Vaccinia Virus Antigens Research Articles

Development of Smallpox Antibody Testing and Surveillance Following Smallpox Vaccination in the Republic of Korea.

Background: Despite its global eradication in 1977, smallpox remains a concern owing to its potential as a biological agent, thereby prompting the ongoing development and utilization of its vaccine. Vaccination with the Vaccinia virus induces immunity against variola virus, the causative agent of smallpox; however, this immunity does not extend to viruses of different genera within the Poxviridae family. In this study, we aimed to assess the efficacy of an enzyme-linked immunosorbent assay (ELISA) method utilizing Vaccinia virus and recombinant A27L antigen for detecting antibodies against smallpox. Methods. An analysis of the serum from 20 individuals pre- and post-vaccination with the CJ strain (CJ50300) revealed neutralizing antibodies, which were confirmed using the plaque reduction neutralization test (PRNT). The ELISA method, validated with a PRNT50 cut-off value of >4, exhibited a sensitivity and specificity of >95% and was particularly reactive with the inactivated virus. Furthermore, adherence to the smallpox vaccination policy revealed significant differences in Orthopoxvirus antibody levels among 300 individuals of different age groups. These findings highlight the reliability and efficacy of the ELISA method in detecting post-vaccination antibodies and contribute significantly to diagnostic methods to prepare for potential smallpox resurgence and bioterrorism threats.

Examination of the cross-reactivity between vaccinia virus Tiantan strain and monkeypox virus

AimTo investigate the cross-reactivity between the sera collected from Vaccinia Virus Tiantan Strain vaccinated rabbits and viral antigens of monkeypox virus. MethodsVaccinia viruses were prepared on chicken embryo fibroblasts (CEF) and Vero cells respectively named as CEF-VTT NVSI-1 and Vero-VTT NVSI-1. Rabbits were inoculated with a total of three doses of adjuvanted 1.3 × 108 PFU CEF-VTT NVSI-1 each dose or adjuvanted 3.9 × 107 PFU Vero-VTT NVSI-1 (Freunds complete adjuvant) via the subcutaneous route. We then performed the enzyme-linked immunosorbent assay (ELISA) and bio-layer interferometry (BLI) for determination of the binding activity and affinity of immune sera to five crucial surface antigens on monkeypox virus including A35, B6R, H3 and to corresponding homologous antigens A33R, B5 and L1R of vaccinia virus. For comparison, plaque reduction neutralizing tests were used to evaluate the neutralization of immune sera against vaccinia virus. ResultsBoth CEF-VTT NVSI-1 and Vero-VTT NVSI-1 vaccinations following planned schedule could induce neutralizing antibody titers greater than 1:2048 in rabbit sera. Binding antibodies targeting monkeypox viral antigens were confirmed by both indirect ELISA and BLI methods. Indirect ELISA for rabbit sera revealed 1:51200 binding antibody titers to A35/B6R/H3 monkeypox virus antigens while BLI tests yielded affinities at 2 × 10−6 to 8 × 10−7 between the sera and the three antigens. Similarly, such sera showed binding strength to vaccinia virus antigens A33R/B5/L1R consistent with that to three preceding monkeypox virus antigens. These results demonstrated the cross-reactivity between the sera of vaccinia virus vaccinated animals and monkeypox virus antigens. Traditional ELISA test and BLI method displayed a high consistency in antigen screening and they were further proved to correlate to the results of plaque reduction neutralizing test, which indicates that BLI could be utilized as an indirect alternative for assessment of neutralizing activity of samples in response to live virus. ConclusionsSera of vaccinia virus-vaccinated rabbits exhibited cross-reactivity with viral antigens of monkeypox virus. Potential in improving the accuracy of antigen discovery while reducing the lengthy work needed for the screening as BLI method possesses, it contributes greatly to the rapid preliminary evaluation of immune response generated by vaccines.

Monkeypox infection elicits strong antibody and B cell response against A35R and H3L antigens

Monkeypox virus (MPXV) resides in two forms; mature and enveloped, and depending on it, distinct proteins are displayed on the viral surface. Here, we expressed two MPXV antigens from the mature, and one from the enveloped form, and tested their reactivity to sera of 11 MPXV recoverees while comparing to sera from recently and past vaccinated individuals. 8 out of 11 recoverees exhibited detectable neutralization levels against Vaccinia Lister. Sera from all recoverees bound strongly to A35R and H3L antigens. Moreover, the responses to A35R were significantly higher within the recoverees compared to both recently and past vaccinated donors. Lastly, A35R- and H3L-specific IgG+ B cells ranging from 0.03-0.46% and 0.11-0.36%, respectively, were detected in all recoverees (A35R), and in 9 out of 11 recoverees (H3L). Therefore, A35R and H3L represent MPXV immune targets and could be used in a heat-inactivated serological ELISA for the identification of recent MPXV infection.

