Vaccinia Virus Protein Research Articles

TRAF2 Facilitates Vaccinia Virus Replication by Promoting Rapid Virus Entry

ABSTRACTTumor necrosis factor receptor (TNFR)-associated factor 2 (TRAF2) is a pivotal intracellular mediator of signaling pathways downstream of TNFR1 and -2 with known pro- and antiviral effects. We investigated its role in the replication of the prototype poxvirus vaccinia virus (VACV). Loss of TRAF2 expression, either through small interfering RNA treatment of HeLa cells or through genetic knockout in murine embryonic fibroblasts (MEFs), led to significant reductions in VACV growth following low-multiplicity infection. In single-cycle infections, there was delayed production of both early and late VACV proteins as well as accelerated virus-induced alterations to cell morphology, indicating that TRAF2 influences early stages of virus replication. Consistent with an early role, uncoating assays showed normal virus attachment but delayed virus entry in the absence of TRAF2. Although alterations to c-Jun N-terminal kinase (JNK) signaling were apparent in VACV-infected TRAF2−/− MEFs, treatment of wild-type cells with a JNK inhibitor did not affect virus entry. Instead, treatment with an inhibitor of endosomal acidification greatly reduced virus entry into TRAF2−/− MEFs, suggesting that VACV is reliant on the endosomal route of entry in the absence of TRAF2. Thus, TRAF2 is a proviral factor for VACV that plays a role in promoting efficient viral entry, most likely via the plasma membrane.IMPORTANCE Tumor necrosis factor receptor-associated factors (TRAFs) are key facilitators of intracellular signaling with roles in innate and adaptive immunity and stress responses. We have discovered that TRAF2 is a proviral factor in vaccinia virus replication in both HeLa cells and mouse embryonic fibroblasts and that its influence is exercised through promotion of efficient virus entry.

Vaccinia virus immune evasion: mechanisms, virulence and immunogenicity

Virus infection of mammalian cells is sensed by pattern recognition receptors and leads to an innate immune response that restricts virus replication and induces adaptive immunity. In response, viruses have evolved many countermeasures that enable them to replicate and be transmitted to new hosts, despite the host innate immune response. Poxviruses, such as vaccinia virus (VACV), have large DNA genomes and encode many proteins that are dedicated to host immune evasion. Some of these proteins are secreted from the infected cell, where they bind and neutralize complement factors, interferons, cytokines and chemokines. Other VACV proteins function inside cells to inhibit apoptosis or signalling pathways that lead to the production of interferons and pro-inflammatory cytokines and chemokines. In this review, these VACV immunomodulatory proteins are described and the potential to create more immunogenic VACV strains by manipulation of the gene encoding these proteins is discussed.

Truncation of gene F5L partially masks rescue of vaccinia virus strain MVA growth on mammalian cells by restricting plaque size

Modified vaccinia virus Ankara (MVA) is a candidate vaccine vector that is severely attenuated due to mutations acquired during several hundred rounds of serial passage in vitro. A previous study used marker rescue to produce a set of MVA recombinants with improved replication on mammalian cells. Here, we extended the characterization of these rescued MVA strains and identified vaccinia virus (VACV) gene F5L as a determinant of plaque morphology but not replication in vitro. F5 joins a growing group of VACV proteins that influence plaque formation more strongly than virus replication and which are disrupted in MVA. These defective genes in MVA confound the interpretation of marker rescue experiments designed to map mutations responsible for the attenuation of this important VACV strain.

Interactions of the Vaccinia Virus A19 Protein

The A19 protein of vaccinia virus (VACV) is conserved among chordopoxviruses, expressed late in infection, packaged in the virus core, and required for a late step in morphogenesis. Multiple-sequence alignments of A19 homologs indicated conservation of a series of lysines and arginines, which could represent a nuclear localization or nucleic acid binding motif, and a pair of CXXC motifs that suggested a zinc finger or redox active sites. The importance of the CXXC motif was confirmed by cysteine-to-serine substitutions, which rendered the altered protein unable to trans-complement infectivity of a null mutant. Nevertheless, the cysteines were not required for function of the poxvirus-specific redox pathway. Epitope-tagged A19 proteins were detected in the nucleus and cytoplasm in both infected and uninfected cells, but this distribution was unaffected by alanine substitutions of the arginine residues, which only partially reduced the ability of the mutated protein to trans-complement infectivity. Viral proteins specifically associated with affinity-purified A19 were identified by mass spectrometry as components of the transcription complex, including RNA polymerase subunits, RAP94 (RNA polymerase-associated protein 94), early transcription factors, capping enzyme, and nucleoside triphosphate phosphohydrolase I, and two core proteins required for morphogenesis. Further studies suggested that the interaction of A19 with the RNA polymerase did not require RAP94 or other intermediate or late viral proteins but was reduced by mutation of cysteines in the putative zinc finger domain. Although A19 was not required for incorporation of the transcription complex in virus particles, the transcriptional activity of A19-deficient virus particles was severely reduced.

