genetic diversity of avian avulavirus 1 (Newcastle disease virus genotypes VIg and VIIb) circulating in wild birds in Kazakhstan.

    PubMed

    Orynbayev, Mukhit B; Fereidouni, Sasan; Sansyzbai, Abylay R; Seidakhmetova, Bakhytkul A; Strochkov, Vitaliy M; Nametov, Askar M; Sadikaliyeva, Sandugash O; Nurgazieva, Asel; Tabynov, Kaissar K; Rametov, Nurkuysa M; Sultankulova, Kulyaisan T

    2018-03-20

    In order to improve current understanding of the molecular epidemiology of avian avulavirus 1 (AAvV-1, formerly avian paramyxovirus 1) in wild birds in Kazakhstan, 860 cloacal swab samples were evaluated. Samples were collected from 37 families of wild birds in nine different regions in the years 2011 and 2014. Overall, 54 positive samples (4.2%) were detected from 17 different families of wild birds, and 16 AAvV-1 isolates were characterized. Three of the isolates contained the fusion protein cleavage site motif RRQKR, and 13 contained KRQKR, which is typical for pathogenic strains of AAvV-1. The AAvV-1 isolates were found to belong to the genotypes VIg and VIIb.

  2. Generation of influenza A viruses as live but replication-incompetent virus vaccines.

    PubMed

    Si, Longlong; Xu, Huan; Zhou, Xueying; Zhang, Ziwei; Tian, Zhenyu; Wang, Yan; Wu, Yiming; Zhang, Bo; Niu, Zhenlan; Zhang, Chuanling; Fu, Ge; Xiao, Sulong; Xia, Qing; Zhang, Lihe; Zhou, Demin

    2016-12-02

    The conversion of life-threatening viruses into live but avirulent vaccines represents a revolution in vaccinology. In a proof-of-principle study, we expanded the genetic code of the genome of influenza A virus via a transgenic cell line containing orthogonal translation machinery. This generated premature termination codon (PTC)-harboring viruses that exerted full infectivity but were replication-incompetent in conventional cells. Genome-wide optimization of the sites for incorporation of multiple PTCs resulted in highly reproductive and genetically stable progeny viruses in transgenic cells. In mouse, ferret, and guinea pig models, vaccination with PTC viruses elicited robust humoral, mucosal, and T cell-mediated immunity against antigenically distinct influenza viruses and even neutralized existing infecting strains. The methods presented here may become a general approach for generating live virus vaccines that can be adapted to almost any virus. Copyright © 2016, American Association for the Advancement of Science.

  3. Vaccines for leishmaniasis in the fore coming 25 years.

    PubMed

    Palatnik-de-Sousa, Clarisa B

    2008-03-25

    Human vaccination against leishmaniasis using live Leishmania was used in Middle East and Russia (1941-1980). First-generation vaccines, composed by killed parasites induce low efficacies (54%) and were tested in humans and dogs Phase III trials in Asia and South America since 1940. Second-generation vaccines using live genetically modified parasites, or bacteria or viruses containing Leishmania genes, recombinant or native fractions are known since the 1990s. Due to the loss of PAMPs, the use of adjuvants increased vaccine efficacies of the purified antigens to 82%, in Phase III dog trials. Recombinant second-generation vaccines and third-generation DNA vaccines showed average values of parasite load reduction of 68% and 59% in laboratory animal models, respectively, but their success in field trials had not yet been reported. This review is focused on vaccine candidates that show any efficacy against leishmaniasis and that are already in different phase trials. A lot of interest though was generated in recent years, by the studies going on in experimental models. The promising candidates may find a place in the forth coming years. Among them most probably are the multiple-gene DNA vaccines that are stable and do not require cold-chain transportation. In the mean time, second-generation vaccines with native antigens and effective adjuvants are likely to be licensed and used in Public Health control programs in the fore coming 25 years. To date, only three vaccines have been licensed for use: one live vaccine for humans in Uzbekistan, one killed vaccine for human immunotherapy in Brazil and a second-generation vaccine for dog prophylaxis in Brazil.