Antibody Recognition of Immunodominant Vaccinia Virus Envelope Proteins.

Vaccinia Virus (VACV) is an enveloped double stranded DNA virus and the active ingredient of the smallpox vaccine. The systematic administration of this vaccine led to the eradication of circulating smallpox (variola virus, VARV) from the human population. As a tribute to its success, global immunization was ended in the late 1970s. The efficacy of the vaccine is attributed to a robust production of protective antibodies against several envelope proteins of VACV, which cross-protect against infection with pathogenic VARV. Since global vaccination was ended, most children and young adults do not possess immunity against smallpox. This is a concern, since smallpox is considered a potential bioweapon. Although the smallpox vaccine is considered the gold standard of all vaccines and the targeted antigens have been widely studied, the epitopes that are targeted by the protective antibodies and their mechanism of binding had been, until recently, poorly characterized. Understanding the precise interaction between the antibodies and their epitopes will be helpful in the design of better vaccines against other diseases. In this review we will discuss the structural basis of recognition of the immunodominant VACV antigens A27, A33, D8, and L1 by protective antibodies and discuss potential implications regarding their protective capacity.

Povidone Iodine Ointment Application to the Vaccination Site Does Not Alter Immunoglobulin G Antibody Response to Smallpox Vaccine

U.S. military personnel deployed to high-risk areas receive the live vaccinia virus (VACV) smallpox vaccine ACAM2000. VACV shedding from the vaccination site can result in autoinoculation and contact transmission. We previously found that the application of povidone iodine ointment (PIO) to the scarification site reduced viral shedding without altering the antibody response, as measured by plaque reduction neutralization or enzyme-linked immunosorbent assays. In this study, we used protein microarray assays to measure the amount of immunoglobulin G antibody bound to (1) ACAM2000 itself and (2) individual VACV antigens that are present within ACAM2000. We assessed antibody binding in sera from primary smallpox vaccinees who applied PIO to the scarification site beginning on day 7 (PIO group) and from those who did not apply PIO (control group). In both cohorts, the postvaccination antibody response-in terms of antibody binding, both to ACAM2000 and to 11 individual VACV antigens-was significantly greater than the prevaccination response (all p < 0.0001). The postvaccination antibody binding levels of vaccinees in the PIO group did not differ from those of control vaccinees. These findings further support the topical application of PIO, starting on day 7, to reduce the viral shedding associated with smallpox vaccination.

Abstract 2829: Patient-derived primary melanoma cells that differentially express melano-specific antigens as a model for vaccines.

Abstract Transformation of pigment producing melanocytes into melanoma is a complex multi-step process involving the generation and/or over expression of various immunotherapeutic antigens. Several of these melanoma associated antigens (MAAs) are directly involved in melanin biosynthesis and melanosome biogenesis. The precise contribution of proteins involved in melanin synthesis to the MAA associated immune response is poorly understood. To this end, we have developed primary cell lines from human patients and xenograft models to analyze melanin synthesis and antigen expression. In vitro, from the perspective of melanin production, these primary cell lines express essential proteins associated with melanin synthesis, namely tyrosinase, Trp-1, Trp-2, and gp100, all of which are immunotherapeutic targets in melanoma. When grafted in nude mice, these cell lines were capable of producing solid pigmented tumors in vivo. However, three out of the five cell lines that produced melanin in vivo failed to produce melanin in vitro, even when cultured with supplementation of either α-melanocyte stimulating hormone (MSH), basic fibroblast growth factor (bFGF), or matrigel. Despite expression of tyrosinase, Trp-1, Trp-2, and gp100, two of the five cell lines failed to grow in nude mice or produce melanin in vitro. These patient derived primary cells and xenografts were also analyzed for expression of various other significant MAAs, which included MART-1 (Melan-A), MAGE-A1, and NY-ESO-1 as well as antigens not classified as MAAs but essential for tumor progression such as CD71 (transferring receptor) and CD146 (MCAM or melanoma cell adhesion molecule). All of the antigens analyzed in our cell lines, such as gp100, NY-ESO-1, MART-1, MAGE-A1, have been the targets of clinical trials but our results clearly indicate that these antigens are differentially expressed by our primary melanoma cells. Therefore, we believe that in order to develop a clinically effective immunotherapeutic vaccine, multiple cell lines expressing a combination of melano-specific antigens and a repertoire of MAAs that allows for minimal immune evasion may be necessary. To this end, we are exploring a vaccinia virus antigen retrieval technology, which incorporates multiple primary cell lines to develop such a pan-antigen melanoma vaccine. Citation Format: Robert Suriano, Andrea L. George, Shilpi Rajoria, Jan Geliebter, Raj K. Tiwari, Marc Wallack. Patient-derived primary melanoma cells that differentially express melano-specific antigens as a model for vaccines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2829. doi:10.1158/1538-7445.AM2013-2829

