Doxycycline Inducible Melanogenic Vaccinia Virus as Theranostic Anti-Cancer Agent.

PubMed

Kirscher, Lorenz; Deán-Ben, Xosé Luis; Scadeng, Miriam; Zaremba, Angelika; Zhang, Qian; Kober, Christina; Fehm, Thomas Felix; Razansky, Daniel; Ntziachristos, Vasilis; Stritzker, Jochen; Szalay, Aladar A

2015-01-01

We reported earlier the diagnostic potential of a melanogenic vaccinia virus based system in magnetic resonance (MRI) and optoacoustic deep tissue imaging (MSOT). Since melanin overproduction lead to attenuated virus replication, we constructed a novel recombinant vaccinia virus strain (rVACV), GLV-1h462, which expressed the key enzyme of melanogenesis (tyrosinase) under the control of an inducible promoter-system. In this study melanin production was detected after exogenous addition of doxycycline in two different tumor xenograft mouse models. Furthermore, it was confirmed that this novel vaccinia virus strain still facilitated signal enhancement as detected by MRI and optoacoustic tomography. At the same time we demonstrated an enhanced oncolytic potential compared to the constitutively melanin synthesizing rVACV system.

DNA and modified vaccinia virus Ankara vaccines encoding multiple cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) are safe but weakly immunogenic in HIV-1-uninfected, vaccinia virus-naive adults.

PubMed

Gorse, Geoffrey J; Newman, Mark J; deCamp, Allan; Hay, Christine Mhorag; De Rosa, Stephen C; Noonan, Elizabeth; Livingston, Brian D; Fuchs, Jonathan D; Kalams, Spyros A; Cassis-Ghavami, Farah L

2012-05-01

We evaluated a DNA plasmid-vectored vaccine and a recombinant modified vaccinia virus Ankara vaccine (MVA-mBN32), each encoding cytotoxic and helper T-lymphocyte epitopes of human immunodeficiency virus type 1 (HIV-1) in a randomized, double-blinded, placebo-controlled trial in 36 HIV-1-uninfected adults using a heterologous prime-boost schedule. HIV-1-specific cellular immune responses, measured as interleukin-2 and/or gamma interferon production, were induced in 1 (4%) of 28 subjects after the first MVA-mBN32 immunization and in 3 (12%) of 25 subjects after the second MVA-mBN32 immunization. Among these responders, polyfunctional T-cell responses, including the production of tumor necrosis factor alpha and perforin, were detected. Vaccinia virus-specific antibodies were induced to the MVA vector in 27 (93%) of 29 and 26 (93%) of 28 subjects after the first and second immunizations with MVA-mBN32. These peptide-based vaccines were safe but were ineffective at inducing HIV-1-specific immune responses and induced much weaker responses than MVA vaccines expressing the entire open reading frames of HIV-1 proteins.

Plasmodium knowlesi Sporozoite Antigen: Expression by Infectious Recombinant Vaccinia Virus

NASA Astrophysics Data System (ADS)

Smith, Geoffrey L.; Godson, G. Nigel; Nussenzweig, Victor; Nussenzweig, Ruth S.; Barnwell, John; Moss, Bernard

1984-04-01

The gene coding for the circumsporozoite antigen of the malaria parasite Plasmodium knowlesi was inserted into the vaccinia virus genome under the control of a defined vaccinia virus promoter. Cells infected with the recombinant virus synthesized polypeptides of 53,000 to 56,000 daltons that reacted with monoclonal antibody against the repeating epitope of the malaria protein. Furthermore, rabbits vaccinated with the recombinant virus produced antibodies that bound specifically to sporozoites. These data provide evidence for expression of a cloned malaria gene in mammalian cells and illustrate the potential of vaccinia virus recombinants as live malaria vaccines.

Vaccinia Virus Entry, Exit, and Interaction with Differentiated Human Airway Epithelia▿

PubMed Central

Vermeer, Paola D.; McHugh, Julia; Rokhlina, Tatiana; Vermeer, Daniel W.; Zabner, Joseph; Welsh, Michael J.

2007-01-01

Variola virus, the causative agent of smallpox, enters and exits the host via the respiratory route. To better understand the pathogenesis of poxvirus infection and its interaction with respiratory epithelia, we used vaccinia virus and examined its interaction with primary cultures of well-differentiated human airway epithelia. We found that vaccinia virus preferentially infected the epithelia through the basolateral membrane and released viral progeny across the apical membrane. Despite infection and virus production, epithelia retained tight junctions, transepithelial electrical conductance, and a steep transepithelial concentration gradient of virus, indicating integrity of the epithelial barrier. In fact, during the first four days of infection, epithelial height and cell number increased. These morphological changes and maintenance of epithelial integrity required vaccinia virus growth factor, which was released basolaterally, where it activated epidermal growth factor 1 receptors. These data suggest a complex interaction between the virus and differentiated airway epithelia; the virus preferentially enters the cells basolaterally, exits apically, and maintains epithelial integrity by stimulating growth factor receptors. PMID:17581984

Vaccinia Virus: A Tool for Research and Vaccine Development

NASA Astrophysics Data System (ADS)

Moss, Bernard

1991-06-01

Vaccinia virus is no longer needed for smallpox immunization, but now serves as a useful vector for expressing genes within the cytoplasm of eukaryotic cells. As a research tool, recombinant vaccinia viruses are used to synthesize biologically active proteins and analyze structure-function relations, determine the targets of humoral- and cell-mediated immunity, and investigate the immune responses needed for protection against specific infectious diseases. When more data on safety and efficacy are available, recombinant vaccinia and related poxviruses may be candidates for live vaccines and for cancer immunotherapy.

Polymeric Cups for Cavitation-mediated Delivery of Oncolytic Vaccinia Virus

PubMed Central

Myers, Rachel; Coviello, Christian; Erbs, Philippe; Foloppe, Johann; Rowe, Cliff; Kwan, James; Crake, Calum; Finn, Seán; Jackson, Edward; Balloul, Jean-Marc; Story, Colin; Coussios, Constantin; Carlisle, Robert

2016-01-01

Oncolytic viruses (OV) could become the most powerful and selective cancer therapies. However, the limited transport of OV into and throughout tumors following intravenous injection means their clinical administration is often restricted to direct intratumoral dosing. Application of physical stimuli, such as focused ultrasound, offers a means of achieving enhanced mass transport. In particular, shockwaves and microstreaming resulting from the instigation of an ultrasound-induced event known as inertial cavitation can propel OV hundreds of microns. We have recently developed a polymeric cup formulation which, when delivered intravenously, provides the nuclei for instigation of sustained inertial cavitation events within tumors. Here we report that exposure of tumors to focused ultrasound after intravenous coinjection of cups and oncolytic vaccinia virus , leads to substantial and significant increases in activity. When cavitation was instigated within SKOV-3 or HepG2 xenografts, reporter gene expression from vaccinia virus was enhanced 1,000-fold (P < 0.0001) or 10,000-fold (P < 0.001), respectively. Similar increases in the number of vaccinia virus genomes recovered from tumors were also observed. In survival studies, the application of cup mediated cavitation to a vaccinia virus expressing a prodrug converting enzyme provided significant (P < 0.05) retardation of tumor growth. This technology could improve the clinical utility of all biological therapeutics including OV. PMID:27375160

Inhibition of vaccinia virus maturation by zinc chloride.

PubMed Central

Katz, E; Margalith, E

1981-01-01

Zinc chloride (0.1 mM) inhibited by 96.4% the growth of vaccinia virus in HeLa cells. Approximately 50% inhibition in formation of particles that sedimented in sucrose gradients similarly to vaccinia virions occurred in the presence of zinc ions. Whereas the synthesis of the viral deoxyribonucleic acid was not affected by zinc chloride, a decrease in the overall synthesis of viral polypeptides and inhibition of the cleavage of precursors to the core polypeptides were observed. Images PMID:7347557

Transcription of a vaccinia virus late promoter template: requirement for the product of the A2L intermediate-stage gene.

PubMed Central

Passarelli, A L; Kovacs, G R; Moss, B

1996-01-01

Evidence is presented that a 26-kDa protein encoded by the vaccinia virus A2L open reading frame, originally shown to be one of three intermediate-stage genes that together can transactivate late-stage gene expression in transfection assays (J. G. Keck, C. J. Baldick, and B. Moss, Cell 61:801-809, 1990), is required for in vitro transcription of a template with a late promoter. The critical step in this analysis was the preparation of an extract containing all the required factors except for the A2L protein. This extract was prepared from cells infected with a recombinant vaccinia virus expressing the bacteriophage T7 RNA polymerase in the presence of the DNA synthesis inhibitor cytosine arabinoside and transfected with plasmids containing the two other known transactivator genes, A1L and G8R, under T7 promoter control. Reaction mixtures made with extracts of these cells had background levels of late transcription activity, unless they were supplemented with extracts of cells transfected with the A2L gene. Active transcription mixtures were also made by mixing extracts from three sets of cells, each transfected with a gene (A1L, A2L, or G8R) encoding a separate factor, indicating the absence of any requirement for their coexpression. To minimize the possibility that the A2L protein functions indirectly by activating another viral or cellular protein, this gene was expressed in insect cells by using a baculovirus vector. The partially purified recombinant protein complemented the activity of A2L-deficient cell extracts. Recombinant A1L, A2L, and G8R proteins, all produced in insect cells, together complemented extracts from mammalian cells containing only viral early proteins, concordant with previous in vivo transfection data. PMID:8676468

Resistance to Two Heterologous Neurotropic Oncolytic Viruses, Semliki Forest Virus and Vaccinia Virus, in Experimental Glioma

PubMed Central

Le Boeuf, Fabrice; Lemay, Chantal; De Silva, Naomi; Diallo, Jean-Simon; Cox, Julie; Becker, Michelle; Choi, Youngmin; Ananth, Abhirami; Sellers, Clara; Breton, Sophie; Roy, Dominic; Falls, Theresa; Brun, Jan; Hemminki, Akseli; Hinkkanen, Ari; Bell, John C.

2013-01-01

Attenuated Semliki Forest virus (SFV) may be suitable for targeting malignant glioma due to its natural neurotropism, but its replication in brain tumor cells may be restricted by innate antiviral defenses. We attempted to facilitate SFV replication in glioma cells by combining it with vaccinia virus, which is capable of antagonizing such defenses. Surprisingly, we found parenchymal mouse brain tumors to be refractory to both viruses. Also, vaccinia virus appears to be sensitive to SFV-induced antiviral interference. PMID:23221568

Vaccinia Virus Encodes a Novel Inhibitor of Apoptosis That Associates with the Apoptosome

PubMed Central

Ryerson, Melissa R.; Richards, Monique M.; Hawkins, Christine J.

2017-01-01

ABSTRACT Apoptosis is an important antiviral host defense mechanism. Here we report the identification of a novel apoptosis inhibitor encoded by the vaccinia virus (VACV) M1L gene. M1L is absent in the attenuated modified vaccinia virus Ankara (MVA) strain of VACV, a strain that stimulates apoptosis in several types of immune cells. M1 expression increased the viability of MVA-infected THP-1 and Jurkat cells and reduced several biochemical hallmarks of apoptosis, such as PARP-1 and procaspase-3 cleavage. Furthermore, ectopic M1L expression decreased staurosporine-induced (intrinsic) apoptosis in HeLa cells. We then identified the molecular basis for M1 inhibitory function. M1 allowed mitochondrial depolarization but blocked procaspase-9 processing, suggesting that M1 targeted the apoptosome. In support of this model, we found that M1 promoted survival in Saccharomyces cerevisiae overexpressing human Apaf-1 and procaspase-9, critical components of the apoptosome, or overexpressing only conformationally active caspase-9. In mammalian cells, M1 coimmunoprecipitated with Apaf-1–procaspase-9 complexes. The current model is that M1 associates with and allows the formation of the apoptosome but prevents apoptotic functions of the apoptosome. The M1 protein features 14 predicted ankyrin (ANK) repeat domains, and M1 is the first ANK-containing protein reported to use this inhibitory strategy. Since ANK-containing proteins are encoded by many large DNA viruses and found in all domains of life, studies of M1 may lead to a better understanding of the roles of ANK proteins in virus-host interactions. IMPORTANCE Apoptosis selectively eliminates dangerous cells such as virus-infected cells. Poxviruses express apoptosis antagonists to neutralize this antiviral host defense. The vaccinia virus (VACV) M1 ankyrin (ANK) protein, a protein with no previously ascribed function, inhibits apoptosis. M1 interacts with the apoptosome and prevents procaspase-9 processing as well as

Nigericin is a potent inhibitor of the early stage of vaccinia virus replication.

PubMed

Myskiw, Chad; Piper, Jessica; Huzarewich, Rhiannon; Booth, Tim F; Cao, Jingxin; He, Runtao

2010-12-01

Poxviruses remain a significant public health concern due to their potential use as bioterrorist agents and the spread of animal borne poxviruses, such as monkeypox virus, to humans. Thus, the identification of small molecule inhibitors of poxvirus replication is warranted. Vaccinia virus is the prototypic member of the Orthopoxvirus genus, which also includes variola and monkeypox virus. In this study, we demonstrate that the carboxylic ionophore nigericin is a potent inhibitor of vaccinia virus replication in several human cell lines. In HeLa cells, we found that the 50% inhibitory concentration of nigericin against vaccinia virus was 7.9 nM, with a selectivity index of 1038. We present data demonstrating that nigericin targets vaccinia virus replication at a post-entry stage. While nigericin moderately inhibits both early vaccinia gene transcription and translation, viral DNA replication and intermediate and late gene expression are severely compromised in the presence of nigericin. Our results demonstrate that nigericin has the potential to be further developed into an effective antiviral to treat poxvirus infections. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Thy1+ Nk Cells from Vaccinia Virus-Primed Mice Confer Protection against Vaccinia Virus Challenge in the Absence of Adaptive Lymphocytes

PubMed Central

Gillard, Geoffrey O.; Bivas-Benita, Maytal; Hovav, Avi-Hai; Grandpre, Lauren E.; Panas, Michael W.; Seaman, Michael S.; Haynes, Barton F.; Letvin, Norman L.

2011-01-01

While immunological memory has long been considered the province of T- and B- lymphocytes, it has recently been reported that innate cell populations are capable of mediating memory responses. We now show that an innate memory immune response is generated in mice following infection with vaccinia virus, a poxvirus for which no cognate germline-encoded receptor has been identified. This immune response results in viral clearance in the absence of classical adaptive T and B lymphocyte populations, and is mediated by a Thy1+ subset of natural killer (NK) cells. We demonstrate that immune protection against infection from a lethal dose of virus can be adoptively transferred with memory hepatic Thy1+ NK cells that were primed with live virus. Our results also indicate that, like classical immunological memory, stronger innate memory responses form in response to priming with live virus than a highly attenuated vector. These results demonstrate that a defined innate memory cell population alone can provide host protection against a lethal systemic infection through viral clearance. PMID:21829360

Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus

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

Schlehofer, J.R.; Ehrbar, M.; zur Hausen, H.

1986-07-15

The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens (initiators) as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. Asmore » reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells.« less

Construction of Poxviruses as Cloning Vectors: Insertion of the Thymidine Kinase Gene from Herpes Simplex Virus into the DNA of Infectious Vaccinia Virus

NASA Astrophysics Data System (ADS)

Panicali, Dennis; Paoletti, Enzo

1982-08-01

We have constructed recombinant vaccinia viruses containing the thymidine kinase gene from herpes simplex virus. The gene was inserted into the genome of a variant of vaccinia virus that had undergone spontaneous deletion as well as into the 120-megadalton genome of the large prototypic vaccinia variant. This was accomplished via in vivo recombination by contransfection of eukaryotic tissue culture cells with cloned BamHI-digested thymidine kinase gene from herpes simplex virus containing flanking vaccinia virus DNA sequences and infectious rescuing vaccinia virus. Pure populations of the recombinant viruses were obtained by replica filter techniques or by growth of the recombinant virus in biochemically selective medium. The herpes simplex virus thymidine kinase gene, as an insert in vaccinia virus, is transcribed in vivo and in vitro, and the fidelity of in vivo transcription into a functional gene product was detected by the phosphorylation of 5-[125I]iodo-2'-deoxycytidine.

The Myristate Moiety and Amino Terminus of Vaccinia Virus L1 Constitute a Bipartite Functional Region Needed for Entry

PubMed Central

Whitbeck, J. Charles; Ponce-de-León, Manuel; Saw, Wan Ting; Cohen, Gary H.; Eisenberg, Roselyn J.

2012-01-01

Vaccinia virus (VACV) L1 is a myristoylated envelope protein which is required for cell entry and the fusion of infected cells. L1 associates with members of the entry-fusion complex (EFC), but its specific role in entry has not been delineated. We recently demonstrated (Foo CH, et al., Virology 385:368–382, 2009) that soluble L1 binds to cells and blocks entry, suggesting that L1 serves as the receptor-binding protein for entry. Our goal is to identify the structural domains of L1 which are essential for its functions in VACV entry. We hypothesized that the myristate and the conserved residues at the N terminus of L1 are critical for entry. To test our hypothesis, we generated mutants in the N terminus of L1 and used a complementation assay to evaluate their ability to rescue infectivity. We also assessed the myristoylation efficiency of the mutants and their ability to interact with the EFC. We found that the N terminus of L1 constitutes a region that is critical for the infectivity of VACV and for myristoylation. At the same time, the nonmyristoylated mutants were incorporated into mature virions, suggesting that the myristate is not required for the association of L1 with the viral membrane. Although some of the mutants exhibited altered structural conformations, two mutants with impaired infectivity were similar in conformation to wild-type L1. Importantly, these two mutants, with changes at A4 and A5, undergo myristoylation. Overall, our results imply dual differential roles for myristate and the amino acids at the N terminus of L1. We propose a myristoyl switch model to describe how L1 functions. PMID:22398293

Expression of the Bacillus anthracis protective antigen gene by baculovirus and vaccinia virus recombinants.

PubMed Central

Iacono-Connors, L C; Schmaljohn, C S; Dalrymple, J M

1990-01-01

The gene encoding Bacillus anthracis protective antigen (PA) was modified by site-directed mutagenesis, subcloned into baculovirus and vaccinia virus plasmid transfer vectors (pAcYM1 and pSC-11, respectively), and inserted via homologous recombinations into baculovirus Autographa californica nuclear polyhedrosis virus or vaccinia virus (strains WR and Connaught). Expression of PA was detected in both systems by immunofluorescence assays with antisera from rabbits immunized with B. anthracis PA. Western blot (immunoblot) analysis showed that the expressed product of both systems was slightly larger (86 kilodaltons) than B. anthracis-produced PA (83.5 kilodaltons). Analysis of trypsin digests of virus-expressed and authentic PA suggested that the size difference was due to the presence of a signal sequence remaining with the virus-expressed protein. Immunization of mice with either recombinant baculovirus-infected Spodoptera frugiperda cells or with vaccinia virus recombinants elicited a high-titer, anti-PA antibody response. Images PMID:2105271

Active vaccination with vaccinia virus A33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response.

PubMed

Paran, Nir; Lustig, Shlomo; Zvi, Anat; Erez, Noam; Israely, Tomer; Melamed, Sharon; Politi, Boaz; Ben-Nathan, David; Schneider, Paula; Lachmi, Batel; Israeli, Ofir; Stein, Dana; Levin, Reuven; Olshevsky, Udy

2013-07-10

Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104-120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox.

Active vaccination with vaccinia virus A33 protects mice against lethal vaccinia and ectromelia viruses but not against cowpoxvirus; elucidation of the specific adaptive immune response

PubMed Central

2013-01-01

Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104–120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope’s critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox. PMID:23842430

Non-plaque-forming virions of Modified Vaccinia virus Ankara express viral genes.

PubMed

Lülf, Anna-Theresa; Freudenstein, Astrid; Marr, Lisa; Sutter, Gerd; Volz, Asisa

2016-12-01

In cell culture infections with vaccinia virus the number of counted virus particles is substantially higher than the number of plaques obtained by titration. We found that standard vaccine preparations of recombinant Modified Vaccinia virus Ankara produce only about 20-30% plaque-forming virions in fully permissive cell cultures. To evaluate the biological activity of the non-plaque-forming particles, we generated recombinant viruses expressing fluorescent reporter proteins under transcriptional control of specific viral early and late promoters. Live cell imaging and automated counting by fluorescent microscopy indicated that virtually all virus particles can enter cells and switch on viral gene expression. Although most of the non-plaque-forming infections are arrested at the level of viral early gene expression, we detected activation of late viral transcription in 10-20% of single infected cells. Thus, non-plaque-forming particles are biologically active, and likely contribute to the immunogenicity of vaccinia virus vaccines. Copyright © 2016 Elsevier Inc. All rights reserved.

A protein-based smallpox vaccine protects mice from vaccinia and ectromelia virus challenges when given as a prime and single boost

PubMed Central

Xiao, Yuhong; Aldaz-Carroll, Lydia; Ortiz, Alexandra M.; Whitbeck, J. Charles; Alexander, Edward; Lou, Huan; Davis, J. Heather L.; Braciale, Thomas J.; Eisenberg, Roselyn J.; Cohen, Gary H.; Isaacs, Stuart N.

2007-01-01

The heightened concern about the intentional release of variola virus has led to the need to develop safer smallpox vaccines. While subunit vaccine strategies are safer than live virus vaccines, subunit vaccines have been hampered by the need for multiple boosts to confer optimal protection. Here we developed a protein-based subunit vaccine strategy that provides rapid protection in mouse models of orthopoxvirus infections after a prime and single boost. Mice vaccinated with vaccinia virus envelope proteins from the mature virus (MV) and extracellular virus (EV) adjuvanted with CpG-ODN and alum were protected from lethal intranasal challenge with vaccinia virus and the mouse-specific ectromelia virus. Organs from mice vaccinated with three proteins (A33, B5 and L1) and then sacrificed after challenge contained significantly lower titers of virus when compared to control groups of mice that were not vaccinated or that received sub-optimal formulations of the vaccine. Sera from groups of mice obtained prior to challenge had neutralizing activity against the MV and also inhibited comet formation indicating anti-EV activity. Long-term partial protection was also seen in mice challenged with vaccinia virus 6 months after initial vaccinations. Thus, this work represents a step toward the development of a practical subunit smallpox vaccine. PMID:17098336

Construction of live vaccines by using genetically engineered poxviruses: biological activity of recombinant vaccinia virus expressing influenza virus hemagglutinin.

PubMed Central

Panicali, D; Davis, S W; Weinberg, R L; Paoletti, E

1983-01-01

Recombinant vaccinia viruses containing the cloned hemagglutinin (HA) gene from influenza virus were constructed. The biological activity of these poxvirus vectors was demonstrated both in vitro and in vivo. Expression of HA in cells infected with recombinant vaccinia was detected by using specific anti-HA antiserum and 125I-labeled protein A, showing that HA synthesized under the regulation of vaccinia virus was antigenic. Immunization of rabbits with these recombinant poxviruses resulted in the production of antibodies reactive with authentic influenza HA as detected by radioimmunoassay, by inhibition of HA erythrocyte agglutination, and by neutralization of influenza virus infectivity. The production of antibodies directed against influenza HA suggested that the HA gene expressed in vaccinia is immunogenic. These data indicate the potential of genetically engineered poxviruses for use as generic live vaccine vehicles that have both human and veterinary applications. Images PMID:6310573

Vaccinia Virus Induces Rapid Necrosis in Keratinocytes by a STAT3-Dependent Mechanism

PubMed Central

He, Yong; Fisher, Robert; Chowdhury, Soma; Sultana, Ishrat; Pereira, Claudia P.; Bray, Mike; Reed, Jennifer L.

2014-01-01

Rationale Humans with a dominant negative mutation in STAT3 are susceptible to severe skin infections, suggesting an essential role for STAT3 signaling in defense against cutaneous pathogens. Methods To focus on innate antiviral defenses in keratinocytes, we used a standard model of cutaneous infection of severe combined immunodeficient mice with the current smallpox vaccine, ACAM-2000. In parallel, early events post-infection with the smallpox vaccine ACAM-2000 were investigated in cultured keratinocytes of human and mouse origin. Results Mice treated topically with a STAT3 inhibitor (Stattic) developed larger vaccinia lesions with higher virus titers and died more rapidly than untreated controls. Cultured human and murine keratinocytes infected with ACAM-2000 underwent rapid necrosis, but when treated with Stattic or with inhibitors of RIP1 kinase or caspase-1, they survived longer, produced higher titers of virus, and showed reduced activation of type I interferon responses and inflammatory cytokines release. Treatment with inhibitors of RIP1 kinase and STAT3, but not caspase-1, also reduced the inflammatory response of keratinocytes to TLR ligands. Vaccinia growth properties in Vero cells, which are known to be defective in some antiviral responses, were unaffected by inhibition of RIP1K, caspase-1, or STAT3. Conclusions Our findings indicate that keratinocytes suppress the replication and spread of vaccinia virus by undergoing rapid programmed cell death, in a process requiring STAT3. These data offer a new framework for understanding susceptibility to skin infection in patients with STAT3 mutations. Interventions which promote prompt necroptosis/pyroptosis of infected keratinocytes may reduce risks associated with vaccination with live vaccinia virus. PMID:25419841

A kinesin-1 binding motif in vaccinia virus that is widespread throughout the human genome

PubMed Central

Dodding, Mark P; Mitter, Richard; Humphries, Ashley C; Way, Michael

2011-01-01

Transport of cargoes by kinesin-1 is essential for many cellular processes. Nevertheless, the number of proteins known to recruit kinesin-1 via its cargo binding light chain (KLC) is still quite small. We also know relatively little about the molecular features that define kinesin-1 binding. We now show that a bipartite tryptophan-based kinesin-1 binding motif, originally identified in Calsyntenin is present in A36, a vaccinia integral membrane protein. This bipartite motif in A36 is required for kinesin-1-dependent transport of the virus to the cell periphery. Bioinformatic analysis reveals that related bipartite tryptophan-based motifs are present in over 450 human proteins. Using vaccinia as a surrogate cargo, we show that regions of proteins containing this motif can function to recruit KLC and promote virus transport in the absence of A36. These proteins interact with the kinesin light chain outside the context of infection and have distinct preferences for KLC1 and KLC2. Our observations demonstrate that KLC binding can be conferred by a common set of features that are found in a wide range of proteins associated with diverse cellular functions and human diseases. PMID:21915095

Characterization of Bunyamwera virus S RNA that is transcribed and replicated by the L protein expressed from recombinant vaccinia virus.

PubMed

Jin, H; Elliott, R M

1993-03-01

Analysis of the 5' termini of Bunyamwera virus S segment mRNAs by cloning and sequence analysis revealed the presence of nonviral, heterogeneous sequences 12 to 17 bases long. This is similar to reports for other members of the family Bunyaviridae and is taken to indicate that mRNA transcription is primed by a "cap-snatching" mechanism. The 3' end of the Bunyamwera virus S mRNA was mapped, by using an RNase protection assay, to 100 to 110 nucleotides upstream of the 3' end of the template. Previously we reported expression of the Bunyamwera virus L (polymerase) protein by recombinant vaccinia virus and demonstrated that the recombinant L protein was functional in terms of RNA synthesis activity in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, J. Virol. 65: 4182-4189, 1991). In the present study we further analyze the RNAs made by using this system and show that positive-sense RNAs contain 5' nonviral sequences. Hence the initiation of mRNA transcription by the recombinant L protein resembles that seen during authentic bunyavirus infection and suggests that the L protein has the endonuclease activity which generates the primers. Some of these positive-sense transcripts terminated at the mRNA termination site, but the majority read through to the end of the template. No primer sequences were found at the 5' terminal of negative-sense RNAs. The recombinant L protein was able to replicate negative-sense RNA supplied by transfected virion-derived nucleocapsids, and both positive- and negative-sense RNAs were synthesized. These results indicate that the recombinant L protein has both transcriptase and replicase activities.

Characterization of Bunyamwera virus S RNA that is transcribed and replicated by the L protein expressed from recombinant vaccinia virus.

PubMed Central

Jin, H; Elliott, R M

1993-01-01

Analysis of the 5' termini of Bunyamwera virus S segment mRNAs by cloning and sequence analysis revealed the presence of nonviral, heterogeneous sequences 12 to 17 bases long. This is similar to reports for other members of the family Bunyaviridae and is taken to indicate that mRNA transcription is primed by a "cap-snatching" mechanism. The 3' end of the Bunyamwera virus S mRNA was mapped, by using an RNase protection assay, to 100 to 110 nucleotides upstream of the 3' end of the template. Previously we reported expression of the Bunyamwera virus L (polymerase) protein by recombinant vaccinia virus and demonstrated that the recombinant L protein was functional in terms of RNA synthesis activity in a nucleocapsid transfection assay (H. Jin and R. M. Elliott, J. Virol. 65: 4182-4189, 1991). In the present study we further analyze the RNAs made by using this system and show that positive-sense RNAs contain 5' nonviral sequences. Hence the initiation of mRNA transcription by the recombinant L protein resembles that seen during authentic bunyavirus infection and suggests that the L protein has the endonuclease activity which generates the primers. Some of these positive-sense transcripts terminated at the mRNA termination site, but the majority read through to the end of the template. No primer sequences were found at the 5' terminal of negative-sense RNAs. The recombinant L protein was able to replicate negative-sense RNA supplied by transfected virion-derived nucleocapsids, and both positive- and negative-sense RNAs were synthesized. These results indicate that the recombinant L protein has both transcriptase and replicase activities. Images PMID:8437222

Studies on the serological relationships between avian pox, sheep pox, goat pox and vaccinia viruses

PubMed Central

Uppal, P. K.; Nilakantan, P. R.

1970-01-01

By using neutralization, complement fixation and immunogel-diffusion tests, it has been demonstrated that cross-reactions occur between various avian pox viruses and between sheep pox and goat pox viruses. No such reactions were demonstrated between avian pox viruses and vaccinia virus or between avian pox and sheep pox and goat pox viruses. Furthermore, no serological relationship was demonstrable between vaccinia virus and sheep pox and goat pox viruses. PMID:4989854

Susceptibility of Vaccinia Virus to Chemical Disinfectants

PubMed Central

de Oliveira, Tércia Moreira Ludolfo; Rehfeld, Izabelle Silva; Coelho Guedes, Maria Isabel Maldonado; Ferreira, Jaqueline Maria Siqueira; Kroon, Erna Geessien; Lobato, Zélia Inês Portela

2011-01-01

Vaccinia virus (VACV) is the cause of bovine vaccinia (BV), an emerging zoonotic disease that affects dairy cows and milkers. Some chemical disinfectants have been used on farms affected by BV to disinfect cow teats and milkers' hands. To date, there is no information about the efficacy of disinfectants against VACV. Therefore, this study aimed to assess the virucidal activity of some active disinfectants commonly used in the field. Sodium hypochlorite, quaternary ammonium combined with chlorhexidine, and quaternary ammonium combined with glutaraldehyde were effective in inactivating the virus at all concentrations tested. Iodine and quaternary ammonium as the only active component were partially effective. The presence of bovine feces as organic matter and light decreased the effectiveness of sodium hypochlorite. These results show that an appropriated disinfection and asepsis of teats and hands may be helpful in the control and prevention of BV and other infections with VACV. PMID:21734141

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

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

Meador, Lydia R.

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

Analysis of variola and vaccinia virus neutralization assays for smallpox vaccines.

PubMed

Hughes, Christine M; Newman, Frances K; Davidson, Whitni B; Olson, Victoria A; Smith, Scott K; Holman, Robert C; Yan, Lihan; Frey, Sharon E; Belshe, Robert B; Karem, Kevin L; Damon, Inger K

2012-07-01

Possible smallpox reemergence drives research for third-generation vaccines that effectively neutralize variola virus. A comparison of neutralization assays using different substrates, variola and vaccinia (Dryvax and modified vaccinia Ankara [MVA]), showed significantly different 90% neutralization titers; Dryvax underestimated while MVA overestimated variola neutralization. Third-generation vaccines may rely upon neutralization as a correlate of protection.

Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures.

PubMed

Ansardi, D C; Porter, D C; Morrow, C D

1991-04-01

The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.

Horizontal study of vaccinia virus infections in an endemic area: epidemiologic, phylogenetic and economic aspects.

PubMed

Assis, Felipe L; Franco-Luiz, Ana Paula M; Paim, Luis M; Oliveira, Graziele P; Pereira, Alexandre F; de Almeida, Gabriel M F; Figueiredo, Leandra B; Tanus, Adriano; Trindade, Giliane S; Ferreira, Paulo P; Kroon, Erna G; Abrahão, Jônatas S

2015-11-01

Vaccinia virus (VACV), the etiological agent of bovine vaccinia (BV), is widespread in Brazil and present in most of the milk-producing regions. We conducted a horizontal study of BV in Bahia, a state of Brazil in which the production of milk is increasing. During 2011, human and bovine clinical samples were collected during outbreaks for BV diagnosis, virus isolation and molecular analysis. We collected data for epidemiological inferences. Vaccinia virus was detected in 87.7% of the analyzed outbreaks, highlighting the effective circulation of VACV in Bahia. The molecular data showed the spreading of group 1 Brazilian VACV to Bahia. We observed a seasonal profile of BV, with its peak in the drier and cooler season. Manual milking was observed in 96 % of the visited properties, showing its importance to viral spread in herds. Under-notification of BV, ineffective animal trade surveillance, and bad milking practices have contributed to the spread of VACV in Brazil.

Can vaccinia virus be replaced by MVA virus for testing virucidal activity of chemical disinfectants?

PubMed

Rabenau, Holger F; Rapp, Ingrid; Steinmann, Jochen

2010-06-23

Vaccinia virus strain Lister Elstree (VACV) is a test virus in the DVV/RKI guidelines as representative of the stable enveloped viruses. Since the potential risk of laboratory-acquired infections with VACV persists and since the adverse effects of vaccination with VACV are described, the replacement of VACV by the modified vaccinia Ankara strain (MVA) was studied by testing the activity of different chemical biocides in three German laboratories. The inactivating properties of different chemical biocides (peracetic acid, aldehydes and alcohols) were tested in a quantitative suspension test according to the DVV/RKI guideline. All tests were performed with a protein load of 10% fetal calf serum with both viruses in parallel using different concentrations and contact times. Residual virus was determined by endpoint dilution method. The chemical biocides exhibited similar virucidal activity against VACV and MVA. In three cases intra-laboratory differences were determined between VACV and MVA - 40% (v/v) ethanol and 30% (v/v) isopropanol are more active against MVA, whereas MVA seems more stable than VACV when testing with 0.05% glutardialdehyde. Test accuracy across the three participating laboratories was high. Remarkably inter-laboratory differences in the reduction factor were only observed in two cases. Our data provide valuable information for the replacement of VACV by MVA for testing chemical biocides and disinfectants. Because MVA does not replicate in humans this would eliminate the potential risk of inadvertent inoculation with vaccinia virus and disease in non-vaccinated laboratory workers.

Role of sulfatide in vaccinia virus infection.

PubMed

Perino, Julien; Foo, Chwan Hong; Spehner, Daniele; Cohen, Gary H; Eisenberg, Roselyn J; Crance, Jean-Marc; Favier, Anne-Laure

2011-07-01

Vaccinia virus (VACV) was used as a surrogate of variola virus (genus Orthopoxvirus), the causative agent of smallpox, to study orthopoxvirus infection. VACV infects cells via attachment and fusion of the viral membrane with the host cell membrane. Glycosphingolipids, expressed in multiple organs, are major components of lipid rafts and have been associated with the infectious route of several pathogens. We demonstrate that the VACV-WR (VACV Western-Reserve strain) displays no binding to Cer (ceramide) or to Gal-Cer (galactosylceramide), but binds to a natural sulfated derivative of these molecules: the Sulf (sulfatide) 3' sulfogalactosylceramide. The interaction between Sulf and VACV-WR resulted in a time-dependent inhibition of virus infection. Virus cell attachment was the crucial step inhibited by Sulf. Electron microscopy showed that SUVs (small unilamellar vesicles) enriched in Sulf bound to VACV particles. Both the A27 and L5 viral membrane proteins were shown to interact with Sulf, indicating that they could be the major viral ligands for Sulf. Soluble Sulf was successful in preventing mortality, but not morbidity, in a lethal mouse model infection with VACV-WR. Together the results suggest that Sulf could play a role as an alternate receptor for VACV-WR and probably other Orthopoxviruses.

Characterization of ectromelia virus deficient in EVM036, the homolog of vaccinia virus F13L, and its application for rapid generation of recombinant viruses.

PubMed

Roscoe, Felicia; Xu, Ren-Huan; Sigal, Luis J

2012-12-01

The orthopoxvirus (OPV) vaccinia virus (VACV) requires an intact F13L gene to produce enveloped virions (EV) and to form plaques in cell monolayers. Simultaneous introduction of an exogenous gene and F13L into F13L-deficient VACV results in expression of the foreign gene and restoration of plaque size. This is used as a method to rapidly generate VACV recombinants without the need for drug selection. However, whether other OPVs require the orthologs of F13L to generate EV and form plaques, whether F13L orthologs and EV are important for OPV pathogenesis in natural hosts, and whether a system based on F13L ortholog deficiency can be used to generate recombinant OPVs other than VACV have not been reported. The F13L ortholog in ectromelia virus (ECTV), the agent of mousepox, is EVM036. We show that ECTV lacking EVM036 formed small plaques and was highly attenuated in vivo but still induced strong antibody responses. Reintroduction of EVM036 in tandem with the DsRed gene resulted in a virus that expressed DsRed in infected cells but was indistinguishable from wild-type ECTV in terms of plaque size and in vivo virulence. Thus, our data show that, like F13L in VACV, EVM036 is required for ECTV plaque formation and that EVM036 and EV are important for ECTV virulence. Our experiments also suggest that OPVs deficient in F13L orthologs could serve as safer anti-OPV vaccines. Further, our results demonstrate that ECTV deficient in EVM036 can be exploited for the rapid generation of fully virulent ECTV expressing foreign genes of interest.

Septins suppress the release of vaccinia virus from infected cells.

PubMed

Pfanzelter, Julia; Mostowy, Serge; Way, Michael

2018-06-19

Septins are conserved components of the cytoskeleton that play important roles in many fundamental cellular processes including division, migration, and membrane trafficking. Septins can also inhibit bacterial infection by forming cage-like structures around pathogens such as Shigella We found that septins are recruited to vaccinia virus immediately after its fusion with the plasma membrane during viral egress. RNA interference-mediated depletion of septins increases virus release and cell-to-cell spread, as well as actin tail formation. Live cell imaging reveals that septins are displaced from the virus when it induces actin polymerization. Septin loss, however, depends on the recruitment of the SH2/SH3 adaptor Nck, but not the activity of the Arp2/3 complex. Moreover, it is the recruitment of dynamin by the third Nck SH3 domain that displaces septins from the virus in a formin-dependent fashion. Our study demonstrates that septins suppress vaccinia release by "entrapping" the virus at the plasma membrane. This antiviral effect is overcome by dynamin together with formin-mediated actin polymerization. © 2018 Pfanzelter et al.

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

PubMed Central

Nitsche, Andreas; Kurth, Andreas; Dunkhorst, Anna; Pänke, Oliver; Sielaff, Hendrik; Junge, Wolfgang; Muth, Doreen; Scheller, Frieder; Stöcklein, Walter; Dahmen, Claudia; Pauli, Georg; Kage, Andreas

2007-01-01

Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method. PMID:17697378

In vitro susceptibility to ST-246 and Cidofovir corroborates the phylogenetic separation of Brazilian Vaccinia virus into two clades.

PubMed

Pires, Mariana A; Rodrigues, Nathália F S; de Oliveira, Danilo B; de Assis, Felipe L; Costa, Galileu B; Kroon, Erna G; Mota, Bruno E F

2018-04-01

The Orthopoxvirus (OPV) genus of the Poxviridae family contains several human pathogens, including Vaccinia virus (VACV), which have been implicating in outbreaks of a zoonotic disease called Bovine Vaccinia in Brazil. So far, no approved treatment exists for OPV infections, but ST-246 and Cidofovir (CDV) are now in clinical development. Therefore, the objective of this work was to evaluate the susceptibility of five strains of Brazilian VACV (Br-VACV) to ST-246 and Cidofovir. The susceptibility of these strains to both drugs was evaluated by plaque reduction assay, extracellular virus's quantification in the presence of ST-246 and one-step growth curve in cells treated with CDV. Besides that, the ORFs F13L and E9L were sequenced for searching of polymorphisms associated with drug resistance. The effective concentration of 50% (EC 50 ) from both drugs varies significantly for different strains (from 0.0054 to 0.051 μM for ST-246 and from 27.14 to 61.23 μM for CDV). ST-246 strongly inhibits the production of extracellular virus for all isolates in concentrations as low as 0.1 μM and it was observed a relevant decrease of progeny production for all Br-VACV after CDV treatment. Sequencing of the F13L and E9L ORFs showed that Br-VACV do not present the polymorphism(s) associated with resistance to ST-246 and CDV. Taken together, our results showed that ST-246 and CDV are effective against diverse, wild VACV strains and that the susceptibility of Br-VACV to these drugs mirrored the phylogenetic split of these isolates into two groups. Thus, both ST-246 and CDV are of great interest as compounds to treat individuals during Bovine Vaccinia outbreaks in Brazil. Copyright © 2018 Elsevier B.V. All rights reserved.

Modulating Vaccinia Virus Immunomodulators to Improve Immunological Memory

PubMed Central

Torres, Alice A.; Smith, Geoffrey L.

2018-01-01

The increasing frequency of monkeypox virus infections, new outbreaks of other zoonotic orthopoxviruses and concern about the re-emergence of smallpox have prompted research into developing antiviral drugs and better vaccines against these viruses. This article considers the genetic engineering of vaccinia virus (VACV) to enhance vaccine immunogenicity and safety. The virulence, immunogenicity and protective efficacy of VACV strains engineered to lack specific immunomodulatory or host range proteins are described. The ultimate goal is to develop safer and more immunogenic VACV vaccines that induce long-lasting immunological memory. PMID:29495547

From Lesions to Viral Clones: Biological and Molecular Diversity amongst Autochthonous Brazilian Vaccinia Virus

PubMed Central

Oliveira, Graziele; Assis, Felipe; Almeida, Gabriel; Albarnaz, Jonas; Lima, Maurício; Andrade, Ana Cláudia; Calixto, Rafael; Oliveira, Cairo; Neto, José Diomedes; Trindade, Giliane; Ferreira, Paulo César; Kroon, Erna Geessien; Abrahão, Jônatas

2015-01-01

Vaccinia virus (VACV) has had an important role for humanity because of its use during the smallpox eradication campaign. VACV is the etiologic agent of the bovine vaccinia (BV), an emerging zoonosis that has been associated with economic, social, veterinary and public health problems, mainly in Brazil and India. Despite the current and historical VACV importance, there is little information about its circulation, prevalence, origins and maintenance in the environment, natural reservoirs and diversity. Brazilian VACV (VACV-BR) are grouped into at least two groups based on genetic and biological diversity: group 1 (G1) and group 2 (G2). In this study, we went to the field and investigated VACV clonal diversity directly from exanthemous lesions, during BV outbreaks. Our results demonstrate that the G1 VACV-BR were more frequently isolated. Furthermore, we were able to co-detect the two variants (G1 and G2) in the same sample. Molecular and biological analysis corroborated previous reports and confirmed the co-circulation of two VACV-BR lineages. The detected G2 clones presented exclusive genetic and biological markers, distinct to reference isolates, including VACV-Western Reserve. Two clones presented a mosaic profile, with both G1 and G2 features based on the molecular analysis of A56R, A26L and C23L genes. Indeed, some SNPs and INDELs in A56R nucleotide sequences were observed among clones of the same virus population, maybe as a result of an increased mutation rate in a mixed population. These results provide information about the diversity profile in VACV populations, highlighting its importance to VACV evolution and maintenance in the environment. PMID:25785515

Vaccinia Virus Recombinants: Expression of VSV Genes and Protective Immunization of Mice and Cattle

NASA Astrophysics Data System (ADS)

Mackett, M.; Yilma, T.; Rose, J. K.; Moss, B.

1985-01-01

Vesicular stomatitis virus (VSV) causes a contagious disease of horses, cattle, and pigs. When DNA copies of messenger RNA's for the G or N proteins of VSV were linked to a vaccinia virus promoter and inserted into the vaccinia genome, the recombinants retained infectivity and synthesized VSV polypeptides. After intradermal vaccination with live recombinant virus expressing the G protein, mice produced VSV-neutralizing antibodies and were protected against lethal encephalitis upon intravenous challenge with VSV. In cattle, the degree of protection against intradermalingually injected VSV was correlated with the level of neutralizing antibody produced following vaccination.

Modified vaccinia virus Ankara encoding influenza virus hemagglutinin induces heterosubtypic immunity in macaques.

PubMed

Florek, Nicholas W; Weinfurter, Jason T; Jegaskanda, Sinthujan; Brewoo, Joseph N; Powell, Tim D; Young, Ginger R; Das, Subash C; Hatta, Masato; Broman, Karl W; Hungnes, Olav; Dudman, Susanne G; Kawaoka, Yoshihiro; Kent, Stephen J; Stinchcomb, Dan T; Osorio, Jorge E; Friedrich, Thomas C

2014-11-01

Current influenza virus vaccines primarily aim to induce neutralizing antibodies (NAbs). Modified vaccinia virus Ankara (MVA) is a safe and well-characterized vector for inducing both antibody and cellular immunity. We evaluated the immunogenicity and protective efficacy of MVA encoding influenza virus hemagglutinin (HA) and/or nucleoprotein (NP) in cynomolgus macaques. Animals were given 2 doses of MVA-based vaccines 4 weeks apart and were challenged with a 2009 pandemic H1N1 isolate (H1N1pdm) 8 weeks after the last vaccination. MVA-based vaccines encoding HA induced potent serum antibody responses against homologous H1 or H5 HAs but did not stimulate strong T cell responses prior to challenge. However, animals that received MVA encoding influenza virus HA and/or NP had high frequencies of virus-specific CD4(+) and CD8(+) T cell responses within the first 7 days of H1N1pdm infection, while animals vaccinated with MVA encoding irrelevant antigens did not. We detected little or no H1N1pdm replication in animals that received vaccines encoding H1 (homologous) HA, while a vaccine encoding NP from an H5N1 isolate afforded no protection. Surprisingly, H1N1pdm viral shedding was reduced in animals vaccinated with MVA encoding HA and NP from an H5N1 isolate. This reduced shedding was associated with cross-reactive antibodies capable of mediating antibody-dependent cellular cytotoxicity (ADCC) effector functions. Our results suggest that ADCC plays a role in cross-protective immunity against influenza. Vaccines optimized to stimulate cross-reactive antibodies with ADCC function may provide an important measure of protection against emerging influenza viruses when NAbs are ineffective. Current influenza vaccines are designed to elicit neutralizing antibodies (NAbs). Vaccine-induced NAbs typically are effective but highly specific for particular virus strains. Consequently, current vaccines are poorly suited for preventing the spread of newly emerging pandemic viruses

Modified Vaccinia Virus Ankara Encoding Influenza Virus Hemagglutinin Induces Heterosubtypic Immunity in Macaques

PubMed Central

Florek, Nicholas W.; Weinfurter, Jason T.; Jegaskanda, Sinthujan; Brewoo, Joseph N.; Powell, Tim D.; Young, Ginger R.; Das, Subash C.; Hatta, Masato; Broman, Karl W.; Hungnes, Olav; Dudman, Susanne G.; Kawaoka, Yoshihiro; Kent, Stephen J.; Stinchcomb, Dan T.

2014-01-01

ABSTRACT Current influenza virus vaccines primarily aim to induce neutralizing antibodies (NAbs). Modified vaccinia virus Ankara (MVA) is a safe and well-characterized vector for inducing both antibody and cellular immunity. We evaluated the immunogenicity and protective efficacy of MVA encoding influenza virus hemagglutinin (HA) and/or nucleoprotein (NP) in cynomolgus macaques. Animals were given 2 doses of MVA-based vaccines 4 weeks apart and were challenged with a 2009 pandemic H1N1 isolate (H1N1pdm) 8 weeks after the last vaccination. MVA-based vaccines encoding HA induced potent serum antibody responses against homologous H1 or H5 HAs but did not stimulate strong T cell responses prior to challenge. However, animals that received MVA encoding influenza virus HA and/or NP had high frequencies of virus-specific CD4+ and CD8+ T cell responses within the first 7 days of H1N1pdm infection, while animals vaccinated with MVA encoding irrelevant antigens did not. We detected little or no H1N1pdm replication in animals that received vaccines encoding H1 (homologous) HA, while a vaccine encoding NP from an H5N1 isolate afforded no protection. Surprisingly, H1N1pdm viral shedding was reduced in animals vaccinated with MVA encoding HA and NP from an H5N1 isolate. This reduced shedding was associated with cross-reactive antibodies capable of mediating antibody-dependent cellular cytotoxicity (ADCC) effector functions. Our results suggest that ADCC plays a role in cross-protective immunity against influenza. Vaccines optimized to stimulate cross-reactive antibodies with ADCC function may provide an important measure of protection against emerging influenza viruses when NAbs are ineffective. IMPORTANCE Current influenza vaccines are designed to elicit neutralizing antibodies (NAbs). Vaccine-induced NAbs typically are effective but highly specific for particular virus strains. Consequently, current vaccines are poorly suited for preventing the spread of newly emerging

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

PubMed

Volz, A; Sutter, G

2017-01-01

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

Modified Vaccinia Ankara Virus Vaccination Provides Long-Term Protection against Nasal Rabbitpox Virus Challenge.

PubMed

Jones, Dorothy I; McGee, Charles E; Sample, Christopher J; Sempowski, Gregory D; Pickup, David J; Staats, Herman F

2016-07-01

Modified vaccinia Ankara virus (MVA) is a smallpox vaccine candidate. This study was performed to determine if MVA vaccination provides long-term protection against rabbitpox virus (RPXV) challenge, an animal model of smallpox. Two doses of MVA provided 100% protection against a lethal intranasal RPXV challenge administered 9 months after vaccination. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Resistance to Human Respiratory Syncytial Virus (RSV) Infection Induced by Immunization of Cotton Rats with a Recombinant Vaccinia Virus Expressing the RSV G Glycoprotein

NASA Astrophysics Data System (ADS)

Elango, Narayanasamy; Prince, Gregory A.; Murphy, Brian R.; Venkatesan, Sundararajan; Chanock, Robert M.; Moss, Bernard

1986-03-01

A cDNA copy of the G glycoprotein gene of human respiratory syncytial virus (RSV) was placed under control of a vaccinia virus promoter and inserted into the thymidine kinase locus of the vaccinia virus genome. The recombinant vaccinia virus retained infectivity and expressed a 93-kDa protein that migrated with the authentic RSV G glycoprotein upon polyacrylamide gel electrophoresis. Glycosylation of the expressed protein and transport to the cell surface were demonstrated in the absence of other RSV proteins. Cotton rats that were inoculated intradermally with the infectious recombinant virus produced serum antibody to the G glycoprotein that neutralized RSV in vitro. Furthermore, the vaccinated animals were resistant to lower respiratory tract infection upon intranasal inoculation with RSV and had reduced titers of RSV in the nose.

Generation of Recombinant Modified Vaccinia Virus Ankara Encoding VP2, NS1, and VP7 Proteins of Bluetongue Virus.

PubMed

Marín-López, Alejandro; Ortego, Javier

2016-01-01

Modified Vaccinia Virus Ankara (MVA) is employed widely as an experimental vaccine vector for its lack of replication in mammalian cells and high expression level of foreign/heterologous genes. Recombinant MVAs (rMVAs) are used as platforms for protein production as well as vectors to generate vaccines against a high number of infectious diseases and other pathologies. The portrait of the virus combines desirable elements such as high-level biological safety, the ability to activate appropriate innate immune mediators upon vaccination, and the capacity to deliver substantial amounts of heterologous antigens. Recombinant MVAs encoding proteins of bluetongue virus (BTV), an Orbivirus that infects domestic and wild ruminants transmitted by biting midges of the Culicoides species, are excellent vaccine candidates against this virus. In this chapter we describe the methods for the generation of rMVAs encoding VP2, NS1, and VP7 proteins of bluetongue virus as a model example for orbiviruses. The protocols included cover the cloning of VP2, NS1, and VP7 BTV-4 genes in a transfer plasmid, the construction of recombinant MVAs, the titration of virus working stocks and the protein expression analysis by immunofluorescence and radiolabeling of rMVA infected cells as well as virus purification.

Identification of vaccinia virus epitope-specific HLA-A*0201-restricted T cells and comparative analysis of smallpox vaccines

PubMed Central

Drexler, Ingo; Staib, Caroline; Kastenmüller, Wolfgang; Stevanović, Stefan; Schmidt, Burkhard; Lemonnier, François A.; Rammensee, Hans-Georg; Busch, Dirk H.; Bernhard, Helga; Erfle, Volker; Sutter, Gerd

2003-01-01

Despite worldwide eradication of naturally occurring variola virus, smallpox remains a potential threat to both civilian and military populations. New, safe smallpox vaccines are being developed, and there is an urgent need for methods to evaluate vaccine efficacy after immunization. Here we report the identification of an immunodominant HLA-A*0201-restricted epitope that is recognized by cytotoxic CD8+ T cells and conserved among Orthopoxvirus species including variola virus. This finding has permitted analysis and monitoring of epitope-specific T cell responses after immunization and demonstration of the identified T cell specificity in an A*0201-positive human donor. Vaccination of transgenic mice allowed us to compare the immunogenicity of several vaccinia viruses including highly attenuated, replication-deficient modified vaccinia virus Ankara (MVA). MVA vaccines elicited levels of CD8+ T cell responses that were comparable to those induced by the replication-competent vaccinia virus strains. Finally, we demonstrate that MVA vaccination is fully protective against a lethal respiratory challenge with virulent vaccinia virus strain Western Reserve. Our data provide a basis to rationally estimate immunogenicity of safe, second-generation poxvirus vaccines and suggest that MVA may be a suitable candidate. PMID:12518065

Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

PubMed Central

Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

2015-01-01

ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex

Modified vaccinia virus Ankara expressing the hemagglutinin of pandemic (H1N1) 2009 virus induces cross-protective immunity against Eurasian 'avian-like' H1N1 swine viruses in mice.