  4. Early exposure to the combined measles-mumps-rubella vaccine and thimerosal-containing vaccines and risk of autism spectrum disorder.

    PubMed

    Uno, Yota; Uchiyama, Tokio; Kurosawa, Michiko; Aleksic, Branko; Ozaki, Norio

    2015-05-15

    This case-control study investigated the relationship between the risk of Autism Spectrum Disorder (ASD) onset, and early exposure to the combined Measles-Mumps-Rubella (MMR) vaccine and thimerosal consumption measured from vaccinations in the highly genetically homogenous Japanese population. Vaccination histories at 1, 3, 6, 12, 18, 24, and 36 months from birth were investigated in ASD cases (189 samples), and controls (224 samples) matching age and sex in each case. Crude odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated to determine relationship between MMR vaccination and ASD. The differences in mean values of the thimerosal dosage between cases and controls were analyzed using an unpaired t-test. MMR vaccination and thimerosal dosage were also investigated using a conditional multiple-regression model. There were no significant differences in MMR vaccination and thimerosal dosage between cases and controls at any age. Furthermore, the ORs (95% CIs) of MMR vaccination and thimerosal dosage associated with ASD in the conditional multiple regression model were, respectively, 0.875 (0.345-2.222) and 1.205 (0.862-1.683) at age 18 months, 0.724 (0.421-1.243) and 1.343 (0.997-1.808) at 24 months, and 1.040 (0.648-1.668) and 0.844 (0.632-1.128) at 36 months. Thus, there were no significant differences. No convincing evidence was found in this study that MMR vaccination and increasing thimerosal dose were associated with an increased risk of ASD onset. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. 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

  6. AAV serotype 2/1-mediated gene delivery of anti-inflammatory interleukin-10 enhances neurogenesis and cognitive function in APP+PS1 mice.

    PubMed

    Kiyota, T; Ingraham, K L; Swan, R J; Jacobsen, M T; Andrews, S J; Ikezu, T

    2012-07-01

    Brain inflammation is a double-edged sword. It is required for brain repair in acute damage, whereas chronic inflammation and autoimmune disorders are neuropathogenic. Certain proinflammatory cytokines and chemokines are closely related to cognitive dysfunction and neurodegeneration. Representative anti-inflammatory cytokines, such as interleukin (IL)-10, can suppress neuroinflammation and have significant therapeutic potentials in ameliorating neurodegenerative disorders such as Alzheimer's disease (AD). Here, we show that adeno-associated virus (AAV) serotype 2/1 hybrid-mediated neuronal expression of the mouse IL-10 gene ameliorates cognitive dysfunction in amyloid precursor protein+ presenilin-1 bigenic mice. AAV2/1 infection of hippocampal neurons resulted in sustained expression of IL-10 without its leakage into the blood, reduced astro/microgliosis, enhanced plasma amyloid-β peptide (Aβ) levels and enhanced neurogenesis. Moreover, increased levels of IL-10 improved spatial learning, as determined by the radial arm water maze. Finally, IL-10-stimulated microglia enhanced proliferation but not differentiation of primary neural stem cells in the co-culture system, whereas IL-10 itself had no effect. Our data suggest that IL-10 gene delivery has a therapeutic potential for a non-Aβ-targeted treatment of AD.

  7. Induction of sustained hypercholesterolemia by single adeno-associated virus-mediated gene transfer of mutant hPCSK9.

    PubMed

    Roche-Molina, Marta; Sanz-Rosa, David; Cruz, Francisco M; García-Prieto, Jaime; López, Sergio; Abia, Rocío; Muriana, Francisco J G; Fuster, Valentín; Ibáñez, Borja; Bernal, Juan A