Reduced Interleukin-4 Receptor α Expression on CD8+ T Cells Correlates with Higher Quality Anti-Viral Immunity

With the hope of understanding how interleukin (IL)-4 and IL-13 modulated quality of anti-viral CD8+ T cells, we evaluated the expression of receptors for these cytokines following a range of viral infections (e.g. pox viruses and influenza virus). Results clearly indicated that unlike other IL-4/IL-13 receptor subunits, IL-4 receptor α (IL-4Rα) was significantly down-regulated on anti-viral CD8+ T cells in a cognate antigen dependent manner. The infection of gene knockout mice and wild-type (WT) mice with vaccinia virus (VV) or VV expressing IL-4 confirmed that IL-4, IL-13 and signal transducer and activator of transcription 6 (STAT6) were required to increase IL-4Rα expression on CD8+ T cells, but not interferon (IFN)-γ. STAT6 dependent elevation of IL-4Rα expression on CD8+ T cells was a feature of poor quality anti-viral CD8+ T cell immunity as measured by the production of IFN-γ and tumor necrosis factor α (TNF-α) in response to VV antigen stimulation in vitro. We propose that down-regulation of IL-4Rα, but not the other IL-4/IL-13 receptor subunits, is a mechanism by which CD8+ T cells reduce responsiveness to IL-4 and IL-13. This can improve the quality of anti-viral CD8+ T cell immunity. Our findings have important implications in understanding anti-viral CD8+ T cell immunity and designing effective vaccines against chronic viral infections.

Structural and Biochemical Characterization of the Vaccinia Virus Envelope Protein D8 and Its Recognition by the Antibody LA5

Smallpox vaccine is considered a gold standard of vaccines, as it is the only one that has led to the complete eradication of an infectious disease from the human population. B cell responses are critical for the protective immunity induced by the vaccine, yet their targeted epitopes recognized in humans remain poorly described. Here we describe the biochemical and structural characterization of one of the immunodominant vaccinia virus (VACV) antigens, D8, and its binding to the monoclonal antibody LA5, which is capable of neutralizing VACV in the presence of complement. The full-length D8 ectodomain was found to form a tetramer. We determined the crystal structure of the LA5 Fab-monomeric D8 complex at a resolution of 2.1 Å, as well as the unliganded structures of D8 and LA5-Fab at resolutions of 1.42 Å and 1.6 Å, respectively. D8 features a carbonic anhydrase (CAH) fold that has evolved to bind to the glycosaminoglycan (GAG) chondroitin sulfate (CS) on host cells. The central positively charged crevice of D8 was predicted to be the CS binding site by automated docking experiments. Furthermore, sequence alignment of various poxvirus D8 orthologs revealed that this crevice is structurally conserved. The D8 epitope is formed by 23 discontinuous residues that are spread across 80% of the D8 protein sequence. Interestingly, LA5 binds with a high-affinity lock-and-key mechanism above this crevice with an unusually large antibody-antigen interface, burying 2,434 Å(2) of protein surface.