PubMed

Castrucci, Maria R; Facchini, Marzia; Di Mario, Giuseppina; Garulli, Bruno; Sciaraffia, Ester; Meola, Monica; Fabiani, Concetta; De Marco, Maria A; Cordioli, Paolo; Siccardi, Antonio; Kawaoka, Yoshihiro; Donatelli, Isabella

2014-05-01

To examine cross-reactivity between hemagglutinin (HA) derived from A/California/7/09 (CA/09) virus and that derived from representative Eurasian "avian-like" (EA) H1N1 swine viruses isolated in Italy between 1999 and 2008 during virological surveillance in pigs. Modified vaccinia virus Ankara (MVA) expressing the HA gene of CA/09 virus (MVA-HA-CA/09) was used as a vaccine to investigate cross-protective immunity against H1N1 swine viruses in mice. Two classical swine H1N1 (CS) viruses and four representative EA-like H1N1 swine viruses previously isolated during outbreaks of respiratory disease in pigs on farms in Northern Italy were used in this study. Female C57BL/6 mice were vaccinated with MVA/HA/CA/09 and then challenged intranasally with H1N1 swine viruses. Cross-reactive antibody responses were determined by hemagglutination- inhibition (HI) and virus microneutralizing (MN) assays of sera from MVA-vaccinated mice. The extent of protective immunity against infection with H1N1 swine viruses was determined by measuring lung viral load on days 2 and 4 post-challenge. Systemic immunization of mice with CA/09-derived HA, vectored by MVA, elicited cross-protective immunity against recent EA-like swine viruses. This immune protection was related to the levels of cross-reactive HI antibodies in the sera of the immunized mice and was dependent on the similarity of the antigenic site Sa of H1 HAs. Our findings suggest that the herd immunity elicited in humans by the pandemic (H1N1) 2009 virus could limit the transmission of recent EA-like swine HA genes into the influenza A virus gene pool in humans. © 2013 The Authors Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

Structural Basis for the Binding of the Neutralizing Antibody, 7D11, to the Poxvirus L1 Protein

DTIC Science & Technology

2007-08-01

pCR- 7D11-vHC and pCR-7D11- vLC , respectively. Crystallization of the complex between L1 and 7D11-Fab VACV L1 protein was expressed and purified as...2005. Vaccinia virus H3L envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in...D.M., Schmaljohn, C., Schmaljohn, A., 2000. DNA vaccination with vaccinia virus L1R and A33R genes protects mice against a lethal poxvirus challenge

Safety and biodistribution of a double-deleted oncolytic vaccinia virus encoding CD40 ligand in laboratory Beagles

PubMed Central

Autio, Karoliina; Knuuttila, Anna; Kipar, Anja; Pesonen, Sari; Guse, Kilian; Parviainen, Suvi; Rajamäki, Minna; Laitinen-Vapaavuori, Outi; Vähä-Koskela, Markus; Kanerva, Anna; Hemminki, Akseli

2014-01-01

We evaluated adverse events, biodistribution and shedding of oncolytic vaccinia virus encoding CD40 ligand in two Beagles, in preparation for a phase 1 trial in canine cancer patients. Dog 1 received one dose of vaccinia virus and was euthanized 24 hours afterwards, while dog 2 received virus four times once weekly and was euthanized 7 days after that. Dogs were monitored for adverse events and underwent a detailed postmortem examination. Blood, saliva, urine, feces, and organs were collected for virus detection. Dog 1 had mild fever and lethargy while dog 2 experienced a possible seizure 5.5 hours after first virus administration. Viral DNA declined quickly in the blood after virus administration in both dogs but was still detectable 1 week later by quantitative polymerase chain reaction. Only samples taken directly after virus infusion contained infectious virus. Small amounts of viral DNA, but no infectious virus, were detected in a few saliva and urine samples. Necropsies did not reveal any relevant pathological changes and virus DNA was detected mainly in the spleen. The dogs in the study did not have cancer, and thus adverse events could be more common and viral load higher in dogs with tumors which allow viral amplification. PMID:27119092

Lister vaccine strain of vaccinia virus armed with the endostatin-angiostatin fusion gene: an oncolytic virus superior to dl1520 (ONYX-015) for human head and neck cancer.

PubMed

Tysome, James R; Wang, Pengju; Alusi, Ghassan; Briat, Arnaud; Gangeswaran, Rathi; Wang, Jiwei; Bhakta, Vipul; Fodor, Istvan; Lemoine, Nick R; Wang, Yaohe

2011-09-01

Oncolytic viral therapy represents a promising strategy for the treatment of head and neck squamous cell carcinoma (HNSCC), with dl1520 (ONYX-015) the most widely used oncolytic adenovirus in clinical trials. This study aimed to determine the effectiveness of the Lister vaccine strain of vaccinia virus as well as a vaccinia virus armed with the endostatin-angiostatin fusion gene (VVhEA) as a novel therapy for HNSCC and to compare them with dl1520. The potency and replication of the Lister strain and VVhEA and the expression and function of the fusion protein were determined in human HNSCC cells in vitro and in vivo. Finally, the efficacy of VVhEA was compared with dl1520 in vivo in a human HNSCC model. The Lister vaccine strain of vaccinia virus was more effective than the adenovirus against all HNSCC cell lines tested in vitro. Although the potency of VVhEA was attenuated in vitro, the expression and function of the endostatin-angiostatin fusion protein was confirmed in HNSCC models both in vitro and in vivo. This novel vaccinia virus (VVhEA) demonstrated superior antitumor potency in vivo compared with both dl1520 and the control vaccinia virus. This study suggests that the Lister strain vaccinia virus armed with an endostatin-angiostatin fusion gene may be a potential therapeutic agent for HNSCC.

Enhanced Efficacy of Cidofovir Combined with Vaccinia Immune Globulin in Treating Progressive Cutaneous Vaccinia Virus Infections in Immunosuppressed Hairless Mice

PubMed Central

Dagley, Ashley; Downs, Brittney; Hagloch, Joseph; Tarbet, E. Bart

2014-01-01

The treatment of progressive vaccinia in individuals has involved antiviral drugs, such as cidofovir (CDV), brincidofovir, and/or tecovirimat, combined with vaccinia immune globulin (VIG). VIG is costly, and its supply is limited, so sparing the use of VIG during treatment is an important objective. VIG sparing was modeled in immunosuppressed mice by maximizing the treatment benefits of CDV combined with VIG to determine the effective treatments that delayed the time to death, reduced cutaneous lesion severity, and/or decreased tissue viral titers. SKH-1 hairless mice immunosuppressed with cyclophosphamide and hairless SCID mice (SHO strain) were infected cutaneously with vaccinia virus. Monotherapy, dual combinations (CDV plus VIG), or triple therapy (topical CDV, parenteral CDV, and VIG) were initiated 2 days postinfection and were given every 3 to 4 days through day 11. The efficacy assessment included survival rate, cutaneous lesion severity, and viral titers. Delays in the time to death and the reduction in lesion severity occurred in the following order of efficacy: triple therapy had greater efficacy than double combinations (CDV plus VIG or topical plus parenteral CDV), which had greater efficacy than VIG alone. Parenteral administration of CDV or VIG was necessary to suppress virus titers in internal organs (liver, lung, and spleen). The skin viral titers were significantly reduced by triple therapy only. The greatest efficacy was achieved by triple therapy. In humans, this regimen should translate to a faster cure rate, thus sparing the amount of VIG used for treatment. PMID:25385098

Functional hyper-IL-6 from vaccinia virus-colonized tumors triggers platelet formation and helps to alleviate toxicity of mitomycin C enhanced virus therapy

PubMed Central

2012-01-01

Background Combination of oncolytic vaccinia virus therapy with conventional chemotherapy has shown promise for tumor therapy. However, side effects of chemotherapy including thrombocytopenia, still remain problematic. Methods Here, we describe a novel approach to optimize combination therapy of oncolytic virus and chemotherapy utilizing virus-encoding hyper-IL-6, GLV-1h90, to reduce chemotherapy-associated side effects. Results We showed that the hyper-IL-6 cytokine was successfully produced by GLV-1h90 and was functional both in cell culture as well as in tumor-bearing animals, in which the cytokine-producing vaccinia virus strain was well tolerated. When combined with the chemotherapeutic mitomycin C, the anti-tumor effect of the oncolytic virotherapy was significantly enhanced. Moreover, hyper-IL-6 expression greatly reduced the time interval during which the mice suffered from chemotherapy-induced thrombocytopenia. Conclusion Therefore, future clinical application would benefit from careful investigation of additional cytokine treatment to reduce chemotherapy-induced side effects. PMID:22236378

Long-lasting stability of vaccinia virus (orthopoxvirus) in food and environmental samples.

PubMed

Essbauer, S; Meyer, H; Porsch-Ozcürümez, M; Pfeffer, M

2007-01-01

Poxviruses are known to remain infectious in the scabs of patients for months to years. The aim of this study was to investigate viral stability in storm water, food or gauze spiked with vaccinia virus strain Munich 1 (VACV M1). Storm water, storm water supplemented with either fetal calf serum (FCS) or potting soil was stored at two different temperatures (refrigerator, room temperature; 4 degrees C/25 degrees C). In addition, we analysed the viability of VACV M1 on the surface of bread, salad, sausages and gauze bandages stored at 4 degrees C. Samples were titrated in MA 104 cells and the presence of viral DNA was demonstrated by orthopoxvirus-specific PCRs. After 2 weeks, reisolation of VACV M1 from all kinds of food, bandage and water samples except for storm water supplemented with potting soil was possible. Viral DNA was detected in almost all samples by PCR. Prolonged experiments with VACV M1-spiked storm water and storm water supplemented with FCS revealed that samples kept at 4.5 degrees C are infectious for up to 166 days. Our data demonstrate that VACV M1 has a longlasting stability in water and food. The results obtained during this study should be taken into account for risk assessment calculations for poxvirus transmission. Implying that variola virus and vaccinia virus behave in a similar way, our data call for sophisticated countermeasures in cases of a variola release in biological warfare.

Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles.

PubMed

Schweneker, Marc; Laimbacher, Andrea S; Zimmer, Gert; Wagner, Susanne; Schraner, Elisabeth M; Wolferstätter, Michael; Klingenberg, Marieken; Dirmeier, Ulrike; Steigerwald, Robin; Lauterbach, Henning; Hochrein, Hubertus; Chaplin, Paul; Suter, Mark; Hausmann, Jürgen

2017-06-01

There are currently no approved therapeutics or vaccines to treat or protect against the severe hemorrhagic fever and death caused by Ebola virus (EBOV). Ebola virus-like particles (EBOV VLPs) consisting of the matrix protein VP40, the glycoprotein (GP), and the nucleoprotein (NP) are highly immunogenic and protective in nonhuman primates against Ebola virus disease (EVD). We have constructed a modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) recombinant coexpressing VP40 and GP of EBOV Mayinga and the NP of Taï Forest virus (TAFV) (MVA-BN-EBOV-VLP) to launch noninfectious EBOV VLPs as a second vaccine modality in the MVA-BN-EBOV-VLP-vaccinated organism. Human cells infected with either MVA-BN-EBOV-VLP or MVA-BN-EBOV-GP showed comparable GP expression levels and transport of complex N-glycosylated GP to the cell surface. Human cells infected with MVA-BN-EBOV-VLP produced large amounts of EBOV VLPs that were decorated with GP spikes but excluded the poxviral membrane protein B5, thus resembling authentic EBOV particles. The heterologous TAFV NP enhanced EBOV VP40-driven VLP formation with efficiency similar to that of the homologous EBOV NP in a transient-expression assay, and both NPs were incorporated into EBOV VLPs. EBOV GP-specific CD8 T cell responses were comparable between MVA-BN-EBOV-VLP- and MVA-BN-EBOV-GP-immunized mice. The levels of EBOV GP-specific neutralizing and binding antibodies, as well as GP-specific IgG1/IgG2a ratios induced by the two constructs, in mice were also similar, raising the question whether the quality rather than the quantity of the GP-specific antibody response might be altered by an EBOV VLP-generating MVA recombinant. IMPORTANCE The recent outbreak of Ebola virus (EBOV), claiming more than 11,000 lives, has underscored the need to advance the development of safe and effective filovirus vaccines. Virus-like particles (VLPs), as well as recombinant viral vectors, have proved to be promising vaccine candidates. Modified

RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

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

Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.

2015-01-15

Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore bemore » added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.« less

How Does Vaccinia Virus Interfere With Interferon?

PubMed

Smith, Geoffrey L; Talbot-Cooper, Callum; Lu, Yongxu

2018-01-01

Interferons (IFNs) are secreted glycoproteins that are produced by cells in response to virus infection and other stimuli and induce an antiviral state in cells bearing IFN receptors. In this way, IFNs restrict virus replication and spread before an adaptive immune response is developed. Viruses are very sensitive to the effects of IFNs and consequently have evolved many strategies to interfere with interferon. This is particularly well illustrated by poxviruses, which have large dsDNA genomes and encode hundreds of proteins. Vaccinia virus is the prototypic poxvirus and expresses many proteins that interfere with IFN and are considered in this review. These proteins act either inside or outside the cell and within the cytoplasm or nucleus. They function by restricting the production of IFN by blocking the signaling pathways leading to transcription of IFN genes, stopping IFNs binding to their receptors, blocking IFN-induced signal transduction leading to expression of interferon-stimulated genes (ISGs), or inhibiting the antiviral activity of ISG products. © 2018 Elsevier Inc. All rights reserved.

Structure and Assembly of Intracellular Mature Vaccinia Virus: Thin-Section Analyses

PubMed Central

Griffiths, Gareth; Roos, Norbert; Schleich, Sybille; Locker, Jacomine Krijnse

2001-01-01

In the preceding study (see accompanying paper), we showed by a variety of different techniques that intracellular mature vaccinia virus (vaccinia IMV) is unexpectedly complex in its structural organization and that this complexity also extends to the underlying viral core, which is highly folded. With that analysis as a foundation, we now present different thin-section electron microscopy approaches for analyzing the IMV and the processes by which it is assembled in infected HeLa cells. We focus on conventional epoxy resin thin sections as well as cryosections to describe key intermediates in the assembly process. We took advantage of streptolysin O's ability to selectively permeabilize the plasma membrane of infected cells to improve membrane contrast, and we used antibodies against bone fide integral membrane proteins of the virus to unequivocally identify membrane profiles in thin sections. All of the images presented here can be rationalized with respect to the model put forward for the assembly of the IMV in the accompanying paper. PMID:11602745

Live-vaccinia virus encapsulation in pH-sensitive polymer increases safety of a reservoir-targeted Lyme disease vaccine by targeting gastrointestinal release.

PubMed

Kern, Aurelie; Zhou, Chensheng W; Jia, Feng; Xu, Qiaobing; Hu, Linden T

2016-08-31

The incidence of Lyme disease has continued to rise despite attempts to control its spread. Vaccination of zoonotic reservoirs of human pathogens has been successfully used to decrease the incidence of rabies in raccoons and foxes. We have previously reported on the efficacy of a vaccinia virus vectored vaccine to reduce carriage of Borrelia burgdorferi in reservoir mice and ticks. One potential drawback to vaccinia virus vectored vaccines is the risk of accidental infection of humans. To reduce this risk, we developed a process to encapsulate vaccinia virus with a pH-sensitive polymer that inactivates the virus until it is ingested and dissolved by stomach acids. We demonstrate that the vaccine is inactive both in vitro and in vivo until it is released from the polymer. Once released from the polymer by contact with an acidic pH solution, the virus regains infectivity. Vaccination with coated vaccinia virus confers protection against B. burgdorferi infection and reduction in acquisition of the pathogen by naïve feeding ticks. Copyright © 2016. Published by Elsevier Ltd.

Generation and Production of Modified Vaccinia Virus Ankara (MVA) as a Vaccine Vector.

PubMed

Pavot, Vincent; Sebastian, Sarah; Turner, Alison V; Matthews, Jake; Gilbert, Sarah C

2017-01-01

The smallpox vaccine based on the vaccinia virus was successfully used to eradicate smallpox, but although very effective, it was a very reactogenic vaccine and responsible for the deaths of one to two people per million vaccinated. Modified Vaccinia virus Ankara (MVA) is an attenuated derivative, also used in the smallpox eradication campaign and now being developed as a recombinant viral vector to produce vaccines against infectious diseases and cancer. MVA can encode one or more foreign antigens and thus can function as a multivalent vaccine. The vector can be used at biosafety level 1, has intrinsic adjuvant properties, and induces humoral and cellular immune responses. Many clinical trials of these new vaccines have been conducted, and the safety of MVA is now well documented. Immunogenicity is influenced by the dose and vaccination regimen, and information on the efficacy of MVA-vectored vaccines is now beginning to accumulate. In this chapter, we provide protocols for generation, isolation, amplification, and purification of recombinant MVA for preclinical and clinical evaluation.

Bioluminescence imaging of a tumor-selective, thymidine kinase-defective vaccinia virus Guang9 strain after intratumoral or intraperitoneal administration in mice

PubMed Central

Ding, Yuedi; Fan, Jun; Deng, Lili; Peng, Ying; Zhang, Jue; Huang, Biao

2017-01-01

Vaccinia virus has been used as an oncolytic virus because of its capacity to preferentially infect tumors rather than normal tissues. The vaccinia Tian Tan strain, used as a vaccine against smallpox for millions of people in China, is a promising candidate for cancer therapy. In this study, we constructed an attenuated Tian Tan strain of Guang9 with a disrupted thymidine kinase gene to enhance tumor selectivity and an inserted firefly luciferase to monitor the viral distribution by in vivo bioluminescence imaging. Living animal imaging confirmed the high specificity of vaccinia Guang9 for tumor targeting after intratumoral and intraperitoneal administration. In addition, the vaccinia Guang9 strain produced higher in vivo luciferase activity and endured longer in immunocompromised nude mice than in immunocompetent C57BL/6 mice, all of which had been tumor-challenged. The luciferase activity and viral titers in excised tissues confirmed these conclusions. These data provide evidence for the safety and efficacy of the clinical application of vaccinia virus, which would be a promising approach for cancer therapy. PMID:29179469

Animal Movement and Establishment of Vaccinia Virus Cantagalo Strain in Amazon Biome, Brazil

PubMed Central

Quixabeira-Santos, Jociane Cristina; Medaglia, Maria Luiza G.; Pescador, Caroline A.

2011-01-01

To understand the emergence of vaccinia virus Cantagalo strain in the Amazon biome of Brazil, during 2008–2010 we conducted a molecular and epidemiologic survey of poxvirus outbreaks. Data indicate that animal movement was the major cause of virus dissemination within Rondônia State, leading to the establishment and spread of this pathogen. PMID:21470472

Vaccinia Virus C9 Ankyrin Repeat/F-Box Protein Is a Newly Identified Antagonist of the Type I Interferon-Induced Antiviral State.

PubMed

Liu, Ruikang; Moss, Bernard

2018-05-01

Type I interferons (IFNs) induce expression of more than 300 cellular genes that provide protection against viruses and other pathogens. For survival, viruses evolved defenses to prevent the IFN response or counteract the IFN-induced antiviral state. However, because viruses and cells coevolved, the dynamic relationship between virus and host is difficult to discern. In the present study, we demonstrated that vaccinia virus with a large deletion near the left end of the genome had a diminished ability to replicate in cells that had been pretreated with beta interferon (IFN-β), suggesting that one or more of the missing 17 open reading frames (ORFs) encode an antagonist of the IFN-induced antiviral state. By systematically deleting groups of ORFs and then individual ORFs, the C9L gene was shown to be required for IFN resistance. Replication of the C9L deletion mutant (vΔC9) was impaired in human cells that had been pretreated with IFN-β. Expression of viral early genes occurred, but subsequent events, including genome uncoating, genome replication, and postreplicative gene expression, were inhibited. Expression of the C9 protein occurred prior to genome replication, consistent with an early role in counteracting the IFN-induced antiviral state. C9 contains six ankyrin repeat motifs and a near C-terminal F-box. Mass spectrometry and immunoblotting identified host proteins that copurified with a functional epitope-tagged C9. The most abundant proteins were components of the SCF (CUL1, SKP1, F-box) and signalosome/deneddylation complexes, which interact with each other, suggesting a possible role in proteolysis of one or more interferon-induced proteins. IMPORTANCE Poxviruses comprise a family of large DNA viruses that replicate in the cytoplasm of vertebrate and insect hosts and cause human and zoonotic diseases. In most cases the primary infection is moderated by innate immune defenses. Vertebrates, including fish, amphibians, reptiles, birds, and mammals, all

Inhibition of Vaccinia virus entry by a broad spectrum antiviral peptide

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

Altmann, S.E.; Jones, J.C.; Schultz-Cherry, S.

2009-06-05

Concerns about the possible use of Variola virus, the causative agent of smallpox, as a weapon for bioterrorism have led to renewed efforts to identify new antivirals against orthopoxviruses. We identified a peptide, EB, which inhibited infection by Vaccinia virus with an EC{sub 50} of 15 muM. A control peptide, EBX, identical in composition to EB but differing in sequence, was inactive (EC{sub 50} > 200 muM), indicating sequence specificity. The inhibition was reversed upon removal of the peptide, and EB treatment had no effect on the physical integrity of virus particles as determined by electron microscopy. Viral adsorption wasmore » unaffected by the presence of EB, and the addition of EB post-entry had no effect on viral titers or on early gene expression. The addition of EB post-adsorption resulted in the inhibition of beta-galactosidase expression from an early viral promoter with an EC{sub 50} of 45 muM. A significant reduction in virus entry was detected in the presence of the peptide when the number of viral cores released into the cytoplasm was quantified. Electron microscopy indicated that 88% of the virions remained on the surface of cells in the presence of EB, compared to 37% in the control (p < 0.001). EB also blocked fusion-from-within, suggesting that virus infection is inhibited at the fusion step. Analysis of EB derivatives suggested that peptide length may be important for the activity of EB. The EB peptide is, to our knowledge, the first known small molecule inhibitor of Vaccinia virus entry.« less

Mucosal Immunization with Newcastle Disease Virus Vector Coexpressing HIV-1 Env and Gag Proteins Elicits Potent Serum, Mucosal, and Cellular Immune Responses That Protect against Vaccinia Virus Env and Gag Challenges.

PubMed

Khattar, Sunil K; Manoharan, Vinoth; Bhattarai, Bikash; LaBranche, Celia C; Montefiori, David C; Samal, Siba K

2015-07-21

Newcastle disease virus (NDV) avirulent strain LaSota was used to coexpress gp160 Env and p55 Gag from a single vector to enhance both Env-specific and Gag-specific immune responses. The optimal transcription position for both Env and Gag genes in the NDV genome was determined by generating recombinant NDV (rNDV)-Env-Gag (gp160 located between the P and M genes and Gag between the HN and L genes), rNDV-Gag-Env (Gag located between the P and M genes and gp160 between the HN and L genes), rNDV-Env/Gag (gp160 followed by Gag located between the P and M genes), and rNDV-Gag/Env (Gag followed by gp160 located between the P and M genes). All the recombinant viruses replicated at levels similar to those seen with parental NDV in embryonated chicken eggs and in chicken fibroblast cells. Both gp160 and Gag proteins were expressed at high levels in cell culture, with gp160 found to be incorporated into the envelope of NDV. The Gag and Env proteins expressed by all the recombinants except rNDV-Env-Gag self-assembled into human immunodeficiency virus type 1 (HIV-1) virus-like particles (VLPs). Immunization of guinea pigs by the intranasal route with these rNDVs produced long-lasting Env- and Gag-specific humoral immune responses. The Env-specific humoral and mucosal immune responses and Gag-specific humoral immune responses were higher in rNDV-Gag/Env and rNDV-Env/Gag than in the other recombinants. rNDV-Gag/Env and rNDV-Env/Gag were also more efficient in inducing cellular as well as protective immune responses to challenge with vaccinia viruses expressing HIV-1 Env and Gag in mice. These results suggest that vaccination with a single rNDV coexpressing Env and Gag represents a promising strategy to enhance immunogenicity and protective efficacy against HIV. A safe and effective vaccine that can induce both systemic and mucosal immune responses is needed to control HIV-1. In this study, we showed that coexpression of Env and Gag proteins of HIV-1 performed using a single

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

PubMed

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

2017-03-01

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

Preliminary analysis of murine cytotoxic T cell responses to the proteins of the flavivirus Kunjin using vaccinia virus expression.

PubMed

Parrish, C R; Coia, G; Hill, A; Müllbacher, A; Westaway, E G; Blanden, R V

1991-07-01

A series of recombinant vaccinia viruses expressing various parts of the entire Kunjin virus (KUN) coding region was used to analyse the cytotoxic T (Tc) cell responses to KUN. CBA/H mice inoculated with KUN or West Nile virus were shown to develop responses to KUN or various vaccinia virus expression constructs in either primary cytotoxic assays, or after secondary stimulation of the Tc cells in vitro with KUN antigens. Tc cells from CBA mice showed the strongest response to target cells infected with recombinant vaccinia viruses expressing parts of the KUN NS3 and NS4A proteins, and only a weak response to the other structural or non-structural proteins. Further analysis of deleted versions of the NS3-NS4A region showed that the main epitope recognized was derived from a sequence of 99 amino acids spanning parts of NS3 and NS4A. No other major epitopes were detected by Tc cells from CBA mice in the remaining 3333 amino acids of the KUN polypeptide.

Effect of Interferon, Polyacrylic Acid, and Polymethacrylic Acid on Tail Lesions in Mice Infected with Vaccinia Virus

PubMed Central

De Clercq, E.; De Somer, P.

1968-01-01

Intravenous inoculation of mice with vaccinia virus produced characteristic lesions of the tail surface which were suppressed by intraperitoneal administration of interferon and polyacrylic acid (PAA). Polymethacrylic acid (PMAA) stimulated the formation of vaccinia virus lesions. For full activity, both interferon and PAA must be given prior to infection. PAA was still significantly effective at small dose levels (3 mg/kg) and achieved protection for at least 4 weeks. Protection increased with increasing molecular weight of the polymer. The mode of action of PAA is discussed. PMID:5676405

SARS-CoV spike protein-expressing recombinant vaccinia virus efficiently induces neutralizing antibodies in rabbits pre-immunized with vaccinia virus.

PubMed

Kitabatake, Masahiro; Inoue, Shingo; Yasui, Fumihiko; Yokochi, Shoji; Arai, Masaaki; Morita, Kouichi; Shida, Hisatoshi; Kidokoro, Minoru; Murai, Fukashi; Le, Mai Quynh; Mizuno, Kyosuke; Matsushima, Kouji; Kohara, Michinori

2007-01-08

A vaccine for severe acute respiratory syndrome (SARS) is being intensively pursued against its re-emergence. We generated a SARS coronavirus (SARS-CoV) spike protein-expressing recombinant vaccinia virus (RVV-S) using highly attenuated strain LC16m8. Intradermal administration of RVV-S into rabbits induced neutralizing (NT) antibodies against SARS-CoV 1 week after administration and the NT titer reached 1:1000 after boost immunization with RVV-S. Significantly, NT antibodies against SARS-CoV were induced by administration of RVV-S to rabbits that had been pre-immunized with LC16m8. RVV-S can induce NT antibodies against SARS-CoV despite the presence of NT antibodies against VV. These results suggest that RVV-S may be a powerful SARS vaccine, including in patients previously immunized with the smallpox vaccine.

Postexposure prevention of progressive vaccinia in SCID mice treated with vaccinia immune globulin.

PubMed

Fisher, R W; Reed, J L; Snoy, P J; Mikolajczyk, M G; Bray, M; Scott, D E; Kennedy, M C

2011-01-01

A recently reported case of progressive vaccinia (PV) in an immunocompromised patient has refocused attention on this condition. Uniformly fatal prior to the licensure of vaccinia immune globulin (VIG) in 1978, PV was still fatal in about half of VIG-treated patients overall, with a greater mortality rate in infants and children. Additional therapies would be needed in the setting of a smallpox bioterror event, since mass vaccination following any variola virus release would inevitably result in exposure of immunocompromised people through vaccination or contact with vaccinees. Well-characterized animal models of disease can support the licensure of new products when human studies are not ethical or feasible, as in the case of PV. We chose vaccinia virus-scarified SCID mice to model PV. As in immunocompromised humans, vaccinia virus-scarified SCID animals develop enlarging primary lesions with minimal or no inflammation, eventual distal virus spread, and lethal outcomes if left untreated. Postexposure treatment with VIG slowed disease progression, caused local lesion regression, and resulted in the healthy survival of most of the mice for more than 120 days. Combination treatment with VIG and topical cidofovir also resulted in long-term disease-free survival of most of the animals, even when initiated 7 days postinfection. These results support the possibility that combination treatments may be effective in humans and support using this SCID model of PV to test new antibody therapies and combination therapies and to provide further insights into the pathogenesis and treatment of PV.

Elements in the Development of a Production Process for Modified Vaccinia Virus Ankara

PubMed Central

Jordan, Ingo; Lohr, Verena; Genzel, Yvonne; Reichl, Udo; Sandig, Volker

2013-01-01

The production of several viral vaccines depends on chicken embryo fibroblasts or embryonated chicken eggs. To replace this logistically demanding substrate, we created continuous anatine suspension cell lines (CR and CR.pIX), developed chemically-defined media, and established production processes for different vaccine viruses. One of the processes investigated in greater detail was developed for modified vaccinia virus Ankara (MVA). MVA is highly attenuated for human recipients and an efficient vector for reactogenic expression of foreign genes. Because direct cell-to-cell spread is one important mechanism for vaccinia virus replication, cultivation of MVA in bioreactors is facilitated if cell aggregates are induced after infection. This dependency may be the mechanism behind our observation that a novel viral genotype (MVA-CR) accumulates with serial passage in suspension cultures. Sequencing of a major part of the genomic DNA of the new strain revealed point mutations in three genes. We hypothesize that these changes confer an advantage because they may allow a greater fraction of MVA-CR viruses to escape the host cells for infection of distant targets. Production and purification of MVA-based vaccines may be simplified by this combination of designed avian cell line, chemically defined media and the novel virus strain. PMID:27694766

Elements in the Development of a Production Process for Modified Vaccinia Virus Ankara.

PubMed

Jordan, Ingo; Lohr, Verena; Genzel, Yvonne; Reichl, Udo; Sandig, Volker

2013-11-01

The production of several viral vaccines depends on chicken embryo fibroblasts or embryonated chicken eggs. To replace this logistically demanding substrate, we created continuous anatine suspension cell lines (CR and CR.pIX), developed chemically-defined media, and established production processes for different vaccine viruses. One of the processes investigated in greater detail was developed for modified vaccinia virus Ankara (MVA). MVA is highly attenuated for human recipients and an efficient vector for reactogenic expression of foreign genes. Because direct cell-to-cell spread is one important mechanism for vaccinia virus replication, cultivation of MVA in bioreactors is facilitated if cell aggregates are induced after infection. This dependency may be the mechanism behind our observation that a novel viral genotype (MVA-CR) accumulates with serial passage in suspension cultures. Sequencing of a major part of the genomic DNA of the new strain revealed point mutations in three genes. We hypothesize that these changes confer an advantage because they may allow a greater fraction of MVA-CR viruses to escape the host cells for infection of distant targets. Production and purification of MVA-based vaccines may be simplified by this combination of designed avian cell line, chemically defined media and the novel virus strain.

Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins.

PubMed

Jancovich, James K; Chapman, Dave; Hansen, Debra T; Robida, Mark D; Loskutov, Andrey; Craciunescu, Felicia; Borovkov, Alex; Kibler, Karen; Goatley, Lynnette; King, Katherine; Netherton, Christopher L; Taylor, Geraldine; Jacobs, Bertram; Sykes, Kathryn; Dixon, Linda K

2018-04-15

African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs. IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant vaccinia

Immunization of Pigs by DNA Prime and Recombinant Vaccinia Virus Boost To Identify and Rank African Swine Fever Virus Immunogenic and Protective Proteins

PubMed Central

2018-01-01

ABSTRACT African swine fever virus (ASFV) causes an acute hemorrhagic fever in domestic pigs, with high socioeconomic impact. No vaccine is available, limiting options for control. Although live attenuated ASFV can induce up to 100% protection against lethal challenge, little is known of the antigens which induce this protective response. To identify additional ASFV immunogenic and potentially protective antigens, we cloned 47 viral genes in individual plasmids for gene vaccination and in recombinant vaccinia viruses. These antigens were selected to include proteins with different functions and timing of expression. Pools of up to 22 antigens were delivered by DNA prime and recombinant vaccinia virus boost to groups of pigs. Responses of immune lymphocytes from pigs to individual recombinant proteins and to ASFV were measured by interferon gamma enzyme-linked immunosorbent spot (ELISpot) assays to identify a subset of the antigens that consistently induced the highest responses. All 47 antigens were then delivered to pigs by DNA prime and recombinant vaccinia virus boost, and pigs were challenged with a lethal dose of ASFV isolate Georgia 2007/1. Although pigs developed clinical and pathological signs consistent with acute ASFV, viral genome levels were significantly reduced in blood and several lymph tissues in those pigs immunized with vectors expressing ASFV antigens compared with the levels in control pigs. IMPORTANCE The lack of a vaccine limits the options to control African swine fever. Advances have been made in the development of genetically modified live attenuated ASFV that can induce protection against challenge. However, there may be safety issues relating to the use of these in the field. There is little information about ASFV antigens that can induce a protective immune response against challenge. We carried out a large screen of 30% of ASFV antigens by delivering individual genes in different pools to pigs by DNA immunization prime and recombinant

N1L is an ectromelia virus virulence factor and essential for in vivo spread upon respiratory infection.

PubMed

Gratz, Meike S; Suezer, Yasemin; Kremer, Melanie; Volz, Asisa; Majzoub, Monir; Hanschmann, Kay-Martin; Kalinke, Ulrich; Schwantes, Astrid; Sutter, Gerd

2011-04-01

The emergence of zoonotic orthopoxvirus infections and the threat of possible intentional release of pathogenic orthopoxviruses have stimulated renewed interest in understanding orthopoxvirus infections and the resulting diseases. Ectromelia virus (ECTV), the causative agent of mousepox, offers an excellent model system to study an orthopoxvirus infection in its natural host. Here, we investigated the role of the vaccinia virus ortholog N1L in ECTV infection. Respiratory infection of mice with an N1L deletion mutant virus (ECTVΔN1L) demonstrated profound attenuation of the mutant virus, confirming N1 as an orthopoxvirus virulence factor. Upon analysis of virus dissemination in vivo, we observed a striking deficiency of ECTVΔN1L spreading from the lungs to the livers or spleens of infected mice. Investigating the immunological mechanism controlling ECTVΔN1L infection, we found the attenuated phenotype to be unaltered in mice deficient in Toll-like receptor (TLR) or RIG-I-like RNA helicase (RLH) signaling as well as in those missing the type I interferon receptor or lacking B cells. However, in RAG-1(-/-) mice lacking mature B and T cells, ECTVΔN1L regained virulence, as shown by increasing morbidity and virus spread to the liver and spleen. Moreover, T cell depletion experiments revealed that ECTVΔN1L attenuation was reversed only by removing both CD4(+) and CD8(+) T cells, so the presence of either cell subset was still sufficient to control the infection. Thus, the orthopoxvirus virulence factor N1 may allow efficient ECTV infection in mice by interfering with host T cell function.

The vaccinia virus E6 protein influences virion protein localization during virus assembly

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

Condit, Richard C., E-mail: condit@mgm.ufl.edu; Moussatche, Nissin

2015-08-15

Vaccinia virus mutants in which expression of the virion core protein gene E6R is repressed are defective in virion morphogenesis. E6 deficient infections fail to properly package viroplasm into viral membranes, resulting in an accumulation of empty immature virions and large aggregates of viroplasm. We have used immunogold electron microscopy and immunofluorescence confocal microscopy to assess the intracellular localization of several virion structural proteins and enzymes during E6R mutant infections. We find that during E6R mutant infections virion membrane proteins and virion transcription enzymes maintain a normal localization within viral factories while several major core and lateral body proteins accumulatemore » in aggregated virosomes. The results support a model in which vaccinia virions are assembled from at least three substructures, the membrane, the viroplasm and a “pre-nucleocapsid”, and that the E6 protein is essential for maintaining proper localization of the seven-protein complex and the viroplasm during assembly. - Highlights: • Mutation of E6 disrupts association of viral membranes with viral core proteins • Mutation of E6 does not perturb viral membrane biosynthesis • Mutation of E6 does not perturb localization of viral transcription enzymes • Mutation of E6 causes mis-localization and aggregation of viral core proteins • Vaccinia assembly uses three subassemblies: membranes, viroplasm, prenucleocapsid.« less

Effect of the Deletion of Genes Encoding Proteins of the Extracellular Virion Form of Vaccinia Virus on Vaccine Immunogenicity and Protective Effectiveness in the Mouse Model

PubMed Central

Meseda, Clement A.; Campbell, Joseph; Kumar, Arunima; Garcia, Alonzo D.; Merchlinsky, Michael; Weir, Jerry P.

2013-01-01

Antibodies to both infectious forms of vaccinia virus, the mature virion (MV) and the enveloped virion (EV), as well as cell-mediated immune response appear to be important for protection against smallpox. EV virus particles, although more labile and less numerous than MV, are important for dissemination and spread of virus in infected hosts and thus important in virus pathogenesis. The importance of the EV A33 and B5 proteins for vaccine induced immunity and protection in a murine intranasal challenge model was evaluated by deletion of both the A33R and B5R genes in a vaccine-derived strain of vaccinia virus. Deletion of either A33R or B5R resulted in viruses with a small plaque phenotype and reduced virus yields, as reported previously, whereas deletion of both EV protein-encoding genes resulted in a virus that formed small infection foci that were detectable and quantifiable only by immunostaining and an even more dramatic decrease in total virus yield in cell culture. Deletion of B5R, either as a single gene knockout or in the double EV gene knockout virus, resulted in a loss of EV neutralizing activity, but all EV gene knockout viruses still induced a robust neutralizing activity against the vaccinia MV form of the virus. The effect of elimination of A33 and/or B5 on the protection afforded by vaccination was evaluated by intranasal challenge with a lethal dose of either vaccinia virus WR or IHD-J, a strain of vaccinia virus that produces relatively higher amounts of EV virus. The results from multiple experiments, using a range of vaccination doses and virus challenge doses, and using mortality, morbidity, and virus dissemination as endpoints, indicate that the absence of A33 and B5 have little effect on the ability of a vaccinia vaccine virus to provide protection against a lethal intranasal challenge in a mouse model. PMID:23785523

Homology between DNA polymerases of poxviruses, herpesviruses, and adenoviruses: nucleotide sequence of the vaccinia virus DNA polymerase gene.

PubMed Central

Earl, P L; Jones, E V; Moss, B

1986-01-01

A 5400-base-pair segment of the vaccinia virus genome was sequenced and an open reading frame of 938 codons was found precisely where the DNA polymerase had been mapped by transfer of a phosphonoacetate-resistance marker. A single nucleotide substitution changing glycine at position 347 to aspartic acid accounts for the drug resistance of the mutant vaccinia virus. The 5' end of the DNA polymerase mRNA was located 80 base pairs before the methionine codon initiating the open reading frame. Correspondence between the predicted Mr 108,577 polypeptide and the 110,000 purified enzyme indicates that little or no proteolytic processing occurs. Extensive homology, extending over 435 amino acids, was found upon comparing the DNA polymerase of vaccinia virus and DNA polymerase of Epstein-Barr virus. A highly conserved sequence of 14 amino acids in the carboxyl-terminal regions of the above DNA polymerases is also present at a similar location in adenovirus DNA polymerase. This structure, which is predicted to form a turn flanked by beta-pleated sheets, may form part of an essential binding or catalytic site that accounts for its presence in DNA polymerases of poxviruses, herpesviruses, and adenoviruses. Images PMID:3012524

Vaccinia virus protein A3 is required for the production of normal immature virions and for the encapsidation of the nucleocapsid protein L4

PubMed Central

Jesus, Desyree Murta; Moussatche, Nissin; McFadden, Baron D.; Nielsen, Casey Paulasue; D’Costa, Susan M.; Condit, Richard C.

2015-01-01

Maturation of the vaccinia virion is an intricate process that results in the organization of the viroplasm contained in immature virions into the lateral bodies, core wall and nucleocapsid observed in the mature particles. It is unclear how this organization takes place and studies with mutants are indispensable in understanding this process. By characterizing an inducible mutant in the A3L gene, we revealed that A3, an inner core wall protein, is important for formation of normal immature viruses and also for the correct localization of L4, a nucleocapsid protein. L4 did not accumulate in the viral factories in the absence of A3 and was not encapsidated in the particles that do not contain A3. These data strengthen our previously suggested hypothesis that A3 and L4 interact and that this interaction is critical for proper formation of the core wall and nucleocapsid. PMID:25765002

Genetically Engineered Vaccinia Viruses As Agents for Cancer Treatment, Imaging, and Transgene Delivery

PubMed Central

Haddad, Dana

2017-01-01

Despite advances in technology, the formidable challenge of treating cancer, especially if advanced, still remains with no significant improvement in survival rates, even with the most common forms of cancer. Oncolytic viral therapies have shown great promise for the treatment of various cancers, with the possible advantages of stronger treatment efficacy compared to conventional therapy due to higher tumor selectivity, and less toxicity. They are able to preferentially and selectively propagate in cancer cells, consequently destroying tumor tissue mainly via cell lysis, while leaving non-cancerous tissues unharmed. Several wild-type and genetically engineered vaccinia virus (VACV) strains have been tested in both preclinical and clinical trials with promising results. Greater understanding and advancements in molecular biology have enabled the generation of genetically engineered oncolytic viruses for safer and more efficacious treatment, including arming VACVs with cytokines and immunostimulatory molecules, anti-angiogenic agents, and enzyme prodrug therapy, in addition to combining VACVs with conventional external and systemic radiotherapy, chemotherapy, immunotherapy, and other virus strains. Furthermore, novel oncolytic vaccinia virus strains have been generated that express reporter genes for the tracking and imaging of viral therapy and monitoring of therapeutic response. Further study is needed to unlock VACVs’ full potential as part of the future of cancer therapy. PMID:28589082

Protective Efficacy of the Conserved NP, PB1, and M1 Proteins as Immunogens in DNA- and Vaccinia Virus-Based Universal Influenza A Virus Vaccines in Mice

PubMed Central

Wang, Wenling; Li, Renqing; Deng, Yao; Lu, Ning; Chen, Hong; Meng, Xin; Wang, Wen; Wang, Xiuping; Yan, Kexia; Qi, Xiangrong; Zhang, Xiangmin; Xin, Wei; Lu, Zhenhua; Li, Xueren; Bian, Tao; Gao, Yingying; Tan, Wenjie

2015-01-01

The conventional hemagglutinin (HA)- and neuraminidase (NA)-based influenza vaccines need to be updated most years and are ineffective if the glycoprotein HA of the vaccine strains is a mismatch with that of the epidemic strain. Universal vaccines targeting conserved viral components might provide cross-protection and thus complement and improve conventional vaccines. In this study, we generated DNA plasmids and recombinant vaccinia viruses expressing the conserved proteins nucleoprotein (NP), polymerase basic 1 (PB1), and matrix 1 (M1) from influenza virus strain A/Beijing/30/95 (H3N2). BALB/c mice were immunized intramuscularly with a single vaccine based on NP, PB1, or M1 alone or a combination vaccine based on all three antigens and were then challenged with lethal doses of the heterologous influenza virus strain A/PR/8/34 (H1N1). Vaccines based on NP, PB1, and M1 provided complete or partial protection against challenge with 1.7 50% lethal dose (LD50) of PR8 in mice. Of the three antigens, NP-based vaccines induced protection against 5 LD50 and 10 LD50 and thus exhibited the greatest protective effect. Universal influenza vaccines based on the combination of NP, PB1, and M1 induced a strong immune response and thus might be an alternative approach to addressing future influenza virus pandemics. PMID:25834017

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

Detection of Vaccinia virus in blood and faeces of experimentally infected cows.

PubMed

Guedes, M I M C; Rehfeld, I S; de Oliveira, T M L; Assis, F L; Matos, A C D; Abrahão, J S; Kroon, E G; Lobato, Z I P

2013-12-01

Bovine vaccinia (BV), a zoonosis caused by Vaccinia virus (VACV), affects dairy cattle and milkers, causing economic, veterinary and human health impacts. Despite such impacts, there are no experimental studies about the pathogenesis of BV in cows to assess whether there is a systemic spread of the virus and whether there are different ways of VACV shedding. Trying to answer some of these questions, a study was proposed using experimental inoculation of VACV in cows. All experimentally infected cows developed lesions compatible with VACV infection in cattle. Two of the six animals presented VACV DNA in blood and faecal samples, starting at the 2nd and the 3rd day post-infection (d.p.i.), respectively, and lasting until the 36th d.p.i., in an intermittent way. This study provides new evidence that VACV can be detected in blood and faeces of infected cows, suggesting that BV could be a systemic disease, and also bringing new information about the epidemiology and pathogenesis of BV. © 2012 Blackwell Verlag GmbH.

A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen

PubMed Central

Vijayan, Aneesh; García-Arriaza, Juan; C. Raman, Suresh; Conesa, José Javier; Chichón, Francisco Javier; Santiago, César; Sorzano, Carlos Óscar S.; Carrascosa, José L.; Esteban, Mariano

2015-01-01

In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive

Efficient and stable production of Modified Vaccinia Ankara virus in two-stage semi-continuous and in continuous stirred tank cultivation systems.

PubMed

Tapia, Felipe; Jordan, Ingo; Genzel, Yvonne; Reichl, Udo

2017-01-01

One important aim in cell culture-based viral vaccine and vector production is the implementation of continuous processes. Such a development has the potential to reduce costs of vaccine manufacturing as volumetric productivity is increased and the manufacturing footprint is reduced. In this work, continuous production of Modified Vaccinia Ankara (MVA) virus was investigated. First, a semi-continuous two-stage cultivation system consisting of two shaker flasks in series was established as a small-scale approach. Cultures of the avian AGE1.CR.pIX cell line were expanded in the first shaker, and MVA virus was propagated and harvested in the second shaker over a period of 8-15 days. A total of nine small-scale cultivations were performed to investigate the impact of process parameters on virus yields. Harvest volumes of 0.7-1 L with maximum TCID50 titers of up to 1.0×109 virions/mL were obtained. Genetic analysis of control experiments using a recombinant MVA virus containing green-fluorescent-protein suggested that the virus was stable over at least 16 d of cultivation. In addition, a decrease or fluctuation of infectious units that may indicate an excessive accumulation of defective interfering particles was not observed. The process was automated in a two-stage continuous system comprising two connected 1 L stirred tank bioreactors. Stable MVA virus titers, and a total production volume of 7.1 L with an average TCID50 titer of 9×107 virions/mL was achieved. Because titers were at the lower range of the shake flask cultivations potential for further process optimization at large scale will be discussed. Overall, MVA virus was efficiently produced in continuous and semi-continuous cultivations making two-stage stirred tank bioreactor systems a promising platform for industrial production of MVA-derived recombinant vaccines and viral vectors.

Recombinant modified vaccinia virus Ankara generating excess early double-stranded RNA transiently activates protein kinase R and triggers enhanced innate immune responses.

PubMed

Wolferstätter, Michael; Schweneker, Marc; Späth, Michaela; Lukassen, Susanne; Klingenberg, Marieken; Brinkmann, Kay; Wielert, Ursula; Lauterbach, Henning; Hochrein, Hubertus; Chaplin, Paul; Suter, Mark; Hausmann, Jürgen

2014-12-01

Double-stranded RNA (dsRNA) is an important molecular pattern associated with viral infection and is detected by various extra- and intracellular recognition molecules. Poxviruses have evolved to avoid producing dsRNA early in infection but generate significant amounts of dsRNA late in infection due to convergent transcription of late genes. Protein kinase R (PKR) is activated by dsRNA and triggers major cellular defenses against viral infection, including protein synthesis shutdown, apoptosis, and type I interferon (IFN-I) production. The poxviral E3 protein binds and sequesters viral dsRNA and is a major antagonist of the PKR pathway. We found that the highly replication-restricted modified vaccinia virus Ankara (MVA) engineered to produce excess amounts of dsRNA early in infection showed enhanced induction of IFN-β in murine and human cells in the presence of an intact E3L gene. IFN-β induction required a minimum overlap length of 300 bp between early complementary transcripts and was strongly PKR dependent. Excess early dsRNA produced by MVA activated PKR early but transiently in murine cells and induced enhanced systemic levels of IFN-α, IFN-γ, and other cytokines and chemokines in mice in a largely PKR-dependent manner. Replication-competent chorioallantois vaccinia virus Ankara (CVA) generating excess early dsRNA also enhanced IFN-I production and was apathogenic in mice even at very high doses but showed no in vitro host range defect. Thus, genetically adjuvanting MVA and CVA to generate excess early dsRNA is an effective method to enhance innate immune stimulation by orthopoxvirus vectors and to attenuate replicating vaccinia virus in vivo. Efficient cellular sensing of pathogen-specific components, including double-stranded RNA (dsRNA), is an important prerequisite of an effective antiviral immune response. The prototype poxvirus vaccinia virus (VACV) and its derivative modified vaccinia virus Ankara (MVA) produce dsRNA as a by-product of viral

Protective Efficacy of the Conserved NP, PB1, and M1 Proteins as Immunogens in DNA- and Vaccinia Virus-Based Universal Influenza A Virus Vaccines in Mice.

PubMed

Wang, Wenling; Li, Renqing; Deng, Yao; Lu, Ning; Chen, Hong; Meng, Xin; Wang, Wen; Wang, Xiuping; Yan, Kexia; Qi, Xiangrong; Zhang, Xiangmin; Xin, Wei; Lu, Zhenhua; Li, Xueren; Bian, Tao; Gao, Yingying; Tan, Wenjie; Ruan, Li

2015-06-01

The conventional hemagglutinin (HA)- and neuraminidase (NA)-based influenza vaccines need to be updated most years and are ineffective if the glycoprotein HA of the vaccine strains is a mismatch with that of the epidemic strain. Universal vaccines targeting conserved viral components might provide cross-protection and thus complement and improve conventional vaccines. In this study, we generated DNA plasmids and recombinant vaccinia viruses expressing the conserved proteins nucleoprotein (NP), polymerase basic 1 (PB1), and matrix 1 (M1) from influenza virus strain A/Beijing/30/95 (H3N2). BALB/c mice were immunized intramuscularly with a single vaccine based on NP, PB1, or M1 alone or a combination vaccine based on all three antigens and were then challenged with lethal doses of the heterologous influenza virus strain A/PR/8/34 (H1N1). Vaccines based on NP, PB1, and M1 provided complete or partial protection against challenge with 1.7 50% lethal dose (LD50) of PR8 in mice. Of the three antigens, NP-based vaccines induced protection against 5 LD50 and 10 LD50 and thus exhibited the greatest protective effect. Universal influenza vaccines based on the combination of NP, PB1, and M1 induced a strong immune response and thus might be an alternative approach to addressing future influenza virus pandemics. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Analysis of canine herpesvirus gB, gC and gD expressed by a recombinant vaccinia virus.

PubMed

Xuan, X; Kojima, A; Murata, T; Mikami, T; Otsuka, H

1997-01-01

The genes encoding the canine herpesvirus (CHV) glycoprotein B (gB), gC and gD homologues have been reported already. However, products of these genes have not been identified yet. Previously, we have identified three CHV glycoproteins, gp 145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gB, gC or gD, the putative genes of gB, gC, and gD of CHV were inserted into the thymidine kinase gene of vaccinia virus LC16mO strain under the control of the early-late promoter for the vaccinia virus 7.5-kilodalton polypeptide. We demonstrated here that gp145/112, gp80 and gp47 were the translation products of the CHV gB, gC and gD genes, respectively. The antigenic authenticity of recombinant gB, gC and gD were confirmed by a panel of MAbs specific for each glycoprotein produced in CHV-infected cells. Immunization of mice with these recombinants produced high titers of neutralizing antibodies against CHV. These results suggest that recombinant vaccinia viruses expressing CHV gB, gC and gD may be useful to develop a vaccine to control CHV infection.

Nucleotide sequence of a cluster of early and late genes in a conserved segment of the vaccinia virus genome.

PubMed Central

Plucienniczak, A; Schroeder, E; Zettlmeissl, G; Streeck, R E

1985-01-01

The nucleotide sequence of a 7.6 kb vaccinia DNA segment from a genomic region conserved among different orthopox virus has been determined. This segment contains a tight cluster of 12 partly overlapping open reading frames most of which can be correlated with previously identified early and late proteins and mRNAs. Regulatory signals used by vaccinia virus have been studied. Presumptive promoter regions are rich in A, T and carry the consensus sequences TATA and AATAA spaced at 20-24 base pairs. Tandem repeats of a CTATTC consensus sequence are proposed to be involved in the termination of early transcription. PMID:2987815

Ectopic expression of vaccinia virus E3 and K3 cannot rescue ectromelia virus replication in rabbit RK13 cells.

PubMed

Hand, Erin S; Haller, Sherry L; Peng, Chen; Rothenburg, Stefan; Hersperger, Adam R

2015-01-01

As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV.

Silk-elastin-like protein polymer matrix for intraoperative delivery of an oncolytic vaccinia virus.

PubMed

Price, Daniel L; Li, Pingdong; Chen, Chun-Hao; Wong, Danni; Yu, Zhenkun; Chen, Nanhai G; Yu, Yong A; Szalay, Aladar A; Cappello, Joseph; Fong, Yuman; Wong, Richard J

2016-02-01

Oncolytic viral efficacy may be limited by the penetration of the virus into tumors. This may be enhanced by intraoperative application of virus immediately after surgical resection. Oncolytic vaccinia virus GLV-1h68 was delivered in silk-elastin-like protein polymer (SELP) in vitro and in vivo in anaplastic thyroid carcinoma cell line 8505c in nude mice. GLV-1h68 in SELP infected and lysed anaplastic thyroid cancer cells in vitro equally as effectively as in phosphate-buffered saline (PBS), and at 1 week retains a thousand fold greater infectious plaque-forming units. In surgical resection models of residual tumor, GLV-1h68 in SELP improves tumor control and shows increased viral β-galactosidase expression as compared to PBS. The use of SELP matrix for intraoperative oncolytic viral delivery protects infectious viral particles from degradation, facilitates sustained viral delivery and transgene expression, and improves tumor control. Such optimization of methods of oncolytic viral delivery may enhance therapeutic outcomes. © 2014 Wiley Periodicals, Inc.