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

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.

A mechanism for induction of a hypoxic response by vaccinia virus.

Viruses have evolved sophisticated strategies to exploit host cell function for their benefit. Here we show that under physiologically normal oxygen levels (normoxia) vaccinia virus (VACV) infection leads to a rapid stabilization of hypoxia-inducible factor (HIF)-1α, its translocation into the nucleus and the activation of HIF-responsive genes, such as vascular endothelial growth factor (VEGF), glucose transporter-1, and pyruvate dehydrogenase kinase-1. HIF-1α stabilization is mediated by VACV protein C16 that binds the human oxygen sensing enzyme prolyl-hydroxylase domain containing protein (PHD)2 and thereby inhibits PHD2-dependent hydroxylation of HIF-1α. The binding between C16 and PHD2 is direct and specific, and ectopic expression of C16 alone induces transcription of HIF-1α responsive genes. Conversely, a VACV strain lacking the gene for C16, C16L, is unable to induce HIF-1α stabilization. Interestingly, the N-terminal region of C16 is predicted to have a PHD2-like structural fold but lacks the catalytic active site residues of PHDs. The induction of a hypoxic response by VACV is reminiscent of the biochemical consequences of solid tumor formation, and illustrates a poxvirus strategy for manipulation of cellular gene expression and biochemistry.

Primary Human Leukocyte Subsets Differentially Express Vaccinia Virus Receptors Enriched in Lipid Rafts

Poxviruses, including vaccinia virus (VV) and canarypox virus (ALVAC), do not indiscriminately infect all cell types of the primary human leukocytes (PHLs) that they encounter but instead demonstrate an extremely strong bias toward infection of monocytes and monocyte lineage cells. We studied the specific molecular events that determine the VV tropism for major PHL subsets including monocytes, B cells, neutrophils, NK cells, and T cells. We found that VV exhibited an extremely strong bias of cell surface protein-dependent binding to monocytes, B cells, and activated T cells to a similar degree and to neutrophils to a much lesser extent. Resting T cells and resting NK cells exhibited only trace amounts of VV binding. Activated T cells, however, became permissive to VV binding, infection, and replication, while activated NK cells still resisted VV binding. VV binding strongly colocalized with lipid rafts on the surfaces of all VV binding-susceptible PHL subsets, even when lipid rafts were relocated to cell uropods upon cell polarization. Immunosera raised against detergent-resistant membranes (DRMs) from monocytes or activated T cells, but not resting T cells, effectively cross-blocked VV binding to and infection of PHL subsets. CD29 and CD98, two lipid raft-associated membrane proteins that had been found to be important for VV entry into HeLa cells, had no effect on VV binding to and infection of primary activated T cells. Our data indicate that PHL subsets express VV protein receptors enriched in lipid rafts and that receptors are cross-presented on all susceptible PHLs.

CD4+ T Cells Provide Intermolecular Help To Generate Robust Antibody Responses in Vaccinia Virus–Vaccinated Humans

Immunization with vaccinia virus elicits a protective Ab response that is almost completely CD4(+) T cell dependent. A recent study in a rodent model observed a deterministic linkage between Ab and CD4(+) T cell responses to particular vaccinia virus proteins suggesting that CD4(+) T cell help is preferentially provided to B cells with the same protein specificity (Sette et al. 2008. Immunity 28: 847-858). However, a causal linkage between Ab and CD4(+) T cell responses to vaccinia or any other large pathogen in humans has yet to be done. In this study, we measured the Ab and CD4(+) T cell responses against four vaccinia viral proteins (A27L, A33R, B5R, and L1R) known to be strongly targeted by humoral and cellular responses induced by vaccinia virus vaccination in 90 recently vaccinated and 7 long-term vaccinia-immunized human donors. Our data indicate that there is no direct linkage between Ab and CD4(+) T cell responses against each individual protein in both short-term and long-term immunized donors. Together with the observation that the presence of immune responses to these four proteins is linked together within donors, our data suggest that in vaccinia-immunized humans, individual viral proteins are not the primary recognition unit of CD4(+) T cell help for B cells. Therefore, we have for the first time, to our knowledge, shown evidence that CD4(+) T cells provide intermolecular (also known as noncognate or heterotypic) help to generate robust Ab responses against four vaccinia viral proteins in humans.