    2015-01-01

    Patients with mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene have hypercholesterolemia and are at high risk of adverse cardiovascular events. We aimed to stably express the pathological human D374Y gain-of-function mutant form of PCSK9 (PCSK9(DY)) in adult wild-type mice to generate a hyperlipidemic and proatherogenic animal model, achieved with a single systemic injection with adeno-associated virus (AAV). We constructed an AAV-based vector to support targeted transfer of the PCSK9(DY) gene to liver. After injection with 3.5×10(10) viral particles, mice in the C57BL/6J, 129/SvPasCrlf, or FVB/NCrl backgrounds developed long-term hyperlipidemia with a strong increase in serum low-density lipoprotein. Macroscopic and histological analysis showed atherosclerotic lesions in the aortas of AAV-PCSK9(DY) mice fed a high-fat-diet. Advanced lesions in these high-fat-diet-fed mice also showed evidence of macrophage infiltration and fibrous cap formation. Hepatic AAV-PCSK9(DY) infection did not result in liver damage or signs of immunologic response. We further tested the use of AAV-PCSK9(DY) to study potential genetic interaction with the ApoE gene. Histological analysis of ApoE(-/-) AAV-PCSK9(DY) mice showed a synergistic response to ApoE deficiency, with aortic lesions twice as extensive in ApoE(-/-) AAV-PCSK9(DY)-transexpressing mice as in ApoE(-/-) AAV-Luc controls without altering serum cholesterol levels. Single intravenous AAV-PCSK9(DY) injection is a fast, easy, and cost-effective approach, resulting in rapid and long-term sustained hyperlipidemia and atherosclerosis. We demonstrate as a proof of concept the synergy between PCSK9(DY) gain-of-function and ApoE deficiency. This methodology could allow testing of the genetic interaction of several mutations without the need for complex and time-consuming backcrosses. © 2014 American Heart Association, Inc.

  8. Evidence for viral virulence as a predominant factor limiting human immunodeficiency virus vaccine efficacy.

    PubMed

    Mooij, P; Bogers, W M; Oostermeijer, H; Koornstra, W; Ten Haaft, P J; Verstrepen, B E; Van Der Auwera, G; Heeney, J L

    2000-05-01

    Current strategies in human immunodeficiency virus type 1 (HIV-1) vaccine development are often based on the production of different vaccine antigens according to particular genetic clades of HIV-1 variants. To determine if virus virulence or genetic distance had a greater impact on HIV-1 vaccine efficacy, we designed a series of heterologous chimeric simian/human immunodeficiency virus (SHIV) challenge experiments in HIV-1 subunit-vaccinated rhesus macaques. Of a total of 22 animals, 10 nonimmunized animals served as controls; the remainder were vaccinated with the CCR5 binding envelope of HIV-1(W6.1D). In the first study, heterologous challenge included two nonpathogenic SHIV chimeras encoding the envelopes of the divergent clade B HIV-1(han2) and HIV-1(sf13) strains. In the second study, all immunized animals were rechallenged with SHIV(89. 6p), a virus closely related to the vaccine strain but highly virulent. Protection from either of the divergent SHIV(sf13) or SHIV(han2) challenges was demonstrated in the majority of the vaccinated animals. In contrast, upon challenge with the more related but virulent SHIV(89.6p), protection was achieved in only one of the previously protected vaccinees. A secondary but beneficial effect of immunization on virus load and CD4(+) T-cell counts was observed despite failure to protect from infection. In addition to revealing different levels of protective immunity, these results suggest the importance of developing vaccine strategies capable of protecting from particularly virulent variants of HIV-1.

  9. Broad protection against influenza infection by vectored immunoprophylaxis in mice

    PubMed Central

    Balazs, Alejandro B.; Bloom, Jesse D.; Hong, Christin M.; Rao, Dinesh S.; Baltimore, David

    2014-01-01

    Neutralizing antibodies that target epitopes conserved among many strains of influenza virus have been recently isolated from humans. Here we demonstrate that adeno-associated viruses (AAV) encoding two such broadly neutralizing antibodies are protective against diverse influenza strains. Serum from mice that received a single intramuscular AAV injection efficiently neutralized all H1, H2 and H5 influenza strains tested. After infection with diverse strains of H1N1 influenza, treated mice showed minimal weight loss and lung inflammation. Protection lasted for at least 11 months after AAV injection. Notably, even immunodeficient and older mice were protected by this method, suggesting that expression of a monoclonal antibody alone is sufficient to protect mice from illness. If translated to humans, this prophylactic approach may be uniquely capable of protecting immunocompromised or elderly patient populations not reliably protected by existing vaccines. PMID:23728362