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

PubMed

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

2011-10-25

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

Enhancement of CD8+ T-cell memory by removal of a vaccinia virus nuclear factor-κB inhibitor

PubMed Central

Ren, Hongwei; Ferguson, Brian J; de Motes, Carlos Maluquer; Sumner, Rebecca P; Harman, Laura E R; Smith, Geoffrey L

2015-01-01

Factors influencing T-cell responses are important for vaccine development but are incompletely understood. Here, vaccinia virus (VACV) protein N1 is shown to impair the development of both effector and memory CD8+ T cells and this correlates with its inhibition of nuclear factor-κB (NF-κB) activation. Infection with VACVs that either have the N1L gene deleted (vΔN1) or contain a I6E mutation (vN1.I6E) that abrogates its inhibition of NF-κB resulted in increased central and memory CD8+ T-cell populations, increased CD8+ T-cell cytotoxicity and lower virus titres after challenge. Furthermore, CD8+ memory T-cell function was increased following infection with vN1.I6E, with more interferon-γ production and greater protection against VACV infection following passive transfer to naive mice, compared with CD8+ T cells from mice infected with wild-type virus (vN1.WT). This demonstrates the importance of NF-κB activation within infected cells for long-term CD8+ T-cell memory and vaccine efficacy. Further, it provides a rationale for deleting N1 from VACV vectors to enhance CD8+ T-cell immunogenicity, while simultaneously reducing virulence to improve vaccine safety. PMID:25382035

Host range, growth property, and virulence of the smallpox vaccine: Vaccinia virus Tian Tan strain

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

Fang Qing; Yang Lin; Zhu Weijun

2005-05-10

Vaccinia Tian Tan (VTT) was used as a vaccine against smallpox in China for millions of people before 1980, yet the biological characteristics of the virus remain unclear. We have characterized VTT with respect to its host cell range, growth properties in vitro, and virulence in vivo. We found that 11 of the 12 mammalian cell lines studied are permissive to VTT infection whereas one, CHO-K1, is non-permissive. Using electron microscopy and sequence analysis, we found that the restriction of VTT replication in CHO-K1 is at a step before viral maturation probably due to the loss of the V025 gene.more » Moreover, VTT is significantly less virulent than vaccinia WR but remains neurovirulent in mice and causes significant body weight loss after intranasal inoculation. Our data demonstrate the need for further attenuation of VTT to serve either as a safer smallpox vaccine or as a live vaccine vector for other pathogens.« less

Serologic and Molecular Evidence of Vaccinia Virus Circulation among Small Mammals from Different Biomes, Brazil

PubMed Central

Miranda, Júlia B.; Borges, Iara A.; Campos, Samantha P.S.; Vieira, Flávia N.; de Ázara, Tatiana M.F.; Marques, Fernanda A.; Costa, Galileu B.; Luis, Ana Paula M.F.; de Oliveira, Jaqueline S.; Ferreira, Paulo César P.; Bonjardim, Cláudio Antônio; da Silva, Silvio L.M.; Eiras, Álvaro E.; Abrahão, Jônatas S.; Kroon, Erna G.; Drumond, Betânia P.; Paglia, Adriano P.

2017-01-01

Vaccinia virus (VACV) is a zoonotic agent that causes a disease called bovine vaccinia, which is detected mainly in milking cattle and humans in close contact with these animals. Even though many aspects of VACV infection have been described, much is still unknown about its circulation in the environment and its natural hosts/reservoirs. To investigate the presence of Orthopoxvirus antibodies or VACV DNA, we captured small rodents and marsupials in 3 areas of Minas Gerais state, Brazil, and tested their samples in a laboratory. A total of 336 animals were tested; positivity ranged from 18.1% to 25.5% in the 3 studied regions located in different biomes, including the Atlantic Forest and the Cerrado. Analysis of nucleotide sequences indicated co-circulation of VACV groups I and II. Our findings reinforce the possible role played by rodents and marsupials in VACV maintenance and its transmission chain. PMID:28518030

The generation of CD8+ T-cell population specific for vaccinia virus epitope involved in the antiviral protection against ectromelia virus challenge.

PubMed

Gierynska, Malgorzata; Szulc-Dabrowska, Lidia; Dzieciatkowski, Tomasz; Golke, Anna; Schollenberger, Ada

2015-12-01

Eradication of smallpox has led to cessation of vaccination programs. This has rendered the human population increasingly susceptible not only to variola virus infection but also to infections with other representatives of Poxviridae family that cause zoonotic variola-like diseases. Thus, new approaches for designing improved vaccine against smallpox are required. Discovering that orthopoxviruses, e.g. variola virus, vaccinia virus, ectromelia virus, share common immunodominant antigen, may result in the development of such a vaccine. In our study, the generation of antigen-specific CD8(+) T cells in mice during the acute and memory phase of the immune response was induced using the vaccinia virus immunodominant TSYKFESV epitope and CpG oligodeoxynucleotides as adjuvants. The role of the generated TSYKFESV-specific CD8(+) T cells was evaluated in mice during ectromelia virus infection using systemic and mucosal model. Moreover, the involvement of dendritic cells subsets in the adaptive immune response stimulation was assessed. Our results indicate that the TSYKFESV epitope/TLR9 agonist approach, delivered systemically or mucosally, generated strong CD8(+) T-cell response when measured 10 days after immunization. Furthermore, the TSYKFESV-specific cell population remained functionally active 2 months post-immunization, and gave cross-protection in virally challenged mice, even though the numbers of detectable antigen-specific T cells decreased. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Recombinant modified vaccinia virus Ankara-based malaria vaccines.

PubMed

Sebastian, Sarah; Gilbert, Sarah C

2016-01-01

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

Real-time PCR to identify variola virus or other human pathogenic orthopox viruses.

PubMed

Scaramozzino, Natale; Ferrier-Rembert, Audrey; Favier, Anne-Laure; Rothlisberger, Corinne; Richard, Stéphane; Crance, Jean-Marc; Meyer, Hermann; Garin, Daniel

2007-04-01

Variola virus (family Poxviridae, genus Orthopoxvirus) and the closely related cowpox, vaccinia, and monkeypox viruses can infect humans. Efforts are mounting to replenish the smallpox vaccine stocks, optimize diagnostic methods for poxviruses, and develop new antivirals against smallpox, because it is feared that variola virus might be used as a weapon of bioterrorism. We developed an assay for the detection of variola virus DNA. The assay is based on TaqMan chemistry targeting the 14-kD protein gene. For the 1st stage of the assay we used genus consensus primers and a mixture of 2 probes (14-kD POX and 14-kD VAR) spanning the 14-kD protein-encoding gene for detection of all human pathogenic orthopoxviruses. We then tested positive samples with the specific orthopoxvirus-specific probe 14-kD POX to identify monkeypox, cowpox, and vaccinia viruses and with the 14-kD VAR probe to identify variola viruses. The assay was established on 4 different PCR cycler platforms. It was assessed in a study with 85 different orthopoxvirus species and strains that included variola, camelpox, cowpox, monkeypox, and vaccinia viruses at concentrations ranging from 100 ng/L to 1 microg/L. The assay detected as little as 0.05 fg of DNA, corresponding to 25 copies of DNA, and enabled differentiation of variola virus from the other orthopoxviruses. This real-time PCR assay provides a rapid method for the early detection and differentiation of smallpox and other human pathogenic orthopoxvirus infections.

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

PubMed

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

2017-06-06

Viruses are used as expression vectors for protein synthesis, immunology research, vaccines, and therapeutics. Advantages of poxvirus vectors include the accommodation of large amounts of heterologous DNA, the presence of a cytoplasmic site of transcription, and high expression levels. On the other hand, competition of approximately 200 viral genes with the target gene for expression and immune recognition may be disadvantageous. We describe a vaccinia virus (VACV) vector that uses an early promoter to express the bacteriophage T7 RNA polymerase; has the A23R intermediate transcription factor gene deleted, thereby restricting virus replication to complementing cells; and has a heterologous gene regulated by a T7 promoter. In noncomplementing cells, viral early gene expression and DNA replication occurred normally but synthesis of intermediate and late proteins was prevented. Nevertheless, the progeny viral DNA provided templates for abundant expression of heterologous genes regulated by a T7 promoter. Selective expression of the Escherichia coli lac repressor gene from an intermediate promoter reduced transcription of the heterologous gene specifically in complementing cells, where large amounts might adversely impact VACV replication. Expression of heterologous proteins mediated by the A23R deletion vector equaled that of a replicating VACV, was higher than that of a nonreplicating modified vaccinia virus Ankara (MVA) vector used for candidate vaccines in vitro and in vivo , and was similarly immunogenic in mice. Unlike the MVA vector, the A23R deletion vector still expresses numerous early genes that can restrict immunogenicity as demonstrated here by the failure of the prototype vector to induce interferon alpha. By deleting immunomodulatory genes, we anticipate further improvements in the system. IMPORTANCE Vaccines provide an efficient and effective way of preventing infectious diseases. Nevertheless, new and better vaccines are needed. Vaccinia virus, which

Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition.

PubMed

Kleinpeter, Patricia; Fend, Laetitia; Thioudellet, Christine; Geist, Michel; Sfrontato, Nathalie; Koerper, Véronique; Fahrner, Catherine; Schmitt, Doris; Gantzer, Murielle; Remy-Ziller, Christelle; Brandely, Renée; Villeval, Dominique; Rittner, Karola; Silvestre, Nathalie; Erbs, Philippe; Zitvogel, Laurence; Quéméneur, Eric; Préville, Xavier; Marchand, Jean-Baptiste

2016-01-01

We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro . Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8 + and CD4 + ). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs.

Vectorization in an oncolytic vaccinia virus of an antibody, a Fab and a scFv against programmed cell death -1 (PD-1) allows their intratumoral delivery and an improved tumor-growth inhibition

PubMed Central

Kleinpeter, Patricia; Fend, Laetitia; Thioudellet, Christine; Geist, Michel; Sfrontato, Nathalie; Koerper, Véronique; Fahrner, Catherine; Schmitt, Doris; Gantzer, Murielle; Remy-Ziller, Christelle; Brandely, Renée; Villeval, Dominique; Rittner, Karola; Silvestre, Nathalie; Erbs, Philippe; Zitvogel, Laurence; Quéméneur, Eric; Préville, Xavier; Marchand, Jean-Baptiste

2016-01-01

ABSTRACT We report here the successful vectorization of a hamster monoclonal IgG (namely J43) recognizing the murine Programmed cell death-1 (mPD-1) in Western Reserve (WR) oncolytic vaccinia virus. Three forms of mPD-1 binders have been inserted into the virus: whole antibody (mAb), Fragment antigen-binding (Fab) or single-chain variable fragment (scFv). MAb, Fab and scFv were produced and assembled with the expected patterns in supernatants of cells infected by the recombinant viruses. The three purified mPD-1 binders were able to block the binding of mPD-1 ligand to mPD-1 in vitro. Moreover, mAb was detected in tumor and in serum of C57BL/6 mice when the recombinant WR-mAb was injected intratumorally (IT) in B16F10 and MCA 205 tumors. The concentration of circulating mAb detected after IT injection was up to 1,900-fold higher than the level obtained after a subcutaneous (SC) injection (i.e., without tumor) confirming the virus tropism for tumoral cells and/or microenvironment. Moreover, the overall tumoral accumulation of the mAb was higher and lasted longer after IT injection of WR-mAb1, than after IT administration of 10 µg of J43. The IT injection of viruses induced a massive infiltration of immune cells including activated lymphocytes (CD8+ and CD4+). Interestingly, in the MCA 205 tumor model, WR-mAb1 and WR-scFv induced a therapeutic control of tumor growth similar to unarmed WR combined to systemically administered J43 and superior to that obtained with an unarmed WR. These results pave the way for next generation of oncolytic vaccinia armed with immunomodulatory therapeutic proteins such as mAbs. PMID:27853644

Silk-elastin-like protein polymer matrix for intraoperative delivery of an oncolytic vaccinia virus

PubMed Central

Price, Daniel L.; Li, Pingdong; Chen, Chun-Hao; Wong, Danni; Yu, Zhenkun; Chen, Nanhai G.; Yu, Yong A.; Szalay, Aladar A.; Cappello, Joseph; Fong, Yuman; Wong, Richard J.

2016-01-01

Background Oncolytic viral efficacy may be limited by the penetration of the virus into tumors. This may be enhanced by intraoperative application of virus immediately after surgical resection. Methods Oncolytic vaccinia virus GLV-1h68 was delivered in silk-elastin-like protein polymer (SELP) in vitro and in vivo in anaplastic thyroid carcinoma cell line 8505c in nude mice. Results GLV-1h68 in SELP infected and lysed anaplastic thyroid cancer cells in vitro equally as effectively as in phosphate-buffered saline (PBS), and at 1 week retains a thousand fold greater infectious plaque-forming units. In surgical resection models of residual tumor, GLV-1h68 in SELP improves tumor control and shows increased viral β-galactosidase expression as compared to PBS. Conclusion The use of SELP matrix for intraoperative oncolytic viral delivery protects infectious viral particles from degradation, facilitates sustained viral delivery and transgene expression, and improves tumor control. Such optimization of methods of oncolytic viral delivery may enhance therapeutic outcomes. PMID:25244076

Species Specificity of Vaccinia Virus Complement Control Protein for the Bovine Classical Pathway Is Governed Primarily by Direct Interaction of Its Acidic Residues with Factor I

PubMed Central

Kumar, Jitendra; Yadav, Viveka Nand; Phulera, Swastik; Kamble, Ashish; Gautam, Avneesh Kumar; Panwar, Hemendra Singh

2017-01-01

ABSTRACT Poxviruses display species tropism—variola virus is a human-specific virus, while vaccinia virus causes repeated outbreaks in dairy cattle. Consistent with this, variola virus complement regulator SPICE (smallpox inhibitor of complement enzymes) exhibits selectivity in inhibiting the human alternative complement pathway and vaccinia virus complement regulator VCP (vaccinia virus complement control protein) displays selectivity in inhibiting the bovine alternative complement pathway. In the present study, we examined the species specificity of VCP and SPICE for the classical pathway (CP). We observed that VCP is ∼43-fold superior to SPICE in inhibiting bovine CP. Further, functional assays revealed that increased inhibitory activity of VCP for bovine CP is solely due to its enhanced cofactor activity, with no effect on decay of bovine CP C3-convertase. To probe the structural basis of this specificity, we utilized single- and multi-amino-acid substitution mutants wherein 1 or more of the 11 variant VCP residues were substituted in the SPICE template. Examination of these mutants for their ability to inhibit bovine CP revealed that E108, E120, and E144 are primarily responsible for imparting the specificity and contribute to the enhanced cofactor activity of VCP. Binding and functional assays suggested that these residues interact with bovine factor I but not with bovine C4(H2O) (a moiety conformationally similar to C4b). Mapping of these residues onto the modeled structure of bovine C4b-VCP-bovine factor I supported the mutagenesis data. Taken together, our data help explain why the vaccine strain of vaccinia virus was able to gain a foothold in domesticated animals. IMPORTANCE Vaccinia virus was used for smallpox vaccination. The vaccine-derived virus is now circulating and causing outbreaks in dairy cattle in India and Brazil. However, the reason for this tropism is unknown. It is well recognized that the virus is susceptible to neutralization by the

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

PubMed Central

Aleshin, SE; Timofeev, AV; Khoretonenko, MV; Zakharova, LG; Pashvykina, GV; Stephenson, JR; Shneider, AM; Altstein, AD

2005-01-01

Background Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol. Results The heterologous prime-boost vaccination protocol, using a VV recombinant and bacterial plasmid, both containing the NS1 TBE virus protein gene under the control of different promoters, achieved a high level of protection in mice against lethal challenge with a highly pathogenic TBE virus strain. No signs of pronounced TBE infection were detected in the surviving animals. Conclusion Heterologous prime-boost vaccination protocols using recombinant VV and bacterial plasmids could be used for the development of flavivirus vaccines. PMID:16076390

CD40L-adjuvanted DNA/modified vaccinia virus Ankara simian immunodeficiency virus SIV239 vaccine enhances SIV-specific humoral and cellular immunity and improves protection against a heterologous SIVE660 mucosal challenge.

PubMed

Kwa, Suefen; Lai, Lilin; Gangadhara, Sailaja; Siddiqui, Mariam; Pillai, Vinod B; Labranche, Celia; Yu, Tianwei; Moss, Bernard; Montefiori, David C; Robinson, Harriet L; Kozlowski, Pamela A; Amara, Rama Rao

2014-09-01

It remains a challenge to develop a successful human immunodeficiency virus (HIV) vaccine that is capable of preventing infection. Here, we utilized the benefits of CD40L, a costimulatory molecule that can stimulate both dendritic cells (DCs) and B cells, as an adjuvant for our simian immunodeficiency virus (SIV) DNA vaccine in rhesus macaques. We coexpressed the CD40L with our DNA/SIV vaccine such that the CD40L is anchored on the membrane of SIV virus-like particle (VLP). These CD40L containing SIV VLPs showed enhanced activation of DCs in vitro. We then tested the potential of DNA/SIV-CD40L vaccine to adjuvant the DNA prime of a DNA/modified vaccinia virus Ankara (MVA) vaccine in rhesus macaques. Our results demonstrated that the CD40L adjuvant enhanced the functional quality of anti-Env antibody response and breadth of anti-SIV CD8 and CD4 T cell responses, significantly delayed the acquisition of heterologous mucosal SIV infection, and improved viral control. Notably, the CD40L adjuvant enhanced the control of viral replication in the gut at the site of challenge that was associated with lower mucosal CD8 immune activation, one of the strong predictors of disease progression. Collectively, our results highlight the benefits of CD40L adjuvant for enhancing antiviral humoral and cellular immunity, leading to enhanced protection against a pathogenic SIV. A single adjuvant that enhances both humoral and cellular immunity is rare and thus underlines the importance and practicality of CD40L as an adjuvant for vaccines against infectious diseases, including HIV-1. Despite many advances in the field of AIDS research, an effective AIDS vaccine that can prevent infection remains elusive. CD40L is a key stimulator of dendritic cells and B cells and can therefore enhance T cell and antibody responses, but its overly potent nature can lead to adverse effects unless used in small doses. In order to modulate local expression of CD40L at relatively lower levels, we expressed

Mucosal Immunization with Newcastle Disease Virus Vector Coexpressing HIV-1 Env and Gag Proteins Elicits Potent Serum, Mucosal, and Cellular Immune Responses That Protect against Vaccinia Virus Env and Gag Challenges

PubMed Central

Khattar, Sunil K.; Manoharan, Vinoth; Bhattarai, Bikash; LaBranche, Celia C.; Montefiori, David C.

2015-01-01

ABSTRACT Newcastle disease virus (NDV) avirulent strain LaSota was used to coexpress gp160 Env and p55 Gag from a single vector to enhance both Env-specific and Gag-specific immune responses. The optimal transcription position for both Env and Gag genes in the NDV genome was determined by generating recombinant NDV (rNDV)-Env-Gag (gp160 located between the P and M genes and Gag between the HN and L genes), rNDV-Gag-Env (Gag located between the P and M genes and gp160 between the HN and L genes), rNDV-Env/Gag (gp160 followed by Gag located between the P and M genes), and rNDV-Gag/Env (Gag followed by gp160 located between the P and M genes). All the recombinant viruses replicated at levels similar to those seen with parental NDV in embryonated chicken eggs and in chicken fibroblast cells. Both gp160 and Gag proteins were expressed at high levels in cell culture, with gp160 found to be incorporated into the envelope of NDV. The Gag and Env proteins expressed by all the recombinants except rNDV-Env-Gag self-assembled into human immunodeficiency virus type 1 (HIV-1) virus-like particles (VLPs). Immunization of guinea pigs by the intranasal route with these rNDVs produced long-lasting Env- and Gag-specific humoral immune responses. The Env-specific humoral and mucosal immune responses and Gag-specific humoral immune responses were higher in rNDV-Gag/Env and rNDV-Env/Gag than in the other recombinants. rNDV-Gag/Env and rNDV-Env/Gag were also more efficient in inducing cellular as well as protective immune responses to challenge with vaccinia viruses expressing HIV-1 Env and Gag in mice. These results suggest that vaccination with a single rNDV coexpressing Env and Gag represents a promising strategy to enhance immunogenicity and protective efficacy against HIV. PMID:26199332

Vaccinia Virus Blocks Stat1-Dependent and Stat1-Independent Gene Expression Induced by Type I and Type II Interferons

PubMed Central

Mann, Brandon A.; Huang, Julia He; Li, Ping; Chang, Hua-Chen; Slee, Roger B.; O'Sullivan, Audrey; Mathur, Anita; Yeh, Norman; Klemsz, Michael J.; Brutkiewicz, Randy R.; Blum, Janice S.

2008-01-01

Blocking the function of Stat (signal transducer and activator of transcription) proteins, which are critical for antiviral responses, has evolved as a common mechanism for pathogen immune evasion. The poxvirus-encoded phosphatase H1 is critical for viral replication, and may play an additional role in the evasion of host defense by dephosphorylating Stat1 and blocking interferon (IFN)-stimulated innate immune responses. Vaccinia virus (VACV) H1 can inhibit the phosphorylation of the transcription factor Stat1 after IFN-γ stimulation of epithelial cells, greatly attenuating IFN-induced biological functions. In this study, we demonstrate that VACV infection is capable of inhibiting the phosphorylation of Stat1 and Stat2 after stimulation of fibroblasts or bone marrow-derived macrophages with either type I or type II IFNs, but did not inhibit the activation of Stat3 or Stat5 in either cell type. By using recombinant proteins for in vitro assays, we observe that variola virus H1 is more active than VACV H1, although it has similar selectivity for Stat targets. Differential effects of VACV infection were observed on the induction of IFN-stimulated genes, with complete inhibition of some genes by VACV infection, while others were less affected. Despite the IFN-γ-induced expression of some genes in VACV-infected cells, IFN-γ was unable to rescue the VACV-mediated inhibition of MHC class II antigen presentation. Moreover, VACV infection can affect the IFN-induced expression of Stat1-dependent and Stat1-independent genes, suggesting that the virus may target additional IFN-activated pathways. Thus, VACV targets multiple signaling pathways in the evasion of antiviral immune responses. PMID:18593332

Enteric Immunization of Mice Against Influenza with Recombinant Vaccinia

NASA Astrophysics Data System (ADS)

Meitin, Catherine A.; Bender, Bradley S.; Small, Parker A., Jr.

1994-11-01

Intrajejunal administration to mice of a recombinant vaccinia virus containing the influenza virus hemagglutinin gene induced IgA antibody in nasal, gut, and vaginal secretions. It also induced IgG antibody in serum and cell-mediated immunity. The immunization provided significant protection against an influenza virus challenge. This work suggests that enteric-coated recombinant vaccinia could be an orally administered, inexpensive, multivalent, temperature-stable, safe, and effective vaccine for children that could be particularly useful in developing nations, where multiple injections are not easily administered. Oral administration of vaccines should also reduce children's fear of shots at the doctor's office.

HIV-1 gp120 and Modified Vaccinia Virus Ankara (MVA) gp140 Boost Immunogens Increase Immunogenicity of a DNA/MVA HIV-1 Vaccine.

PubMed

Shen, Xiaoying; Basu, Rahul; Sawant, Sheetal; Beaumont, David; Kwa, Sue Fen; LaBranche, Celia; Seaton, Kelly E; Yates, Nicole L; Montefiori, David C; Ferrari, Guido; Wyatt, Linda S; Moss, Bernard; Alam, S Munir; Haynes, Barton F; Tomaras, Georgia D; Robinson, Harriet L

2017-12-15

An important goal of human immunodeficiency virus (HIV) vaccine design is identification of strategies that elicit effective antiviral humoral immunity. One novel approach comprises priming with DNA and boosting with modified vaccinia virus Ankara (MVA) expressing HIV-1 Env on virus-like particles. In this study, we evaluated whether the addition of a gp120 protein in alum or MVA-expressed secreted gp140 (MVAgp140) could improve immunogenicity of a DNA prime-MVA boost vaccine. Five rhesus macaques per group received two DNA primes at weeks 0 and 8 followed by three MVA boosts (with or without additional protein or MVAgp140) at weeks 18, 26, and 40. Both boost immunogens enhanced the breadth of HIV-1 gp120 and V1V2 responses, antibody-dependent cellular cytotoxicity (ADCC), and low-titer tier 1B and tier 2 neutralizing antibody responses. However, there were differences in antibody kinetics, linear epitope specificity, and CD4 T cell responses between the groups. The gp120 protein boost elicited earlier and higher peak responses, whereas the MVAgp140 boost resulted in improved antibody durability and comparable peak responses after the final immunization. Linear V3 specific IgG responses were particularly enhanced by the gp120 boost, whereas the MVAgp140 boost also enhanced responses to linear C5 and C2.2 epitopes. Interestingly, gp120, but not the MVAgp140 boost, increased peak CD4 + T cell responses. Thus, both gp120 and MVAgp140 can augment potential protection of a DNA/MVA vaccine by enhancing gp120 and V1/V2 antibody responses, whereas potential protection by gp120, but not MVAgp140 boosts, may be further impacted by increased CD4 + T cell responses. IMPORTANCE Prior immune correlate analyses with humans and nonhuman primates revealed the importance of antibody responses in preventing HIV-1 infection. A DNA prime-modified vaccinia virus Ankara (MVA) boost vaccine has proven to be potent in eliciting antibody responses. Here we explore the ability of boosts with

Pre-clinical efficacy and safety of experimental vaccines based on non-replicating vaccinia vectors against yellow fever.

PubMed

Schäfer, Birgit; Holzer, Georg W; Joachimsthaler, Alexandra; Coulibaly, Sogue; Schwendinger, Michael; Crowe, Brian A; Kreil, Thomas R; Barrett, P Noel; Falkner, Falko G

2011-01-01

Currently existing yellow fever (YF) vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D). Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable. A gene encoding the precursor of the membrane and envelope (prME) protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 10(5) TCID(50). Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles. The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice.

The 3'-to-5' exonuclease activity of vaccinia virus DNA polymerase is essential and plays a role in promoting virus genetic recombination.

PubMed

Gammon, Don B; Evans, David H

2009-05-01

Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3'-to-5' proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3' ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDV(r)) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3'-to-5' exonuclease domain of the viral polymerase. Viruses encoding a CDV(r) mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3' ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3'-to-5' exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.

[Experiments on disinfection of vaccinia virus embedded in scabs and/or at the hand].

PubMed

Schümann, K; Grossgebauer, K

1977-01-01

Vaccinia viruses embedded in rabbit dermal scabs were subjected to physical and chemical disinfection procedures. Scabs were suspended in vitro without saline or in physiological saline, and left for 1 hour at 70 to 90 degrees C. A complete inactivation was achived only in those scab samples which had been incubated at 90 degrees C for 1 hour and suspended in physiological saline. Scabs which had been placed in a disinfecting apparatus (Vacudes 4000) filled with mattrasses consistently proved to be free of infectious vaccinia viruses in each of the chosen programs. In addition scabs were subjected to disinfection by means of chemical disinfecting agents. The scabs had been placed in a chemical disinfecting suspension and left there for 90 minutes. Complete disinfection was obtained with glutaraldehyde 2%, formaldehyde 2%, Lysoformin 2% or 3%, phenol 5% and chloramine T 2%. Complete disinfection was likewise achieved after 3 hours treatment with some alchohols (ethylalcohol 80%, isopropylalcohol 7%, n-propylalcohol 60%), Amocid 5% and formaldehyde 1%.0.5% formaldehyde caused complete disinfection when applied for 6 hours. The only exception was a Quat which did not disinfect fully even after 18 hours application. Concerning the tests to disinfect the hands complete disinfection occurs when using chloramine T (1.5%) or isopropylalcohol (70%) in 2 to 5 minutes. Further tests were performed with scabs which were placed in sick rooms that were terminally disinfected with formaline vapor. It could be confirmed that the usual terminal disinfection with formaldehyde vapor was unable to completely disinfect the scabs. It is necessary to double the amount of formaldehyde (10 g formaldehyde per cubic metre of space) and prolong the period of treatment to 24 hours to achieve a greater degree of disinfection rate.

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

PubMed

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

2005-10-01

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

Rabbitpox virus and vaccinia virus infection of rabbits as a model for human smallpox.

PubMed

Adams, Mathew M; Rice, Amanda D; Moyer, R W

2007-10-01

The threat of smallpox release and use as a bioweapon has encouraged the search for new vaccines and antiviral drugs, as well as development of new small-animal models in which their efficacy can be determined. Here, we reinvestigate a rabbit model in which the intradermal infection of rabbits with very low doses of either rabbitpox virus (RPV) or vaccinia virus Western Reserve (VV-WR) recapitulates many of the clinical features of human smallpox. Following intradermal inoculation with RPV, rabbits develop systemic disease characterized by extensive viremia, numerous secondary lesions on the skin and mucocutaneous tissues, severe respiratory disease, death by 9 days postinfection, and, importantly, natural aerosol transmission between animals. Contrary to previous reports, intradermal infection with VV-WR also resulted in a very similar lethal systemic disease in rabbits, again with natural aerosol transmission between animals. When sentinel and index animals were cohoused, transmission rates approached 100% with either virus, with sentinel animals exhibiting a similar, severe disease. Lower rates of transmission were observed when index and sentinel animals were housed in separate cages. Sentinel animals infected with RPV with one exception succumbed to the disease. However, the majority of VV-WR-infected sentinel animals, while becoming seriously ill, survived. Finally, we tested the efficacy of the drug 1-O-hexadecyloxypropyl-cidofovir in the RPV/rabbit model and found that an oral dose of 5 mg/kg twice a day for 5 days beginning 1 day before infection was able to completely protect rabbits from lethal disease.

Pre-Clinical Efficacy and Safety of Experimental Vaccines Based on Non-Replicating Vaccinia Vectors against Yellow Fever

PubMed Central

Schäfer, Birgit; Holzer, Georg W.; Joachimsthaler, Alexandra; Coulibaly, Sogue; Schwendinger, Michael; Crowe, Brian A.; Kreil, Thomas R.; Barrett, P. Noel; Falkner, Falko G.

2011-01-01

Background Currently existing yellow fever (YF) vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D). Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable. Methodology/Principal Findings A gene encoding the precursor of the membrane and envelope (prME) protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 105 TCID50. Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles. Conclusions/Significance The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice. PMID:21931732

Functional Analysis of Vaccinia Virus B5R Protein: Essential Role in Virus Envelopment Is Independent of a Large Portion of the Extracellular Domain

PubMed Central

Herrera, Elizabeth; del Mar Lorenzo, María; Blasco, Rafael; Isaacs, Stuart N.

1998-01-01

Vaccinia virus has two forms of infectious virions: the intracellular mature virus and the extracellular enveloped virus (EEV). EEV is critical for cell-to-cell and long-range spread of the virus. The B5R open reading frame (ORF) encodes a membrane protein that is essential for EEV formation. Deletion of the B5R ORF results in a dramatic reduction of EEV, and as a consequence, the virus produces small plaques in vitro and is highly attenuated in vivo. The extracellular portion of B5R is composed mainly of four domains that are similar to the short consensus repeats (SCRs) present in complement regulatory proteins. To determine the contribution of these putative SCR domains to EEV formation, we constructed recombinant vaccinia viruses that replaced the wild-type B5R gene with a mutated gene encoding a B5R protein lacking the SCRs. The resulting recombinant viruses produced large plaques, indicating efficient cell-to-cell spread in vitro, and gradient centrifugation of supernatants from infected cells confirmed that EEV was formed. In contrast, phalloidin staining of infected cells showed that the virus lacking the SCR domains was deficient in the induction of thick actin bundles. Thus, the highly conserved SCR domains present in the extracellular portion of the B5R protein are dispensable for EEV formation. This indicates that the B5R protein is a key viral protein with multiple functions in the process of virus envelopment and release. In addition, given the similarity of the extracellular domain to complement control proteins, the B5R protein may be involved in viral evasion from host immune responses. PMID:9420227

Protective effect of surfactant protein d in pulmonary vaccinia virus infection: implication of A27 viral protein.

PubMed

Perino, Julien; Thielens, Nicole M; Crouch, Erika; Spehner, Danièle; Crance, Jean-Marc; Favier, Anne-Laure

2013-03-21

Vaccinia virus (VACV) was used as a surrogate of variola virus (VARV) (genus Orthopoxvirus), the causative agent of smallpox, to study Orthopoxvirus infection. VARV is principally transmitted between humans by aerosol droplets. Once inhaled, VARV first infects the respiratory tract where it could encounter surfactant components, such as soluble pattern recognition receptors. Surfactant protein D (SP-D), constitutively present in the lining fluids of the respiratory tract, plays important roles in innate host defense against virus infection. We investigated the role of SP-D in VACV infection and studied the A27 viral protein involvement in the interaction with SP-D. Interaction between SP-D and VACV caused viral inhibition in a lung cell model. Interaction of SP-D with VACV was mediated by the A27 viral protein. Binding required Ca2+ and interactions were blocked in the presence of excess of SP-D saccharide ligands. A27, which lacks glycosylation, directly interacted with SP-D. The interaction between SP-D and the viral particle was also observed using electron microscopy. Infection of mice lacking SP-D (SP-D-/-) resulted in increased mortality compared to SP-D+/+ mice. Altogether, our data show that SP-D participates in host defense against the vaccinia virus infection and that the interaction occurs with the viral surface protein A27.

Disruption of TNFα/TNFR1 function in resident skin cells impairs host immune response against cutaneous vaccinia virus infection

PubMed Central

Tian, Tian; Dubin, Krista; Jin, Qiushuang; Qureshi, Ali; King, Sandra L.; Liu, Luzheng; Jiang, Xiaodong; Murphy, George F.; Kupper, Thomas S.; Fuhlbrigge, Robert C.

2012-01-01

One strategy adopted by vaccinia virus (VV) to evade the host immune system is to encode homologs of TNF receptors (TNFR) that block TNFα function. The response to VV skin infection under conditions of TNFα deficiency, however, has not been reported. We found that TNFR1−/− mice developed larger primary lesions, numerous satellite lesions and higher skin virus levels after VV scarification. Following their recovery, these TNFR1−/− mice were fully protected against challenge with a lethal intranasal dose of VV, suggesting these mice developed an effective memory immune response. A functional systemic immune response of TNFR1−/− mice was further demonstrated by enhanced production of VV-specific IFNγ and VV-specific CD8+ T cells in spleens and draining lymph nodes. Interestingly, bone marrow (BM) reconstitution studies using WT BM in TNFR1−/− host mice, but not TNFR1−/− BM in WT host mice, reproduced the original results seen in TNFR1−/− mice, indicating that TNFR1 deficiency in resident skin cells, rather than hematopoietic cells, accounts for the impaired cutaneous immune response. Our data suggest that lack of TNFR1 leads to a skin-specific immune deficiency and that resident skin cells play a crucial role in mediating an optimal immune defense to VV cutaneous infection via TNFα/TNFR1 signaling. PMID:22318381

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

PubMed

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

2018-04-24

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

Immunogenicity of recombinant vaccinia virus vaccines co-expressing GP3/GP5 of European PRRSV and Cap protein of PCV2 in pigs.

PubMed

Han, Jicheng; Ma, Haibin; Cao, Liang; Jing, Jie; Xiao, Pengpeng; Sun, Wenchao; Xie, Changzhan; Wen, Shubo; Li, Yiquan; Tian, Mingyao; Lu, Huijun; Jin, Ningyi

2018-02-01

Porcine reproductive and respiratory syndrome (PRRS) is almost always caused by the North American strain of PRRS virus (PRRSV) in China; the European genotype of PRRSV has emerged in China. The mixed infection of PRRSV and Porcine circovirus type 2 virus (PCV2) are always found in pigs and PRRSV-augmented PCV2 replication and serious clinical symptoms. Current vaccines cannot protect mixed European PRRSV and PCV2 infections. Therefore, the development of a safe and effective new vaccine to prevent and control the mixed infection of European PRRSV and PCV2 is both urgent and necessary. In this study, we developed a recombinant vaccinia vaccine co-expressing the GP3 and GP5 proteins of European PRRSV and the ORF2 protein of PCV2 and evaluated the immunogenicity and its protective effects and its inactivated vaccine in pigs. The recombinant vaccinia vaccine and its inactivated vaccine both elicited significant humoral and cellular immune responses with a higher level of specific antibody responses and T-lymphocyte proliferation than the control group. Furthermore, the pigs inoculated with the recombinant vaccinia vaccine were completely protected against challenge with 10 5 TCID 50 of European PRRSV strain LV. These data suggest that the recombinant vaccinia vaccine is a potential candidate vaccine against European PRRSV and PCV2.

Evidence for Protection against Chronic Hepatitis C Virus Infection in Chimpanzees by Immunization with Replicating Recombinant Vaccinia Virus▿

PubMed Central

Youn, Jin-Won; Hu, Yu-Wen; Tricoche, Nancy; Pfahler, Wolfram; Shata, Mohamed Tarek; Dreux, Marlene; Cosset, François-Loic; Folgori, Antonella; Lee, Dong-Hun; Brotman, Betsy; Prince, Alfred M.

2008-01-01

Given the failures of nonreplicating vaccines against chronic hepatitis C virus (HCV) infection, we hypothesized that a replicating viral vector may provide protective immunity. Four chimpanzees were immunized transdermally twice with recombinant vaccinia viruses (rVV) expressing HCV genes. After challenge with 24 50% chimpanzee infective doses of homologous HCV, the two control animals that had received only the parental VV developed chronic HCV infection. All four immunized animals resolved HCV infection. The difference in the rate of chronicity between the immunized and the control animals was close to statistical significance (P = 0.067). Immunized animals developed vigorous gamma interferon enzyme-linked immunospot responses and moderate proliferative responses. To investigate cross-genotype protection, the immunized recovered chimpanzees were challenged with a pool of six major HCV genotypes. During the acute phase after the multigenotype challenge, all animals had high-titer viremia in which genotype 4 dominated (87%), followed by genotype 5 (13%). However, after fluctuating low-level viremia, the viremia finally turned negative or persisted at very low levels. This study suggests the potential efficacy of replicating recombinant vaccinia virus-based immunization against chronic HCV infection. PMID:18753204

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

PubMed

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

2017-10-01

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

Anti-apoptotic Bcl-XL but not Mcl-1 contributes to protection against virus-induced apoptosis.

PubMed

Ohmer, Michaela; Weber, Arnim; Sutter, Gerd; Ehrhardt, Katrin; Zimmermann, Albert; Häcker, Georg

2016-08-18

Infection of mammalian cells with viruses often induces apoptosis. How the recognition of viruses leads to apoptosis of the infected cell and which host cell factors regulate this cell death is incompletely understood. In this study, we focussed on two major anti-apoptotic proteins of the host cell, whose abundance and activity are important for cell survival, the Bcl-2-like proteins Mcl-1 and Bcl-XL. During infection of epithelial cells and fibroblasts with modified vaccinia virus Ankara (MVA), Mcl-1 protein levels dropped but the MVA Bcl-2-like protein F1L could replace Mcl-1 functionally; a similar activity was found in vaccinia virus (VACV)-infected cells. During infection with murine cytomegalovirus (MCMV), Mcl-1-levels were not reduced but a viral Mcl-1-like activity was also generated. Infection of mouse macrophages with any of these viruses, on the other hand, induced apoptosis. Virus-induced macrophage apoptosis was unaltered in the absence of Mcl-1. However, apoptosis was substantially increased in infected Bcl-XL-deficient macrophages or macrophages treated with the Bcl-2/Bcl-XL-inhibitor ABT-737. Genetic loss of Bcl-XL or treatment of macrophages with ABT-737 reduced the generation of infectious VACV. These data show that Mcl-1 is dispensable for the regulation of apoptosis during infection with different large DNA viruses, either because the viruses replace its function (in fibroblasts and epithelial cells) or because the pro-apoptotic activity generated by the infection appears not to be blocked by it (in macrophages). Bcl-XL, on the other hand, can be important to maintain survival of virus-infected cells, and its activity can determine outcome of the infection.

Anti-apoptotic Bcl-XL but not Mcl-1 contributes to protection against virus-induced apoptosis

PubMed Central

Ohmer, Michaela; Weber, Arnim; Sutter, Gerd; Ehrhardt, Katrin; Zimmermann, Albert; Häcker, Georg

2016-01-01

Infection of mammalian cells with viruses often induces apoptosis. How the recognition of viruses leads to apoptosis of the infected cell and which host cell factors regulate this cell death is incompletely understood. In this study, we focussed on two major anti-apoptotic proteins of the host cell, whose abundance and activity are important for cell survival, the Bcl-2-like proteins Mcl-1 and Bcl-XL. During infection of epithelial cells and fibroblasts with modified vaccinia virus Ankara (MVA), Mcl-1 protein levels dropped but the MVA Bcl-2-like protein F1L could replace Mcl-1 functionally; a similar activity was found in vaccinia virus (VACV)-infected cells. During infection with murine cytomegalovirus (MCMV), Mcl-1-levels were not reduced but a viral Mcl-1-like activity was also generated. Infection of mouse macrophages with any of these viruses, on the other hand, induced apoptosis. Virus-induced macrophage apoptosis was unaltered in the absence of Mcl-1. However, apoptosis was substantially increased in infected Bcl-XL-deficient macrophages or macrophages treated with the Bcl-2/Bcl-XL-inhibitor ABT-737. Genetic loss of Bcl-XL or treatment of macrophages with ABT-737 reduced the generation of infectious VACV. These data show that Mcl-1 is dispensable for the regulation of apoptosis during infection with different large DNA viruses, either because the viruses replace its function (in fibroblasts and epithelial cells) or because the pro-apoptotic activity generated by the infection appears not to be blocked by it (in macrophages). Bcl-XL, on the other hand, can be important to maintain survival of virus-infected cells, and its activity can determine outcome of the infection. PMID:27537523

Vaccinia virus Transmission through Experimentally Contaminated Milk Using a Murine Model

PubMed Central

Rehfeld, Izabelle Silva; Guedes, Maria Isabel Maldonado Coelho; Fraiha, Ana Luiza Soares; Costa, Aristóteles Gomes; Matos, Ana Carolina Diniz; Fiúza, Aparecida Tatiane Lino; Lobato, Zélia Inês Portela

2015-01-01

Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV), which affects dairy cattle and humans. Previous studies have detected the presence of viable virus particles in bovine milk samples naturally and experimentally contaminated with VACV. However, it is not known whether milk contaminated with VACV could be a route of viral transmission. However, anti-Orthopoxvirus antibodies were detected in humans from BV endemic areas, whom had no contact with affected cows, which suggest that other VACV transmission routes are possible, such as consumption of contaminated milk and dairy products. Therefore, it is important to study the possibility of VACV transmission by contaminated milk. This study aimed to examine VACV transmission, pathogenesis and shedding in mice orally inoculated with experimentally contaminated milk. Thirty mice were orally inoculated with milk containing 107 PFU/ml of VACV, and ten mice were orally inoculated with uncontaminated milk. Clinical examinations were performed for 30 consecutive days, and fecal samples and oral swabs (OSs) were collected every other day. Mice were euthanized on predetermined days, and tissue and blood samples were collected. Nested-PCR, plaque reduction neutralization test (PRNT), viral isolation, histopathology, and immunohistochemistry (IHC) methods were performed on the collected samples. No clinical changes were observed in the animals. Viral DNA was detected in feces, blood, OSs and tissues, at least in one of the times tested. The lungs displayed moderate to severe interstitial lymphohistiocytic infiltrates, and only the heart, tonsils, tongue, and stomach did not show immunostaining at the IHC analysis. Neutralizing antibodies were detected at the 20th and 30th days post infection in 50% of infected mice. The results revealed that VACV contaminated milk could be a route of viral transmission in mice experimentally infected, showing systemic distribution and shedding through feces and oral mucosa, albeit

Unintentional transfer of vaccinia virus associated with smallpox vaccines: ACAM2000(®) compared with Dryvax(®).

PubMed

Tack, Danielle M; Karem, Kevin L; Montgomery, Jay R; Collins, Limone; Bryant-Genevier, Marthe G; Tiernan, Rosemary; Cano, Maria; Lewis, Paige; Engler, Renata J M; Damon, Inger K; Reynolds, Mary G

2013-07-01

Routine vaccination against smallpox (variola) ceased in the US in 1976. However, in 2002 limited coverage for military personnel and some healthcare workers was reinstituted. In March 2008, ACAM2000® replaced Dryvax® as the vaccine used in the United States against smallpox. Unintentional transfer of vaccinia virus from a vaccination site by autoinoculation or contact transmission, can have significant public health implications. We summarize unintentional virus transfer AEs associated with ACAM2000® since March 2008 and compare with Dryvax®. We identified 309 reports for ACAM2000® with skin or ocular involvement, of which 93 were autoinoculation cases and 20 were contact transmission cases. The rate for reported cases of autoinoculation was 20.6 per 100,000 vaccinations and for contact transmission was 4.4 per 100,000 vaccinations. Eighteen contact transmission cases could be attributed to contact during a sporting activity (45%) or intimate contact (45%). Of the 113 unintentional transfer cases, 6 met the case definition for ocular vaccinia. The most common locations for all autoinoculation and contact cases were arm/elbow/shoulder (35/113; 31%) and face (24/113; 21%). Methods We reviewed 753 reports associated with smallpox in the Vaccine Adverse Event Reporting System and CDC Poxvirus consultation log, reported from March 2008 to August 2010. Reports were classified into categories based upon standard case definitions. Overall, unintentional transfer events for ACAM2000® and Dryvax® are similar. We recommend continued efforts to prevent transfer events and continuing education for healthcare providers focused on recognition of vaccinia lesions, proper sample collection, and laboratory testing to confirm diagnosis.

Development of an animal model of progressive vaccinia in nu/nu mice and the use of bioluminescence imaging for assessment of the efficacy of monoclonal antibodies against vaccinial B5 and L1 proteins.

PubMed

Zaitseva, Marina; Thomas, Antonia; Meseda, Clement A; Cheung, Charles Y K; Diaz, Claudia G; Xiang, Yan; Crotty, Shane; Golding, Hana

2017-08-01

Bioluminescence imaging (BLI) was used to follow dissemination of recombinant vaccinia virus (VACV) expressing luciferase (IHD-J-Luc) in BALB/c nu/nu mice treated post-challenge with monoclonal antibodies (MAbs) against L1 and B5 VACV proteins in a model of Progressive Vaccinia (PV). Areas Under the flux Curve (AUC) were calculated for viral loads in multiple organs in individual mice. Following scarification with 10 5  pfu, IHD-J-Luc VACV undergoes fast replication at the injection site and disseminates rapidly to the inguinal lymph nodes followed by spleen, liver, and axillary lymph nodes within 2-3 days and before primary lesions are visible at the site of scarification. Extension of survival in nude mice treated with a combination of anti-B5 and anti-L1 MAbs 24 h post challenge correlated with a significant reduction in viral load at the site of scarification and delayed systemic dissemination. Nude mice reconstituted with 10 4  T cells prior to challenge with IHD-J-Luc, and treated with MAbs post-challenge, survived infection, cleared the virus from all organs and scarification site, and developed anti-VACV IgG and VACV-specific polyfunctional CD8 + T cells that co-expressed the degranulation marker CD107a, and IFNγ and TNFα cytokines. All T cell reconstituted mice survived intranasal re-challenge with IHD-J-Luc (10 4  pfu) two months after the primary infection. Thus, using BLI to monitor VACV replication in a PV model, we showed that anti-VACV MAbs administered post challenge extended survival of nude mice and protected T cell reconstituted nude mice from lethality by reducing replication at the site of scarification and systemic dissemination of VACV. Published by Elsevier B.V.

Safety and Immunogenicity of Modified Vaccinia Ankara-Bavarian Nordic Smallpox Vaccine in Vaccinia-Naive and Experienced Human Immunodeficiency Virus-Infected Individuals: An Open-Label, Controlled Clinical Phase II Trial.

PubMed

Overton, Edgar Turner; Stapleton, Jack; Frank, Ian; Hassler, Shawn; Goepfert, Paul A; Barker, David; Wagner, Eva; von Krempelhuber, Alfred; Virgin, Garth; Meyer, Thomas Peter; Müller, Jutta; Bädeker, Nicole; Grünert, Robert; Young, Philip; Rösch, Siegfried; Maclennan, Jane; Arndtz-Wiedemann, Nathaly; Chaplin, Paul

2015-04-01

Background.  First- and second-generation smallpox vaccines are contraindicated in individuals infected with human immunodeficiency virus (HIV). A new smallpox vaccine is needed to protect this population in the context of biodefense preparedness. The focus of this study was to compare the safety and immunogenicity of a replication-deficient, highly attenuated smallpox vaccine modified vaccinia Ankara (MVA) in HIV-infected and healthy subjects. Methods.  An open-label, controlled Phase II trial was conducted at 36 centers in the United States and Puerto Rico for HIV-infected and healthy subjects. Subjects received 2 doses of MVA administered 4 weeks apart. Safety was evaluated by assessment of adverse events, focused physical exams, electrocardiogram recordings, and safety laboratories. Immune responses were assessed using enzyme-linked immunosorbent assay (ELISA) and a plaque reduction neutralization test (PRNT). Results.  Five hundred seventy-nine subjects were vaccinated at least once and had data available for analysis. Rates of ELISA seropositivity were comparably high in vaccinia-naive healthy and HIV-infected subjects, whereas PRNT seropositivity rates were higher in healthy compared with HIV-infected subjects. Modified vaccinia Ankara was safe and well tolerated with no adverse impact on viral load or CD4 counts. There were no cases of myo-/pericarditis reported. Conclusions.  Modified vaccinia Ankara was safe and immunogenic in subjects infected with HIV and represents a promising smallpox vaccine candidate for use in immunocompromised populations.

Safety and Immunogenicity of Modified Vaccinia Ankara-Bavarian Nordic Smallpox Vaccine in Vaccinia-Naive and Experienced Human Immunodeficiency Virus-Infected Individuals: An Open-Label, Controlled Clinical Phase II Trial

PubMed Central

Overton, Edgar Turner; Stapleton, Jack; Frank, Ian; Hassler, Shawn; Goepfert, Paul A.; Barker, David; Wagner, Eva; von Krempelhuber, Alfred; Virgin, Garth; Meyer, Thomas Peter; Müller, Jutta; Bädeker, Nicole; Grünert, Robert; Young, Philip; Rösch, Siegfried; Maclennan, Jane; Arndtz-Wiedemann, Nathaly; Chaplin, Paul

2015-01-01

Background. First- and second-generation smallpox vaccines are contraindicated in individuals infected with human immunodeficiency virus (HIV). A new smallpox vaccine is needed to protect this population in the context of biodefense preparedness. The focus of this study was to compare the safety and immunogenicity of a replication-deficient, highly attenuated smallpox vaccine modified vaccinia Ankara (MVA) in HIV-infected and healthy subjects. Methods. An open-label, controlled Phase II trial was conducted at 36 centers in the United States and Puerto Rico for HIV-infected and healthy subjects. Subjects received 2 doses of MVA administered 4 weeks apart. Safety was evaluated by assessment of adverse events, focused physical exams, electrocardiogram recordings, and safety laboratories. Immune responses were assessed using enzyme-linked immunosorbent assay (ELISA) and a plaque reduction neutralization test (PRNT). Results. Five hundred seventy-nine subjects were vaccinated at least once and had data available for analysis. Rates of ELISA seropositivity were comparably high in vaccinia-naive healthy and HIV-infected subjects, whereas PRNT seropositivity rates were higher in healthy compared with HIV-infected subjects. Modified vaccinia Ankara was safe and well tolerated with no adverse impact on viral load or CD4 counts. There were no cases of myo-/pericarditis reported. Conclusions. Modified vaccinia Ankara was safe and immunogenic in subjects infected with HIV and represents a promising smallpox vaccine candidate for use in immunocompromised populations. PMID:26380340

Statistical Approach To Estimate Vaccinia-Specific Neutralizing Antibody Titers Using a High-Throughput Assay▿

PubMed Central

Kennedy, Richard; Pankratz, V. Shane; Swanson, Eric; Watson, David; Golding, Hana; Poland, Gregory A.

2009-01-01

Because of the bioterrorism threat posed by agents such as variola virus, considerable time, resources, and effort have been devoted to biodefense preparation. One avenue of this research has been the development of rapid, sensitive, high-throughput assays to validate immune responses to poxviruses. Here we describe the adaptation of a β-galactosidase reporter-based vaccinia virus neutralization assay to large-scale use in a study that included over 1,000 subjects. We also describe the statistical methods involved in analyzing the large quantity of data generated. The assay and its associated methods should prove useful tools in monitoring immune responses to next-generation smallpox vaccines, studying poxvirus immunity, and evaluating therapeutic agents such as vaccinia virus immune globulin. PMID:19535540

Dogs and Opossums Positive for Vaccinia Virus during Outbreak Affecting Cattle and Humans, São Paulo State, Brazil.

PubMed

Peres, Marina G; Barros, Claudenice B; Appolinário, Camila M; Antunes, João M A P; Mioni, Mateus S R; Bacchiega, Thais S; Allendorf, Susan D; Vicente, Acácia F; Fonseca, Clóvis R; Megid, Jane

2016-02-01

During a vaccinia virus (VACV) outbreak in São Paulo State, Brazil, blood samples were collected from cows, humans, other domestic animals, and wild mammals. Samples from 3 dogs and 3 opossums were positive for VACV by PCR. Results of gene sequencing yielded major questions regarding other mammalian species acting as reservoirs of VACV.

Inhibition of allergic encephalomyelitis in marmosets by vaccination with recombinant vaccinia virus encoding for myelin basic protein.

PubMed

Genain, C P; Gritz, L; Joshi, N; Panicali, D; Davis, R L; Whitaker, J N; Letvin, N L; Hauser, S L

1997-11-01

A primary demyelinating form of experimental allergic encephalomyelitis (EAE) resembling human multiple sclerosis (MS) occurs in Callithrix jacchus marmosets following immunization with human white matter. Participation of a T-cell immune response against myelin basic protein (MBP) in this disease model is supported by observations of increased reactivity against MBP in PBMC and of adoptive transfer of an inflammatory form of EAE by MBP-reactive T-cells. To evaluate the effects of ectopic presentation of MBP on marmoset EAE, animals were vaccinated prior to induction of EAE by subcutaneous injection of attenuated strains of vaccinia virus genetically engineered to contain either the entire coding sequence for human MBP (vT15) or the equine herpes virus glycoprotein gH gene (vAbT249). Vaccination with vT15 was followed by transient cytoplasmic and surface membrane expression of MBP in circulating PBMC (15-45 days). The onset of clinical EAE after immunization (pi) was markedly delayed in vT15-vaccinated animals (37-97 days pi, n = 4) compared to vAbT249-vaccinated controls (14-18 days pi, n = 3). Proliferative responses against MBP but not against vaccinia antigens or phytohemagglutinin were suppressed in protected animals. Thus, development of attenuated live viruses carrying genes for myelin antigens could be useful for induction of immunologic tolerance and for modulation of autoimmune demyelination.