Vaccinia virus protein N2 is a nuclear IRF3 inhibitor that promotes virulence

Vaccinia virus (VACV) expresses many proteins that are non-essential for virus replication but promote virulence by inhibiting components of the host immune response to infection. These immunomodulators include a family of proteins that have, or are predicted to have, a structure related to the B-cell lymphoma (Bcl)-2 protein. Five members of the VACV Bcl-2 family (N1, B14, A52, F1 and K7) have had their crystal structure solved, others have been characterized and a function assigned (C6, A46), and others are predicted to be Bcl-2 proteins but are uncharacterized hitherto (N2, B22, C1). Data presented here show that N2 is a nuclear protein that is expressed early during infection and inhibits the activation of interferon regulatory factor (IRF)3. Consistent with its nuclear localization, N2 inhibits IRF3 downstream of the TANK-binding kinase (TBK)-1 and after IRF3 translocation into the nucleus. A mutant VACV strain Western Reserve lacking the N2L gene (vΔN2) showed normal replication and spread in cultured cells compared to wild-type parental (vN2) and revertant (vN2-rev) viruses, but was attenuated in two murine models of infection. After intranasal infection, the vΔN2 mutant induced lower weight loss and signs of illness, and virus was cleared more rapidly from the infected tissue. In the intradermal model of infection, vΔN2 induced smaller lesions that were resolved more rapidly. In summary, the N2 protein is an intracellular virulence factor that inhibits IRF3 activity in the nucleus.

Vaccinia virus protein K7 is a virulence factor that alters the acute immune response to infection

Vaccinia virus (VACV) encodes many proteins that antagonize the innate immune system including a family of intracellular proteins with a B-cell lymphoma (Bcl)-2-like structure. One of these Bcl-2 proteins called K7 binds Toll-like receptor-adaptor proteins and the DEAD-box RNA helicase DDX3 and thereby inhibits the activation of NF-κB and interferon regulatory factor 3. However, the contribution of K7 to virus virulence is not known. Here a VACV lacking the K7R gene (vΔK7) was constructed and compared with control viruses that included a plaque purified wt (vK7), a revertant with the K7R gene reinserted (vK7-rev) and a frame-shifted virus in which the translational initiation codon was mutated to prevent K7 protein expression (vK7-fs). Data presented show that loss of K7 does not affect virus replication in cell culture or in vivo; however, viruses lacking the K7 protein were less virulent than controls in murine intradermal (i.d.) and intranasal (i.n.) infection models and there was an altered acute immune response to infection. In the i.d. model, vΔK7 induced smaller lesions than controls, and after i.n. infection vΔK7 induced a reduced weight loss and signs of illness, and more rapid clearance of virus from infected tissue. Concomitantly, the intrapulmonary innate immune response to infection with vΔK7 showed increased infiltration of NK cells and CD8+ T-cells, enhanced MHC class II expression by macrophages, and enhanced cytolysis of target cells by NK cells and VACV-specific CD8+ T-cells. Thus protein K7 is a virulence factor that affects the acute immune response to infection.

Biochemical and Biophysical Properties of a Putative Hub Protein Expressed by Vaccinia Virus

H5 is a constitutively expressed, phosphorylated vaccinia virus protein that has been implicated in viral DNA replication, post-replicative gene expression, and virus assembly. For the purpose of understanding the role of H5 in vaccinia biology, we have characterized its biochemical and biophysical properties. Previously, we have demonstrated that H5 is associated with an endoribonucleolytic activity. In this study, we have shown that this cleavage results in a 3'-OH end suitable for polyadenylation of the nascent transcript, corroborating a role for H5 in vaccinia transcription termination. Furthermore, we have shown that H5 is intrinsically disordered, with an elongated rod-shaped structure that preferentially binds double-stranded nucleic acids in a sequence nonspecific manner. The dynamic phosphorylation status of H5 influences this structure and has implications for the role of H5 in multiple processes during virus replication.

Interaction between E3L and K3L proteins of vaccinia virus.