  10. The post-vaccine microevolution of invasive Streptococcus pneumoniae

    PubMed Central

    Cremers, Amelieke J. H.; Mobegi, Fredrick M.; de Jonge, Marien I.; van Hijum, Sacha A. F. T.; Meis, Jacques F.; Hermans, Peter W. M.; Ferwerda, Gerben; Bentley, Stephen D.; Zomer, Aldert L.

    2015-01-01

    The 7-valent pneumococcal conjugated vaccine (PCV7) has affected the genetic population of Streptococcus pneumoniae in pediatric carriage. Little is known however about pneumococcal population genomics in adult invasive pneumococcal disease (IPD) under vaccine pressure. We sequenced and serotyped 349 strains of S. pneumoniae isolated from IPD patients in Nijmegen between 2001 and 2011. Introduction of PCV7 in the Dutch National Immunization Program in 2006 preluded substantial alterations in the IPD population structure caused by serotype replacement. No evidence could be found for vaccine induced capsular switches. We observed that after a temporary bottleneck in gene diversity after the introduction of PCV7, the accessory gene pool re-expanded mainly by genes already circulating pre-PCV7. In the post-vaccine genomic population a number of genes changed frequency, certain genes became overrepresented in vaccine serotypes, while others shifted towards non-vaccine serotypes. Whether these dynamics in the invasive pneumococcal population have truly contributed to invasiveness and manifestations of disease remains to be further elucidated. We suggest the use of whole genome sequencing for surveillance of pneumococcal population dynamics that could give a prospect on the course of disease, facilitating effective prevention and management of IPD. PMID:26492862

  11. Virus like particle-based vaccines against emerging infectious disease viruses.

    PubMed

    Liu, Jinliang; Dai, Shiyu; Wang, Manli; Hu, Zhihong; Wang, Hualin; Deng, Fei

    2016-08-01

    Emerging infectious diseases are major threats to human health. Most severe viral disease outbreaks occur in developing regions where health conditions are poor. With increased international travel and business, the possibility of eventually transmitting infectious viruses between different countries is increasing. The most effective approach in preventing viral diseases is vaccination. However, vaccines are not currently available for numerous viral diseases. Virus-like particles (VLPs) are engineered vaccine candidates that have been studied for decades. VLPs are constructed by viral protein expression in various expression systems that promote the selfassembly of proteins into structures resembling virus particles. VLPs have antigenicity similar to that of the native virus, but are non-infectious as they lack key viral genetic material. VLP vaccines have attracted considerable research interest because they offer several advantages over traditional vaccines. Studies have shown that VLP vaccines can stimulate both humoral and cellular immune responses, which may offer effective antiviral protection. Here we review recent developments with VLP-based vaccines for several highly virulent emerging or re-emerging infectious diseases. The infectious agents discussed include RNA viruses from different virus families, such as the Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Togaviridae families.

  12. Variable Virulence and Efficacy of BCG Vaccine Strains in Mice and Correlation With Genome Polymorphisms

    PubMed Central

    Zhang, Lu; Ru, Huan-wei; Chen, Fu-zeng; Jin, Chun-yan; Sun, Rui-feng; Fan, Xiao-yong; Guo, Ming; Mai, Jun-tao; Xu, Wen-xi; Lin, Qing-xia; Liu, Jun