Combining Oncolytic Vaccinia Virotherapy with Adoptive T Cell Therapy,

Abstract 4042 Background.Immunotherapy with cytotoxic T lymphocytes (CTLs) has proved safe and effective for the treatment of post-transplant viral re-activation and lymphoproliferative diseases. T cells specific for non-viral tumor antigens (TA) have been effective for certain tumors but their efficacy is limited. To improve T cell activity and circumvent tumor evasion T cells have been genetically modified with chimeric antigen receptors (CARs), that consist of extracellular variable domains of antibodies specific for tumor cell surface markers linked via a transmembrane domain to the z chain of the T cell receptor. Although the persistence of CAR-T cell can be improved by the inclusion of signaling domains from various costimulatory molecules, these rarely produce sufficient proliferation and persistence in vivo. An alternative way to promote T cell expansion in vivo is by vaccination. If the T cells carrying the transgenic CAR are naturally specific for a vaccine antigen, then the vaccine could be used in vivo to enhance proliferation and anti-tumor activity.The oncolytic vaccinia virus (OVV) JX-594 produces reduction in tumor bulk, but complete remissions have not been published to date. JX-594 is tumor selective due to its dependency on increased EGFP-Ras pathway and sensitivity to IFN and it potently activates innate immunity by transgenic expression of human GM-CSF and TLR stimulation. We hypothesize that JX-594 could enhance the antitumor efficacy of vaccinia virus (VV)-specific T cells expressing tumor-specific CARs while at the same time reducing tumor mass allowing increased T cell penetration and survival.To identify VV vaccine antigens that induce T cell proliferation in vivo, we selected 6 different VV antigens and asked first if they could activate memory T cells from vaccinated individuals and second if the frequency of these T cells in vivo increased in response to vaccination both in healthy donors and in patients with hepatocellular carcinoma (HCC) who had received OVV treatment. We then determined if VV-specific T cells could be induced to express a CAR specific for human epidermal growth factor receptor 2, HER2 that is expressed on a range of solid tumors. Methods.PBMCs were obtained from healthy donors and patients who had received intratumoral JX-594 injections at the University of California in San Diego, USA, and McMaster University Medical Center, Canada. PBMCs were stimulated with overlapping peptide libraries (20aa overlapping by 15aa) spanning the entire protein sequences of the A10L, D8L, H3L, G5R, B22R, and D8L antigens of VV. Three days after stimulation, cells were transduced with retroviral vector encoding HER2.CAR and then expanded as for non-transduced T cells. After 9 days T cells were tested for their dual specificity and function in ELIspot and cytotoxicity assays. Results.We activated VV-specific T cells from 20/21 healthy donors vaccinated from 1 month to over 40 years previously. VV-specific T cell lines recognized a median of 4 of the 6 VV antigens (range 0 to 6). The frequency of VV-specific T cells increased in response to vaccination in healthy seronegative donors and in patients receiving multiple OVV injections. Transduced VV-specific T cells expressed the HER2.CAR in 40 to 50% of cells and CAR(+) T cells secreted g-IFN in response to stimulation with VV peptides in intracellular cytokine assays, validating their dual specificity. HER2.CAR-transduced VV-specific T cells killed both VV peptide pulsed activated T cells and HER2-expressing tumor cells in an HLA-independent manner.In most donors we were not able to detect VV-specific T cells without prior stimulation. However VV-specific T cells from two healthy donors could be detected in blood and expanded 120 and 90 fold, respectively, after one stimulation. In all the other donors, VV-specific T cells expanded to comparable frequencies at the end of the first stimulation, range 0.1% to 5% (with a median of 1.5%) of the final T cell population as measured by ELISpot assay. Conclusions and future plans.We have identified 6 VV antigens that consistently reactivate and expand T cells after vaccination. T cells specific for these antigens will therefore be suitable hosts for tumor-specific CARs that can be reactivated and expanded in vivo after adoptive transfer until the tumor is eliminated. We will test our combined OVV & adoptive T cell strategy in an immunocompetent murine model prior to evaluation in clinical trials. Disclosures:No relevant conflicts of interest to declare.

Unpolarized Release of Vaccinia Virus and HIV Antigen by Colchicine Treatment Enhances Intranasal HIV Antigen Expression and Mucosal Humoral Responses

The induction of a strong mucosal immune response is essential to building successful HIV vaccines. Highly attenuated recombinant HIV vaccinia virus can be administered mucosally, but even high doses of immunization have been found unable to induce strong mucosal antibody responses. In order to solve this problem, we studied the interactions of recombinant HIV vaccinia virus Tiantan strain (rVTT-gagpol) in mucosal epithelial cells (specifically Caco-2 cell layers) and in BALB/c mice. We evaluated the impact of this virus on HIV antigen delivery and specific immune responses. The results demonstrated that rVTT-gagpol was able to infect Caco-2 cell layers and both the nasal and lung epithelia in BALB/c mice. The progeny viruses and expressed p24 were released mainly from apical surfaces. In BALB/c mice, the infection was limited to the respiratory system and was not observed in the blood. This showed that polarized distribution limited antigen delivery into the whole body and thus limited immune response. To see if this could be improved upon, we stimulated unpolarized budding of the virus and HIV antigens by treating both Caco-2 cells and BALB/c mice with colchicine. We found that, in BALB/c mice, the degree of infection and antigen expression in the epithelia went up. As a result, specific immune responses increased correspondingly. Together, these data suggest that polarized budding limits antigen delivery and immune responses, but unpolarized distribution can increase antigen expression and delivery and thus enhance specific immune responses. This conclusion can be used to optimize mucosal HIV vaccine strategies.

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

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.