L-selectin Is Essential for Delivery of Activated CD8+ T Cells to Virus-Infected Organs for Protective Immunity

PubMed Central

Mohammed, Rebar N.; Watson, H. Angharad; Vigar, Miriam; Ohme, Julia; Thomson, Amanda; Humphreys, Ian R.; Ager, Ann

2016-01-01

Summary Cytotoxic CD8+ T lymphocytes play a critical role in the host response to infection by viruses. The ability to secrete cytotoxic chemicals and cytokines is considered pivotal for eliminating virus. Of equal importance is how effector CD8+ T cells home to virus-infected tissues. L-selectin has not been considered important for effector T cell homing, because levels are low on activated T cells. We report here that, although L-selectin expression is downregulated following T cell priming in lymph nodes, L-selectin is re-expressed on activated CD8+ T cells entering the bloodstream, and recruitment of activated CD8+ T cells from the bloodstream into virus-infected tissues is L-selectin dependent. Furthermore, L-selectin on effector CD8+ T cells confers protective immunity to two evolutionally distinct viruses, vaccinia and influenza, which infect mucosal and visceral organs, respectively. These results connect homing and a function of virus-specific CD8+ T cells to a single molecule, L-selectin. PMID:26804910

Mutational analysis of vaccinia virus E3 protein: the biological functions do not correlate with its biochemical capacity to bind double-stranded RNA.

PubMed

Dueck, Kevin J; Hu, YuanShen Sandy; Chen, Peter; Deschambault, Yvon; Lee, Jocelyn; Varga, Jessie; Cao, Jingxin

2015-05-01

Vaccinia E3 protein has the biochemical capacity of binding to double-stranded RNA (dsRNA). The best characterized biological functions of the E3 protein include its host range function, suppression of cytokine expression, and inhibition of interferon (IFN)-induced antiviral activity. Currently, the role of the dsRNA binding capacity in the biological functions of the E3 protein is not clear. To further understand the mechanism of the E3 protein biological functions, we performed alanine scanning of the entire dsRNA binding domain of the E3 protein to examine the link between its biochemical capacity of dsRNA binding and biological functions. Of the 115 mutants examined, 20 were defective in dsRNA binding. Although the majority of the mutants defective in dsRNA binding also showed defective replication in HeLa cells, nine mutants (I105A, Y125A, E138A, F148A, F159A, K171A, L182A, L183A, and I187/188A) retained the host range function to various degrees. Further examination of a set of representative E3L mutants showed that residues essential for dsRNA binding are not essential for the biological functions of E3 protein, such as inhibition of protein kinase R (PKR) activation, suppression of cytokine expression, and apoptosis. Thus, data described in this communication strongly indicate the E3 protein performs its biological functions via a novel mechanism which does not correlate with its dsRNA binding activity. dsRNAs produced during virus replication are important pathogen-associated molecular patterns (PAMPs) for inducing antiviral immune responses. One of the strategies used by many viruses to counteract such antiviral immune responses is achieved by producing dsRNA binding proteins, such as poxvirus E3 family proteins, influenza virus NS1, and Ebola virus V35 proteins. The most widely accepted model for the biological functions of this class of viral dsRNA binding proteins is that they bind to and sequester viral dsRNA PAMPs; thus, they suppress the related

Purification, crystallization and preliminary diffraction studies of an ectromelia virus glutaredoxin

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

Bacik, John-Paul; Brigley, Angela M.; Channon, Lisa D.

2005-06-01

Ectromelia virus glutaredoxin has been crystallized in the presence of the reducing agent DTT. A diffraction data set has been collected and processed to 1.8 Å resolution. Ectromelia, vaccinia, smallpox and other closely related viruses of the orthopoxvirus genus encode a glutaredoxin gene that is not present in poxviruses outside of this genus. The vaccinia glutaredoxin O2L has been implicated as the reducing agent for ribonucleotide reductase and may thus play an important role in viral deoxyribonucleotide synthesis. As part of an effort to understand nucleotide metabolism by poxviruses, EVM053, the O2L ortholog of the ectromelia virus, has been crystallized.more » EVM053 crystallizes in space group C222{sub 1}, with unit-cell parameters a = 61.98, b = 67.57, c = 108.55 Å. Diffraction data have been processed to 1.8 Å resolution and a self-rotation function indicates that there are two molecules per asymmetric unit.« less

Microbiota is an essential element for mice to initiate a protective immunity against Vaccinia virus.

PubMed

Lima, Maurício T; Andrade, Ana C S P; Oliveira, Graziele P; Calixto, Rafael S; Oliveira, Danilo B; Souza, Éricka L S; Trindade, Giliane S; Nicoli, Jacques R; Kroon, Erna G; Martins, Flaviano S; Abrahão, Jônatas S

2016-02-01

The gastrointestinal tract of vertebrates harbors one of the most complex ecosystems known in microbial ecology and this indigenous microbiota almost always has a profound influence on host-parasite relationships, which can enhance or reduce the pathology of the infection. In this context, the impact of the microbiota during the infection of several viral groups remains poorly studied, including the family Poxviridae. Vaccinia virus (VACV) is a member of this family and is the causative agent of bovine vaccinia, responsible for outbreaks that affect bovines and humans. To determine the influence of the microbiota in the development of the disease caused by VACV, a comparative study using a murine model was performed. Germ-free and conventional, 6- to 7-week-old Swiss NIH mice were infected by tail scarification and intranasally with VACV. Moreover, immunosuppression and microbiota reposition were performed, to establish the interactions among the host's immune system, microbiota and VACV. The data demonstrate that the microbiota is essential for the effective immune response of mice against VACV in intranasal inoculation and to control the virus at the primary site of infection. Furthermore, this study is the first to show that Swiss conventional mice are refractory to the intranasal infection of VACV. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The vaccinia virus I3L gene product is localized to a complex endoplasmic reticulum-associated structure that contains the viral parental DNA.

PubMed

Welsch, Sonja; Doglio, Laura; Schleich, Sibylle; Krijnse Locker, Jacomine

2003-05-01

The vaccinia virus (VV) I3L gene product is a single-stranded DNA-binding protein made early in infection that localizes to the cytoplasmic sites of viral DNA replication (S. C. Rochester and P. Traktman, J. Virol. 72:2917-2926, 1998). Surprisingly, when replication was blocked, the protein localized to distinct cytoplasmic spots (A. Domi and G. Beaud, J. Gen. Virol. 81:1231-1235, 2000). Here these I3L-positive spots were characterized in more detail. By using an anti-I3L peptide antibody we confirmed that the protein localized to the cytoplasmic sites of viral DNA replication by both immunofluorescence and electron microscopy (EM). Before replication had started or when replication was inhibited with hydroxyurea or cytosine arabinoside, I3L localized to distinct cytoplasmic punctate structures of homogeneous size. We show that these structures are not incoming cores or cytoplasmic sites of VV early mRNA accumulation. Instead, morphological and quantitative data indicate that they are specialized sites where the parental DNA accumulates after its release from incoming viral cores. By EM, these sites appeared as complex, electron-dense structures that were intimately associated with the cellular endoplasmic reticulum (ER). By double labeling of cryosections we show that they contain DNA and a viral early protein, the gene product of E8R. Since E8R is a membrane protein that is able to bind to DNA, the localization of this protein to the I3L puncta suggests that they are composed of membranes. The results are discussed in relation to our previous data showing that the process of viral DNA replication also occurs in close association with the ER.

Dynamic monitoring of membrane nanotubes formation induced by vaccinia virus on a high throughput microfluidic chip

NASA Astrophysics Data System (ADS)

Xiao, Min; Xu, Na; Wang, Cheng; Pang, Dai-Wen; Zhang, Zhi-Ling

2017-03-01

Membrane nanotubes (MNTs) are physical connections for intercellular communication and induced by various viruses. However, the formation of vaccinia virus (VACV)-induced MNTs has never been studied. In this report, VACV-induced MNTs formation process was monitored on a microfluidic chip equipped with a series of side chambers, which protected MNTs from fluidic shear stress. MNTs were formed between susceptible cells and be facilitated by VACV infection through three patterns. The formed MNTs varied with cell migration and virus concentration. The length of MNTs was positively correlated with the distance of cell migration. With increasing virus titer, the peak value of the ratio of MNT-carried cell appeared earlier. The immunofluorescence assay indicated that the rearrangement of actin fibers induced by VACV infection may lead to the formation of MNTs. This study presents evidence for the formation of MNTs induced by virus and helps us to understand the relationship between pathogens and MNTs.

Adverse Events Post Smallpox-Vaccination: Insights from Tail Scarification Infection in Mice with Vaccinia virus

PubMed Central

Mota, Bruno E. F.; Gallardo-Romero, Nadia; Trindade, Giliane; Keckler, M. Shannon; Karem, Kevin; Carroll, Darin; Campos, Marco A.; Vieira, Leda Q.; da Fonseca, Flávio G.; Ferreira, Paulo C. P.; Bonjardim, Cláudio A.; Damon, Inger K.; Kroon, Erna G.

2011-01-01

Adverse events upon smallpox vaccination with fully-replicative strains of Vaccinia virus (VACV) comprise an array of clinical manifestations that occur primarily in immunocompromised patients leading to significant host morbidity/mortality. The expansion of immune-suppressed populations and the possible release of Variola virus as a bioterrorist act have given rise to concerns over vaccination complications should more widespread vaccination be reinitiated. Our goal was to evaluate the components of the host immune system that are sufficient to prevent morbidity/mortality in a murine model of tail scarification, which mimics immunological and clinical features of smallpox vaccination in humans. Infection of C57BL/6 wild-type mice led to a strictly localized infection, with complete viral clearance by day 28 p.i. On the other hand, infection of T and B-cell deficient mice (Rag1 −/−) produced a severe disease, with uncontrolled viral replication at the inoculation site and dissemination to internal organs. Infection of B-cell deficient animals (µMT) produced no mortality. However, viral clearance in µMT animals was delayed compared to WT animals, with detectable viral titers in tail and internal organs late in infection. Treatment of Rag1 −/− with rabbit hyperimmune anti-vaccinia serum had a subtle effect on the morbidity/mortality of this strain, but it was effective in reduce viral titers in ovaries. Finally, NUDE athymic mice showed a similar outcome of infection as Rag1 −/−, and passive transfer of WT T cells to Rag1 −/− animals proved fully effective in preventing morbidity/mortality. These results strongly suggest that both T and B cells are important in the immune response to primary VACV infection in mice, and that T-cells are required to control the infection at the inoculation site and providing help for B-cells to produce antibodies, which help to prevent viral dissemination. These insights might prove helpful to better identify

Adverse events post smallpox-vaccination: insights from tail scarification infection in mice with Vaccinia virus.

PubMed

Mota, Bruno E F; Gallardo-Romero, Nadia; Trindade, Giliane; Keckler, M Shannon; Karem, Kevin; Carroll, Darin; Campos, Marco A; Vieira, Leda Q; da Fonseca, Flávio G; Ferreira, Paulo C P; Bonjardim, Cláudio A; Damon, Inger K; Kroon, Erna G

2011-04-15

Adverse events upon smallpox vaccination with fully-replicative strains of Vaccinia virus (VACV) comprise an array of clinical manifestations that occur primarily in immunocompromised patients leading to significant host morbidity/mortality. The expansion of immune-suppressed populations and the possible release of Variola virus as a bioterrorist act have given rise to concerns over vaccination complications should more widespread vaccination be reinitiated. Our goal was to evaluate the components of the host immune system that are sufficient to prevent morbidity/mortality in a murine model of tail scarification, which mimics immunological and clinical features of smallpox vaccination in humans. Infection of C57BL/6 wild-type mice led to a strictly localized infection, with complete viral clearance by day 28 p.i. On the other hand, infection of T and B-cell deficient mice (Rag1(-/-)) produced a severe disease, with uncontrolled viral replication at the inoculation site and dissemination to internal organs. Infection of B-cell deficient animals (µMT) produced no mortality. However, viral clearance in µMT animals was delayed compared to WT animals, with detectable viral titers in tail and internal organs late in infection. Treatment of Rag1(-/-) with rabbit hyperimmune anti-vaccinia serum had a subtle effect on the morbidity/mortality of this strain, but it was effective in reduce viral titers in ovaries. Finally, NUDE athymic mice showed a similar outcome of infection as Rag1(-/-), and passive transfer of WT T cells to Rag1(-/-) animals proved fully effective in preventing morbidity/mortality. These results strongly suggest that both T and B cells are important in the immune response to primary VACV infection in mice, and that T-cells are required to control the infection at the inoculation site and providing help for B-cells to produce antibodies, which help to prevent viral dissemination. These insights might prove helpful to better identify individuals with

Inhibition of DAI-dependent necroptosis by the Z-DNA binding domain of the vaccinia virus innate immune evasion protein, E3.

PubMed

Koehler, Heather; Cotsmire, Samantha; Langland, Jeffrey; Kibler, Karen V; Kalman, Daniel; Upton, Jason W; Mocarski, Edward S; Jacobs, Bertram L

2017-10-24

Vaccinia virus (VACV) encodes an innate immune evasion protein, E3, which contains an N-terminal Z-nucleic acid binding (Zα) domain that is critical for pathogenicity in mice. Here we demonstrate that the N terminus of E3 is necessary to inhibit an IFN-primed virus-induced necroptosis. VACV deleted of the Zα domain of E3 (VACV-E3LΔ83N) induced rapid RIPK3-dependent cell death in IFN-treated L929 cells. Cell death was inhibited by the RIPK3 inhibitor, GSK872, and infection with this mutant virus led to phosphorylation and aggregation of MLKL, the executioner of necroptosis. In 293T cells, induction of necroptosis depended on expression of RIPK3 as well as the host-encoded Zα domain-containing DNA sensor, DAI. VACV-E3LΔ83N is attenuated in vivo, and pathogenicity was restored in either RIPK3- or DAI-deficient mice. These data demonstrate that the N terminus of the VACV E3 protein prevents DAI-mediated induction of necroptosis.

Isolated limb perfusion with melphalan, tumour necrosis factor-alpha and oncolytic vaccinia virus improves tumour targeting and prolongs survival in a rat model of advanced extremity sarcoma.

PubMed

Pencavel, Tim D; Wilkinson, Michelle J; Mansfield, David C; Khan, Aadil A; Seth, Rohit; Karapanagiotou, Eleni M; Roulstone, Victoria; Aguilar, Richard J; Chen, Nanhai G; Szalay, Aladar A; Hayes, Andrew J; Harrington, Kevin J

2015-02-15

Isolated limb perfusion (ILP) is a treatment for advanced extremity sarcoma and in-transit melanoma. Advancing this procedure by investigating the addition of novel agents, such as cancer-selective oncolytic viruses, may improve both the therapeutic efficacy of ILP and the tumour-targeted delivery of oncolytic virotherapy. Standard in vitro assays were used to characterise single agent and combinatorial activities of melphalan, tumour necrosis factor-alpha (TNF-α) and Lister strain vaccinia virus (GLV-1h68) against BN175 rat sarcoma cells. An orthotopic model of advanced extremity sarcoma was used to evaluate survival of animals after ILP with combinations of TNF-α, melphalan and GLV-1h68. We investigated the efficiency of viral tumour delivery by ILP compared to intravenous therapy, the locoregional and systemic biodistribution of virus after ILP, and the effect of mode of administration on antibody response. The combination of melphalan and GLV-1h68 was synergistic in vitro. The addition of virus to standard ILP regimens was well tolerated and demonstrated superior tumour targeting compared to intravenous administration. Triple therapy (melphalan/TNF-α/GLV-1h68) resulted in increased tumour growth delay and enhanced survival compared to other treatment regimens. Live virus was recovered in large amounts from perfused regions, but in smaller amounts from systemic organs. The addition of oncolytic vaccinia virus to existing TNF-α/melphalan-based ILP strategies results in survival advantage in an immunocompetent rat model of advanced extremity sarcoma. Virus administered by ILP has superior tumour targeting compared to intravenous delivery. Further evaluation and clinical translation of this approach is warranted. © 2014 UICC.

INTRACELLULAR FORMS OF POX VIRUSES AS SHOWN BY THE ELECTRON MICROSCOPE (VACCINIA, ECTROMELIA, MOLLUSCUM CONTAGIOSUM)

PubMed Central

Gaylord, William H.; Melnick, Joseph L.

1953-01-01

The intracellular development of three pox viruses has been studied with the electron microscope using thin sections of infected tissue. Cells infected with vaccinia, ectromelia, and molluscum contagiosum viruses all form developmental bodies preliminary to the production of mature virus. Developmental bodies, believed to be virus precursors, are round to oval, slightly larger than mature virus particles, less dense to electrons, and have a more varied morphology. It is suggested as a working hypothesis that the process of maturation of a virus particle takes place as follows. In the earliest form the developmental bodies appear as hollow spheres, imbedded in a very dense cytoplasmic mass constituting an inclusion body, or in a less dense matrix near the nucleus in cells without typical inclusion bodies. The spheres become filled with a homogeneous material of low electron density. A small, dense granule appears in each developmental body and grows in size at the expense of the low density material. Following growth of the granule, particles are found with the dimensions of mature virus and having complex internal structure resembling bars or dumbells. Mature virus is ovoid and very dense to electrons. An "empty" interior may be found within its thick walls. PMID:13069658

Vaccination with Combination DNA and Virus-Like Particles Enhances Humoral and Cellular Immune Responses upon Boost with Recombinant Modified Vaccinia Virus Ankara Expressing Human Immunodeficiency Virus Envelope Proteins.

PubMed

Gangadhara, Sailaja; Kwon, Young-Man; Jeeva, Subbiah; Quan, Fu-Shi; Wang, Baozhong; Moss, Bernard; Compans, Richard W; Amara, Rama Rao; Jabbar, M Abdul; Kang, Sang-Moo

2017-12-19

Heterologous prime boost with DNA and recombinant modified vaccinia virus Ankara (rMVA) vaccines is considered as a promising vaccination approach against human immunodeficiency virus (HIV-1). To further enhance the efficacy of DNA-rMVA vaccination, we investigated humoral and cellular immune responses in mice after three sequential immunizations with DNA, a combination of DNA and virus-like particles (VLP), and rMVA expressing HIV-1 89.6 gp120 envelope proteins (Env). DNA prime and boost with a combination of VLP and DNA vaccines followed by an rMVA boost induced over a 100-fold increase in Env-specific IgG antibody titers compared to three sequential immunizations with DNA and rMVA. Cellular immune responses were induced by VLP-DNA and rMVA vaccinations at high levels in CD8 T cells, CD4 T cells, and peripheral blood mononuclear cells secreting interferon (IFN)-γ, and spleen cells producing interleukin (IL)-2, 4, 5 cytokines. This study suggests that a DNA and VLP combination vaccine with MVA is a promising strategy in enhancing the efficacy of DNA-rMVA vaccination against HIV-1.

Vaccinia immune globulin: current policies, preparedness, and product safety and efficacy.

PubMed

Wittek, Riccardo

2006-05-01

In 1980 the World Health Organization declared that smallpox was eradicated from the world, and routine smallpox vaccination was discontinued. Nevertheless, samples of the smallpox virus (variola virus) were retained for research purposes, not least because of fears that terrorist groups or rogue states might also have kept samples in order to develop a bioweapon. Variola virus represents an effective bioweapon because it is associated with high morbidity and mortality and is highly contagious. Since September 11, 2001, countries around the world have begun to develop policies and preparedness programs to deal with a bioterror attack, including stockpiling of smallpox vaccine. Smallpox vaccine itself may be associated with a number of serious adverse events, which can often be managed with vaccinia immune globulin (VIG). VIG may also be needed as prophylaxis in patients for whom pre-exposure smallpox vaccine is contraindicated (such as those with eczema or pregnant women), although it is currently not licensed in these cases. Two intravenous formulations of VIG (VIGIV Cangene and VIGIV Dynport) have been licensed by the FDA for the management of patients with progressive vaccinia, eczema vaccinatum, severe generalized vaccinia, and extensive body surface involvement or periocular implantation following inadvertent inoculation.

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

PubMed

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

2017-03-01

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

Myxoma and vaccinia viruses exploit different mechanisms to enter and infect human cancer cells

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

Villa, Nancy Y.; Bartee, Eric; Mohamed, Mohamed R.

2010-06-05

Myxoma (MYXV) and vaccinia (VACV) viruses have recently emerged as potential oncolytic agents that can infect and kill different human cancer cells. Although both are structurally similar, it is unknown whether the pathway(s) used by these poxviruses to enter and cause oncolysis in cancer cells are mechanistically similar. Here, we compared the entry of MYXV and VACV-WR into various human cancer cells and observed significant differences: 1 - low-pH treatment accelerates fusion-mediated entry of VACV but not MYXV, 2 - the tyrosine kinase inhibitor genistein inhibits entry of VACV, but not MYXV, 3 - knockdown of PAK1 revealed that itmore » is required for a late stage event downstream of MYXV entry into cancer cells, whereas PAK1 is required for VACV entry into the same target cells. These results suggest that VACV and MYXV exploit different mechanisms to enter into human cancer cells, thus providing some rationale for their divergent cancer cell tropisms.« less

Protection of Mice from Fatal Measles Encephalitis by Vaccination with Vaccinia Virus Recombinants Encoding Either the Hemagglutinin or the Fusion Protein

NASA Astrophysics Data System (ADS)

Drillien, Robert; Spehner, Daniele; Kirn, Andre; Giraudon, Pascale; Buckland, Robin; Wild, Fabian; Lecocq, Jean-Pierre

1988-02-01

Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.

Distinct gene expression profiles in peripheral blood mononuclear cells from patients infected with vaccinia virus, yellow fever 17D virus, or upper respiratory infections.

PubMed

Scherer, Christina A; Magness, Charles L; Steiger, Kathryn V; Poitinger, Nicholas D; Caputo, Christine M; Miner, Douglas G; Winokur, Patricia L; Klinzman, Donna; McKee, Janice; Pilar, Christine; Ward, Patricia A; Gillham, Martha H; Haulman, N Jean; Stapleton, Jack T; Iadonato, Shawn P

2007-08-29

Gene expression in human peripheral blood mononuclear cells was systematically evaluated following smallpox and yellow fever vaccination, and naturally occurring upper respiratory infection (URI). All three infections were characterized by the induction of many interferon stimulated genes, as well as enhanced expression of genes involved in proteolysis and antigen presentation. Vaccinia infection was also characterized by a distinct expression signature composed of up-regulation of monocyte response genes, with repression of genes expressed by B and T-cells. In contrast, the yellow fever host response was characterized by a suppression of ribosomal and translation factors, distinguishing this infection from vaccinia and URI. No significant URI-specific signature was observed, perhaps reflecting greater heterogeneity in the study population and etiological agents. Taken together, these data suggest that specific host gene expression signatures may be identified that distinguish one or a small number of virus agents.

African Swine Fever Virus pB119L Protein Is a Flavin Adenine Dinucleotide-Linked Sulfhydryl Oxidase

PubMed Central

Rodríguez, Irene; Redrejo-Rodríguez, Modesto; Rodríguez, Javier M.; Alejo, Alí; Salas, José; Salas, María L.

2006-01-01

Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway. PMID:16537584

African swine fever virus pB119L protein is a flavin adenine dinucleotide-linked sulfhydryl oxidase.

PubMed

Rodríguez, Irene; Redrejo-Rodríguez, Modesto; Rodríguez, Javier M; Alejo, Alí; Salas, José; Salas, María L

2006-04-01

Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway.

Mucosal Vaccination Overcomes the Barrier to Recombinant Vaccinia Immunization Caused by Preexisting Poxvirus Immunity

NASA Astrophysics Data System (ADS)

Belyakov, Igor M.; Moss, Bernard; Strober, Warren; Berzofsky, Jay A.

1999-04-01

Overcoming preexisting immunity to vaccinia virus in the adult population is a key requirement for development of otherwise potent recombinant vaccinia vaccines. Based on our observation that s.c. immunization with vaccinia induces cellular and antibody immunity to vaccinia only in systemic lymphoid tissue and not in mucosal sites, we hypothesized that the mucosal immune system remains naive to vaccinia and therefore amenable to immunization with recombinant vaccinia vectors despite earlier vaccinia exposure. We show that mucosal immunization of vaccinia-immune BALB/c mice with recombinant vaccinia expressing HIV gp160 induced specific serum antibody and strong HIV-specific cytotoxic T lymphocyte responses. These responses occurred not only in mucosal but also in systemic lymphoid tissue, whereas systemic immunization was ineffective under these circumstances. In this context, intrarectal immunization was more effective than intranasal immunization. Boosting with a second dose of recombinant vaccinia was also more effective via the mucosal route. The systemic HIV-specific cytotoxic T lymphocyte response was enhanced by coadministration of IL-12 at the mucosal site. These results also demonstrate the independent compartmentalization of the mucosal versus systemic immune systems and the asymmetric trafficking of lymphocytes between them. This approach to circumvent previous vaccinia immunity may be useful for induction of protective immunity against infectious diseases and cancer in the sizable populations with preexisting immunity to vaccinia from smallpox vaccination.

Human CD4 T cell epitopes selective for Vaccinia versus Variola virus.

PubMed

Probst, Alicia; Besse, Aurore; Favry, Emmanuel; Imbert, Gilles; Tanchou, Valérie; Castelli, Florence Anne; Maillere, Bernard

2013-04-01

Due to the high degree of sequence identity between Orthopoxvirus species, the specific B and T cell responses raised against these viruses are largely cross-reactive and poorly selective. We therefore searched for CD4 T cell epitopes present in the conserved parts of the Vaccinia genome (VACV) but absent from Variola viruses (VARV), with a view to identifying immunogenic sequences selective for VACV. We identified three long peptide fragments from the B7R, B10R and E7R proteins by in silico comparisons of the poxvirus genomes, and evaluated the recognition of these fragments by VACV-specific T cell lines derived from healthy donors. For the 12 CD4 T cell epitopes identified, we assessed their binding to common HLA-DR allotypes and their capacity to induce peptide-specific CD4 T-cell lines. Four peptides from B7R and B10R displayed a broad binding specificity for HLA-DR molecules and induced multiple T cell lines from healthy donors. Besides their absence from VARV, the two B10R peptide sequences were mutated in the Cowpox virus and completely absent from the Monkeypox genome. This work contributes to the development of differential diagnosis of poxvirus infections. Copyright © 2012 Elsevier Ltd. All rights reserved.

Rapid and Effective Virucidal Activity of Povidone-Iodine Products Against Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and Modified Vaccinia Virus Ankara (MVA).

PubMed

Eggers, Maren; Eickmann, Markus; Zorn, Juergen

2015-12-01

Since the first case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) infection was reported in 2012, the virus has infected more than 1300 individuals in 26 countries, and caused more than 480 deaths. Human-to-human transmission requires close contact, and has typically occurred in the healthcare setting. Improved global awareness, together with improved hygiene practices in healthcare facilities, has been highlighted as key strategies in controlling the spread of MERS-CoV. This study tested the in vitro efficacy of three formulations of povidone iodine (PVP-I: 4% PVP-I skin cleanser, 7.5% PVP-I surgical scrub, and 1% PVP-I gargle/mouthwash) against a reference virus (Modified vaccinia virus Ankara, MVA) and MERS-CoV. According to EN14476, a standard suspension test was used to assess virucidal activity against MVA and large volume plating was used for MERS-CoV. All products were tested under clean (0.3 g/L bovine serum albumin, BSA) and dirty conditions (3.0 g/L BSA + 3.0 mL/L erythrocytes), with application times of 15, 30, and 60 s for MVA, and 15 s for MERS-CoV. The products were tested undiluted, 1:10 and 1:100 diluted against MVA, and undiluted against MERS-CoV. A reduction in virus titer of ≥4 log10 (corresponding to an inactivation of ≥99.99%) was regarded as evidence of virucidal activity. This was achieved versus MVA and MERS-CoV, under both clean and dirty conditions, within 15 s of application of each undiluted PVP-I product. These data indicate that PVP-I-based hand wash products for potentially contaminated skin, and PVP-I gargle/mouthwash for reduction of viral load in the oral cavity and the oropharynx, may help to support hygiene measures to prevent transmission of MERS-CoV. Mundipharma Research GmbH & Co.

Vaccination with recombinant modified vaccinia virus Ankara prevents the onset of intestinal allergy in mice.

PubMed

Bohnen, C; Wangorsch, A; Schülke, S; Nakajima-Adachi, H; Hachimura, S; Burggraf, M; Süzer, Y; Schwantes, A; Sutter, G; Waibler, Z; Reese, G; Toda, M; Scheurer, S; Vieths, S

2013-08-01

Modified vaccinia virus Ankara (MVA)-encoding antigens are considered as safe vaccine candidates for various infectious diseases in humans. Here, we investigated the immune-modulating properties of MVA-encoding ovalbumin (MVA-OVA) on the allergen-specific immune response. The immune-modulating properties of MVA-OVA were investigated using GM-CSF-differentiated BMDCs from C57BL/6 mice. OVA expression upon MVA-OVA infection of BMDCs was monitored. Activation and maturation markers on viable MVA-OVA-infected mDCs were analyzed by flow cytometry. Secretion of INF-γ, IL-2, and IL-10 was determined in a co-culture of BMDCs infected with wtMVA or MVA-OVA and OVA-specific OT-I CD8(+) and OT-II CD4(+ ) T cells. BALB/c mice were vaccinated with wtMVA, MVA-OVA, or PBS, sensitized to OVA/alum and challenged with a diet containing chicken egg white. OVA-specific IgE, IgG1, and IgG2a and cytokine secretion from mesenteric lymph node (MLN) cells were analyzed. Body weight, body temperature, food uptake, intestinal inflammation, and health condition of mice were monitored. Infection with wtMVA and MVA-OVA induced comparable activation of mDCs. MVA-OVA-infected BMDCs expressed OVA and induced enhanced IFN-γ and IL-2 secretion from OVA-specific CD8(+ ) T cells in comparison with OVA, wtMVA, or OVA plus wtMVA. Prophylactic vaccination with MVA-OVA significantly repressed OVA-specific IgE, whereas OVA-specific IgG2a was induced. MVA-OVA vaccination suppressed TH 2 cytokine production in MLN cells and prevented the onset of allergic symptoms and inflammation in a mouse model of OVA-induced intestinal allergy. Modified vaccinia virus Ankara-ovalbumin (MVA-OVA) vaccination induces a strong OVA-specific TH 1- immune response, likely mediated by the induction of IFN-γ and IgG2a. Finally, MVA-based vaccines need to be evaluated for their therapeutic potential in established allergy models. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Single-Virus Fusion Experiments Reveal Proton Influx into Vaccinia Virions and Hemifusion Lag Times

PubMed Central

Schmidt, Florian I.; Kuhn, Phillip; Robinson, Tom; Mercer, Jason; Dittrich, Petra S.

2013-01-01

Recent studies have revealed new insights into the endocytosis of vaccinia virus (VACV). However, the mechanism of fusion between viral and cellular membranes remains unknown. We developed a microfluidic device with a cell-trap array for immobilization of individual cells, with which we analyzed the acid-dependent fusion of single virions. VACV particles incorporating enhanced green fluorescent protein (EGFP) and labeled with self-quenching concentrations of R18 membrane dye were used in combination with total internal reflection fluorescence microscopy to measure the kinetics of R18 dequenching and thus single hemifusion events initiated by a fast low-pH trigger. These studies revealed unexpectedly long lag phases between pH change and hemifusion. In addition, we found that EGFP fluorescence in the virus was quenched upon acidification, indicating that protons could access the virus core, possibly through a proton channel. In a fraction of virus particles, EGFP fluorescence was recovered, presumably after fusion-pore formation and exposure of the core to the physiological pH of the host-cell cytosol. Given that virus-encoded cation channels play a crucial role in the life cycle of many viruses and can serve as antiviral drug targets, further investigations into a potential VACV viroporin are justified. Our findings indicate that the microfluidic device described may be highly beneficial to similar studies requiring fast kinetic measurements. PMID:23870263

De novo Fatty Acid Biosynthesis Contributes Significantly to Establishment of a Bioenergetically Favorable Environment for Vaccinia Virus Infection

PubMed Central

Greseth, Matthew D.; Traktman, Paula

2014-01-01

The poxvirus life cycle, although physically autonomous from the host nucleus, is nevertheless dependent upon cellular functions. A requirement for de novo fatty acid biosynthesis was implied by our previous demonstration that cerulenin, a fatty acid synthase inhibitor, impaired vaccinia virus production. Here we show that additional inhibitors of this pathway, TOFA and C75, reduce viral yield significantly, with partial rescue provided by exogenous palmitate, the pathway's end-product. Palmitate's major role during infection is not for phospholipid synthesis or protein palmitoylation. Instead, the mitochondrial import and β-oxidation of palmitate are essential, as shown by the impact of etomoxir and trimetazidine, which target these two processes respectively. Moreover, the impact of these inhibitors is exacerbated in the absence of exogenous glucose, which is otherwise dispensable for infection. In contrast to glucose, glutamine is essential for productive viral infection, providing intermediates that sustain the TCA cycle (anaplerosis). Cumulatively, these data suggest that productive infection requires the mitochondrial β-oxidation of palmitate which drives the TCA cycle and energy production. Additionally, infection causes a significant rise in the cellular oxygen consumption rate (ATP synthesis) that is ablated by etomoxir. The biochemical progression of the vaccinia life cycle is not impaired in the presence of TOFA, C75, or etomoxir, although the levels of viral DNA and proteins synthesized are somewhat diminished. However, by reversibly arresting infections at the onset of morphogenesis, and then monitoring virus production after release of the block, we determined that virion assembly is highly sensitive to TOFA and C75. Electron microscopic analysis of cells released into C75 revealed fragmented aggregates of viroplasm which failed to be enclosed by developing virion membranes. Taken together, these data indicate that vaccinia infection, and in

De novo fatty acid biosynthesis contributes significantly to establishment of a bioenergetically favorable environment for vaccinia virus infection.

PubMed

Greseth, Matthew D; Traktman, Paula

2014-03-01

The poxvirus life cycle, although physically autonomous from the host nucleus, is nevertheless dependent upon cellular functions. A requirement for de novo fatty acid biosynthesis was implied by our previous demonstration that cerulenin, a fatty acid synthase inhibitor, impaired vaccinia virus production. Here we show that additional inhibitors of this pathway, TOFA and C75, reduce viral yield significantly, with partial rescue provided by exogenous palmitate, the pathway's end-product. Palmitate's major role during infection is not for phospholipid synthesis or protein palmitoylation. Instead, the mitochondrial import and β-oxidation of palmitate are essential, as shown by the impact of etomoxir and trimetazidine, which target these two processes respectively. Moreover, the impact of these inhibitors is exacerbated in the absence of exogenous glucose, which is otherwise dispensable for infection. In contrast to glucose, glutamine is essential for productive viral infection, providing intermediates that sustain the TCA cycle (anaplerosis). Cumulatively, these data suggest that productive infection requires the mitochondrial β-oxidation of palmitate which drives the TCA cycle and energy production. Additionally, infection causes a significant rise in the cellular oxygen consumption rate (ATP synthesis) that is ablated by etomoxir. The biochemical progression of the vaccinia life cycle is not impaired in the presence of TOFA, C75, or etomoxir, although the levels of viral DNA and proteins synthesized are somewhat diminished. However, by reversibly arresting infections at the onset of morphogenesis, and then monitoring virus production after release of the block, we determined that virion assembly is highly sensitive to TOFA and C75. Electron microscopic analysis of cells released into C75 revealed fragmented aggregates of viroplasm which failed to be enclosed by developing virion membranes. Taken together, these data indicate that vaccinia infection, and in

Vaccinia Virus-mediated Expression of Human Erythropoietin in Tumors Enhances Virotherapy and Alleviates Cancer-related Anemia in Mice

PubMed Central

Nguyen, Duong H; Chen, Nanhai G; Zhang, Qian; Le, Ha T; Aguilar, Richard J; Yu, Yong A; Cappello, Joseph; Szalay, Aladar A

2013-01-01

Recombinant human erythropoietin (rhEPO), a glycoprotein hormone regulating red blood cell (RBC) formation, is used for the treatment of cancer-related anemia. The effect of rhEPO on tumor growth, however, remains controversial. Here, we report the construction and characterization of the recombinant vaccinia virus (VACV) GLV-1h210, expressing hEPO. GLV-1h210 was shown to replicate in and kill A549 lung cancer cells in culture efficiently. In mice bearing A549 lung cancer xenografts, treatment with a single intravenous dose of GLV-1h210 resulted in tumor-specific production and secretion of functional hEPO, which exerted an effect on RBC progenitors and precursors in the mouse bone marrow, leading to a significant increase in the number of RBCs and in the level of hemoglobin. Furthermore, virally expressed hEPO, but not exogenously added rhEPO, enhanced virus-mediated green fluorescent protein (GFP) expression in tumors and subsequently accelerated tumor regression when compared with the treatment with the parental virus GLV-1h68 or GLV-1h209 that expressed a nonfunctional hEPO protein. Moreover, intratumorally expressed hEPO caused enlarged tumoral microvessels, likely facilitating virus spreading. Taken together, VACV-mediated intratumorally expressed hEPO not only enhanced oncolytic virotherapy but also simultaneously alleviated cancer-related anemia. PMID:23765443

Prediction of Steps in the Evolution of Variola Virus Host Range

PubMed Central

Smithson, Chad; Purdy, Alex; Verster, Adrian J.; Upton, Chris

2014-01-01

Variola virus, the agent of smallpox, has a severely restricted host range (humans) but a devastatingly high mortality rate. Although smallpox has been eradicated by a World Health Organization vaccination program, knowledge of the evolutionary processes by which human super-pathogens such as variola virus arise is important. By analyzing the evolution of variola and other closely related poxviruses at the level of single nucleotide polymorphisms we detected a hotspot of genome variation within the smallpox ortholog of the vaccinia virus O1L gene, which is known to be necessary for efficient replication of vaccinia virus in human cells. These mutations in the variola virus ortholog and the subsequent loss of the functional gene from camelpox virus and taterapox virus, the two closest relatives of variola virus, strongly suggest that changes within this region of the genome may have played a key role in the switch to humans as a host for the ancestral virus and the subsequent host-range restriction that must have occurred to create the phenotype exhibited by smallpox. PMID:24626337

Prediction of steps in the evolution of variola virus host range.

PubMed

Smithson, Chad; Purdy, Alex; Verster, Adrian J; Upton, Chris

2014-01-01

Variola virus, the agent of smallpox, has a severely restricted host range (humans) but a devastatingly high mortality rate. Although smallpox has been eradicated by a World Health Organization vaccination program, knowledge of the evolutionary processes by which human super-pathogens such as variola virus arise is important. By analyzing the evolution of variola and other closely related poxviruses at the level of single nucleotide polymorphisms we detected a hotspot of genome variation within the smallpox ortholog of the vaccinia virus O1L gene, which is known to be necessary for efficient replication of vaccinia virus in human cells. These mutations in the variola virus ortholog and the subsequent loss of the functional gene from camelpox virus and taterapox virus, the two closest relatives of variola virus, strongly suggest that changes within this region of the genome may have played a key role in the switch to humans as a host for the ancestral virus and the subsequent host-range restriction that must have occurred to create the phenotype exhibited by smallpox.

Recombinant modified vaccinia virus Ankara–simian immunodeficiency virus gag pol elicits cytotoxic T lymphocytes in rhesus monkeys detected by a major histocompatibility complex class I/peptide tetramer

PubMed Central

Seth, Aruna; Ourmanov, Ilnour; Kuroda, Marcelo J.; Schmitz, Jörn E.; Carroll, Miles W.; Wyatt, Linda S.; Moss, Bernard; Forman, Meryl A.; Hirsch, Vanessa M.; Letvin, Norman L.

1998-01-01

The utility of modified vaccinia virus Ankara (MVA) as a vector for eliciting AIDS virus-specific cytotoxic T lymphocytes (CTL) was explored in the simian immunodeficiency virus (SIV)/rhesus monkey model. After two intramuscular immunizations with recombinant MVA-SIVSM gag pol, the monkeys developed a Gag epitope-specific CTL response readily detected in peripheral blood lymphocytes by using a functional killing assay. Moreover, those immunizations also elicited a population of CD8+ T lymphocytes in the peripheral blood that bound a specific major histocompatibility complex class I/peptide tetramer. These Gag epitope-specific CD8+ T lymphocytes also were demonstrated by using both functional and tetramer-binding assays in lymph nodes of the immunized monkeys. These observations suggest that MVA may prove a useful vector for an HIV-1 vaccine. They also suggest that tetramer staining may be a useful technology for monitoring CTL generation in vaccine trials in nonhuman primates and in humans. PMID:9707609

Comparative Proteomics of Human Monkeypox and Vaccinia Intracellular Mature and Extracellular Enveloped Virions

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

Manes, Nathan P.; Estep, Ryan D.; Mottaz, Heather M.

2008-03-07

Orthopoxviruses are the largest and most complex of the animal viruses. In response to the recent emergence of monkeypox in Africa and the threat of smallpox bioterrorism, virulent (monkeypox virus) and benign (vaccinia virus) orthopoxviruses were proteomically compared with the goal of identifying proteins required for pathogenesis. Orthopoxviruses were grown in HeLa cells to two different viral forms (intracellular mature virus and extracellular enveloped virus), purified by sucrose gradient ultracentrifugation, denatured using RapiGest™ surfactant, and digested with trypsin. Unfractionated samples and strong cation exchange HPLC fractions were analyzed by reversed-phase LC-MS/MS, and analyses of the MS/MS spectra using SEQUEST® andmore » X! Tandem resulted in the identification of hundreds of monkeypox, vaccinia, and copurified host proteins. The unfractionated samples were additionally analyzed by LC-MS on an LTQ-Orbitrap™, and the accurate mass and elution time tag approach was used to perform quantitative comparisons. Possible pathophysiological roles of differentially expressed orthopoxvirus genes are discussed.« less

Single-virus fusion experiments reveal proton influx into vaccinia virions and hemifusion lag times.

PubMed

Schmidt, Florian I; Kuhn, Phillip; Robinson, Tom; Mercer, Jason; Dittrich, Petra S

2013-07-16

Recent studies have revealed new insights into the endocytosis of vaccinia virus (VACV). However, the mechanism of fusion between viral and cellular membranes remains unknown. We developed a microfluidic device with a cell-trap array for immobilization of individual cells, with which we analyzed the acid-dependent fusion of single virions. VACV particles incorporating enhanced green fluorescent protein (EGFP) and labeled with self-quenching concentrations of R18 membrane dye were used in combination with total internal reflection fluorescence microscopy to measure the kinetics of R18 dequenching and thus single hemifusion events initiated by a fast low-pH trigger. These studies revealed unexpectedly long lag phases between pH change and hemifusion. In addition, we found that EGFP fluorescence in the virus was quenched upon acidification, indicating that protons could access the virus core, possibly through a proton channel. In a fraction of virus particles, EGFP fluorescence was recovered, presumably after fusion-pore formation and exposure of the core to the physiological pH of the host-cell cytosol. Given that virus-encoded cation channels play a crucial role in the life cycle of many viruses and can serve as antiviral drug targets, further investigations into a potential VACV viroporin are justified. Our findings indicate that the microfluidic device described may be highly beneficial to similar studies requiring fast kinetic measurements. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Endoplasmic Reticulum-Golgi Intermediate Compartment Membranes and Vimentin Filaments Participate in Vaccinia Virus Assembly

PubMed Central

Risco, Cristina; Rodríguez, Juan R.; López-Iglesias, Carmen; Carrascosa, José L.; Esteban, Mariano; Rodríguez, Dolores

2002-01-01

Vaccinia virus (VV) has a complex morphogenetic pathway whose first steps are poorly characterized. We have studied the early phase of VV assembly, when viral factories and spherical immature viruses (IVs) form in the cytoplasm of the infected cell. After freeze-substitution numerous cellular elements are detected around assembling viruses: membranes, ribosomes, microtubules, filaments, and unidentified structures. A double membrane is clearly resolved in the VV envelope for the first time, and freeze fracture reveals groups of tubules interacting laterally on the surface of the viroplasm foci. These data strongly support the hypothesis of a cellular tubulovesicular compartment, related to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC), as the origin of the first VV envelope. Moreover, the cytoskeletal vimentin intermediate filaments are found around viral factories and inside the viroplasm foci, where vimentin and the VV core protein p39 colocalize in the areas where crescents protrude. Confocal microscopy showed that ERGIC elements and vimentin filaments concentrate in the viral factories. We propose that modified cellular ERGIC membranes and vimentin intermediate filaments act coordinately in the construction of viral factories and the first VV form through a unique mechanism of viral morphogenesis from cellular elements. PMID:11799179

Antibodies to the A27 protein of vaccinia virus neutralize and protect against infection but represent a minor component of Dryvax vaccine--induced immunity.

PubMed

He, Yong; Manischewitz, Jody; Meseda, Clement A; Merchlinsky, Michael; Vassell, Russell A; Sirota, Lev; Berkower, Ira; Golding, Hana; Weiss, Carol D

2007-10-01

The smallpox vaccine Dryvax, which consists of replication-competent vaccinia virus, elicits antibodies that play a major role in protection. Several vaccinia proteins generate neutralizing antibodies, but their importance for protection is unknown. We investigated the potency of antibodies to the A27 protein of the mature virion in neutralization and protection experiments and the contributions of A27 antibodies to Dryvax-induced immunity. Using a recombinant A27 protein (rA27), we confirmed that A27 contains neutralizing determinants and that vaccinia immune globulin (VIG) derived from Dryvax recipients contains reactivity to A27. However, VIG neutralization was not significantly reduced when A27 antibodies were removed, and antibodies elicited by an rA27 enhanced the protection conferred by VIG in passive transfer experiments. These findings demonstrate that A27 antibodies do not represent the major fraction of neutralizing activity in VIG and suggest that immunity may be augmented by vaccines and immune globulins that include strong antibody responses to A27.

Molecular characterization of the host defense activity of the barrier to autointegration factor against vaccinia virus.

PubMed

Ibrahim, Nouhou; Wicklund, April; Wiebe, Matthew S

2011-11-01

The barrier to autointegration factor (BAF) is an essential cellular protein with functions in mitotic nuclear reassembly, retroviral preintegration complex stability, and transcriptional regulation. Molecular properties of BAF include the ability to bind double-stranded DNA in a sequence-independent manner, homodimerize, and bind proteins containing a LEM domain. These capabilities allow BAF to compact DNA and assemble higher-order nucleoprotein complexes, the nature of which is poorly understood. Recently, it was revealed that BAF also acts as a potent host defense against poxviral DNA replication in the cytoplasm. Here, we extend these observations by examining the molecular mechanism through which BAF acts as a host defense against vaccinia virus replication and cytoplasmic DNA in general. Interestingly, BAF rapidly relocalizes to transfected DNA from a variety of sources, demonstrating that BAF's activity as a host defense factor is not limited to poxviral infection. BAF's relocalization to cytoplasmic foreign DNA is highly dependent upon its DNA binding and dimerization properties but does not appear to require its LEM domain binding activity. However, the LEM domain protein emerin is recruited to cytoplasmic DNA in a BAF-dependent manner during both transfection and vaccinia virus infection. Finally, we demonstrate that the DNA binding and dimerization capabilities of BAF are essential for its function as an antipoxviral effector, while the presence of emerin is not required. Together, these data provide further mechanistic insight into which of BAF's molecular properties are employed by cells to impair the replication of poxviruses or respond to foreign DNA in general.

42 CFR 102.21 - Smallpox (Vaccinia) Vaccine Injury Table.

Code of Federal Regulations, 2012 CFR

2012-10-01

... of the Table, an autoimmune central nervous system injury. In rare cases, the vaccinia virus is isolated from the central nervous system. Manifestations usually occur abruptly and may include fever... spinal cord (myelitis) such as paralysis or meningismus. Long term central nervous system impairments...

42 CFR 102.21 - Smallpox (Vaccinia) Vaccine Injury Table.

Code of Federal Regulations, 2013 CFR

2013-10-01

... of the Table, an autoimmune central nervous system injury. In rare cases, the vaccinia virus is isolated from the central nervous system. Manifestations usually occur abruptly and may include fever... spinal cord (myelitis) such as paralysis or meningismus. Long term central nervous system impairments...

42 CFR 102.21 - Smallpox (Vaccinia) Vaccine Injury Table.

Code of Federal Regulations, 2011 CFR

2011-10-01

... of the Table, an autoimmune central nervous system injury. In rare cases, the vaccinia virus is isolated from the central nervous system. Manifestations usually occur abruptly and may include fever... spinal cord (myelitis) such as paralysis or meningismus. Long term central nervous system impairments...

42 CFR 102.21 - Smallpox (Vaccinia) Vaccine Injury Table.

Code of Federal Regulations, 2014 CFR

2014-10-01

... of the Table, an autoimmune central nervous system injury. In rare cases, the vaccinia virus is isolated from the central nervous system. Manifestations usually occur abruptly and may include fever... spinal cord (myelitis) such as paralysis or meningismus. Long term central nervous system impairments...

Biophysical analysis of bacterial and viral systems. A shock tube study of bio-aerosols and a correlated AFM/nanosims investigation of vaccinia virus

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

Gates, Sean Damien

2013-05-01

The work presented herein is concerned with the development of biophysical methodology designed to address pertinent questions regarding the behavior and structure of select pathogenic agents. Two distinct studies are documented: a shock tube analysis of endospore-laden bio-aerosols and a correlated AFM/NanoSIMS study of the structure of vaccinia virus.

[Safety and efficacy of an antirabies vaccine consisting of recombinant vaccinia-rabies virus administered orally to the fox, dog and cat].

PubMed

Blancou, J; Artois, M; Brochier, B; Thomas, I; Pastoret, P P; Desmettre, P; Languet, B; Kiény, M P

1989-01-01

One of the most promising ways to control rabies in wildlife seems to be the distribution of bait containing an anti-rabies vaccine. So far, the most widely used vaccines were modified live viruses (SAD strain or derivatives). Nevertheless, these strains retain some pathogenicity for non-target species. A novel vaccine was proposed consisting of genetically modified vaccinia virus (strain Copenhagen, thermosensitive ts 26) expressing the foreign glycoprotein G for the rabies virus (strain ERA). Different doses of this recombinant virus were administered orally to 59 foxes (Vulpes vulpes) and their antibodies were titrated before challenge. Foxes (8/8) resisted 1 month after vaccination with 10(7) plaque forming units (PFU), or 4/4 after 18 months. Seroconversion among dogs was 4/4 after vaccination with 10(9,6) PFU and 4/4 among cats after vaccination with 10(8) PFU. These dogs (4/4) and cats (3/4) resisted the challenge 2-3 months after vaccination. This vaccine thus appears to be potent and safe in these species. Its properties are discussed.

Evaluation of radiation effects against C6 glioma in combination with vaccinia virus-p53 gene therapy

NASA Technical Reports Server (NTRS)

Gridley, D. S.; Andres, M. L.; Li, J.; Timiryasova, T.; Chen, B.; Fodor, I.; Nelson, G. A. (Principal Investigator)

1998-01-01

The primary objective of this study was to evaluate the antitumor effects of recombinant vaccinia virus-p53 (rVV-p53) in combination with radiation therapy against the C6 rat glioma, a p53 deficient tumor that is relatively radioresistant. VV-LIVP, the parental virus (Lister strain), was used as a control. Localized treatment of subcutaneous C6 tumors in athymic mice with either rVV-p53 or VV-LIVP together with tumor irradiation resulted in low tumor incidence and significantly slower tumor progression compared to the agents given as single modalities. Assays of blood and spleen indicated that immune system activation may account, at least partly, for the enhance tumor inhibition seen with combined treatment. No overt signs of treatment-related toxicity were noted.

Linear Epitopes in Vaccinia Virus A27 Are Targets of Protective Antibodies Induced by Vaccination against Smallpox.

PubMed

Kaever, Thomas; Matho, Michael H; Meng, Xiangzhi; Crickard, Lindsay; Schlossman, Andrew; Xiang, Yan; Crotty, Shane; Peters, Bjoern; Zajonc, Dirk M

2016-05-01

Vaccinia virus (VACV) A27 is a target for viral neutralization and part of the Dryvax smallpox vaccine. A27 is one of the three glycosaminoglycan (GAG) adhesion molecules and binds to heparan sulfate. To understand the function of anti-A27 antibodies, especially their protective capacity and their interaction with A27, we generated and subsequently characterized 7 murine monoclonal antibodies (MAbs), which fell into 4 distinct epitope groups (groups I to IV). The MAbs in three groups (groups I, III, and IV) bound to linear peptides, while the MAbs in group II bound only to VACV lysate and recombinant A27, suggesting that they recognized a conformational and discontinuous epitope. Only group I antibodies neutralized the mature virion in a complement-dependent manner and protected against VACV challenge, while a group II MAb partially protected against VACV challenge but did not neutralize the mature virion. The epitope for group I MAbs was mapped to a region adjacent to the GAG binding site, a finding which suggests that group I MAbs could potentially interfere with the cellular adhesion of A27. We further determined the crystal structure of the neutralizing group I MAb 1G6, as well as the nonneutralizing group IV MAb 8E3, bound to the corresponding linear epitope-containing peptides. Both the light and the heavy chains of the antibodies are important in binding to their antigens. For both antibodies, the L1 loop seems to dominate the overall polar interactions with the antigen, while for MAb 8E3, the light chain generally appears to make more contacts with the antigen. Vaccinia virus is a powerful model to study antibody responses upon vaccination, since its use as the smallpox vaccine led to the eradication of one of the world's greatest killers. The immunodominant antigens that elicit the protective antibodies are known, yet for many of these antigens, little information about their precise interaction with antibodies is available. In an attempt to better

Expression of the highly conserved vaccinia virus E6 protein is required for virion morphogenesis

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

Resch, Wolfgang; Weisberg, Andrea S.; Moss, Bernard, E-mail: bmoss@nih.go

2009-04-10

The vaccinia virus E6R gene (VACVWR062) is conserved in all members of the poxvirus family and encodes a protein associated with the mature virion. We confirmed this association and provided evidence for an internal location. An inducible mutant that conditionally expresses E6 was constructed. In the absence of inducer, plaque formation and virus production were severely inhibited in several cell lines, whereas some replication occurred in others. This difference could be due to variation in the stringency of repression, since we could not isolate a stable deletion mutant even in the more 'permissive' cells. Under non-permissive conditions, viral late proteinsmore » were synthesized but processing of core proteins was inefficient, indicative of an assembly block. Transmission electron microscopy of sections of cells infected with the mutant in the absence of inducer revealed morphogenetic defects with crescents and empty immature virions adjacent to dense inclusions of viroplasm. Mature virions were infrequent and cores appeared to have lucent centers.« less

Activity, specificity, and probe design for the smallpox virus protease K7L.

PubMed

Aleshin, Alexander E; Drag, Marcin; Gombosuren, Naran; Wei, Ge; Mikolajczyk, Jowita; Satterthwait, Arnold C; Strongin, Alex Y; Liddington, Robert C; Salvesen, Guy S

2012-11-16

The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.