    2016-01-01

    Bacille Calmette–Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the only vaccine available for tuberculosis (TB) control. However, BCG is not an ideal vaccine and has two major limitations: BCG exhibits highly variable effectiveness against the development of TB both in pediatric and adult populations and can cause disseminated BCG disease in immunocompromised individuals. BCG comprises a number of substrains that are genetically distinct. Whether and how these genetic differences affect BCG efficacy remains largely unknown. In this study, we performed comparative analyses of the virulence and efficacy of 13 BCG strains, representing different genetic lineages, in SCID and BALB/c mice. Our results show that BCG strains of the DU2 group IV (BCG-Phipps, BCG-Frappier, BCG-Pasteur, and BCG-Tice) exhibit the highest levels of virulence, and BCG strains of the DU2 group II (BCG-Sweden, BCG-Birkhaug) are among the least virulent group. These distinct levels of virulence may be explained by strain-specific duplications and deletions of genomic DNA. There appears to be a general trend that more virulent BCG strains are also more effective in protection against Mycobacterium tuberculosis challenge. Our findings have important implications for current BCG vaccine programs and for future TB vaccine development. PMID:26643797

  13. Variable Virulence and Efficacy of BCG Vaccine Strains in Mice and Correlation With Genome Polymorphisms.

    PubMed

    Zhang, Lu; Ru, Huan-Wei; Chen, Fu-Zeng; Jin, Chun-Yan; Sun, Rui-Feng; Fan, Xiao-Yong; Guo, Ming; Mai, Jun-Tao; Xu, Wen-Xi; Lin, Qing-Xia; Liu, Jun

    2016-02-01

    Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the only vaccine available for tuberculosis (TB) control. However, BCG is not an ideal vaccine and has two major limitations: BCG exhibits highly variable effectiveness against the development of TB both in pediatric and adult populations and can cause disseminated BCG disease in immunocompromised individuals. BCG comprises a number of substrains that are genetically distinct. Whether and how these genetic differences affect BCG efficacy remains largely unknown. In this study, we performed comparative analyses of the virulence and efficacy of 13 BCG strains, representing different genetic lineages, in SCID and BALB/c mice. Our results show that BCG strains of the DU2 group IV (BCG-Phipps, BCG-Frappier, BCG-Pasteur, and BCG-Tice) exhibit the highest levels of virulence, and BCG strains of the DU2 group II (BCG-Sweden, BCG-Birkhaug) are among the least virulent group. These distinct levels of virulence may be explained by strain-specific duplications and deletions of genomic DNA. There appears to be a general trend that more virulent BCG strains are also more effective in protection against Mycobacterium tuberculosis challenge. Our findings have important implications for current BCG vaccine programs and for future TB vaccine development.

  14. Safety and durability of effect of contralateral-eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutatons: a follow-on phase 1 trial

    PubMed Central

    Bennett, Jean; Wellman, Jennifer; Marshall, Kathleen A; McCague, Sarah; Ashtari, Manzar; DiStefano-Pappas, Julie; Elci, Okan U; Chung, Daniel C; Sun, Junwei; Wright, J Fraser; Cross, Dominique R; Aravand, Puya; Cyckowski, Laura L; Bennicelli, Jeannette L; Mingozzi, Federico; Auricchio, Alberto; Pierce, Eric A; Ruggiero, Jason; Leroy, Bart P; Simonelli, Francesca; High, Katherine A; Maguire, Albert M

    2017-01-01

    Summary Background Safety and efficacy have been shown in a phase 1 dose-escalation study involving a unilateral subretinal injection of a recombinant adeno-associated virus (AAV) vector containing the RPE65 gene (AAV2-hRPE65v2) in individuals with inherited retinal dystrophy caused by RPE65 mutations. This finding, along with the bilateral nature of the disease and intended use in treatment, prompted us to determine the safety of administration of AAV2-hRPE65v2 to the contralateral eye in patients enrolled in the phase 1 study. Methods In this follow-on phase 1 trial, one dose of AAV2-hRPE65v2 (1·5 × 1011 vector genomes) in a total volume of 300 μL was subretinally injected into the contralateral, previously uninjected, eyes of 11 children and adults (aged 11–46 years at second administration) with inherited retinal dystrophy caused by RPE65 mutations, 1·71–4·58 years after the initial subretinal injection. We assessed safety, immune response, retinal and visual function, functional vision, and activation of the visual cortex from baseline until 3 year follow-up, with observations ongoing. This study is registered with ClinicalTrials.gov, number NCT01208389. Findings No adverse events related to the AAV were reported, and those related to the procedure were mostly mild (dellen formation in three patients and cataracts in two). One patient developed bacterial endophthalmitis and was excluded from analyses. We noted improvements in efficacy outcomes in most patients without significant immunogenicity. Compared with baseline, pooled analysis of ten participants showed improvements in mean mobility and full-field light sensitivity in the injected eye by day 30 that persisted to year 3 (mobility p=0·0003, white light full-field sensitivity p<0·0001), but no significant change was seen in the previously injected eyes over the same time period (mobility p=0·7398, white light full-field sensitivity p=0·6709). Changes in visual acuity from baseline to year 3