Interaction between E3L and K3L proteins of vaccinia virus.

P132 The human dead-box helicase 3 couples IKK-epsilon to IRF3 activation

Introduction The human DEAD-box RNA helicase DDX3 has been implicated in different cellular processes including transcription, splicing, mRNA nuclear export and translation. We and others previously showed that DDX3 is involved in innate immune signalling pathways which trigger the induction of type I interferon through the kinases IKKe or TBK1 [1] , [2] , [3] . We demonstrated that DDX3 interacts with IKKe, and that it contributes to IRF3 activation. Other studies have confirmed a role for DDX3 in IFN-s induction, but placed DDX3 upstream in the MAVS-mediated signalling pathway, or suggested that DDX3 binds to the IFN-s promoter directly. Therefore, our study here aimed at placing DDX3 more firmly in the signalling pathway(s) leading to IFN-s induction, by dissecting its molecular interaction with IKKe and the functional consequences of this interaction. Methods The DDX3, IKKe and IRF3 interaction studies were performed using co-immunoprecipitation (Co-IP) and His pulldown assays. In vitro kinase assays were employed for investigating DDX3 and IRF3 phosphorylation by IKKe. IKKe- phosphorylation sites in DDX3 were identified using Mass Spectrometry. Luciferase reporter assays were used to study effects of phospho-deficient and –mimetic DDX3 mutants on IFN-s promoter induction. Results We show here that DDX3 directly interacts with, and is phosphorylated by IKKe. Phosphorylation of DDX3 occurs mainly in its N-terminal region, which is required for IFN-s induction, and is targeted by the vaccinia virus protein K7 in a viral immune evasion strategy. In agreement with this, we found that K7 specifically inhibits DDX3-phosphorylation by IKKe. Furthermore, we have identified one serine residue in the N-terminus of DDX3 that is crucial for the effect of DDX3 on the IFN-s promoter. We also show that DDX3 directly enhances IKKe activity and phosphorylation of IRF3 in an in vitro reconstitution assay. In addition, we demonstrate that DDX3 directly binds to IRF3, and that this interaction is crucial for the DDX3 effect on IFN-s induction. Phosphorylation of DDX3 by IKKe facilitates the subsequent recruitment of IRF3. IRF3 and IKKe have adjacent and potentially overlapping, but not identical, binding sites in the N-terminus of DDX3. Conclusion Our study has shown that the N-terminus of DDX3 directly interacts with IKKe and IRF3 and that this mediates their activation and functional interaction. The interaction between DDX3 and IRF3 contributes to IRF3 activation and is required for the effect of DDX3 on IFN-s promoter induction. The effects of DDX3 on IKKe activation and IRF3 activation can be uncoupled by mutation of a single serine residue in the N-terminus of DDX3.Our work implicates DDX3 as a bridging adaptor for IKKe and IRF3 in RLH and TLR signalling pathways.

Vaccinia Virus Virion Membrane Biogenesis Protein A11 Associates with Viral Membranes in a Manner That Requires the Expression of Another Membrane Biogenesis Protein, A6

A group of vaccinia virus (VACV) proteins, including A11, L2, and A6, are required for biogenesis of the primary envelope of VACV, specifically, for the acquisition of viral membrane precursors. However, the interconnection among these proteins is unknown and, with the exception of L2, the connection of these proteins with membranes is also unknown. In this study, prompted by the findings that A6 coprecipitated A11 and that the cellular distribution of A11 was dramatically altered by repression of A6 expression, we studied the localization of A11 in cells by using immunofluorescence and cell fractionation analysis. A11 was found to associate with membranes and colocalize with virion membrane proteins in viral replication factories during normal VACV replication. A11 partitioned almost equally between the detergent and aqueous phases upon Triton X-114 phase separation, demonstrating an intrinsic affinity with lipids. However, in the absence of infection or VACV late protein synthesis, A11 did not associate with cellular membranes. Furthermore, when A6 expression was repressed, A11 did not colocalize with any viral membrane proteins or associate with membranes. In contrast, when virion envelope formation was blocked at a later step by repression of A14 expression or by rifampin treatment, A11 colocalized with virion membrane proteins in the factories. Altogether, our data showed that A11 associates with viral membranes during VACV replication, and this association requires A6 expression. This study provides a physical connection between A11 and viral membranes and suggests that A6 regulates A11 membrane association.