A single immunization with modified vaccinia virus Ankara-based influenza virus H7 vaccine affords protection in the influenza A(H7N9) pneumonia ferret model.

PubMed

Kreijtz, Joost H C M; Wiersma, Lidewij C M; De Gruyter, Heidi L M; Vogelzang-van Trierum, Stella E; van Amerongen, Geert; Stittelaar, Koert J; Fouchier, Ron A M; Osterhaus, Albert D M E; Sutter, Gerd; Rimmelzwaan, Guus F

2015-03-01

Since the first reports in early 2013, >440 human cases of infection with avian influenza A(H7N9) have been reported including 122 fatalities. After the isolation of the first A(H7N9) viruses, the nucleotide sequences became publically available. Based on the coding sequence of the influenza virus A/Shanghai/2/2013 hemagglutinin gene, a codon-optimized gene was synthesized and cloned into a recombinant modified vaccinia virus Ankara (MVA). This MVA-H7-Sh2 viral vector was used to immunize ferrets and proved to be immunogenic, even after a single immunization. Subsequently, ferrets were challenged with influenza virus A/Anhui/1/2013 via the intratracheal route. Unprotected animals that were mock vaccinated or received empty vector developed interstitial pneumonia characterized by a marked alveolitis, accompanied by loss of appetite, weight loss, and heavy breathing. In contrast, animals vaccinated with MVA-H7-Sh2 were protected from severe 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.

Immune and histopathological responses in animals vaccinated with recombinant vaccinia viruses that express individual genes of human respiratory syncytial virus.

PubMed

Stott, E J; Taylor, G; Ball, L A; Anderson, K; Young, K K; King, A M; Wertz, G W

1987-12-01

Previous reports have established that vaccinia virus (VV) recombinants expressing G, F, or N protein of respiratory syncytial (RS) virus protect small animals against intranasal challenge with live RS virus. This work demonstrates that a variety of parameters affect the protection induced by recombinant viruses. The route of vaccination, the subtype of challenge virus, and the species used influenced the antibody titers and extent of protection. During these studies, observations were also made on the subclass of antibody generated, and pulmonary histopathological changes induced by challenge after vaccination were noted. The effect of route of inoculation on host response was examined by vaccinating mice intranasally, intraperitoneally, or by scarification with a recombinant VV expressing the RS virus G glycoprotein. Intranasal vaccination induced 25-fold-higher titers of antibody to RS virus in the lung than the intraperitoneal route did, but both routes resulted in complete suppression of virus replication after intranasal challenge 21 days after vaccination. Scarification was a less effective method of vaccination. The antibody induced by recombinant VV in mice was mostly immunoglobulin G2a (IgG2a) with some IgG2b. No antibody to RS virus was detected in the IgA, IgM, IgG1, or IgG3 subclass irrespective of the vaccination route. The G and F glycoproteins were shown to elicit similar subclasses of antibody. However, animals vaccinated with the G and F vectors differed strikingly in their response to challenge by heterologous virus. Mice or cotton rats vaccinated with recombinant VV carrying the G gene of RS virus were protected against challenge only with homologous subtype A virus. Vaccination with a recombinant VV expressing the F glycoprotein induced protection against both homologous and heterologous subtype B virus challenge. The protection induced in mice was greater than that detected in cotton rats, indicating that the host may also affect immunity

Multisubunit DNA-Dependent RNA Polymerases from Vaccinia Virus and Other Nucleocytoplasmic Large-DNA Viruses: Impressions from the Age of Structure.

PubMed

Mirzakhanyan, Yeva; Gershon, Paul D

2017-09-01

The past 17 years have been marked by a revolution in our understanding of cellular multisubunit DNA-dependent RNA polymerases (MSDDRPs) at the structural level. A parallel development over the past 15 years has been the emerging story of the giant viruses, which encode MSDDRPs. Here we link the two in an attempt to understand the specialization of multisubunit RNA polymerases in the domain of life encompassing the large nucleocytoplasmic DNA viruses (NCLDV), a superclade that includes the giant viruses and the biochemically well-characterized poxvirus vaccinia virus. The first half of this review surveys the recently determined structural biology of cellular RNA polymerases for a microbiology readership. The second half discusses a reannotation of MSDDRP subunits from NCLDV families and the apparent specialization of these enzymes by virus family and by subunit with regard to subunit or domain loss, subunit dissociability, endogenous control of polymerase arrest, and the elimination/customization of regulatory interactions that would confer higher-order cellular control. Some themes are apparent in linking subunit function to structure in the viral world: as with cellular RNA polymerases I and III and unlike cellular RNA polymerase II, the viral enzymes seem to opt for speed and processivity and seem to have eliminated domains associated with higher-order regulation. The adoption/loss of viral RNA polymerase proofreading functions may have played a part in matching intrinsic mutability to genome size. Copyright © 2017 American Society for Microbiology.

Development of a Genetically-Engineered Venezuelan Equine Encephalitis virus Vaccine

DTIC Science & Technology

1989-11-13

necrosis. We have evaluated the efficacy of a recombinant vaccinia/VEE virus vaccine (TC-5A) to protect horses against challenge with equine virulent...of horse vaccinees with equine virulent VEE virus 71-180 and vaccinia viruses ........ 27 7. ELISA cross-reactivity of sera from immunized equines ...antibodies in equines after immuniza- tion with TC-83, TC-5A and wild-type vaccinia viruses . .40 5. Body temperature of horses immunized with TC-5A (A

Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B sub 1

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

Aoyama, Toshifumi; Yamano, Shigeru; Gelboin, H.V.

Twelve forms of human hepatic cytochrome P450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P450s were analyzed for their abilities to activate the potent hepatocarcinogen aflatoxin B{sub 1} to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B{sub 1} to mutagenic metabolites as assessed by the production of His revertants of Salmonella typhimurium in the Ames test. The same P450s catalyzed conversion of aflatoxin B{sub 1} to DNA-bound derivatives as judged by an in situ assay in which the radiolabeled carcinogen was incubated with cellsmore » expressing the individual P450 forms. Seven other human P450s, IIC8, IIC9, IID6, IIE1, IIF1, and IIIA5, and IVB1, did not significantly activate aflatoxin B{sub 1} as measured by both the Ames test and the DNA-binding assay. Moreover, polyclonal anti-rat liver P450 antibodies that crossreact with individual human P450s IA2, IIA3, IIIA3, and IIIA4 each inhibited aflatoxin B{sub 1} activation catalyzed by human liver S-9 extracts. Inhibition ranged from as low as 10% with antibody against IIA3 to as high as 65% with antibody against IIIA3 and IIIA4. These results establish that metabolic activation of aflatoxin B{sub 1} in human liver involves the contribution of multiple forms of P450.« less

THE LS-ANTIGEN OF VACCINIA

PubMed Central

Smadel, Joseph E.; Rivers, Thomas M.

1942-01-01

Experimental data are presented which may be interpreted as follows. The heat-labile (L) and heat-stable (S) antigens of vaccinia occur in nature as a complex consisting of a single substance with two serologically active parts, each of which may be degraded independently of the other. PMID:19871173

MVA ROP2 vaccinia virus recombinant as a vaccine candidate for toxoplasmosis.

PubMed

Roque-Reséndiz, J L; Rosales, R; Herion, P

2004-04-01

Toxoplasma gondii is the aetiological agent of toxoplasmosis and is the most frequent and best known of the parasitic diseases. In the United States, a serological survey from the Third National Health and Nutrition Examination Survey found that an estimated 23% of adolescents and adults have laboratory evidence of infection with T. gondii. Although toxoplasmosis is asymptomatic or shows self-limited symptoms in adults, in pregnant women infections can cause severe health problems to the fetus if the parasites are transmitted. Also, in immunodeficient patients, chronic infection with T. gondii can reactivate and produce encephalitis, which is frequently lethal. In addition, in veterinary medicine, T. gondii infection is of economic importance due to abortion and neonatal loss in sheep and goats. Recently, the development of vaccines against toxoplasmosis has progressed considerably. The live attenuated S48 strain of Toxoplasma has been broadly used for veterinary purposes. DNA vaccines containing the full-length of SAG1/P30, ROP2 or ROP1 genes have proved to be a promising candidate to induce protection against toxoplasmosis. Viral vectors have proved to be the best candidates for vaccination in different diseases. A recombinant Herpes virus carrying the ROP2 gene is able to induce protective immunity in cats. In the present work we describe the potential of the MVA ROP2 recombinant vaccinia virus as a vaccine against toxoplasmosis. MVA ROP2 induces antibodies against the ROP2 protein in similar amount and types as the thermo-sensible strain ts-4 of T. gondii, which is able to fully protect mice against challenge with the virulent RH strain of T. gondii. Also, the life-span of mice is increased in MVA ROP2 vaccinated animals. We conclude that MVA ROP2 vaccine can possibly generate an immune response, which could be useful in protection against toxoplasmosis.

Major neutralizing sites on vaccinia virus glycoprotein B5 are exposed differently on variola virus ortholog B6.

PubMed

Aldaz-Carroll, Lydia; Xiao, Yuhong; Whitbeck, J Charles; de Leon, Manuel Ponce; Lou, Huan; Kim, Mikyung; Yu, Jessica; Reinherz, Ellis L; Isaacs, Stuart N; Eisenberg, Roselyn J; Cohen, Gary H

2007-08-01

Immunization against smallpox (variola virus) with Dryvax, a live vaccinia virus (VV), was effective, but now safety is a major concern. To overcome this issue, subunit vaccines composed of VV envelope proteins from both forms of infectious virions, including the extracellular enveloped virion (EV) protein B5, are being developed. However, since B5 has 23 amino acid differences compared with its B6 variola virus homologue, B6 might be a better choice for such a strategy. Therefore, we compared the properties of both proteins using a panel of monoclonal antibodies (MAbs) to B5 that we had previously characterized and grouped according to structural and functional properties. The B6 gene was obtained from the Centers for Disease Control and Prevention, and the ectodomain was cloned and expressed in baculovirus as previously done with B5, allowing us to compare the antigenic properties of the proteins. Polyclonal antibodies to B5 or B6 cross-reacted with the heterologous protein, and 16 of 26 anti-B5 MAbs cross-reacted with B6. Importantly, 10 anti-B5 MAbs did not cross-react with B6. Of these, three have important anti-VV biologic properties, including their ability to neutralize EV infectivity and block comet formation. Here, we found that one of these three MAbs protected mice from a lethal VV challenge by passive immunization. Thus, epitopes that are present on B5 but not on B6 would generate an antibody response that would not recognize B6. Assuming that B6 contains similar variola virus-specific epitopes, our data suggest that a subunit vaccine using the variola virus homologues might exhibit improved protective efficacy against smallpox.

Mutagenic repair of double-stranded DNA breaks in vaccinia virus genomes requires cellular DNA ligase IV activity in the cytosol.

PubMed

Luteijn, Rutger David; Drexler, Ingo; Smith, Geoffrey L; Lebbink, Robert Jan; Wiertz, Emmanuel J H J

2018-06-01

Poxviruses comprise a group of large dsDNA viruses that include members relevant to human and animal health, such as variola virus, monkeypox virus, cowpox virus and vaccinia virus (VACV). Poxviruses are remarkable for their unique replication cycle, which is restricted to the cytoplasm of infected cells. The independence from the host nucleus requires poxviruses to encode most of the enzymes involved in DNA replication, transcription and processing. Here, we use the CRISPR/Cas9 genome engineering system to induce DNA damage to VACV (strain Western Reserve) genomes. We show that targeting CRISPR/Cas9 to essential viral genes limits virus replication efficiently. Although VACV is a strictly cytoplasmic pathogen, we observed extensive viral genome editing at the target site; this is reminiscent of a non-homologous end-joining DNA repair mechanism. This pathway was not dependent on the viral DNA ligase, but critically involved the cellular DNA ligase IV. Our data show that DNA ligase IV can act outside of the nucleus to allow repair of dsDNA breaks in poxvirus genomes. This pathway might contribute to the introduction of mutations within the genome of poxviruses and may thereby promote the evolution of these viruses.

Intratumoral delivery of inactivated modified vaccinia virus Ankara (iMVA) induces systemic antitumor immunity via STING and Batf3-dependent dendritic cells.

PubMed

Dai, Peihong; Wang, Weiyi; Yang, Ning; Serna-Tamayo, Cristian; Ricca, Jacob M; Zamarin, Dmitriy; Shuman, Stewart; Merghoub, Taha; Wolchok, Jedd D; Deng, Liang

2017-05-19

Advanced cancers remain a therapeutic challenge despite recent progress in targeted therapy and immunotherapy. Novel approaches are needed to alter the tumor immunosuppressive microenvironment and to facilitate the recognition of tumor antigens that leads to antitumor immunity. Poxviruses, such as modified vaccinia virus Ankara (MVA), have potential as immunotherapeutic agents. We show that infection of conventional dendritic cells (DCs) with heat- or ultraviolet-inactivated MVA leads to higher levels of interferon induction than MVA alone through the cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase)-STING cytosolic DNA-sensing pathway. Intratumoral injection of inactivated MVA (iMVA) was effective and generated adaptive antitumor immunity in murine melanoma and colon cancer models. iMVA-induced antitumor therapy was less effective in STING- or Batf3-deficient mice than in wild-type mice, indicating that both cytosolic DNA sensing and Batf3-dependent CD103 + /CD8α + DCs are essential for iMVA immunotherapy. The combination of intratumoral delivery of iMVA and systemic delivery of immune checkpoint blockade generated synergistic antitumor effects in bilateral tumor implantation models as well as in a unilateral large established tumor model. Our results suggest that inactivated vaccinia virus could be used as an immunotherapeutic agent for human cancers. Copyright © 2017, American Association for the Advancement of Science.

Establishing elements of a synthetic biology platform for Vaccinia virus production: BioBrick™ design, serum-free virus production and microcarrier-based cultivation of CV-1 cells.

PubMed

Liu, Shuchang; Ruban, Ludmila; Wang, Yaohe; Zhou, Yuhong; Nesbeth, Darren N

2017-02-01

Vaccinia virus (VACV) is an established vector for vaccination and is beginning to prove effective as an oncolytic agent. Industrial production of VACV stands to benefit in future from advances made by synthetic biology in genome engineering and standardisation. The CV-1 cell line can be used for VACV propagation and has been used extensively with the CRISPR/Cas9 system for making precise edits of the VACV genome. Here we take first steps toward establishing a scalable synthetic biology platform for VACV production with CV-1 cells featuring standardised biological tools and serum free cell cultivation. We propose a new BioBrick™ plasmid backbone format for inserting transgenes into VACV. We then test the performance of CV-1 cells in propagation of a conventional recombinant Lister strain VACV, VACVL-15 RFP, in a serum-free process. CV-1 cells grown in 5% foetal bovine serum (FBS) Dulbecco's Modified Eagle Medium (DMEM) were adapted to growth in OptiPRO and VP-SFM brands of serum-free media. Specific growth rates of 0.047 h -1 and 0.044 h -1 were observed for cells adapted to OptiPRO and VP-SFM respectively, compared to 0.035 h -1 in 5% FBS DMEM. Cells adapted to OptiPRO and to 5% FBS DMEM achieved recovery ratios of over 96%, an indication of their robustness to cryopreservation. Cells adapted to VP-SFM showed a recovery ratio of 82%. Virus productivity in static culture, measured as plaque forming units (PFU) per propagator cell, was 75 PFU/cell for cells in 5% FBS DMEM. VP-SFM and OptiPRO adaptation increased VACV production to 150 PFU/cell and 350 PFU/cell respectively. Boosted PFU/cell from OptiPRO-adapted cells persisted when 5% FBS DMEM or OptiPRO medium was observed during the infection step and when titre was measured using cells adapted to 5% FBS DMEM or OptiPRO medium. Finally, OptiPRO-adapted CV-1 cells were successfully cultivated using Cytodex-1 microcarriers to inform future scale up studies.

Expression of Glycoproteins in Wild-Type and Vaccine Strains of Varicella Zoster Virus

DTIC Science & Technology

1990-06-18

gpV vaccinia recombinants 142 XI LIST OF FIGURES ^ig^rfi Page 1 . Structural model of the herpesvirus virion 8 2. A diagram of the VZV and HSV ...gpIV-specific antiserum 139 36. Fc receptor activity in HSV - 1 and VZV glycoprotein recombinant vaccinia viruses 145 37. The gene 14 transcription...subfamily alphaherpesvirinae. The human alphaherpesviruses are comprised of VZV and herpes simplex viruses 1 and 2 ( HSV - 1 and HSV -2). Four other

Smallpox virus plaque phenotypes: genetic, geographical and case fatality relationships.

PubMed

Olson, Victoria A; Karem, Kevin L; Smith, Scott K; Hughes, Christine M; Damon, Inger K

2009-04-01

Smallpox (infection with Orthopoxvirus variola) remains a feared illness more than 25 years after its eradication. Historically, case-fatality rates (CFRs) varied between outbreaks (<1 to approximately 40 %), the reasons for which are incompletely understood. The extracellular enveloped virus (EEV) form of orthopoxvirus progeny is hypothesized to disseminate infection. Investigations with the closely related Orthopoxvirus vaccinia have associated increased comet formation (EEV production) with increased mouse mortality (pathogenicity). Other vaccinia virus genetic manipulations which affect EEV production inconsistently support this association. However, antisera against vaccinia virus envelope protect mice from lethal challenge, further supporting a critical role for EEV in pathogenicity. Here, we show that the increased comet formation phenotypes of a diverse collection of variola viruses associate with strain phylogeny and geographical origin, but not with increased outbreak-related CFRs; within clades, there may be an association of plaque size with CFR. The mechanisms for variola virus pathogenicity probably involves multiple host and pathogen factors.

Major Neutralizing Sites on Vaccinia Virus Glycoprotein B5 Are Exposed Differently on Variola Virus Ortholog B6▿

PubMed Central

Aldaz-Carroll, Lydia; Xiao, Yuhong; Whitbeck, J. Charles; de Leon, Manuel Ponce; Lou, Huan; Kim, Mikyung; Yu, Jessica; Reinherz, Ellis L.; Isaacs, Stuart N.; Eisenberg, Roselyn J.; Cohen, Gary H.

2007-01-01

Immunization against smallpox (variola virus) with Dryvax, a live vaccinia virus (VV), was effective, but now safety is a major concern. To overcome this issue, subunit vaccines composed of VV envelope proteins from both forms of infectious virions, including the extracellular enveloped virion (EV) protein B5, are being developed. However, since B5 has 23 amino acid differences compared with its B6 variola virus homologue, B6 might be a better choice for such a strategy. Therefore, we compared the properties of both proteins using a panel of monoclonal antibodies (MAbs) to B5 that we had previously characterized and grouped according to structural and functional properties. The B6 gene was obtained from the Centers for Disease Control and Prevention, and the ectodomain was cloned and expressed in baculovirus as previously done with B5, allowing us to compare the antigenic properties of the proteins. Polyclonal antibodies to B5 or B6 cross-reacted with the heterologous protein, and 16 of 26 anti-B5 MAbs cross-reacted with B6. Importantly, 10 anti-B5 MAbs did not cross-react with B6. Of these, three have important anti-VV biologic properties, including their ability to neutralize EV infectivity and block comet formation. Here, we found that one of these three MAbs protected mice from a lethal VV challenge by passive immunization. Thus, epitopes that are present on B5 but not on B6 would generate an antibody response that would not recognize B6. Assuming that B6 contains similar variola virus-specific epitopes, our data suggest that a subunit vaccine using the variola virus homologues might exhibit improved protective efficacy against smallpox. PMID:17522205

Comparable polyfunctionality of ectromelia virus- and vaccinia virus-specific murine T cells despite markedly different in vivo replication and pathogenicity.

PubMed

Hersperger, Adam R; Siciliano, Nicholas A; Eisenlohr, Laurence C

2012-07-01

Vaccinia virus (VACV) stimulates long-term immunity against highly pathogenic orthopoxvirus infection of humans (smallpox) and mice (mousepox [ectromelia virus {ECTV}]) despite the lack of a natural host-pathogen relationship with either of these species. Previous research revealed that VACV is able to induce polyfunctional CD8(+) T-cell responses after immunization of humans. However, the degree to which the functional profile of T cells induced by VACV is similar to that generated during natural poxvirus infection remains unknown. In this study, we monitored virus-specific T-cell responses following the dermal infection of C57BL/6 mice with ECTV or VACV. Using polychromatic flow cytometry, we measured levels of degranulation, cytokine expression (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin-2 [IL-2]), and the cytolytic mediator granzyme B. We observed that the functional capacities of T cells induced by VACV and ECTV were of a similar quality in spite of the markedly different replication abilities and pathogenic outcomes of these viruses. In general, a significant fraction (≥50%) of all T-cell responses were positive for at least three functions both during acute infection and into the memory phase. In vivo killing assays revealed that CD8(+) T cells specific for both viruses were equally cytolytic (∼80% target cell lysis after 4 h), consistent with the similar levels of granzyme B and degranulation detected among these cells. Collectively, these data provide a mechanism to explain the ability of VACV to induce protective T-cell responses against pathogenic poxviruses in their natural hosts and provide further support for the use of VACV as a vaccine platform able to induce polyfunctional T cells.

Nucleotide Sequence of the Hantaan Virus S RNA Segment and Expression of Encoded Proteins

DTIC Science & Technology

1987-11-03

human vaccinia vaccination ). A second dose of virus was given in the same ...vaccinia vector. A necessary first step in vaccine investigation woul d be to determine if animals infected with the two HTV recombinant viruses can ...vaccinia virus (Buller et al., 1985). Mice were infected by tail scarification since it is identical to the method used to vaccinate 169 humans

Molecular Characterization of Prostate Cancer Cell Oncolysis by Herpes Simplex Virus ICP0 Mutants

DTIC Science & Technology

2005-10-01

action by the E3L double-stranded RNA 19 binding proteins of vaccinia virus. J Virol 76:5251-9. 20 54. Yokota, S., N. Yokosawa , T. Kubota, T. Suzutani, I...phosphorylation of STATs and janus kinases during an early 23 infection stage. Virology 286:119-124. 25 1 55. Yokota, S.-i., N. Yokosawa , T. Okabayashi, T

Immunogenicity and Protection Against Influenza H7N3 in Mice by Modified Vaccinia Virus Ankara Vectors Expressing Influenza Virus Hemagglutinin or Neuraminidase.

PubMed

Meseda, Clement A; Atukorale, Vajini; Soto, Jackeline; Eichelberger, Maryna C; Gao, Jin; Wang, Wei; Weiss, Carol D; Weir, Jerry P

2018-03-29

Influenza subtypes such as H7 have pandemic potential since they are able to infect humans with severe consequences, as evidenced by the ongoing H7N9 infections in China that began in 2013. The diversity of H7 viruses calls for a broadly cross-protective vaccine for protection. We describe the construction of recombinant modified vaccinia virus Ankara (MVA) vectors expressing the hemagglutinin (HA) or neuraminidase (NA) from three H7 viruses representing both Eurasian and North American H7 lineages - A/mallard/Netherlands/12/2000 (H7N3), A/Canada/rv444/2004 (H7N3), and A/Shanghai/02/2013 (H7N9). These vectors were evaluated for immunogenicity and protective efficacy against H7N3 virus in a murine model of intranasal challenge. High levels of H7-, N3-, and N9-specific antibodies, including neutralizing antibodies, were induced by the MVA-HA and MVA-NA vectors. Mice vaccinated with MVA vectors expressing any of the H7 antigens were protected, suggesting cross-protection among H7 viruses. In addition, MVA vectors expressing N3 but not N9 elicited protection against H7N3 virus challenge. Similar outcomes were obtained when immune sera from MVA vector-immunized mice were passively transferred to naïve mice prior to challenge with the H7N3 virus. The results support the further development of an MVA vector platform as a candidate vaccine for influenza strains with pandemic potential.

Genomic Sequence and Virulence of Clonal Isolates of Vaccinia Virus Tiantan, the Chinese Smallpox Vaccine Strain

PubMed Central

Zhang, Qicheng; Tian, Meijuan; Feng, Yi; Zhao, Kai; Xu, Jing; Liu, Ying; Shao, Yiming

2013-01-01

Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV) as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT) was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV) vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1) viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1) and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs). ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH) strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector. PMID:23593246

Genomic sequence and virulence of clonal isolates of vaccinia virus Tiantan, the Chinese smallpox vaccine strain.

PubMed

Zhang, Qicheng; Tian, Meijuan; Feng, Yi; Zhao, Kai; Xu, Jing; Liu, Ying; Shao, Yiming

2013-01-01

Despite the worldwide eradication of smallpox in 1979, the potential bioterrorism threat from variola virus and the ongoing use of vaccinia virus (VACV) as a vector for vaccine development argue for continued research on VACV. In China, the VACV Tiantan strain (TT) was used in the smallpox eradication campaign. Its progeny strain is currently being used to develop a human immunodeficiency virus (HIV) vaccine. Here we sequenced the full genomes of five TT clones isolated by plaque purification from the TT (752-1) viral stock. Phylogenetic analysis with other commonly used VACV strains showed that TT (752-1) and its clones clustered and exhibited higher sequence diversity than that found in Dryvax clones. The ∼190 kbp genomes of TT appeared to encode 273 open reading frames (ORFs). ORFs located in the middle of the genome were more conserved than those located at the two termini, where many virulence and immunomodulation associated genes reside. Several patterns of nucleotide changes including point mutations, insertions and deletions were identified. The polymorphisms in seven virulence-associated proteins and six immunomodulation-related proteins were analyzed. We also investigated the neuro- and skin- virulence of TT clones in mice and rabbits, respectively. The TT clones exhibited significantly less virulence than the New York City Board of Health (NYCBH) strain, as evidenced by less extensive weight loss and morbidity in mice as well as produced smaller skin lesions and lower incidence of putrescence in rabbits. The complete genome sequences, ORF annotations, and phenotypic diversity yielded from this study aid our understanding of the Chinese historic TT strain and are useful for HIV vaccine projects employing TT as a vector.

Effects of Postchallenge Administration of ST-246 on Dissemination of IHD-J-Luc Vaccinia Virus in Normal Mice and in Immune-Deficient Mice Reconstituted with T Cells

PubMed Central

Shotwell, Elisabeth; Scott, John; Cruz, Stephanie; King, Lisa R.; Manischewitz, Jody; Diaz, Claudia G.; Jordan, Robert A.; Grosenbach, Douglas W.; Golding, Hana

2013-01-01

Whole-body bioimaging was used to study dissemination of vaccinia virus (VACV) in normal and in immune deficient (nu−/nu−) mice protected from lethality by postchallenge administration of ST-246. Total fluxes were recorded in the liver, spleen, lungs, and nasal cavities of live mice after intranasal infection with a recombinant IHD-J-Luc VACV expressing luciferase. Areas under the flux curve were calculated for individual mice to assess viral loads. Treatment for 2 to 5 days of normal BALB/c mice with ST-246 at 100 mg/kg starting 24 h postchallenge conferred 100% protection and reduced viral loads in four organs compared to control mice. Mice also survived after 5 days of treatment with ST-246 at 30 mg/kg, and yet the viral loads and poxes were higher in these mice compared to 100-mg/kg treatment group. Nude mice were not protected by ST-246 alone or by 10 million adoptively transferred T cells. In contrast, nude mice that received T cells and 7-day treatment with ST-246 survived infection and exhibited reduced viral loads compared to nonreconstituted and ST-246-treated mice after ST-246 was stopped. Similar protection of nude mice was achieved using adoptively transferred 1.0 and 0.1 million, but not 0.01 million, purified T cells or CD4+ or CD8+ T cells in conjunction with ST-246 treatment. These data suggest that ST-246 protects immunocompetent mice from lethality and reduces viral dissemination in internal organs and poxvirus lesions. Furthermore, immune-deficient animals with partial T cell reconstitution can control virus replication after a course of ST-246 and survive lethal vaccinia virus challenge. PMID:23468500

Specificity and 6-Month Durability of Immune Responses Induced by DNA and Recombinant Modified Vaccinia Ankara Vaccines Expressing HIV-1 Virus-Like Particles

PubMed Central

Goepfert, Paul A.; Elizaga, Marnie L.; Seaton, Kelly; Tomaras, Georgia D.; Montefiori, David C.; Sato, Alicia; Hural, John; DeRosa, Stephen C.; Kalams, Spyros A.; McElrath, M. Juliana; Keefer, Michael C.; Baden, Lindsey R.; Lama, Javier R.; Sanchez, Jorge; Mulligan, Mark J.; Buchbinder, Susan P.; Hammer, Scott M.; Koblin, Beryl A.; Pensiero, Michael; Butler, Chris; Moss, Bernard; Robinson, Harriet L.; Donastorg, Yeycy; Qin, Li; Lawrence, Dale; Cardinali, Massimo; Bae, Jin; Holt, Renée; Redinger, Huguette; Johannessen, Jan; Broder, Gail; Moody-White, Jerri; McKay, Butch; Calazans, Gabriela; Bentley, Carter; Kakinami, Lisa; Skibinski, Katie; Estep, Scharla; Tseng, Jenny; Swenson, Molly; Madenwald, Tamra; Overton, Edgar Turner; Edupuganti, Srilatha; Rouphael, Nadine; Whitaker, Jennifer; Hay, C Mhorag; Bunce, Catherine A; Gonzales, Pedro; Hurtado, Juan Carlos; Dolin, Raphael; Mayer, Ken; Walsh, Steven; Johnson, Jennifer

2014-01-01

Background. Clade B DNA and recombinant modified vaccinia Ankara (MVA) vaccines producing virus-like particles displaying trimeric membrane-bound envelope glycoprotein (Env) were tested in a phase 2a trial in human immunodeficiency virus (HIV)–uninfected adults for safety, immunogenicity, and 6-month durability of immune responses. Methods. A total of 299 individuals received 2 doses of JS7 DNA vaccine and 2 doses of MVA/HIV62B at 0, 2, 4, and 6 months, respectively (the DDMM regimen); 3 doses of MVA/HIV62B at 0, 2, and 6 months (the MMM regimen); or placebo injections. Results. At peak response, 93.2% of the DDMM group and 98.4% of the MMM group had binding antibodies for Env. These binding antibodies were more frequent and of higher magnitude for the transmembrane subunit (gp41) than the receptor-binding subunit (gp120) of Env. For both regimens, response rates were higher for CD4+ T cells (66.4% in the DDMM group and 43.1% in the MMM group) than for CD8+ T cells (21.8% in the DDMM group and 14.9% in the MMM group). Responding CD4+ and CD8+ T cells were biased toward Gag, and >70% produced 2 or 3 of the 4 cytokines evaluated (ie, interferon γ, interleukin 2, tumor necrosis factor α, and granzyme B). Six months after vaccination, the magnitudes of antibodies and T-cell responses had decreased by <3-fold. Conclusions. DDMM and MMM vaccinations with virus-like particle–expressing immunogens elicited durable antibody and T-cell responses. PMID:24403557

Bovine Vaccinia: Insights into the Disease in Cattle

PubMed Central

Rehfeld, Izabelle Silva; Lobato, Zélia Inês Portela

2018-01-01

Bovine vaccinia (BV), caused by Vaccinia virus (VACV), is a zoonosis characterized by exanthematous lesions in the teats of dairy cows and the hands of milkers and is an important public health issue. Severe VACV-induced lesions in the teats and udder of cows and buffaloes could lead to mastitis and other secondary infections, thereby reducing productivity and resulting in economic losses to the dairy industry. In Brazil, BV re-emerged in the late 1990s and is now endemic in most of the Brazilian territory. In the last 15 years, much effort has been made to know more about this disease and its epidemiology, etiologic agents, and interactions with the host and the environment. In this review, we describe the known dynamics of VACV infection in cattle and the viral shedding routes, as well as the relevance of BV for animal and public health. PMID:29522489

Protective effects of a Modified Vaccinia Ankara-based vaccine candidate against Crimean-Congo Haemorrhagic Fever virus require both cellular and humoral responses.

PubMed

Dowall, Stuart D; Graham, Victoria A; Rayner, Emma; Hunter, Laura; Watson, Robert; Taylor, Irene; Rule, Antony; Carroll, Miles W; Hewson, Roger

2016-01-01

Crimean-Congo Haemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. There is no approved vaccine currently available against CCHF. The most promising candidate, which has previously been shown to confer protection in the small animal model, is a modified Vaccinia Ankara virus vector expressing the CCHF viral glycoprotein (MVA-GP). It has been shown that MVA-GP induces both humoral and cellular immunogenicity. In the present study, sera and T-lymphocytes were passively and adoptively transferred into recipient mice prior to challenge with CCHF virus. Results demonstrated that mediators from both arms of the immune system were required to demonstrate protective effects against lethal challenge.

Distinct Gene Expression Profiles in Peripheral Blood Mononuclear Cells from Patients Infected with Vaccinia Virus, Yellow Fever 17D Virus, or Upper Respiratory Infections Running Title: PBMC Expression Response to Viral Agents

PubMed Central

Scherer, Christina A.; Magness, Charles L.; Steiger, Kathryn V.; Poitinger, Nicholas D.; Caputo, Christine M.; Miner, Douglas G.; Winokur, Patricia L.; Klinzman, Donna; McKee, Janice; Pilar, Christine; Ward, Patricia A.; Gillham, Martha H.; Haulman, N. Jean; Stapleton, Jack T.; Iadonato, Shawn P.

2007-01-01

Gene expression in human peripheral blood mononuclear cells was systematically evaluated following smallpox and yellow fever vaccination, and naturally occurring upper respiratory infection (URI). All three infections were characterized by the induction of many interferon stimulated genes, as well as enhanced expression of genes involved in proteolysis and antigen presentation. Vaccinia infection was also characterized by a distinct expression signature composed of up-regulation of monocyte response genes, with repression of genes expressed by B and T-cells. In contrast, the yellow fever host response was characterized by a suppression of ribosomal and translation factors, distinguishing this infection from vaccinia and URI. No significant URI-specific signature was observed, perhaps reflecting greater heterogeneity in the study population and etiological agents. Taken together, these data suggest that specific host gene expression signatures may be identified that distinguish one or a small number of virus agents. PMID:17651872

Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins.

PubMed

Hyun, Seong-In; Weisberg, Andrea; Moss, Bernard

2017-08-01

The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes. IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights into

Deletion of the Vaccinia Virus I2 Protein Interrupts Virion Morphogenesis, Leading to Retention of the Scaffold Protein and Mislocalization of Membrane-Associated Entry Proteins

PubMed Central

Hyun, Seong-In; Weisberg, Andrea

2017-01-01

ABSTRACT The I2L open reading frame of vaccinia virus (VACV) encodes a conserved 72-amino-acid protein with a putative C-terminal transmembrane domain. Previous studies with a tetracycline-inducible mutant demonstrated that I2-deficient virions are defective in cell entry. The purpose of the present study was to determine the step of replication or entry that is affected by loss of the I2 protein. Fluorescence microscopy experiments showed that I2 colocalized with a major membrane protein of immature and mature virions. We generated a cell line that constitutively expressed I2 and allowed construction of the VACV I2L deletion mutant vΔI2. As anticipated, vΔI2 was unable to replicate in cells that did not express I2. Unexpectedly, morphogenesis was interrupted at a stage after immature virion formation, resulting in the accumulation of dense spherical particles instead of brick-shaped mature virions with well-defined core structures. The abnormal particles retained the D13 scaffold protein of immature virions, were severely deficient in the transmembrane proteins that comprise the entry fusion complex (EFC), and had increased amounts of unprocessed membrane and core proteins. Total lysates of cells infected with vΔI2 also had diminished EFC proteins due to instability attributed to their hydrophobicity and failure to be inserted into viral membranes. A similar instability of EFC proteins had previously been found with unrelated mutants blocked earlier in morphogenesis that also accumulated viral membranes retaining the D13 scaffold. We concluded that I2 is required for virion morphogenesis, release of the D13 scaffold, and the association of EFC proteins with viral membranes. IMPORTANCE Poxviruses comprise a large family that infect vertebrates and invertebrates, cause disease in both in humans and in wild and domesticated animals, and are being engineered as vectors for vaccines and cancer therapy. In addition, investigations of poxviruses have provided insights

Induction of Both Local Immune Response in Mice and Protection in a Rabbit Model by Intranasal Immunization with Modified Vaccinia Ankara Virus Expressing a Secreted Form of Bovine Herpesvirus 1 Glycoprotein D.

PubMed

Del Medico Zajac, María Paula; Zanetti, Flavia Adriana; Esusy, María Soledad; Federico, Carlos Rodolfo; Zabal, Osvaldo; Valera, Alejandro Rafael; Calamante, Gabriela

In this study, we evaluated the immunogenicity and efficacy of mucosal delivery of a recombinant modified vaccinia Ankara virus (MVA) expressing the secreted version of bovine herpesvirus type 1 (BoHV-1) glycoprotein D (MVA-gDs) without addition of adjuvant in two animal models. First, we demonstrated the capability of MVA-gDs of inducing both local and systemic anti-gD humoral immune response after intranasal immunization of mice. Then, we confirmed that two doses of MVA-gDs administered intranasally to rabbits induced systemic anti-gD antibodies and conferred protection against BoHV-1 challenge. Our results show the potential of using MVA as a vector for the rational design of veterinary vaccines capable of inducing specific and protective immune responses both at local and systemic level.

Myristoylation increases the CD8+T-cell response to a GFP prototype antigen delivered by modified vaccinia virus Ankara.

PubMed

Marr, Lisa; Lülf, Anna-Theresa; Freudenstein, Astrid; Sutter, Gerd; Volz, Asisa

2016-04-01

Activation of CD8(+)T-cells is an essential part of immune responses elicited by recombinant modified vaccinia virus Ankara (MVA). Strategies to enhance T-cell responses to antigens may be particularly necessary for broadly protective immunization against influenza A virus infections or for candidate vaccines targeting chronic infections and cancer. Here, we tested recombinant MVAs that targeted a model antigen, GFP, to different localizations in infected cells. In vitro characterization demonstrated that GFP accumulated in the nucleus (MVA-nls-GFP), associated with cellular membranes (MVA-myr-GFP) or was equally distributed throughout the cell (MVA-GFP). On vaccination, we found significantly higher levels of GFP-specific CD8(+)T-cells in MVA-myr-GFP-vaccinated BALB/c mice than in those immunized with MVA-GFP or MVA-nls-GFP. Thus, myristoyl modification may be a useful strategy to enhance CD8(+)T-cell responses to MVA-delivered target antigens.

[Behavior of Orf virus in permissive and nonpermissive systems].

PubMed

Büttner, M; Czerny, C P; Schumm, M

1995-04-01

Dogs were immunized i.m. with attenuated poxvirus vaccines (vaccinia virus, Orf-virus) and a bovine herpesvirus-1 (BHV-1) vaccine. After intradermal (i.d.) application of the vaccine viruses a specific delayed type hypersensitivity (DTH) reaction of the skin occurred only with vaccinia virus. The i.d. application of Orf-virus caused a short-term, non-specific inflammatory reaction of the skin, even in dogs not immunized with Orf-virus. Out of 30 sera from Orf-virus immunized beagles (n = 4) only eight were found reactive to Orf-virus in a competition ELISA. Three sera from dogs not Orf-virus immunized but skin-tested with the virus contained low antibody titers. Using indirect immunofluorescence (IIF) in flow cytometry, the existence of Orf-virus antigens was examined on the surface and in the cytoplasm of permissive (BFK and Vero)- and questionable permissive MDCK cells. The canine kidney MDCK cell line was found to be non-permissive for Orf-virus replication; the occurrence of an Orf-(ecthyma contagiosum) like disease in dogs is unlikely.

Three-Year Durability of Immune Responses Induced by HIV-DNA and HIV-Modified Vaccinia Virus Ankara and Effect of a Late HIV-Modified Vaccinia Virus Ankara Boost in Tanzanian Volunteers.

PubMed

Joachim, Agricola; Munseri, Patricia J; Nilsson, Charlotta; Bakari, Muhammad; Aboud, Said; Lyamuya, Eligius F; Tecleab, Teghesti; Liakina, Valentina; Scarlatti, Gabriella; Robb, Merlin L; Earl, Patricia L; Moss, Bernard; Wahren, Britta; Mhalu, Fred; Ferrari, Guido; Sandstrom, Eric; Biberfeld, Gunnel

2017-08-01

We explored the duration of immune responses and the effect of a late third HIV-modified vaccinia virus Ankara (MVA) boost in HIV-DNA primed and HIV-MVA boosted Tanzanian volunteers. Twenty volunteers who had previously received three HIV-DNA and two HIV-MVA immunizations were given a third HIV-MVA immunization 3 years after the second HIV-MVA boost. At the time of the third HIV-MVA, 90% of the vaccinees had antibodies to HIV-1 subtype C gp140 (median titer 200) and 85% to subtype B gp160 (median titer 100). The majority of vaccinees had detectable antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies, 70% against CRF01_AE virus-infected cells (median titer 239) and 84% against CRF01_AE gp120-coated cells (median titer 499). A high proportion (74%) of vaccinees had IFN-γ ELISpot responses, 63% to Gag and 42% to Env, 3 years after the second HIV-MVA boost. After the third HIV-MVA, there was an increase in Env-binding antibodies and ADCC-mediating antibodies relative to the response seen at the time of the third HIV-MVA vaccination, p < .0001 and p < .05, respectively. The frequency of IFN-γ ELISpot responses increased to 95% against Gag or Env and 90% to both Gag and Env, p = .064 and p = .002, respectively. In conclusion, the HIV-DNA prime/HIV-MVA boost regimen elicited potent antibody and cellular immune responses with remarkable durability, and a third HIV-MVA immunization significantly boosted both antibody and cellular immune responses relative to the levels detected at the time of the third HIV-MVA, but not to higher levels than after the second HIV-MVA.

Protective effects of a Modified Vaccinia Ankara-based vaccine candidate against Crimean-Congo Haemorrhagic Fever virus require both cellular and humoral responses

PubMed Central

Dowall, Stuart D.; Graham, Victoria A.; Rayner, Emma; Hunter, Laura; Watson, Robert; Taylor, Irene; Rule, Antony; Carroll, Miles W.; Hewson, Roger

2016-01-01

Crimean-Congo Haemorrhagic Fever (CCHF) is a severe tick-borne disease, endemic in many countries in Africa, the Middle East, Eastern Europe and Asia. There is no approved vaccine currently available against CCHF. The most promising candidate, which has previously been shown to confer protection in the small animal model, is a modified Vaccinia Ankara virus vector expressing the CCHF viral glycoprotein (MVA-GP). It has been shown that MVA-GP induces both humoral and cellular immunogenicity. In the present study, sera and T-lymphocytes were passively and adoptively transferred into recipient mice prior to challenge with CCHF virus. Results demonstrated that mediators from both arms of the immune system were required to demonstrate protective effects against lethal challenge. PMID:27272940

Vaccinia virus recombinants encoding the truncated structural gene region of Venezuelan equine encephalitis virus (VEEV) give solid protection against peripheral challenge but only partial protection against airborne challenge with virulent VEEV.

PubMed

Phillpotts, R J; Lescott, T L; Jacobs, S C

2000-10-01

Vaccinia virus (VV) recombinants that contain the genes encoding the Venezuelan equine encephalitis virus (VEEV) structural gene region (C-E3-E2-6 K-E1) solidly protect mice against peripheral challenge with virulent VEEV, but provide only partial protection against airborne challenge. To improve upon these results we focussed on the principal antigens involved in protection. VV recombinants encoding the structural genes E3-E2-6 K-E1, E3-E2-6 K or 6 K-E1 were prepared and evaluated for their ability to protect Balb/c mice after a single dorsal scarification with 10(8) PFU against peripheral or airborne challenge with virulent VEEV. The antibody response was also examined. Our experiments provide new evidence that truncates of the VEEV structural region (E3-E2-6 K-E1, E3-E2-6 K), cloned and expressed in VV, protect against challenge with virulent virus. They also confirm the important role of E2 in protection. However, we were unable to improve upon previously reported levels of protection against airborne challenge. A substantial level of circulating antibodies and the presence of local IgA (not always induced by mucosal immunization) (Greenway et al., 1992) appear essential for protection against the airborne virus. Current VV-VEEV recombinants seem unable to elicit this level of immune response and further improvements are therefore required to increase the immunogenicity of VV-VEEV vaccines.

Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins.

PubMed

Beissert, Tim; Koste, Lars; Perkovic, Mario; Walzer, Kerstin C; Erbar, Stephanie; Selmi, Abderraouf; Diken, Mustafa; Kreiter, Sebastian; Türeci, Özlem; Sahin, Ugur

2017-12-01

Among nucleic acid-based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdown. To reduce pattern recognition receptor stimulation and unleash suppressed saRNA translation, this study co-delivered non-replicating mRNA encoding vaccinia virus immune evasion proteins E3, K3, and B18. It was shown that E3 is far superior to K3 or B18 as a highly potent blocker of PKR activation and of interferon (IFN)-β upregulation. B18, in contrast, is superior in controlling OAS1, a key IFN-inducible gene involved in viral RNA degradation. By combining all three vaccinia proteins, the study achieved significant suppression of PKR and IFN pathway activation in vitro and enhanced expression of saRNA-encoded genes of interest both in vitro and in vivo. This approach promises to overcome key hurdles of saRNA gene delivery. Its application may improve the bioavailability of the encoded protein, and reduce the effective dose and correspondingly the cost of goods of manufacture in the various fields where saRNA utilization is envisioned.

Improvement of In Vivo Expression of Genes Delivered by Self-Amplifying RNA Using Vaccinia Virus Immune Evasion Proteins

PubMed Central

Beissert, Tim; Koste, Lars; Perkovic, Mario; Walzer, Kerstin C.; Erbar, Stephanie; Selmi, Abderraouf; Diken, Mustafa; Kreiter, Sebastian; Türeci, Özlem; Sahin, Ugur

2017-01-01

Among nucleic acid–based delivery platforms, self-amplifying RNA (saRNA) vectors are of increasing interest for applications such as transient expression of recombinant proteins and vaccination. saRNA is safe and, due to its capability to amplify intracellularly, high protein levels can be produced from even minute amounts of transfected templates. However, it is an obstacle to full exploitation of this platform that saRNA induces a strong innate host immune response. In transfected cells, pattern recognition receptors sense double-stranded RNA intermediates and via activation of protein kinase R (PKR) and interferon signaling initiate host defense measures including a translational shutdown. To reduce pattern recognition receptor stimulation and unleash suppressed saRNA translation, this study co-delivered non-replicating mRNA encoding vaccinia virus immune evasion proteins E3, K3, and B18. It was shown that E3 is far superior to K3 or B18 as a highly potent blocker of PKR activation and of interferon (IFN)-β upregulation. B18, in contrast, is superior in controlling OAS1, a key IFN-inducible gene involved in viral RNA degradation. By combining all three vaccinia proteins, the study achieved significant suppression of PKR and IFN pathway activation in vitro and enhanced expression of saRNA-encoded genes of interest both in vitro and in vivo. This approach promises to overcome key hurdles of saRNA gene delivery. Its application may improve the bioavailability of the encoded protein, and reduce the effective dose and correspondingly the cost of goods of manufacture in the various fields where saRNA utilization is envisioned. PMID:28877647

Weak vaccinia virus-induced NK cell regulation of CD4 T cells is associated with reduced NK cell differentiation and cytolytic activity.

PubMed

Hatfield, Steven D; Daniels, Keith A; O'Donnell, Carey L; Waggoner, Stephen N; Welsh, Raymond M

2018-06-01

Natural killer (NK) cells control antiviral adaptive immune responses in mice during some virus infections, but the universality of this phenomenon remains unknown. Lymphocytic choriomeningitis virus (LCMV) infection of mice triggered potent cytotoxic activity of NK cells (NK LCMV ) against activated CD4 T cells, tumor cells, and allogeneic lymphocytes. In contrast, NK cells activated by vaccinia virus (VACV) infection (NK VACV ) exhibited weaker cytolytic activity against each of these target cells. Relative to NK LCMV cells, NK VACV cells exhibited a more immature (CD11b - CD27 + ) phenotype, and lower expression levels of the activation marker CD69, cytotoxic effector molecules (perforin, granzyme B), and the transcription factor IRF4. NK VACV cells expressed higher levels of the inhibitory molecule NKG2A than NK LCMV cells. Consistent with this apparent lethargy, NK VACV cells only weakly constrained VACV-specific CD4 T-cell responses. This suggests that NK cell regulation of adaptive immunity, while universal, may be limited with viruses that poorly activate NK cells. Published by Elsevier Inc.

Gene Therapy Using Therapeutic and Diagnostic Recombinant Oncolytic Vaccinia Virus GLV-1h153 for Management of Colorectal Peritoneal Carcinomatosis

PubMed Central

Eveno, Clarisse; Mojica, Kelly; Ady, Justin W.; Thorek, Daniel L.J.; Longo, Valerie; Belin, Laurence J.; Gholami, Sepideh; Johnsen, Clark; Zanzonico, Pat; Chen, Nanhai; Yu, Tony; Szalay, Aladar A.; Fong, Yuman

2015-01-01

Background Peritoneal carcinomatosis (PC) is a terminal progression of colorectal cancer (CRC). Poor response to cytoreductive surgery and chemotherapy, coupled with the inability to reliably track disease progression using established diagnostic methods make this a deadly disease. This paper examines the effectiveness of the oncolytic vaccinia virus GLV-1h153 as a therapeutic and diagnostic vehicle. We believe that viral expression of the human sodium iodide transporter (hNIS) can provide both real-time monitoring of viral therapy and effective treatment of colorectal peritoneal carcinomatosis (CRPC). Methods Infectivity and cytotoxic effect of GLV-1h153 on CRC cell lines was assayed in-vitro. Viral replication was examined by standard viral plaque assays. Orthotopic CRPC xenografts were generated in athymic nude mice, and subsequently administered GLV-1h153 intraperitoneally. Reduction of tumor burden was assessed by mass. Orthotopic tumors were visualized by SPECT/CT after Iodine (131I) administration and by fluorescence optical imaging. Results GLV-1h153 infected and killed CRC cells in a time and concentration dependent manner. Viral replication demonstrated greater than a 2.35 log increase in titer over 4 days. Intraperitoneal treatment of orthotopic CRPC xenografts resulted in a significant reduction of tumor burden. Infection of orthotopic xenografts was both therapeutic and facilitated monitoring by 131I-SPECT/CT via expression of hNIS in infected tissue. Conclusions GLV-1h153 effectively kills CRC in-vitro and dramatically reduces tumor burden in-vivo. We demonstrate that GLV-1h153 can be used as an agent to provide accurate delineation of tumor burden in-vivo. These findings indicate that GLV-1h153 has significant potential for use as theragnostic agent in the treatment of CRPC. PMID:25616946

Vaccination of horses with a recombinant modified vaccinia Ankara virus (MVA) expressing African horse sickness (AHS) virus major capsid protein VP2 provides complete clinical protection against challenge.

PubMed

Alberca, Berta; Bachanek-Bankowska, Katarzyna; Cabana, Marta; Calvo-Pinilla, Eva; Viaplana, Elisenda; Frost, Lorraine; Gubbins, Simon; Urniza, Alicia; Mertens, Peter; Castillo-Olivares, Javier

2014-06-17

African horse sickness virus (AHSV) is an arthropod-borne pathogen that infects all species of equidae and causes high mortality in horses. Previously, a recombinant modified vaccinia Ankara (MVA) virus expressing the protein VP2 of AHSV serotype 4 was shown to induce virus neutralising antibodies in horses and protected interferon alpha receptor gene knock-out mice (IFNAR -/-) against virulent AHSV challenge. This study builds on the previous work, examining the protective efficacy of MVA-VP2 vaccination in the natural host of AHSV infection. A study group of 4 horses was vaccinated twice with a recombinant MVA virus expressing the major capsid protein (VP2) of AHSV serotype 9. Vaccinated animals and a control group of unvaccinated horses were then challenged with a virulent strain of AHSV-9. The vaccinated animals were completely protected against clinical disease and also against viraemia as measured by standard end-point dilution assays. In contrast, all control horses presented viraemia after challenge and succumbed to the infection. These results demonstrate the potential of recombinant MVA viruses expressing the outer capsid VP2 of AHSV as a protective vaccine against AHSV infection in the field. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

Genome of Horsepox Virus

PubMed Central

Tulman, E. R.; Delhon, G.; Afonso, C. L.; Lu, Z.; Zsak, L.; Sandybaev, N. T.; Kerembekova, U. Z.; Zaitsev, V. L.; Kutish, G. F.; Rock, D. L.

2006-01-01

Here we present the genomic sequence of horsepox virus (HSPV) isolate MNR-76, an orthopoxvirus (OPV) isolated in 1976 from diseased Mongolian horses. The 212-kbp genome contained 7.5-kbp inverted terminal repeats and lacked extensive terminal tandem repetition. HSPV contained 236 open reading frames (ORFs) with similarity to those in other OPVs, with those in the central 100-kbp region most conserved relative to other OPVs. Phylogenetic analysis of the conserved region indicated that HSPV is closely related to sequenced isolates of vaccinia virus (VACV) and rabbitpox virus, clearly grouping together these VACV-like viruses. Fifty-four HSPV ORFs likely represented fragments of 25 orthologous OPV genes, including in the central region the only known fragmented form of an OPV ribonucleotide reductase large subunit gene. In terminal genomic regions, HSPV lacked full-length homologues of genes variably fragmented in other VACV-like viruses but was unique in fragmentation of the homologue of VACV strain Copenhagen B6R, a gene intact in other known VACV-like viruses. Notably, HSPV contained in terminal genomic regions 17 kbp of OPV-like sequence absent in known VACV-like viruses, including fragments of genes intact in other OPVs and approximately 1.4 kb of sequence present only in cowpox virus (CPXV). HSPV also contained seven full-length genes fragmented or missing in other VACV-like viruses, including intact homologues of the CPXV strain GRI-90 D2L/I4R CrmB and D13L CD30-like tumor necrosis factor receptors, D3L/I3R and C1L ankyrin repeat proteins, B19R kelch-like protein, D7L BTB/POZ domain protein, and B22R variola virus B22R-like protein. These results indicated that HSPV contains unique genomic features likely contributing to a unique virulence/host range phenotype. They also indicated that while closely related to known VACV-like viruses, HSPV contains additional, potentially ancestral sequences absent in other VACV-like viruses. PMID:16940536

Safety mechanism assisted by the repressor of tetracycline (SMART) vaccinia virus vectors for vaccines and therapeutics.