  15. Specific Genetic Immunotherapy Induced by Recombinant Vaccine Alpha-Fetoprotein-Heat Shock Protein 70 Complex

    NASA Astrophysics Data System (ADS)

    Wang, Xiaoping; Lin, Huanping; Wang, Qiaoxia

    Purposes: To construct a recombinant vaccine alpha-fetoprotein (AFP)-heat shock protein (HSP70) complex, and study its ability to induce specific CTL response and its protective effect against AFP-producing tumor. Material/Methods: A recombinant vaccine was constructed by conjugating mouse alpha-fetoprotein to heat shock protein 70. By way of intracutaneous injection, mice were primed and boosted with recombinant vaccine mAFP/HSP70, whereas single mAFP or HSP70 injection as controls. The ELISPOT and ELISA were used to measure the frequency of cells producing the cytokine IFN-γ in splenocytes and the level of anti-AFP antibody of serum from immunized mice respectively. In vivo tumor challenge were carried out to assess the immune effect of the recombinant vaccine. Results: By recombinant mAFP/HSP70 vaccine immunization, the results of ELISPOT and ELISA showed that the number of splenic cells producing IFN-γ and the level of anti-AFP antibody of serum were significantly higher in mAFP/HSP70 group than those in mAFP and HSP70 groups (108.50±11.70 IFN-γ spots/106 cells vs 41.60±10.40 IFN-γ spots/106 cells, 7.32±3.14 IFN-γ spots/106 cells, P<0.01; 156.32±10.42 μg/mL vs 66.52±7.35 μg/mL, 5.73±2.89 μg/mL, P<0.01). The tumor volume in mAFP/HSP70 group was significantly smaller than that in mAFP and HSP70 groups (42.44±7.14 mm3 vs 392.23±12.46 mm3, 838.63±13.84 mm3, P<0.01). Conclusions: The study further confirmed the function of heat shock protein 70's immune adjuvant. Sequential immunization with recombinant mAFP/HSP70 vaccine could generate effective antitumor immunity on AFP-producing tumor. The recombined mAFP/HSP70 vaccine may be suitable for serving as an immunotherapy for hepatocellular carcinoma.

  16. Conditional genetic deletion of PTEN after a spinal cord injury enhances regenerative growth of CST axons and motor function recovery in mice

    PubMed Central

    Danilov, Camelia A.; Steward, Oswald

    2015-01-01

    Previous studies indicate that conditional genetic deletion of phosphatase and tensin homolog (PTEN) in neonatal mice enhances the ability of axons to regenerate following spinal cord injury (SCI) in adults. Here, we assessed whether deleting PTEN in adult neurons post-SCI is also effective, and whether enhanced regenerative growth is accompanied by enhanced recovery of voluntary motor function. PTENloxP/loxP mice received moderate contusion injuries at cervical level 5 (C5). One group received unilateral injections of adeno-associated virus expressing CRE (AAV-CRE) into the sensorimotor cortex; controls received a vector expressing green fluorescent protein (AAV-GFP) or injuries only (no vector injections). Forelimb function was tested for 14 weeks post-SCI using a grip strength meter (GSM) and a hanging task. The corticospinal tract (CST) was traced by injecting mini-ruby BDA into the sensorimotor cortex. Forelimb gripping ability was severely impaired immediately post-SCI but recovered slowly over time. The extent of recovery was significantly greater in PTEN-deleted mice in comparison to either the AAV-GFP group or the injury only group. BDA tract tracing revealed significantly higher numbers of BDA-labeled axons in caudal segments in the PTEN-deleted group compared to control groups. In addition, in the PTEN-deleted group, there were exuberant collaterals extending from the main tract rostral to the lesion, into and around the scar tissue at the injury site. These results indicate that PTEN deletion in adult mice shortly post-SCI can enhance regenerative growth of CST axons and forelimb motor function recovery. PMID:25704959