Vaccinia virus protein C4 inhibits NF-κB activation and promotes virus virulence

Vaccinia virus (VACV) strain Western Reserve protein C4 has been characterized and its function and contribution to virus virulence assessed. Bioinformatic analysis showed that C4 is conserved in six orthopoxvirus species and shares 43 % amino acid identity with VACV protein C16, a known virulence factor. A recombinant VACV expressing a C-terminally tagged version of C4 showed that, like C16, this 37 kDa protein is expressed early during infection and localizes to both the cytoplasm and the nucleus. Functional assays using a firefly luciferase reporter plasmid under the control of a nuclear factor kappa B (NF-κB)-dependent promoter demonstrated that C4 inhibits NF-κB activation at, or downstream of, the inhibitor of kappa kinase (IKK) complex. Consistent with this, C4 inhibited interleukin-1β-induced translocation of p65 into the nucleus. A VACV lacking the C4L gene (vΔC4) showed no significant differences from wild-type virus in growth kinetics or spread in cell culture, but had reduced virulence in a murine intranasal model of infection. vΔC4-infected mice exhibited fewer symptoms, lost less weight and recovered 7 days earlier than animals infected with control viruses expressing C4. Furthermore, bronchoalveolar lavage fluid from vΔC4-infected mice had increased cell numbers at day 5 post-infection, which correlated with reduced lung virus titres from this time onward. C4 represents the ninth VACV protein to inhibit NF-κB activation and remarkably, in every case examined, loss of each protein individually caused an alteration in virus virulence, despite the presence of other NF-κB inhibitors.

Adjuvant-like Effect of Vaccinia Virus 14K Protein: A Case Study with Malaria Vaccine Based on the Circumsporozoite Protein

Development of subunit vaccines for malaria that elicit a strong, long-term memory response is an intensive area of research, with the focus on improving the immunogenicity of a circumsporozoite (CS) protein-based vaccine. In this study, we found that a chimeric protein, formed by fusing vaccinia virus protein 14K (A27) to the CS of Plasmodium yoelii, induces strong effector memory CD8(+) T cell responses in addition to high-affinity Abs when used as a priming agent in the absence of any adjuvant, followed by an attenuated vaccinia virus boost expressing CS in murine models. Moreover, priming with the chimeric protein improved the magnitude and polyfunctionality of cytokine-secreting CD8(+) T cells. This fusion protein formed oligomers/aggregates that led to activation of STAT-1 and IFN regulatory factor-3 in human macrophages, indicating a type I IFN response, resulting in NO, IL-12, and IL-6 induction. Furthermore, this vaccination regimen inhibited the liver stage development of the parasite, resulting in sterile protection. In summary, we propose a novel approach in designing CS based pre-erythrocytic vaccines against Plasmodium using the adjuvant-like effect of the immunogenic vaccinia virus protein 14K.

Protein B5 is required on extracellular enveloped vaccinia virus for repulsion of superinfecting virions

Vaccinia virus (VACV) spreads across cell monolayers fourfold faster than predicted from its replication kinetics. Early after infection, infected cells repulse some superinfecting extracellular enveloped virus (EEV) particles by the formation of actin tails from the cell surface, thereby causing accelerated spread to uninfected cells. This strategy requires the expression of two viral proteins, A33 and A36, on the surface of infected cells and upon contact with EEV this complex induces actin polymerization. Here we have studied this phenomenon further and investigated whether A33 and A36 expression in cell lines causes an increase in VACV plaque size, whether these proteins are able to block superinfection by EEV, and which protein(s) on the EEV surface are required to initiate the formation of actin tails from infected cells. Data presented show that VACV plaque size was not increased by expression of A33 and A36, and these proteins did not block entry of the majority of EEV binding to these cells. In contrast, expression of proteins A56 and K2 inhibited entry of both EEV and intracellular mature virus. Lastly, VACV protein B5 was required on EEV to induce the formation of actin tails at the surface of cells expressing A33 and A36, and B5 short consensus repeat 4 is critical for this induction.