PubMed

Grigg, Patricia; Titong, Allison; Jones, Leslie A; Yilma, Tilahun D; Verardi, Paulo H

2013-09-17

Replication-competent viruses, such as Vaccinia virus (VACV), are powerful tools for the development of oncolytic viral therapies and elicit superior immune responses when used as vaccine and immunotherapeutic vectors. However, severe complications from uncontrolled viral replication can occur, particularly in immunocompromised individuals or in those with other predisposing conditions. VACVs constitutively expressing interferon-γ (IFN-γ) replicate in cell culture indistinguishably from control viruses; however, they replicate in vivo to low or undetectable levels, and are rapidly cleared even in immunodeficient animals. In an effort to develop safe and highly effective replication-competent VACV vectors, we established a system to inducibly express IFN-γ. Our SMART (safety mechanism assisted by the repressor of tetracycline) vectors are designed to express the tetracycline repressor under a constitutive VACV promoter and IFN-γ under engineered tetracycline-inducible promoters. Immunodeficient SCID mice inoculated with VACVs not expressing IFN-γ demonstrated severe weight loss, whereas those given VACVs expressing IFN-γ under constitutive VACV promoters showed no signs of infection. Most importantly, mice inoculated with a VACV expressing the IFN-γ gene under an inducible promoter remained healthy in the presence of doxycycline, but exhibited severe weight loss in the absence of doxycycline. In this study, we developed a safety mechanism for VACV based on the conditional expression of IFN-γ under a tightly controlled tetracycline-inducible VACV promoter for use in vaccines and oncolytic cancer therapies.

Modified Vaccinia virus Ankara-based vaccines in the era of personalized immunotherapy of cancer.

PubMed

Bendjama, Kaïdre; Quemeneur, Eric

2017-09-02

While interest in immunotherapies is renewed by the successful introduction of immune checkpoint blocking agent in the clinic, advances in genome sequencing are opening new possibilities in the design of increasingly personalized vaccines. Personalization of medicine can now be realistically contemplated at the single patient level. Unlike the previous generation of cancer vaccines, neoantigen directed vaccines would target truly specific tumor antigens resulting from acquired tumor genome mutations. Immune response induced by this next generation vaccine would not be subject to self-tolerance and will likely result to enhanced efficacy. Nevertheless, this new technologies can hold to their promises only if sponsors manage to meet several scientific, technical, logistical and regulatory challenges. In particular manufacturers will have to design, manufacture, and deliver to the patient a new pharmaceutical grade in a matters of weeks. In this paper, we briefly review current technologies currently tried at the translation of personalized vaccines and explore the possibilities offered by the Modified Vaccinia virus Ankara in this next wave of cancer vaccines.

Laboratory-acquired vaccinia virus infection in a recently immunized person--Massachusetts, 2013.

PubMed

Hsu, Christopher H; Farland, Julien; Winters, Thomas; Gunn, Julia; Caron, Donna; Evans, Jennifer; Osadebe, Lynda; Bethune, Leon; McCollum, Andrea M; Patel, Nishi; Wilkins, Kimberly; Davidson, Whitni; Petersen, Brett; Barry, M Anita

2015-05-01

On November 26, 2013, the CDC poxvirus laboratory was notified by the Boston Public Health Commission (BPHC) of an inadvertent inoculation of a recently vaccinated (ACAM2000 smallpox vaccine) laboratory worker with wild type vaccinia virus (VACV) Western Reserve. A joint investigation by CDC and BPHC confirmed orthopoxvirus infection in the worker, who had reported a needle stick in his thumb while inoculating a mouse with VACV. He experienced a non-tender, red rash on his arm, diagnosed at a local emergency department as cellulitis. He subsequently developed a necrotic lesion on his thumb, diagnosed as VACV infection. Three weeks after the injury, the thumb lesion was surgically debrided and at 2 months post-injury, the skin lesion had resolved. The investigation confirmed that the infection was the first reported VACV infection in the United States in a laboratory worker vaccinated according to the Advisory Committee on Immunization Practices (ACIP) recommendations. The incident prompted the academic institution to outline biosafety measures for working with biologic agents, such as biosafety training of laboratory personnel, vaccination (if appropriate), and steps in incident reporting. Though vaccination has been shown to be an effective measure in protecting personnel in the laboratory setting, this case report underscores the importance of proper safety measures and incident reporting.

Effective Induction of Simian Immunodeficiency Virus-Specific Cytotoxic T Lymphocytes in Macaques by Using a Multiepitope Gene and DNA Prime-Modified Vaccinia Virus Ankara Boost Vaccination Regimen

PubMed Central

Hanke, Tomas; Samuel, Rachel V.; Blanchard, Tom J.; Neumann, Veronica C.; Allen, Todd M.; Boyson, Jon E.; Sharpe, Sally A.; Cook, Nicola; Smith, Geoffrey L.; Watkins, David I.; Cranage, Martin P.; McMichael, Andrew J.

1999-01-01

DNA and modified vaccinia virus Ankara (MVA) are vaccine vehicles suitable and safe for use in humans. Here, by using a multicytotoxic T-lymphocyte (CTL) epitope gene and a DNA prime-MVA boost vaccination regimen, high levels of CTLs specific for a single simian immunodeficiency virus (SIV) gag-derived epitope were elicited in rhesus macaques. These vaccine-induced CTLs were capable of killing SIV-infected cells in vitro. Fluorescence-activated cell sorter analysis using soluble tetrameric major histocompatibility complex-peptide complexes showed that the vaccinated animals had 1 to 5% circulating CD8+ lymphocytes specific for the vaccine epitope, frequencies comparable to those in SIV-infected monkeys. Upon intrarectal challenge with pathogenic SIVmac251, no evidence for protection was observed in at least two of the three vaccinated animals. This study does not attempt to define correlates of protective immunity nor design a protective vaccine against immunodeficiency viruses, but it demonstrates clearly that the DNA prime-MVA boost regimen is an effective protocol for induction of CTLs in macaques. It also shows that powerful tools for studying the role of CTLs in the control of SIV and human immunodeficiency virus infections are now available: epitope-based vaccines, a protocol for an effective induction of CTLs in primates, and a simple and sensitive method for quantitation of epitope-specific T cells. The advantages of the DNA prime-MVA boost regimen as well as the correlations of tetramer staining of peripheral blood lymphocytes with CTL killing in vitro and postchallenge control of viremia are discussed. PMID:10438842

Attenuated and Replication-Competent Vaccinia Virus Strains M65 and M101 with Distinct Biology and Immunogenicity as Potential Vaccine Candidates against Pathogens

PubMed Central

Sánchez-Sampedro, Lucas; Gómez, Carmen Elena; Mejías-Pérez, Ernesto; Pérez-Jiménez, Eva; Oliveros, Juan Carlos

2013-01-01

Replication-competent poxvirus vectors with an attenuation phenotype and with a high immunogenic capacity of the foreign expressed antigen are being pursued as novel vaccine vectors against different pathogens. In this investigation, we have examined the replication and immunogenic characteristics of two vaccinia virus (VACV) mutants, M65 and M101. These mutants were generated after 65 and 101 serial passages of persistently infected Friend erythroleukemia (FEL) cells. In cultured cells of different origins, the mutants are replication competent and have growth kinetics similar to or slightly reduced in comparison with those of the parental Western Reserve (WR) virus strain. In normal and immune-suppressed infected mice, the mutants showed different levels of attenuation and pathogenicity in comparison with WR and modified vaccinia Ankara (MVA) strains. Wide genome analysis after deep sequencing revealed selected genomic deletions and mutations in a number of viral open reading frames (ORFs). Mice immunized in a DNA prime/mutant boost regimen with viral vectors expressing the LACK (Leishmania homologue for receptors of activated C kinase) antigen of Leishmania infantum showed protection or a delay in the onset of cutaneous leishmaniasis. Protection was similar to that triggered by MVA-LACK. In immunized mice, both polyfunctional CD4+ and CD8+ T cells with an effector memory phenotype were activated by the two mutants, but the DNA-LACK/M65-LACK protocol preferentially induced CD4+ whereas DNA-LACK/M101-LACK preferentially induced CD8+ T cell responses. Altogether, our findings showed the adaptive changes of the WR genome during long-term virus-host cell interaction and how the replication competency of M65 and M101 mutants confers distinct biological properties and immunogenicity in mice compared to those of the MVA strain. These mutants could have applicability for understanding VACV biology and as potential vaccine vectors against pathogens and tumors. PMID

Hazard Characterization of Modified Vaccinia Virus Ankara Vector: What Are the Knowledge Gaps?

PubMed Central

Okeke, Malachy I.; Okoli, Arinze S.; Offor, Collins; Oludotun, Taiwo G.; Tryland, Morten; Bøhn, Thomas; Moens, Ugo

2017-01-01

Modified vaccinia virus Ankara (MVA) is the vector of choice for human and veterinary applications due to its strong safety profile and immunogenicity in vivo. The use of MVA and MVA-vectored vaccines against human and animal diseases must comply with regulatory requirements as they pertain to environmental risk assessment, particularly the characterization of potential adverse effects to humans, animals and the environment. MVA and recombinant MVA are widely believed to pose low or negligible risk to ecosystem health. However, key aspects of MVA biology require further research in order to provide data needed to evaluate the potential risks that may occur due to the use of MVA and MVA-vectored vaccines. The purpose of this paper is to identify knowledge gaps in the biology of MVA and recombinant MVA that are of relevance to its hazard characterization and discuss ongoing and future experiments aimed at providing data necessary to fill in the knowledge gaps. In addition, we presented arguments for the inclusion of uncertainty analysis and experimental investigation of verifiable worst-case scenarios in the environmental risk assessment of MVA and recombinant MVA. These will contribute to improved risk assessment of MVA and recombinant MVA vaccines. PMID:29109380

Hazard Characterization of Modified Vaccinia Virus Ankara Vector: What Are the Knowledge Gaps?

PubMed

Okeke, Malachy I; Okoli, Arinze S; Diaz, Diana; Offor, Collins; Oludotun, Taiwo G; Tryland, Morten; Bøhn, Thomas; Moens, Ugo

2017-10-29

Modified vaccinia virus Ankara (MVA) is the vector of choice for human and veterinary applications due to its strong safety profile and immunogenicity in vivo. The use of MVA and MVA-vectored vaccines against human and animal diseases must comply with regulatory requirements as they pertain to environmental risk assessment, particularly the characterization of potential adverse effects to humans, animals and the environment. MVA and recombinant MVA are widely believed to pose low or negligible risk to ecosystem health. However, key aspects of MVA biology require further research in order to provide data needed to evaluate the potential risks that may occur due to the use of MVA and MVA-vectored vaccines. The purpose of this paper is to identify knowledge gaps in the biology of MVA and recombinant MVA that are of relevance to its hazard characterization and discuss ongoing and future experiments aimed at providing data necessary to fill in the knowledge gaps. In addition, we presented arguments for the inclusion of uncertainty analysis and experimental investigation of verifiable worst-case scenarios in the environmental risk assessment of MVA and recombinant MVA. These will contribute to improved risk assessment of MVA and recombinant MVA vaccines.

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

PubMed

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

2017-04-01

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

Vaccinia virus proteins A36 and F12/E2 show strong preferences for different kinesin light chain isoforms.

PubMed

Gao, William N D; Carpentier, David C J; Ewles, Helen A; Lee, Stacey-Ann; Smith, Geoffrey L

2017-08-01

Vaccinia virus (VACV) utilizes microtubule-mediated trafficking at several stages of its life cycle, of which virus egress is the most intensely studied. During egress VACV proteins A36, F12 and E2 are involved in kinesin-1 interactions; however, the roles of these proteins remain poorly understood. A36 forms a direct link between virions and kinesin-1, yet in its absence VACV egress still occurs on microtubules. During a co-immunoprecipitation screen to seek an alternative link between virions and kinesin, A36 was found to bind isoform KLC1 rather than KLC2. The F12/E2 complex associates preferentially with the C-terminal tail of KLC2, to a region that overlaps the binding site of cellular 14-3-3 proteins. F12/E2 displaces 14-3-3 from KLC and, unlike 14-3-3, does not require phosphorylation of KLC for its binding. The region determining the KLC1 specificity of A36 was mapped to the KLC N-terminal heptad repeat region that is responsible for its association with kinesin heavy chain. Despite these differing binding properties F12/E2 can co-operatively enhance A36 association with KLC, particularly when using a KLC1-KLC2 chimaera that resembles several KLC1 spliceforms and can bind A36 and F12/E2 efficiently. This is the first example of a pathogen encoding multiple proteins that co-operatively associate with kinesin-1. © 2017 The Authors. Traffic published by John Wiley & Sons Ltd.

Re-Assembly and Analysis of an Ancient Variola Virus Genome.

PubMed

Smithson, Chad; Imbery, Jacob; Upton, Chris

2017-09-08

We report a major improvement to the assembly of published short read sequencing data from an ancient variola virus (VARV) genome by the removal of contig-capping sequencing tags and manual searches for gap-spanning reads. The new assembly, together with camelpox and taterapox genomes, permitted new dates to be calculated for the last common ancestor of all VARV genomes. The analysis of recently sequenced VARV-like cowpox virus genomes showed that single nucleotide polymorphisms (SNPs) and amino acid changes in the vaccinia virus (VACV)-Cop-O1L ortholog, predicted to be associated with VARV host specificity and virulence, were introduced into the lineage before the divergence of these viruses. A comparison of the ancient and modern VARV genome sequences also revealed a measurable drift towards adenine + thymine (A + T) richness.

Regression of Established Human Papillomavirus Type 16 (HPV-16) Immortalized Tumors In Vivo by Vaccinia Viruses Expressing Different Forms of HPV-16 E7 Correlates with Enhanced CD8+ T-Cell Responses That Home to the Tumor Site

PubMed Central

Lamikanra, Abigail; Pan, Zhen-Kun; Isaacs, Stuart N.; Wu, Tzyy-Choou; Paterson, Yvonne

2001-01-01

Using vaccinia virus as a live vector, we show that the expression of human papillomavirus type 16 (HPV-16) E7 fused to a nonhemolytic portion of the Listeria monocytogenes virulence factor, listeriolysin O (LLO), induces an immune response that causes the regression of established HPV-16 immortalized tumors in C57BL/6 mice. The vaccinia virus construct expressing LLO fused to E7 (VacLLOE7) was compared with two previously described vaccinia virus constructs: one that expresses unmodified E7 (VacE7) and another that expresses E7 in a form designed to direct it to intracellular lysosomal compartments and improve major histocompatibility complex class II-restricted responses (VacSigE7LAMP-1). C57BL/6 mice bearing established HPV-16 immortalized tumors of 5 or 8 mm were treated with each of these vaccines. Fifty percent of the mice treated with VacLLOE7 remained tumor free 2 months after tumor inoculation, whereas 12 to 25% of the mice were tumor free after treatment with VacSigE7LAMP-1 (depending on the size of the tumor). No mice were tumor free in the group given VacE7. Compared to VacE7, VacSigE7LAMP-1 and VacLLOE7 resulted in increased numbers of H2-Db-specific tetramer-positive CD8+ T cells in mouse spleens that produced gamma interferon and tumor necrosis factor alpha upon stimulation with RAHYNIVTF peptide. In addition, the highest frequency of tetramer-positive T cells was seen in the tumor sites of mice treated with VacLLOE7. An increased efficiency of E7-specific lysis by splenocytes from mice immunized with VacLLOE7 was also observed. These results indicate that the fusion of E7 with LLO not only enhances antitumor therapy by improving the tumoricidal function of E7-specific CD8+ T cells but may also increase the number of antigen-specific CD8+ T cells in the tumor, the principle site of antigen expression. PMID:11559797

DNA Packaging Mutant: Repression of the Vaccinia Virus A32 Gene Results in Noninfectious, DNA-Deficient, Spherical, Enveloped Particles

PubMed Central

Cassetti, Maria Cristina; Merchlinsky, Michael; Wolffe, Elizabeth J.; Weisberg, Andrea S.; Moss, Bernard

1998-01-01

The vaccinia virus A32 open reading frame was predicted to encode a protein with a nucleoside triphosphate-binding motif and a mass of 34 kDa. To investigate the role of this protein, we constructed a mutant in which the original A32 gene was replaced by an inducible copy. The recombinant virus, vA32i, has a conditional lethal phenotype: infectious virus formation was dependent on isopropyl-β-d-thiogalactopyranoside (IPTG). Under nonpermissive conditions, the mutant synthesized early- and late-stage viral proteins, as well as viral DNA that was processed into unit-length genomes. Electron microscopy of cells infected in the absence of IPTG revealed normal-appearing crescents and immature virus particles but very few with nucleoids. Instead of brick-shaped mature particles with defined core structures, there were numerous electron-dense, spherical particles. Some of these spherical particles were wrapped with cisternal membranes, analogous to intracellular and extracellular enveloped virions. Mutant viral particles, purified by sucrose density gradient centrifugation, had low infectivity and transcriptional activity, and the majority were spherical and lacked DNA. Nevertheless, the particle preparation contained representative membrane proteins, cleaved and uncleaved core proteins, the viral RNA polymerase, the early transcription factor and several enzymes, suggesting that incorporation of these components is not strictly coupled to DNA packaging. PMID:9621036

Real-Time PCR Assay To Detect Smallpox Virus

PubMed Central

Sofi Ibrahim, M.; Kulesh, David A.; Saleh, Sharron S.; Damon, Inger K.; Esposito, Joseph J.; Schmaljohn, Alan L.; Jahrling, Peter B.

2003-01-01

We developed a highly sensitive and specific assay for the rapid detection of smallpox virus DNA on both the Smart Cycler and LightCycler platforms. The assay is based on TaqMan chemistry with the orthopoxvirus hemagglutinin gene used as the target sequence. With genomic DNA purified from variola virus Bangladesh 1975, the limit of detection was estimated to be approximately 25 copies on both machines. The assay was evaluated in a blinded study with 322 coded samples that included genomic DNA from 48 different isolates of variola virus; 25 different strains and isolates of camelpox, cowpox, ectromelia, gerbilpox, herpes, monkeypox, myxoma, rabbitpox, raccoonpox, skunkpox, vaccinia, and varicella-zoster viruses; and two rickettsial species at concentrations mostly ranging from 100 fg/μl to 1 ng/μl. Contained within those 322 samples were variola virus DNA, obtained from purified viral preparations, at concentrations of 1 fg/μl to 1 ng/μl. On the Smart Cycler platform, 2 samples with false-positive results were detected among the 116 samples not containing variola virus tested; i.e., the overall specificity of the assay was 98.3%. On the LightCycler platform, five samples with false-positive results were detected (overall specificity, 95.7%). Of the 206 samples that contained variola virus DNA ranging in concentrations from 100 fg/μl to 1 ng/μl, 8 samples were considered negative on the Smart Cycler platform and 1 sample was considered negative on the LightCycler platform. Thus, the clinical sensitivities were 96.1% for the Smart Cycler instrument and 99.5% for the LightCycler instrument. The vast majority of these samples were derived from virus-infected cell cultures and variola virus-infected tissues; thus, the DNA material contained both viral DNA and cellular DNA. Of the 43 samples that contained purified variola virus DNA ranging in concentration from 1 fg/μl to 1 ng/μl, the assay correctly detected the virus in all 43 samples on both the Smart Cycler

Further Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and its Interaction with ICP8, the Major DNA-Binding Protein of Herpes Simplex Virus

DTIC Science & Technology

1994-01-01

HSV envelopment and egress . Gross structures of the genomes of tbe buman herpesviruses . Layout of genes in the genome of HSV - 1 ........... . A... HSV - 1 capsid maturation . Seletion of recombinant vaccinia viruses Protein fusion and purification system . Generation of tbe recombinant plasmid...with purified HSV -I virions Effect of detergent treatment on the association of the UL37 protein with purified HSV - 1 vIrIons

A Single Dose of Modified Vaccinia Ankara expressing Ebola Virus Like Particles Protects Nonhuman Primates from Lethal Ebola Virus Challenge.

PubMed

Domi, Arban; Feldmann, Friederike; Basu, Rahul; McCurley, Nathanael; Shifflett, Kyle; Emanuel, Jackson; Hellerstein, Michael S; Guirakhoo, Farshad; Orlandi, Chiara; Flinko, Robin; Lewis, George K; Hanley, Patrick W; Feldmann, Heinz; Robinson, Harriet L; Marzi, Andrea

2018-01-16

Ebola virus (EBOV), isolate Makona, was the causative agent of the West African epidemic devastating predominantly Guinea, Liberia and Sierra Leone from 2013-2016. While several experimental vaccine and treatment approaches have been accelerated through human clinical trials, there is still no approved countermeasure available against this disease. Here, we report the construction and preclinical efficacy testing of a novel recombinant modified vaccinia Ankara (MVA)-based vaccine expressing the EBOV-Makona glycoprotein GP and matrix protein VP40 (MVA-EBOV). GP and VP40 form EBOV-like particles and elicit protective immune responses. In this study, we report 100% protection against lethal EBOV infection in guinea pigs after prime/boost vaccination with MVA-EBOV. Furthermore, this MVA-EBOV protected macaques from lethal disease after a single dose or prime/boost vaccination. The vaccine elicited a variety of antibody responses to both antigens, including neutralizing antibodies and antibodies with antibody-dependent cellular cytotoxic activity specific for GP. This is the first report that a replication-deficient MVA vector can confer full protection against lethal EBOV challenge after a single dose vaccination in macaques.

Virus inactivation under the photodynamic effect of phthalocyanine zinc(II) complexes.

PubMed

Remichkova, Mimi; Mukova, Luchia; Nikolaeva-Glomb, Lubomira; Nikolova, Nadya; Doumanova, Lubka; Mantareva, Vanya; Angelov, Ivan; Kussovski, Veselin; Galabov, Angel S

2017-03-01

Various metal phthalocyanines have been studied for their capacity for photodynamic effects on viruses. Two newly synthesized water-soluble phthalocyanine Zn(II) complexes with different charges, cationic methylpyridyloxy-substituted Zn(II)- phthalocyanine (ZnPcMe) and anionic sulfophenoxy-substituted Zn(II)-phthalocyanine (ZnPcS), were used for photoinactivation of two DNA-containing enveloped viruses (herpes simplex virus type 1 and vaccinia virus), two RNA-containing enveloped viruses (bovine viral diarrhea virus and Newcastle disease virus) and two nude viruses (the enterovirus Coxsackie B1, a RNA-containing virus, and human adenovirus 5, a DNA virus). These two differently charged phthalocyanine complexes showed an identical marked virucidal effect against herpes simplex virus type 1, which was one and the same at an irradiation lasting 5 or 20 min (Δlog=3.0 and 4.0, respectively). Towards vaccinia virus this effect was lower, Δlog=1.8 under the effect of ZnPcMe and 2.0 for ZnPcS. Bovine viral diarrhea virus manifested a moderate sensitivity to ZnPcMe (Δlog=1.8) and a pronounced one to ZnPcS at 5- and 20-min irradiation (Δlog=5.8 and 5.3, respectively). The complexes were unable to inactivate Newcastle disease virus, Coxsackievirus B1 and human adenovirus type 5.

Vaccinia Virus Protein C6 Inhibits Type I IFN Signalling in the Nucleus and Binds to the Transactivation Domain of STAT2.

PubMed

Stuart, Jennifer H; Sumner, Rebecca P; Lu, Yongxu; Snowden, Joseph S; Smith, Geoffrey L

2016-12-01

The type I interferon (IFN) response is a crucial innate immune signalling pathway required for defense against viral infection. Accordingly, the great majority of mammalian viruses possess means to inhibit this important host immune response. Here we show that vaccinia virus (VACV) strain Western Reserve protein C6, is a dual function protein that inhibits the cellular response to type I IFNs in addition to its published function as an inhibitor of IRF-3 activation, thereby restricting type I IFN production from infected cells. Ectopic expression of C6 inhibits the induction of interferon stimulated genes (ISGs) in response to IFNα treatment at both the mRNA and protein level. C6 inhibits the IFNα-induced Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway at a late stage, downstream of STAT1 and STAT2 phosphorylation, nuclear translocation and binding of the interferon stimulated gene factor 3 (ISGF3) complex to the interferon stimulated response element (ISRE). Mechanistically, C6 associates with the transactivation domain of STAT2 and this might explain how C6 inhibits the type I IFN signalling very late in the pathway. During virus infection C6 reduces ISRE-dependent gene expression despite the presence of the viral protein phosphatase VH1 that dephosphorylates STAT1 and STAT2. The ability of a cytoplasmic replicating virus to dampen the immune response within the nucleus, and the ability of viral immunomodulators such as C6 to inhibit multiple stages of the innate immune response by distinct mechanisms, emphasizes the intricacies of host-pathogen interactions and viral immune evasion.

Expression of the A56 and K2 proteins is sufficient to inhibit vaccinia virus entry and cell fusion.

PubMed

Wagenaar, Timothy R; Moss, Bernard

2009-02-01

Many animal viruses induce cells to fuse and form syncytia. For vaccinia virus, this phenomenon is associated with mutations affecting the A56 and K2 proteins, which form a multimer (A56/K2) on the surface of infected cells. Recent evidence that A56/K2 interacts with the entry/fusion complex (EFC) and that the EFC is necessary for syncytium formation furnishes a strong connection between virus entry and cell fusion. Among the important remaining questions are whether A56/K2 can prevent virus entry as well as cell-cell fusion and whether these two viral proteins are sufficient as well as necessary for this. To answer these questions, we transiently and stably expressed A56 and K2 in uninfected cells. Uninfected cells expressing A56 and K2 exhibited resistance to fusing with A56 mutant virus-infected cells, whereas expression of A56 or K2 alone induced little or no resistance, which fits with the need for both proteins to bind the EFC. Furthermore, transient or stable expression of A56/K2 interfered with virus entry and replication as determined by inhibition of early expression of a luciferase reporter gene, virus production, and plaque formation. The specificity of this effect was demonstrated by restoring entry after enzymatically removing a chimeric glycophosphatidylinositol-anchored A56/K2 or by binding a monoclonal antibody to A56. Importantly, the antibody disrupted the interaction between A56/K2 and the EFC without disrupting the A56-K2 interaction itself. Thus, we have shown that A56/K2 is sufficient to prevent virus entry and fusion as well as formation of syncytia through interaction with the EFC.

Modified Vaccinia Virus Ankara Preferentially Targets Antigen Presenting Cells In Vitro, Ex Vivo and In Vivo.

PubMed

Altenburg, Arwen F; van de Sandt, Carolien E; Li, Bobby W S; MacLoughlin, Ronan J; Fouchier, Ron A M; van Amerongen, Geert; Volz, Asisa; Hendriks, Rudi W; de Swart, Rik L; Sutter, Gerd; Rimmelzwaan, Guus F; de Vries, Rory D

2017-08-17

Modified Vaccinia virus Ankara (MVA) is a promising vaccine vector with an excellent safety profile. However, despite extensive pre-clinical and clinical testing, surprisingly little is known about the cellular tropism of MVA, especially in relevant animal species. Here, we performed in vitro, ex vivo and in vivo experiments with recombinant MVA expressing green fluorescent protein (rMVA-GFP). In both human peripheral blood mononuclear cells and mouse lung explants, rMVA-GFP predominantly infected antigen presenting cells. Subsequent in vivo experiments performed in mice, ferrets and non-human primates indicated that preferential targeting of dendritic cells and alveolar macrophages was observed after respiratory administration, although subtle differences were observed between the respective animal species. Following intramuscular injection, rMVA-GFP was detected in interdigitating cells between myocytes, but also in myocytes themselves. These data are important in advancing our understanding of the basis for the immunogenicity of MVA-based vaccines and aid rational vaccine design and delivery strategies.

Oral vaccination of wildlife using a vaccinia-rabies-glycoprotein recombinant virus vaccine (RABORAL V-RG®): a global review.

PubMed

Maki, Joanne; Guiot, Anne-Laure; Aubert, Michel; Brochier, Bernard; Cliquet, Florence; Hanlon, Cathleen A; King, Roni; Oertli, Ernest H; Rupprecht, Charles E; Schumacher, Caroline; Slate, Dennis; Yakobson, Boris; Wohlers, Anne; Lankau, Emily W

2017-09-22

RABORAL V-RG ® is an oral rabies vaccine bait that contains an attenuated ("modified-live") recombinant vaccinia virus vector vaccine expressing the rabies virus glycoprotein gene (V-RG). Approximately 250 million doses have been distributed globally since 1987 without any reports of adverse reactions in wildlife or domestic animals since the first licensed recombinant oral rabies vaccine (ORV) was released into the environment to immunize wildlife populations against rabies. V-RG is genetically stable, is not detected in the oral cavity beyond 48 h after ingestion, is not shed by vaccinates into the environment, and has been tested for thermostability under a range of laboratory and field conditions. Safety of V-RG has been evaluated in over 50 vertebrate species, including non-human primates, with no adverse effects observed regardless of route or dose. Immunogenicity and efficacy have been demonstrated under laboratory and field conditions in multiple target species (including fox, raccoon, coyote, skunk, raccoon dog, and jackal). The liquid vaccine is packaged inside edible baits (i.e., RABORAL V-RG, the vaccine-bait product) which are distributed into wildlife habitats for consumption by target species. Field application of RABORAL V-RG has contributed to the elimination of wildlife rabies from three European countries (Belgium, France and Luxembourg) and of the dog/coyote rabies virus variant from the United States of America (USA). An oral rabies vaccination program in west-central Texas has essentially eliminated the gray fox rabies virus variant from Texas with the last case reported in a cow during 2009. A long-term ORV barrier program in the USA using RABORAL V-RG is preventing substantial geographic expansion of the raccoon rabies virus variant. RABORAL V-RG has also been used to control wildlife rabies in Israel for more than a decade. This paper: (1) reviews the development and historical use of RABORAL V-RG; (2) highlights wildlife rabies control

Towards a universal vaccine for avian influenza: Protective efficacy of modified Vaccinia virus Ankara and Adenovirus vaccines expressing conserved influenza antigens in chickens challenged with low pathogenic avian influenza virus

PubMed Central

Boyd, Amy C.; Ruiz-Hernandez, Raul; Peroval, Marylene Y.; Carson, Connor; Balkissoon, Devanand; Staines, Karen; Turner, Alison V.; Hill, Adrian V.S.; Gilbert, Sarah C.; Butter, Colin

2013-01-01

Current vaccines targeting surface proteins can drive antigenic variation resulting either in the emergence of more highly pathogenic viruses or of antigenically distinct viruses that escape control by vaccination and thereby persist in the host population. Influenza vaccines typically target the highly mutable surface proteins and do not provide protection against heterologous challenge. Vaccines which induce immune responses against conserved influenza epitopes may confer protection against heterologous challenge. We report here the results of vaccination with recombinant modified Vaccinia virus Ankara (MVA) and Adenovirus (Ad) expressing a fusion construct of nucleoprotein and matrix protein (NP + M1). Prime and boost vaccination regimes were trialled in different ages of chicken and were found to be safe and immunogenic. Interferon-γ (IFN-γ) ELISpot was used to assess the cellular immune response post secondary vaccination. In ovo Ad prime followed by a 4 week post hatch MVA boost was identified as the most immunogenic regime in one outbred and two inbred lines of chicken. Following vaccination, one inbred line (C15I) was challenged with low pathogenic avian influenza (LPAI) H7N7 (A/Turkey/England/1977). Birds receiving a primary vaccination with Ad-NP + M1 and a secondary vaccination with MVA-NP + M1 exhibited reduced cloacal shedding as measured by plaque assay at 7 days post infection compared with birds vaccinated with recombinant viruses containing irrelevant antigen. This preliminary indication of efficacy demonstrates proof of concept in birds; induction of T cell responses in chickens by viral vectors containing internal influenza antigens may be a productive strategy for the development of vaccines to induce heterologous protection against influenza in poultry. PMID:23200938

Cooperative vaccinia infection demonstrated at the single-cell level using FluidFM.

PubMed

Stiefel, Philipp; Schmidt, Florian I; Dörig, Pablo; Behr, Pascal; Zambelli, Tomaso; Vorholt, Julia A; Mercer, Jason

2012-08-08

The mechanisms used by viruses to enter and replicate within host cells are subjects of intense investigation. These studies are ultimately aimed at development of new drugs that interfere with these processes. Virus entry and infection are generally monitored by dispensing bulk virus suspensions on layers of cells without accounting for the fate of each virion. Here, we take advantage of the recently developed FluidFM to deposit single vaccinia virions onto individual cells in a controlled manner. While the majority of virions were blocked prior to early gene expression, infection of individual cells increased in a nondeterministic fashion with respect to the number of viruses placed. Microscopic analyses of several stages of the virus lifecycle indicated that this was the result of cooperativity between virions during early stages of infection. These findings highlight the importance of performing controlled virus infection experiments at the single cell level.

Reversible Inactivation and Desiccation Tolerance of Silicified Viruses

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

Laidler, James J.; Shugart, Jessica A.; Cady, Sherry L.

2013-11-19

Long-distance host-independent virus dispersal is poorly understood, especially for viruses found in isolated ecosystems. To demonstrate a possible dispersal mechanism, we show that bacteriophage T4, archaeal virus SSV-K and Vaccinia are reversibly inactivated by mineralization in silica under conditions similar to volcanic hot springs. By contrast, bacteriophage PRD1 is not silicified. Moreover silicification provides viruses with remarkable desiccation resistance, which could allow extensive aerial dispersal.

Molecular Smallpox Vaccine Delivered by Alphavirus Replicons Elicits Protective Immunity in Mice and Non-human Primates

PubMed Central

Hooper, Jay W.; Ferro, Anthony M.; Golden, Joseph W.; Silvera, Peter; Dudek, Jeanne; Alterson, Kim; Custer, Max; Rivers, Bryan; Morris, John; Owens, Gary; Smith, Jonathan F.; Kamrud, Kurt I.

2009-01-01

Naturally occurring smallpox was eradicated as a result of successful vaccination campaigns during the 1960s and 70s. Because of its highly contagious nature and high mortality rate, smallpox has significant potential as a biological weapon. Unfortunately, the current vaccine for orthopoxviruses is contraindicated for large portions of the population. Thus, there is a need for new, safe, and effective orthopoxvirus vaccines. Alphavirus replicon vectors, derived from strains of Venezuelan equine encephalitis virus, are being used to develop alternatives to the current smallpox vaccine. Here, we demonstrated that virus-like replicon particles (VRP) expressing the vaccinia virus A33R, B5R, A27L, and L1R genes elicited protective immunity in mice comparable to vaccination with live-vaccinia virus. Furthermore, cynomolgus macaques vaccinated with a combination of the four poxvirus VRPs (4pox-VRP) developed antibody responses to each antigen. These antibody responses were able to neutralize and inhibit the spread of both vaccinia virus and monkeypox virus. Macaques vaccinated with 4pox-VRP, flu HA VRP (negative control), or live-vaccinia virus (positive control) were challenged intravenously with 5 × 106 PFU of monkeypox virus 1 month after the second VRP vaccination. Four of the six negative control animals succumbed to monkeypox and the remaining two animals demonstrated either severe or grave disease. Importantly, all 10 macaques vaccinated with the 4pox-VRP vaccine survived without developing severe disease. These findings revealed that a single-boost VRP smallpox vaccine shows promise as a safe alternative to the currently licensed live-vaccinia virus smallpox vaccine. PMID:19833247

Co-administration of recombinant major envelope proteins (rA27L and rH3L) of buffalopox virus provides enhanced immunogenicity and protective efficacy in animal models.

PubMed

Kumar, Amit; Yogisharadhya, Revanaiah; Venkatesan, Gnanavel; Bhanuprakash, Veerakyathappa; Pandey, Awadh Bihari; Shivachandra, Sathish Bhadravati

2017-05-01

Buffalopox virus (BPXV) and other vaccinia-like viruses (VLVs) are causing an emerging/re-emerging zoonosis affecting buffaloes, cattle and humans in India and other countries. A27L and H3L are immuno-dominant major envelope proteins of intracellular mature virion (IMV) of orthopoxviruses (OPVs) and are highly conserved with an ability to elicit neutralizing antibodies. In the present study, two recombinant proteins namely; rA27L ( 21 S to E 110 ; ∼30 kDa) and rH3L( 1 M to I 280 ; ∼50 kDa) of BPXV-Vij/96 produced from Escherichia coli were used in vaccine formulation. A combined recombinant subunit vaccine comprising rA27L and rH3L antigens (10 μg of each) was used for active immunization of adult mice (20μg/dose/mice) with or without adjuvant (FCA/FIA) by intramuscular route. Immune responses revealed a gradual increase in antigen specific serum IgG as well as neutralizing antibody titers measured by using indirect-ELISA and serum neutralization test (SNT) respectively, which were higher as compared to that elicited by individual antigens. Suckling mice passively administered with combined anti-A27L and anti-H3L sera showed a complete (100%) pre-exposure protection upon challenge with virulent BPXV. Conclusively, this study highlights the potential utility of rA27L and rH3L proteins as safer candidate prophylactic antigens in combined recombinant subunit vaccine for buffalopox as well as passive protective efficacy of combined sera in employing better pre-exposure protection against virulent BPXV. Copyright © 2017 Elsevier B.V. All rights reserved.

Variola virus immune evasion proteins.

PubMed

Dunlop, Lance R; Oehlberg, Katherine A; Reid, Jeremy J; Avci, Dilek; Rosengard, Ariella M

2003-09-01

Variola virus, the causative agent of smallpox, encodes approximately 200 proteins. Over 80 of these proteins are located in the terminal regions of the genome, where proteins associated with host immune evasion are encoded. To date, only two variola proteins have been characterized. Both are located in the terminal regions and demonstrate immunoregulatory functions. One protein, the smallpox inhibitor of complement enzymes (SPICE), is homologous to a vaccinia virus virulence factor, the vaccinia virus complement-control protein (VCP), which has been found experimentally to be expressed early in the course of vaccinia infection. Both SPICE and VCP are similar in structure and function to the family of mammalian complement regulatory proteins, which function to prevent inadvertent injury to adjacent cells and tissues during complement activation. The second variola protein is the variola virus high-affinity secreted chemokine-binding protein type II (CKBP-II, CBP-II, vCCI), which binds CC-chemokine receptors. The vaccinia homologue of CKBP-II is secreted both early and late in infection. CKBP-II proteins are highly conserved among orthopoxviruses, sharing approximately 85% homology, but are absent in eukaryotes. This characteristic sets it apart from other known virulence factors in orthopoxviruses, which share sequence homology with known mammalian immune regulatory gene products. Future studies of additional variola proteins may help illuminate factors associated with its virulence, pathogenesis and strict human tropism. In addition, these studies may also assist in the development of targeted therapies for the treatment of both smallpox and human immune-related diseases.

Preventing vaccinia virus class-I epitopes presentation by HSV-ICP47 enhances the immunogenicity of a TAP-independent cancer vaccine epitope.

PubMed

Raafat, Nermin; Sadowski-Cron, Charlotte; Mengus, Chantal; Heberer, Michael; Spagnoli, Giulio C; Zajac, Paul

2012-09-01

Herpes simplex virus protein ICP47, encoded by US12 gene, strongly downregulates major histocompatibility complex (MHC) class-I antigen restricted presentation by blocking transporter associated with antigen processing (TAP) protein. To decrease viral vector antigenic immunodominance and MHC class-I driven clearance, we engineered recombinant vaccinia viruses (rVV) expressing ICP47 alone (rVV-US12) or together with endoplasmic reticulum (ER)-targeted Melan-A/MART-1(27-35) model tumor epitope (rVV-MUS12). In this study, we show that antigen presenting cells (APC), infected with rVV-US12, display a decreased ability to present TAP dependent MHC class-I restricted viral antigens to CD8+ T-cells. While HLA class-I cell surface expression is strongly downregulated, other important immune related molecules such as CD80, CD44 and, most importantly, MHC class-II are unaffected. Characterization of rVV-MUS12 infected cells demonstrates that over-expression of a TAP-independent peptide, partially compensates for ICP47 induced surface MHC class-I downregulation (30% vs. 70% respectively). Most importantly, in conditions where clearance of infected APC by virus-specific CTL represents a limiting factor, a significant enhancement of CTL responses to the tumor epitope can be detected in cultures stimulated with rVV-MUS12, as compared to those stimulated by rVV-MART alone. Such reagents could become of high relevance in multiple boost protocols required for cancer immunotherapy, to limit vector-specific responsiveness. Copyright © 2011 UICC.

Efficient Processing of the Immunodominant, HLA-A*0201-Restricted Human Immunodeficiency Virus Type 1 Cytotoxic T-Lymphocyte Epitope despite Multiple Variations in the Epitope Flanking Sequences

PubMed Central

Brander, Christian; Yang, Otto O.; Jones, Norman G.; Lee, Yun; Goulder, Philip; Johnson, R. Paul; Trocha, Alicja; Colbert, David; Hay, Christine; Buchbinder, Susan; Bergmann, Cornelia C.; Zweerink, Hans J.; Wolinsky, Steven; Blattner, William A.; Kalams, Spyros A.; Walker, Bruce D.

1999-01-01

Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences. PMID:10559335

Photodynamic therapy augments the efficacy of oncolytic vaccinia virus against primary and metastatic tumours in mice

PubMed Central

Gil, M; Bieniasz, M; Seshadri, M; Fisher, D; Ciesielski, M J; Chen, Y; Pandey, R K; Kozbor, D

2011-01-01

Background: Therapies targeted towards the tumour vasculature can be exploited for the purpose of improving the systemic delivery of oncolytic viruses to tumours. Photodynamic therapy (PDT) is a clinically approved treatment for cancer that is known to induce potent effects on tumour vasculature. In this study, we examined the activity of PDT in combination with oncolytic vaccinia virus (OVV) against primary and metastatic tumours in mice. Methods: The effect of 2-[1-hexyloxyethyl-]-2-devinyl pyropheophorbide-a (HPPH)-sensitised-PDT on the efficacy of oncolytic virotherapy was investigated against subcutaneously implanted syngeneic murine NXS2 neuroblastoma and human FaDu head and neck squamous cell carcinoma xenografts in nude mice. Treatment efficacy was evaluated by monitoring tumour growth and survival. The effects of combination treatment on vascular function were examined using magnetic resonance imaging (MRI) and immunohistochemistry, whereas viral replication in tumour cells was analysed by a standard plaque assay. Normal tissue phototoxicity following PDT-OV treatment was studied using the mouse foot response assay. Results: Combination of PDT with OVV resulted in inhibition of primary and metastatic tumour growth compared with either monotherapy. PDT-induced vascular disruption resulted in higher intratumoural viral titres compared with the untreated tumours. Five days after delivery of OVV, there was a loss of blood flow to the interior of tumour that was associated with infiltration of neutrophils. Administration of OVV did not result in any additional photodynamic damage to normal mouse foot tissue. Conclusion: These results provide evidence into the usefulness of PDT as a means of enhancing intratumoural replication and therapeutic efficacy of OV. PMID:21989183

Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies

PubMed Central

Chervyakova, Olga V.; Zaitsev, Valentin L.; Iskakov, Bulat K.; Tailakova, Elmira T.; Strochkov, Vitaliy M.; Sultankulova, Kulyaisan T.; Sandybayev, Nurlan T.; Stanbekova, Gulshan E.; Beisenov, Daniyar K.; Abduraimov, Yergali O.; Mambetaliyev, Muratbay; Sansyzbay, Abylay R.; Kovalskaya, Natalia Y.; Nemchinov, Lev. G.; Hammond, Rosemarie W.

2016-01-01

The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. PMID:27338444

Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies.

PubMed

Chervyakova, Olga V; Zaitsev, Valentin L; Iskakov, Bulat K; Tailakova, Elmira T; Strochkov, Vitaliy M; Sultankulova, Kulyaisan T; Sandybayev, Nurlan T; Stanbekova, Gulshan E; Beisenov, Daniyar K; Abduraimov, Yergali O; Mambetaliyev, Muratbay; Sansyzbay, Abylay R; Kovalskaya, Natalia Y; Nemchinov, Lev G; Hammond, Rosemarie W

2016-06-07

The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins.

Preferential Targeting of Conserved Gag Regions after Vaccination with a Heterologous DNA Prime-Modified Vaccinia Virus Ankara Boost HIV-1 Vaccine Regimen.

PubMed

Bauer, Asli; Podola, Lilli; Mann, Philipp; Missanga, Marco; Haule, Antelmo; Sudi, Lwitiho; Nilsson, Charlotta; Kaluwa, Bahati; Lueer, Cornelia; Mwakatima, Maria; Munseri, Patricia J; Maboko, Leonard; Robb, Merlin L; Tovanabutra, Sodsai; Kijak, Gustavo; Marovich, Mary; McCormack, Sheena; Joseph, Sarah; Lyamuya, Eligius; Wahren, Britta; Sandström, Eric; Biberfeld, Gunnel; Hoelscher, Michael; Bakari, Muhammad; Kroidl, Arne; Geldmacher, Christof

2017-09-15

Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function, and specificity of Gag-specific T cells induced by a DNA-prime modified vaccinia virus Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding a subtype B and AB-recombinant Gag p37 and two vaccinations with MVA-CMDR encoding subtype A Gag p55 Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced gamma interferon-positive (IFN-γ + ) Gag-specific T-cell responses were dominated by CD4 + T cells ( P < 0.001 compared to CD8 + T cells) that coexpressed interleukin-2 (IL-2) (66.4%) and/or tumor necrosis factor alpha (TNF-α) (63.7%). A median of 3 antigenic regions were targeted with a higher-magnitude median response to Gag p24 regions, more conserved between prime and boost, compared to those of regions within Gag p15 (not primed) and Gag p17 (less conserved; P < 0.0001 for both). Four regions within Gag p24 each were targeted by 45% to 74% of vaccinees upon restimulation with DNA-SMI-Gag matched peptides. The response rate to individual antigenic regions correlated with the sequence homology between the MVA- and DNA Gag-encoded immunogens ( P = 0.04, r 2 = 0.47). In summary, after the first MVA-CMDR boost, the sequence-mismatched DNA-prime MVA-boost vaccine strategy induced a Gag-specific T-cell response that was dominated by polyfunctional CD4 + T cells and that targeted multiple antigenic regions within the conserved Gag p24 protein. IMPORTANCE Genetic diversity is a major challenge for the design of vaccines against variable viruses. While including multiple variants for a

The detection of Vaccinia virus confirms the high circulation of Orthopoxvirus in buffaloes living in geographical isolation, Marajó Island, Brazilian Amazon.

PubMed

Franco-Luiz, Ana Paula Moreira; Fagundes Pereira, Alexandre; de Oliveira, Cairo Henrique Sousa; Barbosa, José Diomedes; Oliveira, Danilo Bretas; Bonjardim, Cláudio Antônio; Ferreira, Paulo César Peregrino; de Souza Trindade, Giliane; Abrahão, Jônatas Santos; Kroon, Erna Geessien

2016-06-01

In Brazil, serologic evidence of Orthopoxvirus (OPV) circulation showed positivity around 20% in cattle, humans, monkeys and rodents. Although OPV seropositivity has been described in buffalo herds in southeastern Brazil, no Vaccinia virus (VACV) (member of genus OPV) outbreaks in buffalo herds have been described in this country. This study aimed to investigate the detection of anti-OPV antibodies and to study the OPV genome in Brazilian buffalo herds. Our results demonstrated a high OPV seropositivity in buffalo herds on Marajó Island and molecular data confirmed the circulation of VACV. The geographical isolation conditionmight be a sine qua non condition to explain our results. Copyright © 2016. Published by Elsevier Ltd.

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

PubMed

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

2018-04-01

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

Mapping vaccinia virus DNA replication origins at nucleotide level by deep sequencing.

PubMed

Senkevich, Tatiana G; Bruno, Daniel; Martens, Craig; Porcella, Stephen F; Wolf, Yuri I; Moss, Bernard

2015-09-01

Poxviruses reproduce in the host cytoplasm and encode most or all of the enzymes and factors needed for expression and synthesis of their double-stranded DNA genomes. Nevertheless, the mode of poxvirus DNA replication and the nature and location of the replication origins remain unknown. A current but unsubstantiated model posits only leading strand synthesis starting at a nick near one covalently closed end of the genome and continuing around the other end to generate a concatemer that is subsequently resolved into unit genomes. The existence of specific origins has been questioned because any plasmid can replicate in cells infected by vaccinia virus (VACV), the prototype poxvirus. We applied directional deep sequencing of short single-stranded DNA fragments enriched for RNA-primed nascent strands isolated from the cytoplasm of VACV-infected cells to pinpoint replication origins. The origins were identified as the switching points of the fragment directions, which correspond to the transition from continuous to discontinuous DNA synthesis. Origins containing a prominent initiation point mapped to a sequence within the hairpin loop at one end of the VACV genome and to the same sequence within the concatemeric junction of replication intermediates. These findings support a model for poxvirus genome replication that involves leading and lagging strand synthesis and is consistent with the requirements for primase and ligase activities as well as earlier electron microscopic and biochemical studies implicating a replication origin at the end of the VACV genome.

Single virus particle mass detection using microresonators with nanoscale thickness

NASA Astrophysics Data System (ADS)

Gupta, A.; Akin, D.; Bashir, R.

2004-03-01

In this letter, we present the microfabrication and application of arrays of silicon cantilever beams as microresonator sensors with nanoscale thickness to detect the mass of individual virus particles. The dimensions of the fabricated cantilever beams were in the range of 4-5 μm in length, 1-2 μm in width and 20-30 nm in thickness. The virus particles we used in the study were vaccinia virus, which is a member of the Poxviridae family and forms the basis of the smallpox vaccine. The frequency spectra of the cantilever beams, due to thermal and ambient noise, were measured using a laser Doppler vibrometer under ambient conditions. The change in resonant frequency as a function of the virus particle mass binding on the cantilever beam surface forms the basis of the detection scheme. We have demonstrated the detection of a single vaccinia virus particle with an average mass of 9.5 fg. These devices can be very useful as components of biosensors for the detection of airborne virus particles.

Vaccinia virus decreases major histocompatibility complex (MHC) class II antigen presentation, T-cell priming, and peptide association with MHC class II

PubMed Central

Rehm, Kristina E; Connor, Ramsey F; Jones, Gwendolyn J B; Yimbu, Kenneth; Mannie, Mark D; Roper, Rachel L

2009-01-01

Vaccinia virus (VACV) is the current live virus vaccine used to protect humans against smallpox and monkeypox, but its use is contraindicated in several populations because of its virulence. It is therefore important to elucidate the immune evasion mechanisms of VACV. We found that VACV infection of antigen-presenting cells (APCs) significantly decreased major histocompatibility complex (MHC) II antigen presentation and decreased synthesis of 13 chemokines and cytokines, suggesting a potent viral mechanism for immune evasion. In these model systems, responding T cells were not directly affected by virus, indicating that VACV directly affects the APC. VACV significantly decreased nitric oxide production by peritoneal exudate cells and the RAW macrophage cell line in response to lipopolysaccharide (LPS) and interferon (IFN)-γ, decreased class II MHC expression on APCs, and induced apoptosis in macrophages and dendritic cells. However, VACV decreased antigen presentation by 1153 B cells without apparent apoptosis induction, indicating that VACV differentially affects B lymphocytes and other APCs. We show that the key mechanism of VACV inhibition of antigen presentation may be its reduction of antigenic peptide loaded into the cleft of MHC class II molecules. These data indicate that VACV evades the host immune response by impairing critical functions of the APC. PMID:20067538

Development and Evaluation of Adeno-HTLV-III Hybrid Virus and Non-Cytopathic HTLV-III Mutant for Vaccine Use.

DTIC Science & Technology

1987-10-28

34" HIV will be tested. The recombinant Adeno-HIV virus is being developed as part of this proposal. The vaccines will be tested in two species of monkeys...vaccinia-HIV viruses was investigated (Table 4). All the vaccinated monkeys developed antibodies to HIV envelope antigen. However, no neutralizing antibody...Table 5). Epstein Barr virus transformed autologous B cells infected with recon- binant vaccinia-HIV virus may be used as targets. 30 chimpanzees from

Modified Vaccinia Virus Ankara Vector Induces Specific Cellular and Humoral Responses in the Female Reproductive Tract, the Main HIV Portal of Entry.

PubMed

Marlin, Romain; Nugeyre, Marie-Thérèse; Tchitchek, Nicolas; Parenti, Matteo; Hocini, Hakim; Benjelloun, Fahd; Cannou, Claude; Dereuddre-Bosquet, Nathalie; Levy, Yves; Barré-Sinoussi, Françoise; Scarlatti, Gabriella; Le Grand, Roger; Menu, Elisabeth

2017-09-01

The female reproductive tract (FRT) is one of the major mucosal invasion sites for HIV-1. This site has been neglected in previous HIV-1 vaccine studies. Immune responses in the FRT after systemic vaccination remain to be characterized. Using a modified vaccinia virus Ankara (MVA) as a vaccine model, we characterized specific immune responses in all compartments of the FRT of nonhuman primates after systemic vaccination. Memory T cells were preferentially found in the lower tract (vagina and cervix), whereas APCs and innate lymphoid cells were mainly located in the upper tract (uterus and fallopian tubes). This compartmentalization of immune cells in the FRT was supported by transcriptomic analyses and a correlation network. Polyfunctional MVA-specific CD8 + T cells were detected in the blood, lymph nodes, vagina, cervix, uterus, and fallopian tubes. Anti-MVA IgG and IgA were detected in cervicovaginal fluid after a second vaccine dose. Thus, systemic vaccination with an MVA vector elicits cellular and Ab responses in the FRT. Copyright © 2017 by The American Association of Immunologists, Inc.

Changes in the mitochondrial network during ectromelia virus infection of permissive L929 cells.

PubMed

Gregorczyk, Karolina P; Szulc-Dąbrowska, Lidia; Wyżewski, Zbigniew; Struzik, Justyna; Niemiałtowski, Marek

2014-01-01

Mitochondria are extremely important organelles in the life of a cell. Recent studies indicate that mitochondria also play a fundamental role in the cellular innate immune mechanisms against viral infections. Moreover, mitochondria are able to alter their shape continuously through fusion and fission. These tightly regulated processes are activated or inhibited under physiological or pathological (e.g. viral infection) conditions to help restore homeostasis. However, many types of viruses, such as orthopoxviruses, have developed various strategies to evade the mitochondrial-mediated antiviral innate immune responses. Moreover, orthopoxviruses exploit the mitochondria for their survival. Such viral activity has been reported during vaccinia virus (VACV) infection. Our study shows that the Moscow strain of ectromelia virus (ECTV-MOS), an orthopoxvirus, alters the mitochondrial network in permissive L929 cells. Upon infection, the branching structure of the mitochondrial network collapses and becomes disorganized followed by destruction of mitochondrial tubules during the late stage of infection. Small, discrete mitochondria co-localize with progeny virions, close to the cell membrane. Furthermore, clustering of mitochondria is observed around viral factories, particularly between the nucleus and viroplasm. Our findings suggest that ECTV-MOS modulates mitochondrial cellular distribution during later stages of the replication cycle, probably enabling viral replication and/or assembly as well as transport of progeny virions inside the cell. However, this requires further investigation.