  17. A study of vaccine-induced immune pressure on breakthrough infections in the Phambili phase 2b HIV-1 vaccine efficacy trial

    PubMed Central

    Rolland, M.; Magaret, C.A.; Rademeyer, C.; Fiore-Gartland, A.; Edlefsen, P.T.; DeCamp, A.; Ahmed, H.; Ngandu, N.; Larsen, B.B.; Frahm, N.; Marais, J.; Thebus, R.; Geraghty, D.; Hural, J.; Corey, L.; Kublin, J.; Gray, G.; McElrath, M.J.; Mullins, J.I.; Gilbert, P.B.; Williamson, C.

    2016-01-01

    Introduction The Merck Adenovirus-5 Gag/Pol/Nef HIV-1 subtype-B vaccine evaluated in predominately subtype B epidemic regions (Step Study), while not preventing infection, exerted vaccine-induced immune pressure on HIV-1 breakthrough infections. Here we investigated if the same vaccine exerted immune pressure when tested in the Phambili Phase 2b study in a subtype C epidemic. Materials and methods A sieve analysis, which compares breakthrough viruses from placebo and vaccine arms, was performed on 277 near full-length genomes generated from 23 vaccine and 20 placebo recipients. Vaccine coverage was estimated by computing the percentage of 9-mers that were exact matches to the vaccine insert. Results There was significantly greater protein distances from the vaccine immunogen sequence in Gag (p = 0.045) and Nef (p = 0.021) in viruses infecting vaccine recipients compared to placebo recipients. Twenty-seven putative sites of vaccine-induced pressure were identified (p < 0.05) in Gag (n = 10), Pol (n = 7) and Nef (n = 10), although they did not remain significant after adjustment for multiple comparisons. We found the epitope sieve effect in Step was driven by HLA A*02:01; an allele which was found in low frequency in Phambili participants compared to Step participants. Furthermore, the coverage of the vaccine against subtype C Phambili viruses was 31%, 46% and 14% for Gag, Pol and Nef, respectively, compared to subtype B Step virus coverage of 56%, 61% and 26%, respectively. Discussion This study presents evidence of sieve effects in Gag and Nef; however could not confirm effects on specific amino acid sites. We propose that this weaker signal of vaccine immune pressure detected in the Phambili study compared to the Step study may have been influenced by differences in host genetics (HLA allele frequency) and reduced impact of vaccine-induced immune responses due to mismatch between the viral subtype in the vaccine and infecting subtypes. PMID:27756485

  18. Systems vaccinology: probing humanity's diverse immune systems with vaccines.

    PubMed

    Pulendran, Bali

    2014-08-26

    Homo sapiens are genetically diverse, but dramatic demographic and socioeconomic changes during the past century have created further diversification with respect to age, nutritional status, and the incidence of associated chronic inflammatory disorders and chronic infections. These shifting demographics pose new challenges for vaccination, as emerging evidence suggests that age, the metabolic state, and chronic infections can exert major influences on the immune system. Thus, a key public health challenge is learning how to reprogram suboptimal immune systems to induce effective vaccine immunity. Recent advances have applied systems biological analysis to define molecular signatures induced early after vaccination that correlate with and predict the later adaptive immune responses in humans. Such "systems vaccinology" approaches offer an integrated picture of the molecular networks driving vaccine immunity, and are beginning to yield novel insights about the immune system. Here we discuss the promise of systems vaccinology in probing humanity's diverse immune systems, and in delineating the impact of genes, the environment, and the microbiome on protective immunity induced by vaccination. Such insights will be critical in reengineering suboptimal immune systems in immunocompromised populations.