Increased Interaction between Vaccinia Virus Proteins A33 and B5 Is Detrimental to Infectious Extracellular Enveloped Virion Production

Two mechanisms exist for the incorporation of B5 into extracellular virions, one of which is dependent on A33. In the companion to this paper (W. M. Chan and B. M. Ward, J. Virol. 86:8210-8220, 2012), we show that the lumenal domain of A33 is sufficient for interaction with the coiled-coil domain of B5 and capable of directing B5-green fluorescent protein (GFP) into extracellular virions. Here, we have created a panel of charge-to-alanine mutations in the lumenal domain of A33 to map the B5 interaction site. While none of these mutations abolished the interaction with B5, a subset displayed an increased interaction with both B5 and B5-GFP. Both B5 and B5-GFP recombinant viruses expressing these mutant proteins in place of normal A33 had a small-plaque phenotype. The increased interaction of the mutant proteins was detected during infection, suggesting that normally the interaction is either weak or transient. In addition, the increased A33-B5 interaction was detected on virions produced by recombinant viruses and correlated with reduced target cell binding. Taken together, these results show that both B5 and B5-GFP interact with A33 during infection and that the duration of this interaction needs to be regulated for the production of fully infectious extracellular virions.

Modulation of the Myxoma Virus Plaque Phenotype by Vaccinia Virus Protein F11

Vaccinia virus (VACV) produces large plaques consisting of a rapidly expanding ring of infected cells surrounding a lytic core, whereas myxoma virus (MYXV) produces small plaques that resemble a focus of transformed cells. This is odd, because bioinformatics suggests that MYXV carries homologs of nearly all of the genes regulating Orthopoxvirus attachment, entry, and exit. So why does MYXV produce foci? One notable difference is that MYXV-infected cells produce few of the actin microfilaments that promote VACV exit and spread. This suggested that although MYXV carries homologs of the required genes (A33R, A34R, A36R, and B5R), they are dysfunctional. To test this, we produced MYXV recombinants expressing these genes, but we could not enhance actin projectile formation even in cells expressing all four VACV proteins. Another notable difference between these viruses is that MYXV lacks a homolog of the F11L gene. F11 inhibits the RhoA-mDia signaling that maintains the integrity of the cortical actin layer. We constructed an MYXV strain encoding F11L and observed that, unlike wild-type MYXV, the recombinant virus disrupted actin stress fibers and produced plaques up to 4-fold larger than those of controls, and these plaques expanded ∼6-fold faster. These viruses also grew to higher titers in multistep growth conditions, produced higher levels of actin projectiles, and promoted infected cell movement, although neither process was to the extent of that observed in VACV-infected cells. Thus, one reason for why MYXV produces small plaques is that it cannot spread via actin filaments, although the reason for this deficiency remains obscure. A second reason is that leporipoxviruses lack vaccinia's capacity to disrupt cortical actin.

Abstract 1569: Multivalent melanoma vaccine using primary human melanoma cells and a vaccinia virus based antigen retrieval system

Abstract Melanoma is the fifth most diagnosed cancer in men and seventh most diagnosed cancer in women and it is estimated that there will be over 70,000 new cases and 8,000 deaths in 2011. Prevention of both primary and secondary melanomas requires the development of a melanoma specific multi-valent vaccine. We have been involved in developing a vaccinia virus based vaccine consisting of melanoma cell membrane antigens and integrated vaccinia viral proteins, which serve as a potent adjuvant. This melanoma vaccine, referred to as viral membrane oncolysate (VMO), is composed of five primary cell lines derived from patient tumors each independently infected with vaccinia virus and has been shown to induce anti-cancer immune response in clinical trials. Notable results were obtained in node positive subsets of patients that showed a positive response in survival distribution function suggesting that this preparation is an active therapeutic immunogen but the mechanism of immune activation needs to be elucidated. In order for us to define the immunogens that impart the clinical efficacy of the this melanoma VMO vaccine, it is essential to characterize the primary cell lines with respect to their phenotypic characteristics as well as to define the melanoma antigen repertoire so that effective vaccine strategies can be designed that do not rely exclusively on tumor derived primary cells. Our study reveals that all the five primary cell lines expressed antigens, such as gp100, CD146, and tyrosinase, similar to several commercially available melanoma cell lines although at varying concentrations. In addition, the five primary cell lines demonstrated varying migratory/invasive properties using a Boyden Chamber assay and their tumorigenic potential is being tested in nude mice. The cells were also characterized with respect to BRAF mutations. We observed several similarities between the antigenic profile of the primary and established cell lines as well as some unique characteristics. We conclude that using a combination of defined antigens from primary tumor cells is useful in designing a pan-antigen immunotherapeutic vaccine. This melanoma pan antigen vaccine can be an effective tool in primary prevention of melanoma presumably having the highest clinical efficacy when combined with unique antigens from primary cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1569. doi:1538-7445.AM2012-1569