Preclinical Testing Oncolytic Vaccinia Virus Strain GLV-5b451 Expressing an Anti-VEGF Single-Chain Antibody for Canine Cancer Therapy

PubMed Central

Adelfinger, Marion; Bessler, Simon; Frentzen, Alexa; Cecil, Alexander; Langbein-Laugwitz, Johanna; Gentschev, Ivaylo; Szalay, Aladar A.

2015-01-01

Virotherapy on the basis of oncolytic vaccinia virus (VACV) strains is a novel approach for canine cancer therapy. Here we describe, for the first time, the characterization and the use of VACV strain GLV-5b451 expressing the anti-vascular endothelial growth factor (VEGF) single-chain antibody (scAb) GLAF-2 as therapeutic agent against different canine cancers. Cell culture data demonstrated that GLV-5b451 efficiently infected and destroyed all four tested canine cancer cell lines including: mammary carcinoma (MTH52c), mammary adenoma (ZMTH3), prostate carcinoma (CT1258), and soft tissue sarcoma (STSA-1). The GLV-5b451 virus-mediated production of GLAF-2 antibody was observed in all four cancer cell lines. In addition, this antibody specifically recognized canine VEGF. Finally, in canine soft tissue sarcoma (CSTS) xenografted mice, a single systemic administration of GLV-5b451 was found to be safe and led to anti-tumor effects resulting in the significant reduction and substantial long-term inhibition of tumor growth. A CD31-based immuno-staining showed significantly decreased neo-angiogenesis in GLV-5b451-treated tumors compared to the controls. In summary, these findings indicate that GLV-5b451 has potential for use as a therapeutic agent in the treatment of CSTS. PMID:26205404

Role of Metalloproteases in Vaccinia Virus Epitope Processing for Transporter Associated with Antigen Processing (TAP)-independent Human Leukocyte Antigen (HLA)-B7 Class I Antigen Presentation*

PubMed Central

Lorente, Elena; García, Ruth; Mir, Carmen; Barriga, Alejandro; Lemonnier, François A.; Ramos, Manuel; López, Daniel

2012-01-01

The transporter associated with antigen processing (TAP) translocates the viral proteolytic peptides generated by the proteasome and other proteases in the cytosol to the endoplasmic reticulum lumen. There, they complex with nascent human leukocyte antigen (HLA) class I molecules, which are subsequently recognized by the CD8+ lymphocyte cellular response. However, individuals with nonfunctional TAP complexes or tumor or infected cells with blocked TAP molecules are able to present HLA class I ligands generated by TAP-independent processing pathways. Herein, using a TAP-independent polyclonal vaccinia virus-polyspecific CD8+ T cell line, two conserved vaccinia-derived TAP-independent HLA-B*0702 epitopes were identified. The presentation of these epitopes in normal cells occurs via complex antigen-processing pathways involving the proteasome and/or different subsets of metalloproteinases (amino-, carboxy-, and endoproteases), which were blocked in infected cells with specific chemical inhibitors. These data support the hypothesis that the abundant cellular proteolytic systems contribute to the supply of peptides recognized by the antiviral cellular immune response, thereby facilitating immunosurveillance. These data may explain why TAP-deficient individuals live normal life spans without any increased susceptibility to viral infections. PMID:22298786

Postchallenge Administration of Brincidofovir Protects Healthy and Immune-Deficient Mice Reconstituted with Limited Numbers of T Cells from Lethal Challenge with IHD-J-Luc Vaccinia Virus

PubMed Central

McCullough, Kevin Tyler; Cruz, Stephanie; Thomas, Antonia; Diaz, Claudia G.; Keilholz, Laurie; Grossi, Irma M.; Trost, Lawrence C.; Golding, Hana

2015-01-01

ABSTRACT Protection from lethality by postchallenge administration of brincidofovir (BCV, CMX001) was studied in normal and immune-deficient (nude, nu/nu) BALB/c mice infected with vaccinia virus (VACV). Whole-body bioluminescence imaging was used to record total fluxes in the nasal cavity, lungs, spleen, and liver and to enumerate pox lesions on tails of mice infected via the intranasal route with 105 PFU of recombinant IHD-J-Luc VACV expressing luciferase. Areas under the flux curve (AUCs) were calculated for individual mice to assess viral loads. A three-dose regimen of 20 mg/kg BCV administered every 48 h starting either on day 1 or day 2 postchallenge protected 100% of mice. Initiating BCV treatment earlier was more efficient in reducing viral loads and in providing protection from pox lesion development. All BCV-treated mice that survived challenge were also protected from rechallenge with IHD-J-Luc or WRvFire VACV without additional treatment. In immune-deficient mice, BCV protected animals from lethality and reduced viral loads while animals were on the drug. Viral recrudescence occurred within 4 to 9 days, and mice succumbed ∼10 to 20 days after treatment termination. Nude mice reconstituted with 105 T cells prior to challenge with 104 PFU of IHD-J-Luc and treated with BCV postchallenge survived the infection, cleared the virus from all organs, and survived rechallenge with 105 PFU of IHD-J-Luc VACV without additional BCV treatment. Together, these data suggest that BCV protects immunocompetent and partially T cell-reconstituted immune-deficient mice from lethality, reduces viral dissemination in organs, prevents pox lesion development, and permits generation of VACV-specific memory. IMPORTANCE Mass vaccination is the primary element of the public health response to a smallpox outbreak. In addition to vaccination, however, antiviral drugs are required for individuals with uncertain exposure status to smallpox or for whom vaccination is contraindicated

Variola virus immune evasion design: expression of a highly efficient inhibitor of human complement.

PubMed

Rosengard, Ariella M; Liu, Yu; Nie, Zhiping; Jimenez, Robert

2002-06-25

Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans and has no other animal reservoir. Variola causes the contagious disease smallpox, which has a 30-40% mortality rate. Conversely, the prototype orthopoxvirus, vaccinia, causes no disease in immunocompetent humans and was used in the global eradication of smallpox, which ended in 1977. However, the threat of smallpox persists because clandestine stockpiles of variola still exist. Although variola and vaccinia share remarkable DNA homology, the strict human tropism of variola suggests that its proteins are better suited than those of vaccinia to overcome the human immune response. Here, we demonstrate the functional advantage of a variola complement regulatory protein over that of its vaccinia homologue. Because authentic variola proteins are not available for study, we molecularly engineered and characterized the smallpox inhibitor of complement enzymes (SPICE), a homologue of a vaccinia virulence factor, vaccinia virus complement control protein (VCP). SPICE is nearly 100-fold more potent than VCP at inactivating human C3b and 6-fold more potent at inactivating C4b. SPICE is also more human complement-specific than is VCP. By inactivating complement components, SPICE serves to inhibit the formation of the C3/C5 convertases necessary for complement-mediated viral clearance. SPICE provides the first evidence that variola proteins are particularly adept at overcoming human immunity, and the decreased function of VCP suggests one reason why the vaccinia virus vaccine was associated with relatively low mortality. Disabling SPICE may be therapeutically useful if smallpox reemerges.

Variola virus immune evasion design: Expression of a highly efficient inhibitor of human complement

PubMed Central

Rosengard, Ariella M.; Liu, Yu; Nie, Zhiping; Jimenez, Robert

2002-01-01

Variola virus, the most virulent member of the genus Orthopoxvirus, specifically infects humans and has no other animal reservoir. Variola causes the contagious disease smallpox, which has a 30–40% mortality rate. Conversely, the prototype orthopoxvirus, vaccinia, causes no disease in immunocompetent humans and was used in the global eradication of smallpox, which ended in 1977. However, the threat of smallpox persists because clandestine stockpiles of variola still exist. Although variola and vaccinia share remarkable DNA homology, the strict human tropism of variola suggests that its proteins are better suited than those of vaccinia to overcome the human immune response. Here, we demonstrate the functional advantage of a variola complement regulatory protein over that of its vaccinia homologue. Because authentic variola proteins are not available for study, we molecularly engineered and characterized the smallpox inhibitor of complement enzymes (SPICE), a homologue of a vaccinia virulence factor, vaccinia virus complement control protein (VCP). SPICE is nearly 100-fold more potent than VCP at inactivating human C3b and 6-fold more potent at inactivating C4b. SPICE is also more human complement-specific than is VCP. By inactivating complement components, SPICE serves to inhibit the formation of the C3/C5 convertases necessary for complement-mediated viral clearance. SPICE provides the first evidence that variola proteins are particularly adept at overcoming human immunity, and the decreased function of VCP suggests one reason why the vaccinia virus vaccine was associated with relatively low mortality. Disabling SPICE may be therapeutically useful if smallpox reemerges. PMID:12034872

ACAM2000 clonal Vero cell culture vaccinia virus (New York City Board of Health strain)--a second-generation smallpox vaccine for biological defense.

PubMed

Monath, Thomas P; Caldwell, Joseph R; Mundt, Wolfgang; Fusco, Joan; Johnson, Casey S; Buller, Mark; Liu, Jian; Gardner, Bridget; Downing, Greg; Blum, Paul S; Kemp, Tracy; Nichols, Richard; Weltzin, Richard

2004-10-01

The threat of smallpox as a biological weapon has spurred efforts to create stockpiles of vaccine for emergency preparedness. In lieu of preparing vaccine in animal skin (the original method), we cloned vaccinia virus (New York City Board of Health strain, Dryvax by plaque purification and amplified the clone in cell culture. The overarching goal was to produce a modern vaccine that was equivalent to the currently licensed Dryvax in its preclinical and clinical properties, and could thus reliably protect humans against smallpox. A variety of clones were evaluated, and many were unacceptably virulent in animal models. One clonal virus (ACAM1000) was selected and produced at clinical grade in MRC-5 human diploid cells. ACAM1000 was comparable to Dryvax in immunogenicity and protective activity but was less neurovirulent for mice and nonhuman primates. To meet requirements for large quantities of vaccine after the events of September 11th 2001, the ACAM1000 master virus seed was used to prepare vaccine (designated ACAM2000) at large scale in Vero cells under serum-free conditions. The genomes of ACAM1000 and ACAM2000 had identical nucleotide sequences, and the vaccines had comparable biological phenotypes. ACAM1000 and ACAM2000 were evaluated in three Phase 1 clinical trials. The vaccines produced major cutaneous reactions and evoked neutralizing antibody and cell-mediated immune responses in the vast majority of subjects and had a reactogenicity profile similar to that of Dryvax.

Humoral Immunity to Primary Smallpox Vaccination: Impact of Childhood versus Adult Immunization on Vaccinia Vector Vaccine Development in Military Populations.

PubMed

Slike, Bonnie M; Creegan, Matthew; Marovich, Mary; Ngauy, Viseth

2017-01-01

Modified Vaccinia virus has been shown to be a safe and immunogenic vector platform for delivery of HIV vaccines. Use of this vector is of particular importance to the military, with the implementation of a large scale smallpox vaccination campaign in 2002 in active duty and key civilian personnel in response to potential bioterrorist activities. Humoral immunity to smallpox vaccination was previously shown to be long lasting (up to 75 years) and protective. However, using vaccinia-vectored vaccine delivery for other diseases on a background of anti-vector antibodies (i.e. pre-existing immunity) may limit their use as a vaccine platform, especially in the military. In this pilot study, we examined the durability of vaccinia antibody responses in adult primary vaccinees in a healthy military population using a standard ELISA assay and a novel dendritic cell neutralization assay. We found binding and neutralizing antibody (NAb) responses to vaccinia waned after 5-10 years in a group of 475 active duty military, born after 1972, who were vaccinated as adults with Dryvax®. These responses decreased from a geometric mean titer (GMT) of 250 to baseline (<20) after 10-20 years post vaccination. This contrasted with a comparator group of adults, ages 35-49, who were vaccinated with Dryvax® as children. In the childhood vaccinees, titers persisted for >30 years with a GMT of 210 (range 112-3234). This data suggests limited durability of antibody responses in adult vaccinees compared to those vaccinated in childhood and further that adult vaccinia recipients may benefit similarly from receipt of a vaccinia based vaccine as those who are vaccinia naïve. Our findings may have implications for the smallpox vaccination schedule and support the ongoing development of this promising viral vector in a military vaccination program.

Humoral Immunity to Primary Smallpox Vaccination: Impact of Childhood versus Adult Immunization on Vaccinia Vector Vaccine Development in Military Populations

PubMed Central

Slike, Bonnie M.; Creegan, Matthew

2017-01-01

Modified Vaccinia virus has been shown to be a safe and immunogenic vector platform for delivery of HIV vaccines. Use of this vector is of particular importance to the military, with the implementation of a large scale smallpox vaccination campaign in 2002 in active duty and key civilian personnel in response to potential bioterrorist activities. Humoral immunity to smallpox vaccination was previously shown to be long lasting (up to 75 years) and protective. However, using vaccinia-vectored vaccine delivery for other diseases on a background of anti-vector antibodies (i.e. pre-existing immunity) may limit their use as a vaccine platform, especially in the military. In this pilot study, we examined the durability of vaccinia antibody responses in adult primary vaccinees in a healthy military population using a standard ELISA assay and a novel dendritic cell neutralization assay. We found binding and neutralizing antibody (NAb) responses to vaccinia waned after 5–10 years in a group of 475 active duty military, born after 1972, who were vaccinated as adults with Dryvax®. These responses decreased from a geometric mean titer (GMT) of 250 to baseline (<20) after 10–20 years post vaccination. This contrasted with a comparator group of adults, ages 35–49, who were vaccinated with Dryvax® as children. In the childhood vaccinees, titers persisted for >30 years with a GMT of 210 (range 112–3234). This data suggests limited durability of antibody responses in adult vaccinees compared to those vaccinated in childhood and further that adult vaccinia recipients may benefit similarly from receipt of a vaccinia based vaccine as those who are vaccinia naïve. Our findings may have implications for the smallpox vaccination schedule and support the ongoing development of this promising viral vector in a military vaccination program. PMID:28046039

Transforming growth factor alpha, Shope fibroma growth factor, and vaccinia growth factor can replace myxoma growth factor in the induction of myxomatosis in rabbits.

PubMed

Opgenorth, A; Nation, N; Graham, K; McFadden, G

1993-02-01

The epidermal growth factor (EGF) homologues encoded by vaccinia virus, myxoma virus, and malignant rabbit fibroma virus have been shown to contribute to the pathogenicity of virus infection upon inoculation of susceptible hosts. However, since the primary structures of these growth factors and the disease profiles induced by different poxvirus genera vary substantially, the degree to which the various EGF homologues perform similar roles in viral pathogenesis remains unclear. In order to determine whether different EGF-like growth factors can perform qualitatively similar functions in the induction of myxomatosis in rabbits, we created recombinant myxoma virus variants in which the native growth factor, myxoma growth factor (MGF), was disrupted and replaced with either vaccinia virus growth factor, Shope fibroma growth factor, or rat transforming growth factor alpha. Unlike the control virus containing an inactivated MGF gene, which caused marked attenuation of the disease syndrome and substantially less proliferation of the epithelial cell layers in the conjunctiva and respiratory tract, the recombinant myxoma virus strains expressing heterologous growth factors produced infections which were both clinically and histopathologically indistinguishable from wild-type myxomatosis. We conclude that these poxviral and cellular EGF-like growth factors, which are diverse with respect to primary structure and origin, have similar biological functions in the context of myxoma virus pathogenesis and are mitogenic for the same target cells.

Daily ingestion of the probiotic Lactobacillus paracasei ST11 decreases Vaccinia virus dissemination and lethality in a mouse model.

PubMed

Dos Santos Pereira Andrade, A C; Lima, M Teixeira; Oliveira, G Pereira; Calixto, R Silva; de Sales E Souza, É Lorenna; da Glória de Souza, D; de Almeida Leite, C M; Ferreira, J M Siqueira; Kroon, E G; de Oliveira, D Bretas; Dos Santos Martins, F; Abrahão, J S

2017-02-07

Vaccinia virus (VACV) is an important pathogen. Although studies have shown relationships between probiotics and viruses, the effect of probiotics on VACV infection is unknown. Therefore, this work aims to investigate the probiotics effects on VACV infection. Mice were divided into four groups, two non-infected groups, one receiving the probiotic, the other one not receiving it, and two groups infected intranasally with VACV Western Reserve (VACV-WR) receiving or not receiving the probiotic. Viral titres in organs and cytokine production in the lungs were analysed. Lung samples were also subjected to histological analysis. The intake of probiotic results in reduction in viral spread with a significant decrease of VACV titer on lung, liver and brain of treated group. In addition,treatment with the probiotic results in attenuated mice lung inflammation showing fewer lesions on histological findings and decreased lethality in mice infected with VACV. The ingestion of Lactobacillus paracasei ST11 (LPST11) after VACV infection resulted in 2/9 animal lethality compared with 4/9 in the VACV group. This is the first study on probiotics and VACV interactions, providing not only information about this interaction, but also proposing a model for future studies involving probiotics and other poxvirus.

Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

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

Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert

Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

Binding of undamaged double stranded DNA to vaccinia virus uracil-DNA glycosylase

DOE PAGES

Schormann, Norbert; Banerjee, Surajit; Ricciardi, Robert; ...

2015-06-02

Background: Uracil-DNA glycosylases are evolutionarily conserved DNA repair enzymes. However, vaccinia virus uracil-DNA glycosylase (known as D4), also serves as an intrinsic and essential component of the processive DNA polymerase complex during DNA replication. In this complex D4 binds to a unique poxvirus specific protein A20 which tethers it to the DNA polymerase. At the replication fork the DNA scanning and repair function of D4 is coupled with DNA replication. So far, DNA-binding to D4 has not been structurally characterized. Results: This manuscript describes the first structure of a DNA-complex of a uracil-DNA glycosylase from the poxvirus family. This alsomore » represents the first structure of a uracil DNA glycosylase in complex with an undamaged DNA. In the asymmetric unit two D4 subunits bind simultaneously to complementary strands of the DNA double helix. Each D4 subunit interacts mainly with the central region of one strand. DNA binds to the opposite side of the A20-binding surface on D4. In comparison of the present structure with the structure of uracil-containing DNA-bound human uracil-DNA glycosylase suggests that for DNA binding and uracil removal D4 employs a unique set of residues and motifs that are highly conserved within the poxvirus family but different in other organisms. Conclusion: The first structure of D4 bound to a truly non-specific undamaged double-stranded DNA suggests that initial binding of DNA may involve multiple non-specific interactions between the protein and the phosphate backbone.« less

Transmission of vaccinia virus, possibly through sexual contact, to a woman at high risk for adverse complications.

PubMed

Said, Maria A; Haile, Charles; Palabindala, Venkataraman; Barker, Naomi; Myers, Robert; Thompson, Ruth; Wilson, Lucy; Allan-Martinez, Frances; Montgomery, Jay; Monroe, Benjamin; Tack, Danielle; Reynolds, Mary; Damon, Inger; Blythe, David

2013-12-01

Severe adverse events, including eczema vaccinatum (EV), can result after smallpox vaccination. Persons at risk for EV include those with underlying dermatologic conditions, such as atopic dermatitis. We investigated a case of vaccinia infection, possibly acquired during sexual contact with a recently vaccinated military service member, in a female Maryland resident with atopic dermatitis. The U.S. Department of Defense's Vaccine Healthcare Centers Network (VHCN) and the Centers for Disease Control and Prevention (CDC) worked in conjunction with the patient's physician and the Maryland Department of Health and Mental Hygiene (DHMH) to confirm the diagnosis, ensure treatment, and prevent further transmission. Specimens collected from the patient were tested at the DHMH laboratories and were positive by real-time polymerase chain reaction for nonvariola orthopoxvirus. Testing at the CDC verified the presence of vaccinia-specific DNA signatures. Continuing spread of the patient's lesions led to the administration of vaccinia immune globulin and strict infection control measures to prevent tertiary transmission to vulnerable family members, also with atopic dermatitis. VHCN contacted the service member to reinforce vaccination site care and hygiene. This case underscores the importance of prevaccination education for those receiving the smallpox vaccine to protect contacts at risk for developing severe adverse reactions. Reprint & Copyright © 2013 Association of Military Surgeons of the U.S.

A new nucleoside analog, 9-[[2-hydroxy-1-(hydroxymethyl)ethoxyl]methyl]guanine, highly active in vitro against herpes simplex virus types 1 and 2.

PubMed Central

Smith, K O; Galloway, K S; Kennell, W L; Ogilvie, K K; Radatus, B K

1982-01-01

A novel nucleoside analog, 9-[[2-hydroxy-1-(hydroxymethyl)ethoxy]methyl]-guanine (BIOLF-62), was found to have potent antiviral activity against herpes simplex virus types 1 and 2 at concentrations well below cytotoxic levels. For example, the Patton strain of herpes simplex virus type 1 was susceptible at concentrations 140- to 2,900-fold below that which inhibited cell division by 50%, depending upon the cell line used for assay. Different herpesvirus strains varied considerably in their susceptibility to the drug, as did results obtained with the same virus strain in different cell lines. BIOLF-62 compared favorably with 5-iodo-2'-deoxyuridine and acyclovir with respect to ratios of viral to cell inhibitory drug concentrations. Patterns of drug resistance to herpesvirus mutants suggested that the primary mode of action of BIOLF-62 is different from that of known antiviral compounds. Human adenovirus type 2, varicella-zoster virus, and Epstein-Barr virus were inhibited by this drug but at concentrations within the cell inhibitory range. Vaccinia virus and human cytomegalovirus were not inhibited at high drug concentrations. PMID:6289741

Intratumoral INF-γ triggers an antiviral state in GL261 tumor cells: a major hurdle to overcome for oncolytic vaccinia virus therapy of cancer.

PubMed

Kober, Christina; Weibel, Stephanie; Rohn, Susanne; Kirscher, Lorenz; Szalay, Aladar A

2015-01-01

Oncolytic vaccinia virus (VACV) therapy is an alternative treatment option for glioblastoma multiforme. Here, we used a comparison of different tumor locations and different immunologic and genetic backgrounds to determine the replication efficacy and oncolytic potential of the VACV LIVP 1.1.1, an attenuated wild-type isolate of the Lister strain, in murine GL261 glioma models. With this approach, we expected to identify microenvironmental factors, which may be decisive for failure or success of oncolytic VACV therapy. We found that GL261 glioma cells implanted subcutaneously or orthotopically into Balb/c athymic, C57BL/6 athymic, or C57BL/6 wild-type mice formed individual tumors that respond to oncolytic VACV therapy with different outcomes. Surprisingly, only Balb/c athymic mice with subcutaneous tumors supported viral replication. We identified intratumoral IFN-γ expression levels that upregulate MHCII expression on GL261 cells in C57BL/6 wild-type mice associated with a non-permissive status of the tumor cells. Moreover, this IFN-γ-induced tumor cell phenotype was reversible.

Smallpox and live-virus vaccination in transplant recipients.

PubMed

Fishman, Jay A

2003-07-01

Recent bioterrorism raises the specter of reemergence of smallpox as a clinical entity. The mortality of variola major infection ('typical smallpox') was approximately 30% in past outbreaks. Programs for smallpox immunization for healthcare workers have been proposed. Atypical forms of smallpox presenting with flat or hemorrhagic skin lesions are most common in individuals with immune deficits with historic mortality approaching 100%. Smallpox vaccination, even after exposure, is highly effective. Smallpox vaccine contains a highly immunogenic live virus, vaccinia. Few data exist for the impact of variola or safety of vaccinia in immunocompromised hosts. Both disseminated infection by vaccinia and person-to-person spread after vaccination are uncommon. When it occurs, secondary vaccinia has usually affected individuals with pre-existing skin conditions (atopic dermatitis or eczema) or with other underlying immune deficits. Historically, disseminated vaccinia infection was uncommon but often fatal even in the absence of the most severe form of disease, "progressive vaccinia". Some responded to vaccinia immune globulin. Smallpox exposure would be likely to cause significant mortality among immunocompromised hosts. In the absence of documented smallpox exposures, immunocompromised hosts should not be vaccinated against smallpox. Planning for bioterrorist events must include consideration of uniquely susceptible hosts.

Comparison of the Transcription and Replication Strategies of Marburg Virus and Ebola Virus by Using Artificial Replication Systems

PubMed Central

Mühlberger, Elke; Weik, Michael; Volchkov, Viktor E.; Klenk, Hans-Dieter; Becker, Stephan

1999-01-01

The members of the family Filoviridae, Marburg virus (MBGV) and Ebola virus (EBOV), are very similar in terms of morphology, genome organization, and protein composition. To compare the replication and transcription strategies of both viruses, an artificial replication system based on the vaccinia virus T7 expression system was established for EBOV. Specific transcription and replication of an artificial monocistronic minireplicon was demonstrated by reporter gene expression and detection of the transcribed and replicated RNA species. As it was shown previously for MBGV, three of the four EBOV nucleocapsid proteins, NP, VP35, and L, were essential and sufficient for replication. In contrast to MBGV, EBOV-specific transcription was dependent on the presence of the fourth nucleocapsid protein, VP30. When EBOV VP30 was replaced by MBGV VP30, EBOV-specific transcription was observed but with lower efficiency. Exchange of NP, VP35, and L between the two replication systems did not lead to detectable reporter gene expression. It was further observed that neither MBGV nor EBOV were able to replicate the heterologous minigenomes. A chimeric minigenome, however, containing the EBOV leader and the MBGV trailer was encapsidated, replicated, transcribed, and packaged by both viruses. PMID:9971816

Homosubtypic and heterosubtypic antibodies against highly pathogenic avian influenza H5N1 recombinant proteins in H5N1 survivors and non-H5N1 subjects.

PubMed

Noisumdaeng, Pirom; Pooruk, Phisanu; Prasertsopon, Jarunee; Assanasen, Susan; Kitphati, Rungrueng; Auewarakul, Prasert; Puthavathana, Pilaipan

2014-04-01

Six recombinant vaccinia viruses containing HA, NA, NP, M or NS gene insert derived from a highly pathogenic avian influenza H5N1 virus, and the recombinant vaccinia virus harboring plasmid backbone as the virus control were constructed. The recombinant proteins were characterized for their expression and subcellular locations in TK(-) cells. Antibodies to the five recombinant proteins were detected in all 13 sequential serum samples collected from four H5N1 survivors during four years of follow-up; and those directed to rVac-H5 HA and rVac-NA proteins were found in higher titers than those directed to the internal proteins as revealed by indirect immunofluorescence assay. Although all 28 non-H5N1 subjects had no neutralizing antibodies against H5N1 virus, they did have cross-reactive antibodies to those five recombinant proteins. A significant increase in cross-reactive antibody titer to rVac-H5 HA and rVac-NA was found in paired blood samples from patients infected with the 2009 pandemic virus. Copyright © 2014 Elsevier Inc. All rights reserved.

Identification and Characterization of the UL37 Protein of Herpes Simplex Virus Type 1 and Demonstration that it Interacts with ICP8, the Major DNA Binding Protein of Herpes Simplex Virus

DTIC Science & Technology

1992-10-20

Identification of ORFs HSV DNA binding proteins • 1 3 3 5 7 7 11 17 18 22 reps and its role in HSV replication 23 Biochemical properties . . 23...Figure 1 . 2. 3 • 4. 5. 6. 7. 8. Structural model of the herpesvirus virion Schematic diagram of HSV pathogenesis . Diagram of the main...vaccinia virus- 13. Autoradiogram of an immunoblot of HSV - 1 -infected cell proteins harvested at various times postinfec- 85 tioD probed with anti-UL42

Maturation of the Hepatitis A Virus Capsid Protein VP1 Is Not Dependent on Processing by the 3Cpro Proteinase

PubMed Central

Martin, Annette; Bénichou, Danièle; Chao, Shih-Fong; Cohen, Lisette M.; Lemon, Stanley M.

1999-01-01

Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the family Picornaviridae, the primary cleavage of the HAV polyprotein is mediated by 3Cpro, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3Cpro, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3Cpro proteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3Cpro that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3Cpro recognition criteria and found that a substitution that ablates the presumed 3Cpro dipeptide recognition sequence at Glu764-Ser765 abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg764 substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3Cpro processing and may thus be uniquely dependent on a cellular proteinase. PMID:10400711

Viral Vectors for Use in the Development of Biodefense Vaccines

DTIC Science & Technology

2005-06-17

vaccinia virus, and Venezuelan equine encephalitis virus, as vaccine vectors has enabled researchers to develop effective means for countering the...biowarfare. The use of viruses, for example adenovirus, vaccinia virus, and Venezuelan equine encephalitis virus, as vaccine -vectors has enabled researchers to... vaccines . . . . . . . . . . . . . . . . . . . 1298 2.1.3. Vaccinia virus-vectored Venezuelan equine encephalitis vaccines

Oncolytic vaccinia virus combined with radiotherapy induces apoptotic cell death in sarcoma cells by down-regulating the inhibitors of apoptosis

PubMed Central

Wilkinson, Michelle J.; Smith, Henry G.; McEntee, Gráinne; Kyula-Currie, Joan; Mansfield, David C.; Khan, Aadil A.; Roulstone, Victoria

2016-01-01

Advanced extremity melanoma and sarcoma present a significant therapeutic challenge, requiring multimodality therapy to treat or even palliate disease. These aggressive tumours are relatively chemo-resistant, therefore new treatment approaches are urgently required. We have previously reported on the efficacy of oncolytic virotherapy (OV) delivered by isolated limb perfusion. In this report, we have improved therapeutic outcomes by combining OV with radiotherapy. In vitro, the combination of oncolytic vaccinia virus (GLV-1h68) and radiotherapy demonstrated synergistic cytotoxicity. This effect was not due to increased viral replication, but mediated through induction of intrinsic apoptosis. GLV-1h68 therapy downregulated the anti-apoptotic BCL-2 proteins (MCL-1 and BCL-XL) and the downstream inhibitors of apoptosis, resulting in cleavage of effector caspases 3 and 7. In an in vivo ILP model, the combination of OV and radiotherapy significantly delayed tumour growth and prolonged survival compared to single agent therapy. These data suggest that the virally-mediated down-regulation of anti-apoptotic proteins may increase the sensitivity of tumour cells to the cytotoxic effects of ionizing radiation. Oncolytic virotherapy represents an exciting candidate for clinical development when delivered by ILP. Its ability to overcome anti-apoptotic signals within tumour cells points the way to further development in combination with conventional anti-cancer therapies. PMID:27783991

Immunization with a Recombinant Vaccinia Virus That Encodes Nonstructural Proteins of the Hepatitis C Virus Suppresses Viral Protein Levels in Mouse Liver

PubMed Central

Sekiguchi, Satoshi; Kimura, Kiminori; Chiyo, Tomoko; Ohtsuki, Takahiro; Tobita, Yoshimi; Tokunaga, Yuko; Yasui, Fumihiko; Tsukiyama-Kohara, Kyoko; Wakita, Takaji; Tanaka, Toshiyuki; Miyasaka, Masayuki; Mizuno, Kyosuke; Hayashi, Yukiko; Hishima, Tsunekazu; Matsushima, Kouji; Kohara, Michinori

2012-01-01

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid–polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29(+/−)/MxCre(+/−) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine. PMID:23284733

Immunization with a recombinant vaccinia virus that encodes nonstructural proteins of the hepatitis C virus suppresses viral protein levels in mouse liver.

PubMed

Sekiguchi, Satoshi; Kimura, Kiminori; Chiyo, Tomoko; Ohtsuki, Takahiro; Tobita, Yoshimi; Tokunaga, Yuko; Yasui, Fumihiko; Tsukiyama-Kohara, Kyoko; Wakita, Takaji; Tanaka, Toshiyuki; Miyasaka, Masayuki; Mizuno, Kyosuke; Hayashi, Yukiko; Hishima, Tsunekazu; Matsushima, Kouji; Kohara, Michinori

2012-01-01

Chronic hepatitis C, which is caused by infection with the hepatitis C virus (HCV), is a global health problem. Using a mouse model of hepatitis C, we examined the therapeutic effects of a recombinant vaccinia virus (rVV) that encodes an HCV protein. We generated immunocompetent mice that each expressed multiple HCV proteins via a Cre/loxP switching system and established several distinct attenuated rVV strains. The HCV core protein was expressed consistently in the liver after polyinosinic acid-polycytidylic acid injection, and these mice showed chronic hepatitis C-related pathological findings (hepatocyte abnormalities, accumulation of glycogen, steatosis), liver fibrosis, and hepatocellular carcinoma. Immunization with one rVV strain (rVV-N25), which encoded nonstructural HCV proteins, suppressed serum inflammatory cytokine levels and alleviated the symptoms of pathological chronic hepatitis C within 7 days after injection. Furthermore, HCV protein levels in liver tissue also decreased in a CD4 and CD8 T-cell-dependent manner. Consistent with these results, we showed that rVV-N25 immunization induced a robust CD8 T-cell immune response that was specific to the HCV nonstructural protein 2. We also demonstrated that the onset of chronic hepatitis in CN2-29((+/-))/MxCre((+/-)) mice was mainly attributable to inflammatory cytokines, (tumor necrosis factor) TNF-α and (interleukin) IL-6. Thus, our generated mice model should be useful for further investigation of the immunological processes associated with persistent expression of HCV proteins because these mice had not developed immune tolerance to the HCV antigen. In addition, we propose that rVV-N25 could be developed as an effective therapeutic vaccine.

Survival of viral biowarfare agents in disinfected waters.

PubMed

Wade, Mary Margaret; Chambers, Amanda E; Insalaco, Joseph M; Zulich, Alan W

2010-01-01

Protecting civilian and military water supplies has received more attention since the United States began its war on terror in 2001. Both chlorine and bromine are used by branches of the U.S. military for disinfecting water supplies; however, limited data exists as to the effectiveness of these additives when used against viral biowarfare agents. The present study sought to evaluate the survival of selected viral biothreat agents in disinfected water. Disinfected water samples were spiked with vaccinia virus strain WR and Venezuelan equine encephalitis (VEE) virus strain TC-83 each separately to a final concentration of approximately 1 × 10(6) PFU/mL, and survival was assessed by plaque assay. Both viruses were inactivated by 1 mg/L free available chlorine (FAC) and 2mg/L total bromine within one hour. In conclusion, these results demonstrate that both chlorine and bromine are effective disinfectants against vaccinia virus and VEE strain TC-83 at the concentrations tested.

Survival of Viral Biowarfare Agents in Disinfected Waters

PubMed Central

Wade, Mary Margaret; Chambers, Amanda E.; Insalaco, Joseph M.; Zulich, Alan W.

2010-01-01

Protecting civilian and military water supplies has received more attention since the United States began its war on terror in 2001. Both chlorine and bromine are used by branches of the U.S. military for disinfecting water supplies; however, limited data exists as to the effectiveness of these additives when used against viral biowarfare agents. The present study sought to evaluate the survival of selected viral biothreat agents in disinfected water. Disinfected water samples were spiked with vaccinia virus strain WR and Venezuelan equine encephalitis (VEE) virus strain TC-83 each separately to a final concentration of approximately 1 × 106 PFU/mL, and survival was assessed by plaque assay. Both viruses were inactivated by 1 mg/L free available chlorine (FAC) and 2mg/L total bromine within one hour. In conclusion, these results demonstrate that both chlorine and bromine are effective disinfectants against vaccinia virus and VEE strain TC-83 at the concentrations tested. PMID:21197430

Recombinant Vaccinia Viruses Coding Transgenes of Apoptosis-Inducing Proteins Enhance Apoptosis But Not Immunogenicity of Infected Tumor Cells

PubMed Central

Tkachenko, Anastasiya; Richter, Vladimir

2017-01-01

Genetic modifications of the oncolytic vaccinia virus (VV) improve selective tumor cell infection and death, as well as activation of antitumor immunity. We have engineered a double recombinant VV, coding human GM-CSF, and apoptosis-inducing protein apoptin (VV-GMCSF-Apo) for comparing with the earlier constructed double recombinant VV-GMCSF-Lact, coding another apoptosis-inducing protein, lactaptin, which activated different cell death pathways than apoptin. We showed that both these recombinant VVs more considerably activated a set of critical apoptosis markers in infected cells than the recombinant VV coding GM-CSF alone (VV-GMCSF-dGF): these were phosphatidylserine externalization, caspase-3 and caspase-7 activation, DNA fragmentation, and upregulation of proapoptotic protein BAX. However, only VV-GMCSF-Lact efficiently decreased the mitochondrial membrane potential of infected cancer cells. Investigating immunogenic cell death markers in cancer cells infected with recombinant VVs, we demonstrated that all tested recombinant VVs were efficient in calreticulin and HSP70 externalization, decrease of cellular HMGB1, and ATP secretion. The comparison of antitumor activity against advanced MDA-MB-231 tumor revealed that both recombinants VV-GMCSF-Lact and VV-GMCSF-Apo efficiently delay tumor growth. Our results demonstrate that the composition of GM-CSF and apoptosis-inducing proteins in the VV genome is very efficient tool for specific killing of cancer cells and for activation of antitumor immunity. PMID:28951871

Virus-Like Particles Displaying Trimeric Simian Immunodeficiency Virus (SIV) Envelope gp160 Enhance the Breadth of DNA/Modified Vaccinia Virus Ankara SIV Vaccine-Induced Antibody Responses in Rhesus Macaques.

PubMed

Iyer, Smita S; Gangadhara, Sailaja; Victor, Blandine; Shen, Xiaoying; Chen, Xuemin; Nabi, Rafiq; Kasturi, Sudhir P; Sabula, Michael J; Labranche, Celia C; Reddy, Pradeep B J; Tomaras, Georgia D; Montefiori, David C; Moss, Bernard; Spearman, Paul; Pulendran, Bali; Kozlowski, Pamela A; Amara, Rama Rao

2016-10-01

The encouraging results of the RV144 vaccine trial have spurred interest in poxvirus prime-protein boost human immunodeficiency virus (HIV) vaccine modalities as a strategy to induce protective immunity. Because vaccine-induced protective immunity is critically determined by HIV envelope (Env) conformation, significant efforts are directed toward generating soluble trimeric Env immunogens that assume native structures. Using the simian immunodeficiency virus (SIV)-macaque model, we tested the immunogenicity and efficacy of sequential immunizations with DNA (D), modified vaccinia virus Ankara (MVA) (M), and protein immunogens, all expressing virus-like particles (VLPs) displaying membrane-anchored trimeric Env. A single VLP protein boost displaying trimeric gp160 adjuvanted with nanoparticle-encapsulated Toll-like receptor 4/7/8 (TLR4/7/8) agonists, administered 44 weeks after the second MVA immunization, induced up to a 3-fold increase in Env-specific IgG binding titers in serum and mucosa. Importantly, the VLP protein boost increased binding antibody against scaffolded V1V2, antibody-dependent phagocytic activity against VLP-coated beads, and antibody breadth and neutralizing antibody titers against homologous and heterologous tier 1 SIVs. Following 5 weekly intrarectal SIVmac251 challenges, two of seven DNA/MVA and VLP (DM+VLP)-vaccinated animals were completely protected compared to productive infection in all seven DM-vaccinated animals. Vaccinated animals demonstrated stronger acute viral pulldown than controls, but a trend for higher acute viremia was observed in the DM+VLP group, likely due to a slower recall of Gag-specific CD8 T cells. Our findings support immunization with VLPs containing trimeric Env as a strategy to augment protective antibody but underscore the need for optimal engagement of CD8 T cells to achieve robust early viral control. The development of an effective HIV vaccine remains a global necessity for preventing HIV infection and reducing

Role of L-Particles during Herpes Simplex Virus Infection.

PubMed

Heilingloh, Christiane S; Krawczyk, Adalbert

2017-01-01

Infection of eukaryotic cells with α-herpesviruses results in the formation and secretion of infectious heavy particles (virions; H-particles) and non-infectious light particles (L-particles). Herpes simplex virus type 1 (HSV-1) H-particles consist of a genome-containing capsid surrounded by tegument proteins and a glycoprotein-rich lipid bilayer. Non-infectious L-particles are composed mainly of envelope and tegument proteins and are devoid of capsids and viral DNA. L-particles were first described in the early nineties and from then on investigated for their formation and role during virus infection. The development and secretion of L-particles occur simultaneously to the assembly of complete viral particles. HSV-1 L-particles are assembled by budding of condensed tegument into Golgi-delivered vesicles and are capable of delivering their functional content to non-infected cells. Thereby, HSV-1 L-particles contribute to viral pathogenesis within the infected host by enhancing virion infectivity and providing immune evasion functions. In this review we discuss the emergence of HSV-1 L-particles during virus replication and their biological functions described thus far.

HIV-1 adenoviral vector vaccines expressing multi-trimeric BAFF and 4-1BBL enhance T cell mediated anti-viral immunity.

PubMed

Kanagavelu, Saravana; Termini, James M; Gupta, Sachin; Raffa, Francesca N; Fuller, Katherine A; Rivas, Yaelis; Philip, Sakhi; Kornbluth, Richard S; Stone, Geoffrey W

2014-01-01

Adenoviral vectored vaccines have shown considerable promise but could be improved by molecular adjuvants. Ligands in the TNF superfamily (TNFSF) are potential adjuvants for adenoviral vector (Ad5) vaccines based on their central role in adaptive immunity. Many TNFSF ligands require aggregation beyond the trimeric state (multi-trimerization) for optimal biological function. Here we describe Ad5 vaccines for HIV-1 Gag antigen (Ad5-Gag) adjuvanted with the TNFSF ligands 4-1BBL, BAFF, GITRL and CD27L constructed as soluble multi-trimeric proteins via fusion to Surfactant Protein D (SP-D) as a multimerization scaffold. Mice were vaccinated with Ad5-Gag combined with Ad5 expressing one of the SP-D-TNFSF constructs or single-chain IL-12p70 as adjuvant. To evaluate vaccine-induced protection, mice were challenged with vaccinia virus expressing Gag (vaccinia-Gag) which is known to target the female genital tract, a major route of sexually acquired HIV-1 infection. In this system, SP-D-4-1BBL or SP-D-BAFF led to significantly reduced vaccinia-Gag replication when compared to Ad5-Gag alone. In contrast, IL-12p70, SP-D-CD27L and SP-D-GITRL were not protective. Histological examination following vaccinia-Gag challenge showed a dramatic lymphocytic infiltration into the uterus and ovaries of SP-D-4-1BBL and SP-D-BAFF-treated animals. By day 5 post challenge, proinflammatory cytokines in the tissue were reduced, consistent with the enhanced control over viral replication. Splenocytes had no specific immune markers that correlated with protection induced by SP-D-4-1BBL and SP-D-BAFF versus other groups. IL-12p70, despite lack of anti-viral efficacy, increased the total numbers of splenic dextramer positive CD8+ T cells, effector memory T cells, and effector Gag-specific CD8+ T cells, suggesting that these markers are poor predictors of anti-viral immunity in this model. In conclusion, soluble multi-trimeric 4-1BBL and BAFF adjuvants led to strong protection from vaccinia

Binding affinity of the L-742,001 inhibitor to the endonuclease domain of A/H1N1/PA influenza virus variants: Molecular simulation approaches

NASA Astrophysics Data System (ADS)

Nguyen, Hung; Nguyen, Hoang Linh; Linh, Huynh Quang; Nguyen, Minh Tho

2018-01-01

The steered molecular dynamics (SMD), molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and free energy perturbation (FEP) methods were used to determine the binding affinity of the L-742,001 inhibitor to the endonuclease domain of the A/H1N1/PA influenza viruses (including wild type (WT) and three mutations I79L, E119D and F105S for both pH1N1 PA and PR8 PA viruses). Calculated results showed that the L-742,001 inhibitor not only binds to the PR8 PAs (1934 A influenza virus) better than to the pH1N1 PAs (2009 A influenza virus) but also more strongly interacts with the WT endonuclease domain than with three mutant variants for both pH1N1 PA and PR8 PA viruses. The binding affinities obtained by the SMD, MM-PBSA and FEP methods attain high correlation with available experimental data. Here the FEP method appears to provide a more accurate determination of the binding affinity than the SMD and MM-PBSA counterparts.

Inactivation of Viruses by Benzalkonium Chloride

PubMed Central

Armstrong, J. A.; Froelich, E. J.

1964-01-01

Benzalkonium chloride (as Roccal or Zephiran) was found to inactivate influenza, measles, canine distemper, rabies, fowl laryngotracheitis, vaccinia, Semliki Forest, feline pneumonitis, meningopneumonitis, and herpes simplex viruses after 10 min of exposure at 30 C or at room temperature. Poliovirus and encephalomyocarditis virus were not inactivated under the same conditions. It was concluded that all viruses tested were sensitive except members of the picorna group. The literature was reviewed. PMID:4288740

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

PubMed

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

2008-03-01

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

Identification of Novel 5,6-Dimethoxyindan-1-one Derivatives as Antiviral Agents.

PubMed

Patil, Siddappa A; Patil, Vikrant; Patil, Renukadevi; Beaman, Kenneth; Patil, Shivaputra A

2017-01-01

Discovery of novel antiviral agents is essential because viral infection continues to threaten human life globally. Various heterocyclic small molecules have been developed as antiviral agents. The 5,6-dimethoxyindan-1-on nucleus is of considerable interest as this ring is the key constituent in a range of bioactive compounds, both naturally occurring and synthetic, and often of considerable complexity. The main purpose of this research was to discover and develop small molecule heterocycles as broad-spectrum of antiviral agents. A focused small set of 5,6-dimethoxyindan-1-one analogs (6-8) along with a thiopene derivative (9) was screened for selected viruses (Vaccinia virus - VACA, Human papillomavirus - HPV, Zika virus - ZIKV, Dengue virus - DENV, Measles virus - MV, Poliovirus 3 - PV, Rift Valley fever virus - RVFV, Tacaribe virus - TCRV, Venezuelan equine encephalitis virus - VEEV, Herpes simplex virus 1 -HSV-1 and Human cytomegalovirus - HCMV) using the National Institute of Allergy and Infectious Diseases (NIAID)'s Division of Microbiology and Infectious Diseases (DMID) antiviral screening program. These molecules demonstrated moderate to excellent antiviral activity towards variety of viruses. The 5,6-dimethoxyindan-1-one analog (7) demonstrated high efficacy towards vaccinia virus (EC50: <0.05 µM) and was nearly 232 times more potent than the standard drug Cidofovir (EC50: 11.59 µM) in primary assay whereas it demonstrated moderate activity (EC50: >30.00 µM) in secondary plaque reduction assay. The thiophene analog (9) has shown very good viral inhibition towards several viruses such as Human papillomavirus, Measles virus, Rift Valley fever virus, Tacaribe virus and Herpes simplex virus 1. Our research identified a novel 5,6-dimethoxyindan-1-one analog (compound 7), as a potent antiviral agent for vaccinia virus, and heterocyclic chalcone analog (compound 9) as a broad spectrum antiviral agent. Copyright© Bentham Science Publishers; For any queries

Monocytes Phenotype and Cytokine Production in Human Immunodeficiency Virus-1 Infected Patients Receiving a Modified Vaccinia Ankara-Based HIV-1 Vaccine: Relationship to CD300 Molecules Expression.

PubMed

Vitallé, Joana; Zenarruzabeitia, Olatz; Terrén, Iñigo; Plana, Montserrat; Guardo, Alberto C; Leal, Lorna; Peña, José; García, Felipe; Borrego, Francisco

2017-01-01

A modified vaccinia Ankara-based HIV-1 vaccine clade B (MVA-B) has been tested for safety and immunogenicity in low-risk human immunodeficiency virus (HIV)-uninfected individuals and as a therapeutic vaccine in HIV-1-infected individuals on combined antiretroviral therapy (cART). As a therapeutic vaccine, MVA-B was safe and broadly immunogenic; however, patients still showed a viral rebound upon treatment interruption. Monocytes are an important part of the viral reservoir and several studies suggest that they are partly responsible for the chronic inflammation observed in cART-treated HIV-infected people. The CD300 family of receptors has an important role in several diseases, including viral infections. Monocytes express CD300a, c, e, and f molecules and lipopolysaccharide (LPS) and other stimuli regulate their expression. However, the expression and function of CD300 receptors on monocytes in HIV infection is still unknown. In this work, we investigated for the first time the expression of CD300 molecules and the cytokine production in response to LPS on monocytes from HIV-1-infected patients before and after vaccination with MVA-B. Our results showed that CD300 receptors expression on monocytes from HIV-1-infected patients correlates with markers of HIV infection progression and immune inflammation. Specifically, we observed a positive correlation between the expression of CD300e and CD300f receptors on monocytes with the number of CD4+ T cells of HIV-1-infected patients before vaccination. We also saw a positive correlation between the expression of the inhibitory receptor CD300f and the expression of CD163 on monocytes from HIV-1-infected individuals before and after vaccination. In addition, monocytes exhibited a higher cytokine production in response to LPS after vaccination, almost at the same levels of monocytes from healthy donors. Furthermore, we also described a correlation in the expression of CD300e and CD300f receptors with TNF-α production in

Vaccinia Virus Immunomodulator A46: A Lipid and Protein-Binding Scaffold for Sequestering Host TIR-Domain Proteins

PubMed Central

Radakovics, Katharina; Smith, Terry K.; Bobik, Nina; Round, Adam; Djinović-Carugo, Kristina; Usón, Isabel

2016-01-01

Vaccinia virus interferes with early events of the activation pathway of the transcriptional factor NF-kB by binding to numerous host TIR-domain containing adaptor proteins. We have previously determined the X-ray structure of the A46 C-terminal domain; however, the structure and function of the A46 N-terminal domain and its relationship to the C-terminal domain have remained unclear. Here, we biophysically characterize residues 1–83 of the N-terminal domain of A46 and present the X-ray structure at 1.55 Å. Crystallographic phases were obtained by a recently developed ab initio method entitled ARCIMBOLDO_BORGES that employs tertiary structure libraries extracted from the Protein Data Bank; data analysis revealed an all β-sheet structure. This is the first such structure solved by this method which should be applicable to any protein composed entirely of β-sheets. The A46(1–83) structure itself is a β-sandwich containing a co-purified molecule of myristic acid inside a hydrophobic pocket and represents a previously unknown lipid-binding fold. Mass spectrometry analysis confirmed the presence of long-chain fatty acids in both N-terminal and full-length A46; mutation of the hydrophobic pocket reduced the lipid content. Using a combination of high resolution X-ray structures of the N- and C-terminal domains and SAXS analysis of full-length protein A46(1–240), we present here a structural model of A46 in a tetrameric assembly. Integrating affinity measurements and structural data, we propose how A46 simultaneously interferes with several TIR-domain containing proteins to inhibit NF-κB activation and postulate that A46 employs a bipartite binding arrangement to sequester the host immune adaptors TRAM and MyD88. PMID:27973613

Immunomodulator-based enhancement of anti smallpox immune responses.

PubMed

Martínez, Osmarie; Miranda, Eric; Ramírez, Maite; Santos, Saritza; Rivera, Carlos; Vázquez, Luis; Sánchez, Tomás; Tremblay, Raymond L; Ríos-Olivares, Eddy; Otero, Miguel

2015-01-01

The current live vaccinia virus vaccine used in the prevention of smallpox is contraindicated for millions of immune-compromised individuals. Although vaccination with the current smallpox vaccine produces protective immunity, it might result in mild to serious health complications for some vaccinees. Thus, there is a critical need for the production of a safe virus-free vaccine against smallpox that is available to everyone. For that reason, we investigated the impact of imiquimod and resiquimod (Toll-like receptors agonists), and the codon-usage optimization of the vaccinia virus A27L gene in the enhancement of the immune response, with intent of producing a safe, virus-free DNA vaccine coding for the A27 vaccinia virus protein. We analyzed the cellular-immune response by measuring the IFN-γ production of splenocytes by ELISPOT, the humoral-immune responses measuring total IgG and IgG2a/IgG1 ratios by ELISA, and the TH1 and TH2 cytokine profiles by ELISA, in mice immunized with our vaccine formulation. The proposed vaccine formulation enhanced the A27L vaccine-mediated production of IFN-γ on mouse spleens, and increased the humoral immunity with a TH1-biased response. Also, our vaccine induced a TH1 cytokine milieu, which is important against viral infections. These results support the efforts to find a new mechanism to enhance an immune response against smallpox, through the implementation of a safe, virus-free DNA vaccination platform.

Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

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

Zheng Min; Guangxi Center for Animal Disease Control and Prevention, Nanning 530001; College of Animal Science and Veterinary Medicine, Jilin University, Changchun 130062

Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AALmore » and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.« less

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

Meseda, Clement A.; Srinivasan, Kumar; Wise, Jasen

Highlights: • Heme oxygenase-1 (HO-1) induction inhibited vaccinia virus infection of macrophages. • Reduced infectivity inversely correlated with increased expression of non-coding RNAs. • The regulation of HO-1 and ncRNAs suggests a novel host defense response against vaccinia virus infection. - Abstract: Small nuclear RNAs (snRNAs) are <200 nucleotide non-coding uridylate-rich RNAs. Although the functions of many snRNAs remain undetermined, a population of snRNAs is produced during the early phase of infection of cells by vaccinia virus. In the present study, we demonstrate a direct correlation between expression of the cytoprotective enzyme heme oxygenase-1 (HO-1), suppression of selective snRNA expression,more » and inhibition of vaccinia virus infection of macrophages. Hemin induced HO-1 expression, completely reversed virus-induced host snRNA expression, and suppressed vaccinia virus infection. This involvement of specific virus-induced snRNAs and associated gene clusters suggests a novel HO-1-dependent host-defense pathway in poxvirus infection.« less

A novel mechanism of RNase L inhibition: Theiler's virus L* protein prevents 2-5A from binding to RNase L

PubMed Central

Drappier, Melissa; Elliott, Ruth; Zhang, Rong; Weiss, Susan R.; Silverman, Robert H.

2018-01-01

The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler’s murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2’-5’ oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L in vivo. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A. PMID:29652922

Vaccination with recombinant Modified Vaccinia Ankara (MVA) viruses expressing single African horse sickness virus VP2 antigens induced cross-reactive virus neutralising antibodies (VNAb) in horses when administered in combination.