  19. Oral vaccination against plague using Yersinia pseudotuberculosis.

    PubMed

    Demeure, Christian E; Derbise, Anne; Carniel, Elisabeth

    2017-04-01

    Yersinia pestis, the agent of plague, is among the deadliest bacterial pathogens affecting humans, and is a potential biological weapon. Because antibiotic resistant strains of Yersinia pestis have been observed or could be engineered for evil use, vaccination against plague might become the only means to reduce mortality. Although plague is re-emerging in many countries, a vaccine with worldwide license is currently lacking. The vaccine strategy described here is based on an oral vaccination with an attenuated strain of Yersinia pseudotuberculosis. Indeed, this species is genetically almost identical to Y. pestis, but has a much lower pathogenicity and a higher genomic stability. Gradual modifications of the wild-type Yersinia pseudotuberculosis strain IP32953 were performed to generate a safe and immunogenic vaccine. Genes coding for three essential virulence factors were deleted from this strain. To increase cross-species immunogenicity, an F1-encapsulated Y. pseudotuberculosis strain was then generated. For this, the Y. pestis caf operon, which encodes F1, was inserted first on a plasmid, and subsequently into the chromosome. The successive steps achieved to reach maximal vaccine potential are described, and how each step affected bacterial virulence and the development of a protective immune response is discussed. The final version of the vaccine, named VTnF1, provides a highly efficient and long-lasting protection against both bubonic and pneumonic plague after a single oral vaccine dose. Since a Y. pestis strain deprived of F1 exist or could be engineered, we also analyzed the protection conferred by the vaccine against such strain and found that it also confers full protection against the two forms of plague. Thus, the properties of VTnF1 makes it one of the most efficient candidate vaccine for mass vaccination in tropical endemic areas as well as for populations exposed to bioterrorism. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  20. Identification of the Mechanisms Causing Reversion to Virulence in an Attenuated SARS-CoV for the Design of a Genetically Stable Vaccine.

    PubMed

    Jimenez-Guardeño, Jose M; Regla-Nava, Jose A; Nieto-Torres, Jose L; DeDiego, Marta L; Castaño-Rodriguez, Carlos; Fernandez-Delgado, Raul; Perlman, Stanley; Enjuanes, Luis

    2015-10-01

    A SARS-CoV lacking the full-length E gene (SARS-CoV-∆E) was attenuated and an effective vaccine. Here, we show that this mutant virus regained fitness after serial passages in cell culture or in vivo, resulting in the partial duplication of the membrane gene or in the insertion of a new sequence in gene 8a, respectively. The chimeric proteins generated in cell culture increased virus fitness in vitro but remained attenuated in mice. In contrast, during SARS-CoV-∆E passage in mice, the virus incorporated a mutated variant of 8a protein, resulting in reversion to a virulent phenotype. When the full-length E protein was deleted or its PDZ-binding motif (PBM) was mutated, the revertant viruses either incorporated a novel chimeric protein with a PBM or restored the sequence of the PBM on the E protein, respectively. Similarly, after passage in mice, SARS-CoV-∆E protein 8a mutated, to now encode a PBM, and also regained virulence. These data indicated that the virus requires a PBM on a transmembrane protein to compensate for removal of this motif from the E protein. To increase the genetic stability of the vaccine candidate, we introduced small attenuating deletions in E gene that did not affect the endogenous PBM, preventing the incorporation of novel chimeric proteins in the virus genome. In addition, to increase vaccine biosafety, we introduced additional attenuating mutations into the nsp1 protein. Deletions in the carboxy-terminal region of nsp1 protein led to higher host interferon responses and virus attenuation. Recombinant viruses including attenuating mutations in E and nsp1 genes maintained their attenuation after passage in vitro and in vivo. Further, these viruses fully protected mice against challenge with the lethal parental virus, and are therefore safe and stable vaccine candidates for protection against SARS-CoV.