PubMed

Manning, Nicola Mary; Bachanek-Bankowska, Katarzyna; Mertens, Peter Paul Clement; Castillo-Olivares, Javier

2017-10-20

African horse sickness is a lethal viral disease of equids transmitted by biting midges of the Genus Culicoides. The disease is endemic to sub-Saharan Africa but outbreaks of high mortality and economic impact have occurred in the past in non-endemic regions of Africa, Asia and Southern Europe. Vaccination is critical for the control of this disease but only live attenuated vaccines are currently available. However, there are bio-safety concerns over the use of this type of vaccines, especially in non-endemic countries, and live attenuated vaccines do not have DIVA (Differentiation of Infected from Vaccinated Animals) capacity. In addition, large scale manufacturing of live attenuated vaccines of AHSV represents a significant environmental and health risk and level 3 bio-safety containment facilities are required for their production. A variety of different technologies have been investigated over the years to develop alternative AHSV vaccines, including the use of viral vaccine vectors such Modified Vaccinia Ankara virus (MVA). In previous studies we demonstrated that recombinant MVA expressing outer capsid protein AHSV-VP2 induced virus neutralising antibodies and protection against virulent challenge both in a mouse model and in the horse. However, AHSV-VP2 is antigenically variable and determines the existence of 9 different AHSV serotypes. Immunity against AHSV is serotype-specific and there is limited cross-reactivity between certain AHSV serotypes: 1 and 2, 3 and 7, 5 and 8, 6 and 9. In Africa, multiple serotypes circulate simultaneously and a polyvalent attenuated vaccine comprising different AHSV serotypes is used. We investigated the potential of a polyvalent AHSV vaccination strategy based on combinations of MVA-VP2 viruses each expressing a single VP2 antigen from a specific serotype. We showed that administration of 2 different recombinant MVA viruses, each expressing a single VP2 protein from AHSV serotype 4 or 9, denoted respectively as MVA-VP2

A modified vaccinia Ankara vaccine vector expressing a mosaic H5 hemagglutinin reduces viral shedding in rhesus macaques.

PubMed

Florek, Nicholas W; Kamlangdee, Attapon; Mutschler, James P; Kingstad-Bakke, Brock; Schultz-Darken, Nancy; Broman, Karl W; Osorio, Jorge E; Friedrich, Thomas C

2017-01-01

The rapid antigenic evolution of influenza viruses requires frequent vaccine reformulations. Due to the economic burden of continuous vaccine reformulation and the threat of new pandemics, there is intense interest in developing vaccines capable of eliciting broadly cross-reactive immunity to influenza viruses. We recently constructed a "mosaic" hemagglutinin (HA) based on subtype 5 HA (H5) and designed to stimulate cellular and humoral immunity to multiple influenza virus subtypes. Modified vaccinia Ankara (MVA) expressing this H5 mosaic (MVA-H5M) protected mice against multiple homosubtypic H5N1 strains and a heterosubtypic H1N1 virus. To assess its potential as a human vaccine we evaluated the ability of MVA-H5M to provide heterosubtypic immunity to influenza viruses in a non-human primate model. Rhesus macaques received an initial dose of either MVA-H5M or plasmid DNA encoding H5M, followed by a boost of MVA-H5M, and then were challenged, together with naïve controls, with the heterosubtypic virus A/California/04/2009 (H1N1pdm). Macaques receiving either vaccine regimen cleared H1N1pdm challenge faster than naïve controls. Vaccination with H5M elicited antibodies that bound H1N1pdm HA, but did not neutralize the H1N1pdm challenge virus. Plasma from vaccinated macaques activated NK cells in the presence of H1N1pdm HA, suggesting that vaccination elicited cross-reactive antibodies capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Although HA-specific T cell responses to the MVA-H5M vaccine were weak, responses after challenge were stronger in vaccinated macaques than in control animals. Together these data suggest that mosaic HA antigens may provide a means for inducing broadly cross-reactive immunity to influenza viruses.

A modified vaccinia Ankara vaccine vector expressing a mosaic H5 hemagglutinin reduces viral shedding in rhesus macaques

PubMed Central

Mutschler, James P.; Kingstad-Bakke, Brock; Schultz-Darken, Nancy; Broman, Karl W.; Osorio, Jorge E.

2017-01-01

The rapid antigenic evolution of influenza viruses requires frequent vaccine reformulations. Due to the economic burden of continuous vaccine reformulation and the threat of new pandemics, there is intense interest in developing vaccines capable of eliciting broadly cross-reactive immunity to influenza viruses. We recently constructed a “mosaic” hemagglutinin (HA) based on subtype 5 HA (H5) and designed to stimulate cellular and humoral immunity to multiple influenza virus subtypes. Modified vaccinia Ankara (MVA) expressing this H5 mosaic (MVA-H5M) protected mice against multiple homosubtypic H5N1 strains and a heterosubtypic H1N1 virus. To assess its potential as a human vaccine we evaluated the ability of MVA-H5M to provide heterosubtypic immunity to influenza viruses in a non-human primate model. Rhesus macaques received an initial dose of either MVA-H5M or plasmid DNA encoding H5M, followed by a boost of MVA-H5M, and then were challenged, together with naïve controls, with the heterosubtypic virus A/California/04/2009 (H1N1pdm). Macaques receiving either vaccine regimen cleared H1N1pdm challenge faster than naïve controls. Vaccination with H5M elicited antibodies that bound H1N1pdm HA, but did not neutralize the H1N1pdm challenge virus. Plasma from vaccinated macaques activated NK cells in the presence of H1N1pdm HA, suggesting that vaccination elicited cross-reactive antibodies capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Although HA-specific T cell responses to the MVA-H5M vaccine were weak, responses after challenge were stronger in vaccinated macaques than in control animals. Together these data suggest that mosaic HA antigens may provide a means for inducing broadly cross-reactive immunity to influenza viruses. PMID:28771513

ST-246 inhibits in vivo poxvirus dissemination, virus shedding, and systemic disease manifestation.

PubMed

Berhanu, Aklile; King, David S; Mosier, Stacie; Jordan, Robert; Jones, Kevin F; Hruby, Dennis E; Grosenbach, Douglas W

2009-12-01

Orthopoxvirus infections, such as smallpox, can lead to severe systemic disease and result in considerable morbidity and mortality in immunologically naïve individuals. Treatment with ST-246, a small-molecule inhibitor of virus egress, has been shown to provide protection against severe disease and death induced by several members of the poxvirus family, including vaccinia, variola, and monkeypox viruses. Here, we show that ST-246 treatment not only results in the significant inhibition of vaccinia virus dissemination from the site of inoculation to distal organs, such as the spleen and liver, but also reduces the viral load in organs targeted by the dissemination. In mice intranasally infected with vaccinia virus, virus shedding from the nasal and lung mucosa was significantly lower (approximately 22- and 528-fold, respectively) upon ST-246 treatment. Consequently, virus dissemination from the nasal site of replication to the lung also was dramatically reduced, as evidenced by a 179-fold difference in virus levels in nasal versus bronchoalveolar lavage. Furthermore, in ACAM2000-immunized mice, vaccination site swabs showed that ST-246 treatment results in a major (approximately 3,900-fold by day 21) reduction in virus detected at the outside surfaces of lesions. Taken together, these data suggest that ST-246 would play a dual protective role if used during a smallpox bioterrorist attack. First, ST-246 would provide therapeutic benefit by reducing the disease burden and lethality in infected individuals. Second, by reducing virus shedding from those prophylactically immunized with a smallpox vaccine or harboring variola virus infection, ST-246 could reduce the risk of virus transmission to susceptible contacts.

Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes

PubMed Central

Mühlberger, Elke; Lötfering, Beate; Klenk, Hans-Dieter; Becker, Stephan

1998-01-01

This paper describes the first reconstituted replication system established for a member of the Filoviridae, Marburg virus (MBGV). MBGV minigenomes containing the leader and trailer regions of the MBGV genome and the chloramphenicol acetyltransferase (CAT) gene were constructed. In MBGV-infected cells, these minigenomes were replicated and encapsidated and could be passaged. Unlike most other members of the order Mononegavirales, filoviruses possess four proteins presumed to be components of the nucleocapsid (NP, VP35, VP30, and L). To determine the protein requirements for replication and transcription, a reverse genetic system was established for MBGV based on the vaccinia virus T7 expression system. Northern blot analysis of viral RNA revealed that three nucleocapsid proteins (NP, VP35, and L) were essential and sufficient for transcription as well as replication and encapsidation. These data indicate that VP35, rather than VP30, is the functional homologue of rhabdo- and paramyxovirus P proteins. The reconstituted replication system was profoundly affected by the NP-to-VP35 expression ratio. To investigate whether CAT gene expression was achieved entirely by mRNA or in part by full-length plus-strand minigenomes, a copy-back minireplicon containing the CAT gene but lacking MBGV-specific transcriptional start sites was employed in the artificial replication system. This construct was replicated without accompanying CAT activity. It was concluded that the CAT activity reflected MBGV-specific transcription and not replication. PMID:9765419

Cell lines that support replication of a novel herpes simplex virus 1 U{sub L}31 deletion mutant can properly target U{sub L}34 protein to the nuclear rim in the absence of U{sub L}31

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

Liang Li; Tanaka, Michiko; Kawaguchi, Yasushi

2004-11-10

Previous results indicated that the herpes simplex virus 1 (HSV-1) U{sub L}31 gene is necessary and sufficient for localization of the U{sub L}34 protein exclusively to the nuclear membrane of infected Hep2 cells. In the current studies, a bacterial artificial chromosome containing the entire HSV-1 strain F genome was used to construct a recombinant viral genome in which a gene encoding kanamycin resistance was inserted in place of 262 codons of the 306 codon U{sub L}31 open reading frame. The deletion virus produced virus titers approximately 10- to 50-fold lower in rabbit skin cells, more than 2000-fold lower in Veromore » cells, and more than 1500-fold lower in CV1 cells, compared to a virus bearing a restored U{sub L}31 gene. The replication of the U{sub L}31 deletion virus was restored on U{sub L}31-complementing cell lines derived either from rabbit skin cells or CV1 cells. Confocal microscopy indicated that the majority of U{sub L}34 protein localized aberrantly in the cytoplasm and nucleoplasm of Vero cells and CV1 cells, whereas U{sub L}34 protein localized at the nuclear membrane in rabbit skin cells, and U{sub L}31 complementing CV1 cells infected with the U{sub L}31 deletion virus. We conclude that rabbit skin cells encode a function that allows proper localization of U{sub L}34 protein to the nuclear membrane. We speculate that this function partially complements that of U{sub L}31 and may explain why U{sub L}31 is less critical for replication in rabbit skin cells as opposed to Vero and CV1 cells.« less

Sheeppox virus SPPV14 encodes a Bcl-2-like cell death inhibitor that counters a distinct set of mammalian proapoptotic proteins.

PubMed

Okamoto, Toru; Campbell, Stephanie; Mehta, Ninad; Thibault, John; Colman, Peter M; Barry, Michele; Huang, David C S; Kvansakul, Marc

2012-11-01

Many viruses express inhibitors of programmed cell death (apoptosis), thereby countering host defenses that would otherwise rapidly clear infected cells. To counter this, viruses such as adenoviruses and herpesviruses express recognizable homologs of the mammalian prosurvival protein Bcl-2. In contrast, the majority of poxviruses lack viral Bcl-2 (vBcl-2) homologs that are readily identified by sequence similarities. One such virus, myxoma virus, which is the causative agent of myxomatosis, expresses a virulence factor that is a potent inhibitor of apoptosis. In spite of the scant sequence similarity to Bcl-2, myxoma virus M11L adopts an almost identical 3-dimensional fold. We used M11L as bait in a sequence similarity search for other Bcl-2-like proteins and identified six putative vBcl-2 proteins from poxviruses. Some are potent inhibitors of apoptosis, in particular sheeppox virus SPPV14, which inhibited cell death induced by multiple agents. Importantly, SPPV14 compensated for the loss of antiapoptotic F1L in vaccinia virus and acts to directly counter the cell death mediators Bax and Bak. SPPV14 also engages a unique subset of the death-promoting BH3-only ligands, including Bim, Puma, Bmf, and Hrk. This suggests that SPPV14 may have been selected for specific biological roles as a virulence factor for sheeppox virus.

Microarray Analysis Reveals Characteristic Changes of Host Cell Gene Expression in Response to Attenuated Modified Vaccinia Virus Ankara Infection of Human HeLa Cells

PubMed Central

Guerra, Susana; López-Fernández, Luis A.; Conde, Raquel; Pascual-Montano, Alberto; Harshman, Keith; Esteban, Mariano

2004-01-01

The potential use of the modified vaccinia virus Ankara (MVA) strain as a live recombinant vector to deliver antigens and elicit protective immune responses against infectious diseases demands a comprehensive understanding of the effect of MVA infection on human host gene expression. We used microarrays containing more than 15,000 human cDNAs to identify gene expression changes in human HeLa cell cultures at 2, 6, and 16 h postinfection. Clustering of the 410 differentially regulated genes identified 11 discrete gene clusters with altered expression patterns after MVA infection. Clusters 1 and 2 (accounting for 16.59% [68 of 410] of the genes) contained 68 transcripts showing a robust induction pattern that was maintained during the course of infection. Changes in cellular gene transcription detected by microarrays after MVA infection were confirmed for selected genes by Northern blot analysis and by real-time reverse transcription-PCR. Upregulated transcripts in clusters 1 and 2 included 20 genes implicated in immune responses, including interleukin 1A (IL-1A), IL-6, IL-7, IL-8, and IL-15 genes. MVA infection also stimulated the expression of NF-κB and components of the NF-κB signal transduction pathway, including p50 and TRAF-interacting protein. A marked increase in the expression of histone family members was also induced during MVA infection. Expression of the Wiskott-Aldrich syndrome family members WAS, WASF1, and the small GTP-binding protein RAC-1, which are involved in actin cytoskeleton reorganization, was enhanced after MVA infection. This study demonstrates that MVA infection triggered the induction of groups of genes, some of which may be involved in host resistance and immune modulation during virus infection. PMID:15140980

Natural protection from zoonosis by alpha-gal epitopes on virus particles in xenotransmission.

PubMed

Kim, Na Young; Jung, Woon-Won; Oh, Yu-Kyung; Chun, Taehoon; Park, Hong-Yang; Lee, Hoon-Taek; Han, In-Kwon; Yang, Jai Myung; Kim, Young Bong

2007-03-01

Clinical transplantation has become one of the preferred treatments for end-stage organ failure, and one of the novel approaches being pursued to overcome the limited supply of human organs involves the use of organs from other species. The pig appears to be a near ideal animal due to proximity to humans, domestication, and ability to procreate. The presence of Gal-alpha1,3-Gal residues on the surfaces of pig cells is a major immunological obstacle to xenotransplantation. Alpha1,3galactosyltransferase (alpha1,3GT) catalyzes the synthesis of Gal alpha 1-3Gal beta 1-4GlcNAc-R (alpha-gal epitope) on the glycoproteins and glycolipids of non-primate mammals, but this does not occur in humans. Moreover, the alpha-gal epitope causes hyperacute rejection of pig organs in humans, and thus, the elimination of this antigen from pig tissues is highly desirable. Recently, concerns have been raised that the risk of virus transmission from such pigs may be increased due to the absence of alpha-gal on their viral particles. In this study, transgenic cells expressing alpha1,3GT were selected using 1.25 mg/ml neomycin. The development of HeLa cells expressing alpha1,3GT now allows accurate studies to be conducted on the function of the alpha-gal epitope in xenotransmission. The expressions of alpha-gal epitopes on HeLa/alpha-gal cells were demonstrated by flow cytometry and confocal microscopy using cells stained with IB4-fluorescein isothiocyanate lectin. Vaccinia viruses propagated in HeLa/alpha-gal cells also expressed alpha-gal on their viral envelopes and were more sensitive to inactivation by human sera than vaccinia virus propagated in HeLa cells. Moreover, neutralization of vaccinia virus was inhibited in human serum by 10 mm ethylene glycol bis(beta-aminoethylether)tetraacetic acid (EDTA) treatment. Our data indicated that alpha-gal epitopes are one of the major barriers to zoonosis via xenotransmission.

Artificial ribonucleases inactivate a wide range of viruses using their ribonuclease, membranolytic, and chaotropic-like activities.

PubMed

Fedorova, Antonina A; Goncharova, Elena P; Koroleva, Lyudmila S; Burakova, Ekatherina A; Ryabchikova, Elena I; Bichenkova, Elena V; Silnikov, Vladimir N; Vlassov, Valentin V; Zenkova, Marina A

2016-09-01

Artificial ribonucleases (aRNases) are small compounds catalysing RNA cleavage. Recently we demonstrated that aRNases readily inactivate various viruses in vitro. Here, for three series of aRNases (1,4-diazabicyclo [2.2.2]octane-based and peptide-like compounds) we show that apart from ribonuclease activity the aRNases display chaotropic-like and membranolytic activities. The levels of membranolytic and chaotropic-like activities correlate well with the efficiency of various viruses inactivation (enveloped, non-enveloped, RNA-, DNA-containing). We evaluated the impact of these activities on the efficiency of virus inactivation and found: i) the synergism between membranolytic and chaotropic-like activities is sufficient for the inactivation of enveloped viruses (influenza A, encephalitis, vaccinia viruses) for 1,4-diazabicyclo [2.2.2]octane based aRNases, ii) the inactivation of non-enveloped viruses (encephalomyocarditis, acute bee paralysis viruses) is totally dependent on the synergism of chaotropic-like and ribonuclease activities, iii) ribonuclease activity plays a leading role in the inactivation of RNA viruses by aRNases Dp12F6, Dtr12 and K-D-1, iv) peptide-like aRNases (L2-3, K-2) being effective virus killers have a more specific mode of action. Obtained results clearly demonstrate that aRNases represent a new class of broad-spectrum virus-inactivating agents. Copyright © 2016 Elsevier B.V. All rights reserved.

Immunogenicity of HILDA/LIF either in a soluble or in a membrane anchored form expressed in vivo by recombinant vaccinia viruses.

PubMed

Taupin, J L; Acres, B; Dott, K; Schmitt, D; Kieny, M P; Gualde, N; Moreau, J F

1993-09-01

Insertion of various cDNAs in the genome of the vaccinia virus (VV) enables the in vivo and in vitro study of the functional role and/or the immunogenicity of the virally encoded recombinant proteins. We have prepared a recombinant VV expressing the cDNA of the human cytokine HILDA/LIF (human interleukin for DA cells/leukaemia inhibitory factor), and used this virus to immunize mice against this protein, which is very homologous to its murine counterpart (approximately 80% homology). We also constructed and expressed by the same system a chimeric gene encoding the HILDA/LIF protein fused to the 37 COOH-terminal amino-acids of the human decay accelerating factor (DAF). This sequence proved to be sufficient for the targeting of the fusion protein to the cell membrane, where it is linked to the phosphatidylinositols. Both recombinant VVs induced cytokine-specific antibodies in mice as analysed with an ELISA where the recombinant HILDA/LIF was plastic-coated and a cytofluorometric assay where the LIF-DAF molecule was present at the cell surface of stably transfected P815. In the latter case HILDA/LIF remained biologically active suggesting that it was expressed in its native form. The LIF-DAF fusion protein was found to exhibit a better capacity to elicit an antibody response against the native form of the cytokine as detected in cytofluorometric assays. Whatever the recombinant virus used to immunize the mice, the MoAbs obtained were positive either in the ELISA or in the cytofluorometric assays but one, which suggested that the plastic coating induced a conformational change of HILDA/LIF.

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

PubMed Central

2018-01-01

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

Immunomodulator-Based Enhancement of Anti Smallpox Immune Responses

PubMed Central

Martínez, Osmarie; Miranda, Eric; Ramírez, Maite; Santos, Saritza; Rivera, Carlos; Vázquez, Luis; Sánchez, Tomás; Tremblay, Raymond L.; Ríos-Olivares, Eddy; Otero, Miguel

2015-01-01

Background The current live vaccinia virus vaccine used in the prevention of smallpox is contraindicated for millions of immune-compromised individuals. Although vaccination with the current smallpox vaccine produces protective immunity, it might result in mild to serious health complications for some vaccinees. Thus, there is a critical need for the production of a safe virus-free vaccine against smallpox that is available to everyone. For that reason, we investigated the impact of imiquimod and resiquimod (Toll-like receptors agonists), and the codon-usage optimization of the vaccinia virus A27L gene in the enhancement of the immune response, with intent of producing a safe, virus-free DNA vaccine coding for the A27 vaccinia virus protein. Methods We analyzed the cellular-immune response by measuring the IFN-γ production of splenocytes by ELISPOT, the humoral-immune responses measuring total IgG and IgG2a/IgG1 ratios by ELISA, and the TH1 and TH2 cytokine profiles by ELISA, in mice immunized with our vaccine formulation. Results The proposed vaccine formulation enhanced the A27L vaccine-mediated production of IFN-γ on mouse spleens, and increased the humoral immunity with a TH1-biased response. Also, our vaccine induced a TH1 cytokine milieu, which is important against viral infections. Conclusion These results support the efforts to find a new mechanism to enhance an immune response against smallpox, through the implementation of a safe, virus-free DNA vaccination platform. PMID:25875833

Infection cycles of large DNA viruses: Emerging themes and underlying questions

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

Mutsafi, Yael, E-mail: yael.mutsafi@weizmann.ac.il; Fridmann-Sirkis, Yael; Milrot, Elad

The discovery of giant DNA viruses and the recent realization that such viruses are diverse and abundant blurred the distinction between viruses and cells. These findings elicited lively debates on the nature and origin of viruses as well as on their potential roles in the evolution of cells. The following essay is, however, concerned with new insights into fundamental structural and physical aspects of viral replication that were derived from studies conducted on large DNA viruses. Specifically, the entirely cytoplasmic replication cycles of Mimivirus and Vaccinia are discussed in light of the highly limited trafficking of large macromolecules in themore » crowded cytoplasm of cells. The extensive spatiotemporal order revealed by cytoplasmic viral factories is described and contended to play an important role in promoting the efficiency of these ‘nuclear-like’ organelles. Generation of single-layered internal membrane sheets in Mimivirus and Vaccinia, which proceeds through a novel membrane biogenesis mechanism that enables continuous supply of lipids, is highlighted as an intriguing case study of self-assembly. Mimivirus genome encapsidation was shown to occur through a portal different from the ‘stargate’ portal that is used for genome release. Such a ‘division of labor’ is proposed to enhance the efficacy of translocation processes of very large viral genomes. Finally, open questions concerning the infection cycles of giant viruses to which future studies are likely to provide novel and exciting answers are discussed. - Highlights: • The discovery of giant DNA viruses blurs the distinction between viruses and cells. • Mimivirus and Vaccinia replicate exclusively in their host cytoplasm. • Mimivirus genome is delivered through a unique portal coined the Stargate. • Generation of Mimivirus internal membrane proceeds through a novel pathway.« less

Vergleich von rekombinanten Vaccinia- und DNA-Vektoren zur Tumorimmuntherapie im C57BL/6-Mausmodell

NASA Astrophysics Data System (ADS)

Johnen, Heiko

2002-10-01

antigenpräsentierender Zellen verantwortlich sein. Durch die Modifikation einer Methode zur intrazellulären IFN-gamma Färbung konnten in vakzinierten Mäusen tumorantigenspezifische CTL sensitiv und quantitativ detektiert werden. Die so bestimmte CTL-Frequenz, nicht jedoch die humorale Antwort, korrelierte mit der in vivo Wirksamkeit der verschiedenen Vakzinen: DNA vakzinierte Tiere entwickeln starke tumorantigenspezifische CTL-Antworten, wohingegen in MVA-vakzinierten Tieren überwiegend gegen virale Epitope gerichtete CD4 und CD8-T-Zellen detektiert wurden. Die Wirksamkeit der pCI-DNA-Vakzine spricht für die Weiterentwicklung in weiteren präklinischen Mausmodellen, beispielsweise unter Verwendung von MUC1 oder HLA-A2 transgenen Mäusen. Die Methoden zur Detektion Tumorantigen-spezifischer CTL in 96-Loch-Mikrotiterplatten können dabei zur systematischen Suche nach im Menschen immundominanten T-Zell-Epitopen im Muzin-Molekül genutzt werden. Der durchgeführte Vergleich der auf den Vektoren pCI und MVA basierenden Vakzinen und die Analyse neuerer Publikationen führen zu dem Ergebniss, daß vor allem DNA-Vakzinen in Zukunft eine wichtige Rolle bei der Entwicklung von aktiven Tumorimpfstoffen spielen werden. Rekombinante MVA-Viren, eventuell in Kombination mit DNA- oder anderen Vektoren, haben sich dagegen in zahlreichen Studien als wirksame Impfstoffe zur Kontrolle von durch Pathogene hervorgerufenen Infektionserkrankungen erwiesen. In this study, tumor vaccines based on the plasmid pCI, the attenuated vaccinia virus strain modified vaccinia virus Ankara (MVA) and MVA-infected dendritic cells were constructed and characterized by sequencing, Western blot and flow cytometric analysis. The efficiency to induce tumor immunity in vivo was compared in several C57BL/6 mouse tumor models. Naked DNA Vaccination based on the eukaryotic expression vector pCI did induce very effective, antigen-specific and long-term protection against tumor cell lines expressing mucin, CEA or

Deletion of the Vaccinia Virus Gene A46R, Encoding for an Inhibitor of TLR Signalling, Is an Effective Approach to Enhance the Immunogenicity in Mice of the HIV/AIDS Vaccine Candidate NYVAC-C

PubMed Central

Perdiguero, Beatriz; Gómez, Carmen Elena; Di Pilato, Mauro; Sorzano, Carlos Oscar S.; Delaloye, Julie; Roger, Thierry; Calandra, Thierry; Pantaleo, Giuseppe; Esteban, Mariano

2013-01-01

Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-ΔA46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates. PMID:24069354

RNase L targets distinct sites in influenza A virus RNAs.

PubMed

Cooper, Daphne A; Banerjee, Shuvojit; Chakrabarti, Arindam; García-Sastre, Adolfo; Hesselberth, Jay R; Silverman, Robert H; Barton, David J

2015-03-01

Influenza A virus (IAV) infections are influenced by type 1 interferon-mediated antiviral defenses and by viral countermeasures to these defenses. When IAV NS1 protein is disabled, RNase L restricts virus replication; however, the RNAs targeted for cleavage by RNase L under these conditions have not been defined. In this study, we used deep-sequencing methods to identify RNase L cleavage sites within host and viral RNAs from IAV PR8ΔNS1-infected A549 cells. Short hairpin RNA knockdown of RNase L allowed us to distinguish between RNase L-dependent and RNase L-independent cleavage sites. RNase L-dependent cleavage sites were evident at discrete locations in IAV RNA segments (both positive and negative strands). Cleavage in PB2, PB1, and PA genomic RNAs suggests that viral RNPs are susceptible to cleavage by RNase L. Prominent amounts of cleavage mapped to specific regions within IAV RNAs, including some areas of increased synonymous-site conservation. Among cellular RNAs, RNase L-dependent cleavage was most frequent at precise locations in rRNAs. Our data show that RNase L targets specific sites in both host and viral RNAs to restrict influenza virus replication when NS1 protein is disabled. RNase L is a critical component of interferon-regulated and double-stranded-RNA-activated antiviral host responses. We sought to determine how RNase L exerts its antiviral activity during influenza virus infection. We enhanced the antiviral activity of RNase L by disabling a viral protein, NS1, that inhibits the activation of RNase L. Then, using deep-sequencing methods, we identified the host and viral RNAs targeted by RNase L. We found that RNase L cleaved viral RNAs and rRNAs at very precise locations. The direct cleavage of IAV RNAs by RNase L highlights an intimate battle between viral RNAs and an antiviral endonuclease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

A recombinant modified vaccinia ankara vaccine encoding Epstein-Barr Virus (EBV) target antigens: a phase I trial in UK patients with EBV-positive cancer.

PubMed

Taylor, Graham S; Jia, Hui; Harrington, Kevin; Lee, Lip Wai; Turner, James; Ladell, Kristin; Price, David A; Tanday, Manjit; Matthews, Jen; Roberts, Claudia; Edwards, Ceri; McGuigan, Lesley; Hartley, Andrew; Wilson, Steve; Hui, Edwin P; Chan, Anthony T C; Rickinson, Alan B; Steven, Neil M

2014-10-01

Epstein-Barr virus (EBV) is associated with several cancers in which the tumor cells express EBV antigens EBNA1 and LMP2. A therapeutic vaccine comprising a recombinant vaccinia virus, MVA-EL, was designed to boost immunity to these tumor antigens. A phase I trial was conducted to demonstrate the safety and immunogenicity of MVA-EL across a range of doses. Sixteen patients in the United Kingdom (UK) with EBV-positive nasopharyngeal carcinoma (NPC) received three intradermal vaccinations of MVA-EL at 3-weekly intervals at dose levels between 5 × 10(7) and 5 × 10(8) plaque-forming units (pfu). Blood samples were taken at screening, after each vaccine cycle, and during the post-vaccination period. T-cell responses were measured using IFNγ ELISpot assays with overlapping EBNA1/LMP2 peptide mixes or HLA-matched epitope peptides. Polychromatic flow cytometry was used to characterize functionally responsive T-cell populations. Vaccination was generally well tolerated. Immunity increased after vaccination to at least one antigen in 8 of 14 patients (7/14, EBNA1; 6/14, LMP2), including recognition of epitopes that vary between EBV strains associated with different ethnic groups. Immunophenotypic analysis revealed that vaccination induced differentiation and functional diversification of responsive T-cell populations specific for EBNA1 and LMP2 within the CD4 and CD8 compartments, respectively. MVA-EL is safe and immunogenic across diverse ethnicities and thus suitable for use in trials against different EBV-positive cancers globally as well as in South-East Asia where NPC is most common. The highest dose (5 × 10(8) pfu) is recommended for investigation in current phase IB and II trials. ©2014 American Association for Cancer Research.

Use of Bioclimatic Factors to Determine Potential Niche of Vaccinia Virus, an Emerging and Zoonotic Pathogen

NASA Astrophysics Data System (ADS)

Quiner, C. A.; Nakazawa, Y.

2017-12-01

Emerging and understudied pathogens often lack information that most commonly used analytical tools require, such as negative controls or baseline data making public health control of emerging pathogens challenging. In lieu of opportunities to collect more data from larger outbreaks or formal epidemiological studies, new analytical strategies, merging case data with publically available datasets, can be used to understand transmission patterns and drivers of disease emergence. Zoonotic infections with Vaccinia virus (VACV) were first reported in Brazil in 1999, VACV is an emerging zoonotic Orthopoxvirus, which primarily infects dairy cattle and farmers in close contact with infected cows. Prospective studies of emerging pathogens could provide critical data that would inform public health planning and response to outbreaks. By using the location of 87-recorded outbreaks and publicly available bioclimatic data we demonstrate one such approach. Using an Ecological Niche Model (ENM), we identify the environmental conditions under which VACV outbreaks have occurred, and determine additional locations in two affected South American countries that may be susceptible to transmission. Further, we show how suitability for the virus responds to different levels of various environmental factors and highlight the most important climatic factors in determining its transmission. The final ENM predicted all areas where Brazilian outbreaks occurred, two out of five Colombian outbreaks and identified new regions within Brazil that are suitable for transmission based on bioclimatic factors. Further, the most important factors in determining transmission suitability are precipitation of the wettest quarter, annual precipitation, mean temperature of the coldest quarter and mean diurnal range. The analyses here provide a means by which to study patterns of an emerging infectious disease, and regions that are potentially at risk for it, in spite of the paucity of critical data. Policy

Expression and regulation of glycoprotein C gene of herpes simplex virus 1 resident in a clonal L-cell line.

PubMed Central

Arsenakis, M; Tomasi, L F; Speziali, V; Roizman, B; Campadelli-Fiume, G

1986-01-01

Ltk- cells were transfected with a plasmid containing the entire domain of glycoprotein C (gC), a true gamma or gamma 2 gene of herpes simplex virus 1 (HSV-1) and the methotrexate-resistant mouse dihydrofolate reductase mutant gene. The resulting methotrexate-resistant cell line was cloned; of the 39 clonal lines tested only 1, L3153(28), expressed gC after infection with HSV-1(MP), a gC- mutant, and none expressed gC constitutively. The induction of gC was optimal at multiplicities ranging between 0.5 and 2 PFU per cell, and the quantities produced were equivalent to or higher than those made by methotrexate-resistant gC- L cells infected with wild-type (gC+) virus. The gC gene resident in the L3153(28) cells was regulated as a beta gene inasmuch as the amounts of gC made in infected L3153(28) cells exposed to concentrations of phosphonoacetate that inhibited viral DNA synthesis were higher than those made in the absence of the drug, gC was induced at both permissive and nonpermissive temperatures by the DNA- mutant tsHA1 carrying a lesion in the gene specifying the major DNA-binding protein and which does not express gamma 2 genes at the nonpermissive temperature, and gC was induced only at the permissive temperature in cells infected with ts502 containing a mutation in the alpha 4 gene. The gC induced in L3153(28) cells was made earlier and processed faster to the mature form than that induced in a gC- clone of methotrexate-resistant cells infected with wild-type virus. Unlike virus stocks made in gC- cells, HSV-1(MP) made in L3153(28) cells was susceptible to neutralization by anti-gC monoclonal antibody. Images PMID:3009854

Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium.

PubMed

Rodríguez, Dolores; González-Aseguinolaza, Gloria; Rodríguez, Juan R; Vijayan, Aneesh; Gherardi, Magdalena; Rueda, Paloma; Casal, J Ignacio; Esteban, Mariano

2012-01-01

With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs) carrying the CD8(+) T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS), and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8(+) T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV) vectors from the Western Reserve (WR) and modified virus Ankara (MVA) strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria.

Subclinical bovine vaccinia: An important risk factor in the epidemiology of this zoonosis in cattle.

PubMed

Rehfeld, Izabelle Silva; Matos, Ana Carolina Diniz; Guedes, Maria Isabel Maldonado Coelho; Costa, Aristóteles Gomes; Fraiha, Ana Luiza Soares; Lobato, Zélia Inês Portela

2017-10-01

Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus (VACV) that mainly affects lactating cows and dairy farm milkers. The epidemiological role(s) of other cattle categories such as dry cows, bulls, and heifers in BV remains unclear. This study was performed to investigate VACV in affected dairy cattle herds and perifocal farms during an outbreak in Brazil. Crusts from lesions of cows' teats were collected from all farms with BV outbreaks. Milk, feces, blood, and serum were collected from symptomatic and asymptomatic lactating cows. Blood and serum were also sampled from other cattle categories (calves, heifers, dry cows, and bulls). The samples were tested for VACV by PCR, and to confirm VACV viability, VACV-positive samples were inoculated in BSC-40 cells and stained using immunoperoxidase. Neutralizing antibodies were investigated using plaque reduction neutralization test. Viral DNA was detected in milk, blood, and feces samples of symptomatic and asymptomatic dairy cows and in blood samples from other cattle categories on farms with and without confirmed BV outbreak. In affected farms, viable virus was identified in feces and milk samples from lactating cows and in blood samples from asymptomatic dry cows. Viable VACV was also identified in feces from lactating cows and one bull's blood sample from perifocal farms. Neutralizing antibodies were detected in 81.6% of the herds affected by BV and in 53.8% of the herds on perifocal farms. The presented data indicate a potential source of viral dissemination, which contributes to the persistence and spread of VACV in the environment. Copyright © 2017 Elsevier Ltd. All rights reserved.

Inactivation of viruses in bubbling processes utilized for personal bioaerosol monitoring.

PubMed

Agranovski, I E; Safatov, A S; Borodulin, A I; Pyankov, O V; Petrishchenko, V A; Sergeev, A N; Agafonov, A P; Ignatiev, G M; Sergeev, A A; Agranovski, V

2004-12-01

A new personal bioaerosol sampler has recently been developed and evaluated for sampling of viable airborne bacteria and fungi under controlled laboratory conditions and in the field. The operational principle of the device is based on the passage of air through porous medium immersed in liquid. This process leads to the formation of bubbles within the filter as the carrier gas passes through and thus provides effective mechanisms for aerosol removal. As demonstrated in previous studies, the culturability of sampled bacterium and fungi remained high for the entire 8-h sampling period. The present study is the first step of the evaluation of the new sampler for monitoring of viable airborne viruses. It focuses on the investigation of the inactivation rate of viruses in the bubbling process during 4 h of continuous operation. Four microbes were used in this study, influenza, measles, mumps, and vaccinia viruses. It was found that the use of distilled water as the collection fluid was associated with a relatively high decay rate. A significant improvement was achieved by utilizing virus maintenance fluid prepared by using Hank's solution with appropriate additives. The survival rates of the influenza, measles, and mumps viruses were increased by 1.4 log, 0.83 log, and 0.82 log, respectively, after the first hour of operation compared to bubbling through the sterile water. The same trend was observed throughout the entire 4-h experiment. There was no significant difference observed only for the robust vaccinia virus.

Inactivation of Viruses in Bubbling Processes Utilized for Personal Bioaerosol Monitoring

PubMed Central

Agranovski, I. E.; Safatov, A. S.; Borodulin, A. I.; Pyankov, O. V.; Petrishchenko, V. A.; Sergeev, A. N.; Agafonov, A. P.; Ignatiev, G. M.; Sergeev, A. A.; Agranovski, V.

2004-01-01

A new personal bioaerosol sampler has recently been developed and evaluated for sampling of viable airborne bacteria and fungi under controlled laboratory conditions and in the field. The operational principle of the device is based on the passage of air through porous medium immersed in liquid. This process leads to the formation of bubbles within the filter as the carrier gas passes through and thus provides effective mechanisms for aerosol removal. As demonstrated in previous studies, the culturability of sampled bacterium and fungi remained high for the entire 8-h sampling period. The present study is the first step of the evaluation of the new sampler for monitoring of viable airborne viruses. It focuses on the investigation of the inactivation rate of viruses in the bubbling process during 4 h of continuous operation. Four microbes were used in this study, influenza, measles, mumps, and vaccinia viruses. It was found that the use of distilled water as the collection fluid was associated with a relatively high decay rate. A significant improvement was achieved by utilizing virus maintenance fluid prepared by using Hank's solution with appropriate additives. The survival rates of the influenza, measles, and mumps viruses were increased by 1.4 log, 0.83 log, and 0.82 log, respectively, after the first hour of operation compared to bubbling through the sterile water. The same trend was observed throughout the entire 4-h experiment. There was no significant difference observed only for the robust vaccinia virus. PMID:15574888

Mutagenic analysis of herpes simplex virus type 1 glycoprotein L reveals the importance of an arginine-rich region for function

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

Klyachkin, Yuri M.; Geraghty, Robert J.

2008-04-25

Herpes simplex virus type 1 (HSV-1) glycoproteins H and L (gH and gL) are required for virus-induced membrane fusion. Expression of gH at the virion or infected cell surface is mediated by the chaperone-like activity of gL. We have previously shown that a region between amino acids 155 and 161 is critical for gL chaperone-like activity. Here, we conducted Ala substitution mutagenesis of residues in this region and found that substitution of Cys160, Arg156, Arg158, or Arg156/158/159 with Ala resulted in a gL mutant that bound gH but displayed a reduced ability in gH trafficking and membrane fusion. Substitution ofmore » Arg156 with another positively charged amino acid, Lys, restored function. Substitution of Arg158 with Lys restored function in gH trafficking and cell fusion but not virus entry. These results indicate that an arginine-rich region of gL is critical for function.« less

Characterization of an RNA aptamer against HPV-16 L1 virus-like particles.

PubMed

Leija-Montoya, Ana Gabriela; Benítez-Hess, María Luisa; Toscano-Garibay, Julia Dolores; Alvarez-Salas, Luis Marat

2014-10-01

The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3'end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection.

Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles

PubMed Central

Leija-Montoya, Ana Gabriela; Benítez-Hess, María Luisa; Toscano-Garibay, Julia Dolores

2014-01-01

The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3′end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection. PMID:25111024

Links between Human LINE-1 Retrotransposons and Hepatitis Virus-Related Hepatocellular Carcinoma.

PubMed

Honda, Tomoyuki

2016-01-01

Hepatocellular carcinoma (HCC) accounts for approximately 80% of liver cancers, the third most frequent cause of cancer mortality. The most prevalent risk factors for HCC are infections by hepatitis B or hepatitis C virus. Findings suggest that hepatitis virus-related HCC might be a cancer in which LINE-1 retrotransposon, often termed L1, activity plays a potential role. Firstly, hepatitis viruses can suppress host defense factors that also control L1 mobilization. Secondly, many recent studies also have indicated that hypomethylation of L1 affects the prognosis of HCC patients. Thirdly, endogenous L1 retrotransposition was demonstrated to activate oncogenic pathways in HCC. Fourthly, several L1 chimeric transcripts with host or viral genes are found in hepatitis virus-related HCC. Such lines of evidence suggest a linkage between L1 retrotransposons and hepatitis virus-related HCC. Here, I briefly summarize current understandings of the association between hepatitis virus-related HCC and L1. Then, I discuss potential mechanisms of how hepatitis viruses drive the development of HCC via L1 retrotransposons. An increased understanding of the contribution of L1 to hepatitis virus-related HCC may provide unique insights related to the development of novel therapeutics for this disease.

Links between human LINE-1 retrotransposons and hepatitis virus-related hepatocellular carcinoma

NASA Astrophysics Data System (ADS)

Honda, Tomoyuki

2016-05-01

Hepatocellular carcinoma (HCC) accounts for approximately 80% of liver cancers, the third most frequent cause of cancer mortality. The most prevalent risk factors for HCC are infections by hepatitis B or hepatitis C virus. Findings suggest that hepatitis virus-related HCC might be a cancer in which LINE-1 retrotransposons, often termed L1, activity plays a potential role. Firstly, hepatitis viruses can suppress host defense factors that also control L1 mobilization. Secondly, many recent studies also have indicated that hypomethylation of L1 affects the prognosis of HCC patients. Thirdly, endogenous L1 retrotransposition was demonstrated to activate oncogenic pathways in HCC. Fourthly, several L1 chimeric transcripts with host or viral genes are found in hepatitis virus-related HCC. Such lines of evidence suggest a linkage between L1 retrotransposons and hepatitis virus-related HCC. Here, I briefly summarize current understandings of the association between hepatitis virus-related HCC and L1. Then, I discuss potential mechanisms of how hepatitis viruses drive the development of HCC via L1 retrotransposons. An increased understanding of the contribution of L1 to hepatitis virus-related HCC may provide unique insights related to the development of novel therapeutics for this disease.

The neutralizing antibody response to the vaccinia virus A28 protein is specifically enhanced by its association with the H2 protein

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

Shinoda, Kaori; Wyatt, Linda S.; Moss, Bernard, E-mail: bmoss@niaid.nih.go

2010-09-15

The vaccinia virus (VACV) entry-fusion complex (EFC) is composed of at least nine membrane proteins. Immunization of mice with individual EFC genes induced corresponding protein-binding antibody but failed to protect against VACV intranasal challenge and only DNA encoding A28 elicited low neutralizing antibody. Because the A28 and H2 proteins interact, we determined the effect of immunizing with both genes simultaneously. This procedure greatly enhanced the amount of antibody that bound intact virions, neutralized infectivity, and provided partial protection against respiratory challenge. Neither injection of A28 and H2 plasmids at different sites or mixing A28 and H2 sera enhanced neutralizing antibody.more » The neutralizing antibody could be completely removed by binding to the A28 protein alone and the epitope was located in the C-terminal segment. These data suggest that the interaction of H2 with A28 stabilizes the immunogenic form of A28, mimicking an exposed region of the entry-fusion complex on infectious virions.« less

Nano-mechanical Resonantor Sensors for Virus Detection

NASA Astrophysics Data System (ADS)

Bashir, Rashid

2005-03-01

Micro and nanoscale cantilever beams can be used as highly sensitive mass detectors. Scaling down the area of the cantilever allows a decrease in minimum detectable mass limit while scaling down the thickness allows the resonant frequencies to be within measurable range. We have fabricated arrays of silicon cantilever beams as nanomechanical resonant sensors to detect the mass of individual virus particles. The dimensions of the fabricated cantilever beams were in the range of 4-5 μm in length, 1-2 μm in width and 20-30 nm in thickness. The virus particles we used in the study were vaccinia virus, which is a member of the Poxviridae family and forms the basis of the smallpox vaccine. The frequency spectra of the cantilever beams, due to thermal and ambient noise, were measured using a laser Doppler vibrometer under ambient conditions. The change in resonant frequency as a function of the virus particle mass binding on the cantilever beam surface forms the basis of the detection scheme. We have demonstrated the detection of a single vaccinia virus particle with an average mass of 9.5 fg. Specific capture of the antigens requires attachment of antibodies, which can be in the same range of thickness as these cantilever sensors, and can alter their mechanical properties. We have attached protein layers on both sides of 30nm thick cantilever beams and we show that the resonant frequencies can increase or decrease upon the attachment of protein layers to the cantilevers. In certain cases, the increase in spring constant out-weighs the increase in mass and the resonant frequencies can increase upon the attachment of the protein layers. These devices can be very useful as components of biosensors for the detection of air-borne virus particles.

Recombinant sheep pox virus proteins elicit neutralizing antibodies

USDA-ARS?s Scientific Manuscript database

The aim of this study was to evaluate the immunogenicity and neutralizing activity of bacterially-expressed sheep pox virus (SPPV) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins from vaccinia...

N-alpha-Cocoyl-L-arginine ethyl ester, DL-pyroglutamic acid salt, as an inactivator of hepatitis B surface antigen.

PubMed Central

Sugimoto, Y; Toyoshima, S

1979-01-01

N-alpha-Cocoyl-L-arginine ethyl ester, DL-pyroglutamic acid salt (CAE), exhibited a strong inactivating effect on hepatitis B surface antigen. Concentrations of CAE required for 50 and 100% inactivation of the antigen were 0.01 to 0.025% and 0.025 to 0.05% respectively. CAE completely inactivated hepatitis B surface antigen at the lowest concentration compared with various compounds including about 500 amino acid derivatives, sodium hypochlorite, 2,4,4'-trichloro-2'-hydroxydiphenyl ether, and some detergents. Furthermore, CAE inactivated vaccinia virus, herpes simplex virus, and influenza virus, whereas poliovirus was not inactivated at all. The results suggest that the inactivating effects of CAE are related to interaction with lipid-containing viral envelopes. PMID:228595

CD70 encoded by modified vaccinia virus Ankara enhances CD8 T-cell-dependent protective immunity in MHC class II-deficient mice.

PubMed

Bathke, Barbara; Pätzold, Juliane; Kassub, Ronny; Giessel, Raphael; Lämmermann, Kerstin; Hinterberger, Maria; Brinkmann, Kay; Chaplin, Paul; Suter, Mark; Hochrein, Hubertus; Lauterbach, Henning

2017-12-27

The immunological outcome of infections and vaccinations is largely determined during the initial first days in which antigen-presenting cells instruct T cells to expand and differentiate into effector and memory cells. Besides the essential stimulation of the T-cell receptor complex a plethora of co-stimulatory signals not only ensures a proper T-cell activation but also instils phenotypic and functional characteristics in the T cells appropriate to fight off the invading pathogen. The tumour necrosis factor receptor/ligand pair CD27/CD70 gained a lot of attention because of its key role in regulating T-cell activation, survival, differentiation and maintenance, especially in the course of viral infections and cancer. We sought to investigate the role of CD70 co-stimulation for immune responses induced by the vaccine vector modified vaccinia virus Ankara-Bavarian Nordic ® (MVA-BN ® ). Short-term blockade of CD70 diminished systemic CD8 T-cell effector and memory responses in mice. The dependence on CD70 became even more apparent in the lungs of MHC class II-deficient mice. Importantly, genetically encoded CD70 in MVA-BN ® not only increased CD8 T-cell responses in wild-type mice but also substituted for CD4 T-cell help. MHC class II-deficient mice that were immunized with recombinant MVA-CD70 were fully protected against a lethal virus infection, whereas MVA-BN ® -immunized mice failed to control the virus. These data are in line with CD70 playing an important role for vaccine-induced CD8 T-cell responses and prove the potency of integrating co-stimulatory molecules into the MVA-BN ® backbone. © 2017 The Authors. Immunology Published by John Wiley & Sons Ltd.

Genetic Variability of Myxoma Virus Genomes

PubMed Central

Braun, Christoph; Thürmer, Andrea; Daniel, Rolf; Schultz, Anne-Kathrin; Bulla, Ingo; Schirrmeier, Horst; Mayer, Dietmar; Neubert, Andreas

2016-01-01

ABSTRACT Myxomatosis is a recurrent problem on rabbit farms throughout Europe despite the success of vaccines. To identify gene variations of field and vaccine strains that may be responsible for changes in virulence, immunomodulation, and immunoprotection, the genomes of 6 myxoma virus (MYXV) strains were sequenced: German field isolates Munich-1, FLI-H, 2604, and 3207; vaccine strain MAV; and challenge strain ZA. The analyzed genomes ranged from 147.6 kb (strain MAV) to 161.8 kb (strain 3207). All sequences were affected by several mutations, covering 24 to 93 open reading frames (ORFs) and resulted in amino acid substitutions, insertions, or deletions. Only strains Munich-1 and MAV revealed the deletion of 10 ORFs (M007L to M015L) and 11 ORFs (M007L to M008.1L and M149R to M008.1R), respectively. Major differences were observed in the 27 immunomodulatory proteins encoded by MYXV. Compared to the reference strain Lausanne, strains FLI-H, 2604, 3207, and ZA showed the highest amino acid identity (>98.4%). In strains Munich-1 and MAV, deletion of 5 and 10 ORFs, respectively, was observed, encoding immunomodulatory proteins with ankyrin repeats or members of the family of serine protease inhibitors. Furthermore, putative immunodominant surface proteins with homology to vaccinia virus (VACV) were investigated in the sequenced strains. Only strain MAV revealed above-average frequencies of amino acid substitutions and frameshift mutations. Finally, we performed recombination analysis and found signs of recombination in vaccine strain MAV. Phylogenetic analysis showed a close relationship of strain MAV and the MSW strain of Californian MYXV. However, in a challenge model, strain MAV provided full protection against lethal challenges with strain ZA. IMPORTANCE Myxoma virus (MYXV) is pathogenic for European rabbits and two North American species. Due to sophisticated strategies in immune evasion and oncolysis, MYXV is an important model virus for immunological and

Vaccine Efficacy against Malaria by the Combination of Porcine Parvovirus-Like Particles and Vaccinia Virus Vectors Expressing CS of Plasmodium

PubMed Central

Rodríguez, Dolores; González-Aseguinolaza, Gloria; Rodríguez, Juan R.; Vijayan, Aneesh; Gherardi, Magdalena; Rueda, Paloma; Casal, J. Ignacio; Esteban, Mariano

2012-01-01

With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs) carrying the CD8+ T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS), and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8+ T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV) vectors from the Western Reserve (WR) and modified virus Ankara (MVA) strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria. PMID:22529915

Co-expression of HIV-1 virus-like particles and granulocyte-macrophage colony stimulating factor by GEO-D03 DNA vaccine.

PubMed

Hellerstein, Michael; Xu, Yongxian; Marino, Tracie; Lu, Shan; Yi, Hong; Wright, Elizabeth R; Robinson, Harriet L

2012-11-01

Here, we report on GEO-D03, a DNA vaccine that co-expresses non-infectious HIV-1 virus-like particles (VLPs) and the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). The virus-like particles display the native gp160 form of the HIV-1 Envelope glycoprotein (Env) and are designed to elicit antibody against the natural form of Env on virus and virus-infected cells. The DNA-expressed HIV Gag, Pol and Env proteins also have the potential to elicit virus-specific CD4 and CD8 T cells. The purpose of the co-expressed GM-CSF is to target a cytokine that recruits, expands and differentiates macrophages and dendritic cells to the site of VLP expression. The GEO-D03 DNA vaccine is currently entered into human trials as a prime for a recombinant modified vaccinia Ankara (MVA) boost. In preclinical studies in macaques using an SIV prototype vaccine, this vaccination regimen elicited both anti-viral T cells and antibody, and provided 70% protection against acquisition during 12 weekly rectal exposures with a heterologous SIV. Higher avidity of the Env-specific Ab for the native form of the Env in the challenge virus correlated with lower likelihood of SIV infection.

African swine fever virus encodes two genes which share significant homology with the two largest subunits of DNA-dependent RNA polymerases.

PubMed Central

Yáñez, R J; Boursnell, M; Nogal, M L; Yuste, L; Viñuela, E

1993-01-01

A random sequencing strategy applied to two large SalI restriction fragments (SB and SD) of the African swine fever virus (ASFV) genome revealed that they might encode proteins similar to the two largest RNA polymerase subunits of eukaryotes, poxviruses and Escherichia coli. After further mapping by dot-blot hybridization, two large open reading frames (ORFs) were completely sequenced. The first ORF (NP1450L) encodes a protein of 1450 amino acids with extensive similarity to the largest subunit of RNA polymerases. The second one (EP1242L) codes for a protein of 1242 amino acids similar to the second largest RNA polymerase subunit. Proteins NP1450L and EP1242L are more similar to the corresponding subunits of eukaryotic RNA polymerase II than to those of vaccinia virus, the prototype poxvirus, which shares many functional characteristics with ASFV. ORFs NP1450L and EP1242L are mainly expressed late in ASFV infection, after the onset of DNA replication. Images PMID:8506138

One More Piece in the VACV Ecological Puzzle: Could Peridomestic Rodents Be the Link between Wildlife and Bovine Vaccinia Outbreaks in Brazil?

PubMed Central

Abrahão, Jônatas S.; Guedes, Maria Isabel M.; Trindade, Giliane S.; Fonseca, Flávio G.; Campos, Rafael K.; Mota, Bruno F.; Lobato, Zélia I. P.; Silva-Fernandes, André T.; Rodrigues, Gisele O. L.; Lima, Larissa S.; Ferreira, Paulo C. P.; Bonjardim, Cláudio A.; Kroon, Erna G.

2009-01-01

Background Despite the fact that smallpox eradication was declared by the World Health Organization (WHO) in 1980, other poxviruses have emerged and re-emerged, with significant public health and economic impacts. Vaccinia virus (VACV), a poxvirus used during the WHO smallpox vaccination campaign, has been involved in zoonotic infections in Brazilian rural areas (Bovine Vaccinia outbreaks – BV), affecting dairy cattle and milkers. Little is known about VACV's natural hosts and its epidemiological and ecological characteristics. Although VACV was isolated and/or serologically detected in Brazilian wild animals, the link between wildlife and farms has not yet been elucidated. Methodology/Principal Findings In this study, we describe for the first time, to our knowledge, the isolation of a VACV (Mariana virus - MARV) from a mouse during a BV outbreak. Genetic data, in association with biological assays, showed that this isolate was the same etiological agent causing exanthematic lesions observed in the cattle and human inhabitants of a particular BV-affected area. Phylogenetic analysis grouped MARV with other VACV isolated during BV outbreaks. Conclusion/Significance These data provide new biological and epidemiological information on VACV and lead to an interesting question: could peridomestic rodents be the link between wildlife and BV outbreaks? PMID:19838293