Bi‐specific MHC Heterodimers for Characterization of Cross‐reactive T Cells

T cell cross‐reactivity describes the phenomenon whereby a single T cell can recognize two or more different peptide antigens presented in complex with MHC proteins. Cross‐reactive T cells have previously been characterized at the population level by cytokine secretion and MHC tetramer staining assays, but single‐cell analysis is difficult or impossible using these methods. In this study, we describe development of a novel peptide‐MHC heterodimer specific for cross‐reactive T cells. MHC‐peptide monomers were independently conjugated to hydrazide or aldehyde‐containing cross‐linkers using thiol‐maleimide coupling at cysteine residues introduced into recombinant MHC heavy chain proteins. Hydrazone formation provided bi‐specific MHC heterodimers carrying two different peptides. Using this approach we prepared heterodimers of the murine class I MHC protein H‐2K(b) carrying peptides from lymphocytic choriomeningitis virus and vaccinia virus, and used these to identify cross‐reactive CD8+ T cells recognizing both lymphocytic choriomeningitis virus and vaccinia virus antigens. A similar strategy could be used to develop reagents to analyze cross‐reactive T cell responses in humans.

Bi-specific MHC heterodimers for the characterization of cross-reactive T cells (67.13)

Abstract T cell cross-reactivity describes the phenomenon whereby a single T cell can recognize two or more different peptide antigens presented in complex withMHCproteins. Cross-reactive T cells have previously been characterized at the population level by cytokine secretion and MHC tetramer staining assays, but single-cell analysis is difficult or impossible using these methods. In this study, we describe development of a novel peptide-MHC heterodimer specific for cross-reactive T cells. MHC-peptide monomers were independently conjugated to hydrazide or aldehyde-containing cross-linkers using thiol-maleimide coupling at cysteine residues introduced into recombinant MHC heavy chain proteins. Hydrazone formation provided bi-specific MHC heterodimers carrying two different peptides. Using this approach we prepared heterodimers of the murine class I MHC protein H-2Kb carrying peptides from lymphocytic choriomeningitis virus and vaccinia virus, and used these to identify crossreactive CD8[|#1#|] T cells recognizing both lymphocytic choriomeningitis virus and vaccinia virus antigens. A similar strategy could be used to develop reagents to analyze cross-reactive T cell responses in humans.

Capturing the Natural Diversity of the Human Antibody Response against Vaccinia Virus

The eradication of smallpox (variola) and the subsequent cessation of routine vaccination have left modern society vulnerable to bioterrorism employing this devastating contagious disease. The existing, licensed vaccines based on live vaccinia virus (VACV) are contraindicated for a substantial number of people, and prophylactic vaccination of large populations is not reasonable when there is little risk of exposure. Consequently, there is an emerging need to develop efficient and safe therapeutics to be used shortly before or after exposure, either alone or in combination with vaccination. We have characterized the human antibody response to smallpox vaccine (VACV Lister) in immunized volunteers and isolated a large number of VACV-specific antibodies that recognize a variety of different VACV antigens. Using this broad antibody panel, we have generated a fully human, recombinant analogue to plasma-derived vaccinia immunoglobulin (VIG), which mirrors the diversity and specificity of the human antibody immune response and offers the advantage of unlimited supply and reproducible specificity and activity. The recombinant VIG was found to display a high specific binding activity toward VACV antigens, potent in vitro VACV neutralizing activity, and a highly protective efficacy against VACV challenge in the mouse tail lesion model when given either prophylactically or therapeutically. Altogether, the results suggest that this compound has the potential to be used as an effective postexposure prophylaxis or treatment of disease caused by orthopoxviruses.

Generation and characterization of a large panel of murine monoclonal antibodies against vaccinia virus

Vaccinia virus (VACV), the vaccine for smallpox, induces an antibody response that is largely responsible for conferring protection. Here, we studied the antibody response to VACV by generating and characterizing B cell hybridomas from a mouse immunized with VACV. Antibodies from 66 hybridomas were found to recognize 11 VACV antigens (D8, A14, WR148, D13, H3, A56, A33, C3, B5, A10 and F13), 10 of which were previously recognized as major antigens in smallpox vaccine by a microarray of VACV proteins produced with a prokaryotic expression system. VACV C3 protein, which was not detected as a target of antibody response by the proteome array, was recognized by two hybridomas, suggesting that selection of hybridomas based on immune recognition of infected cells has the advantage of detecting additional antibody response to native VACV antigens. In addition, these monoclonal antibodies are valuable reagents for studying poxvirus biology and protective mechanism of smallpox vaccine.

Bi-specific MHC Heterodimers for Characterization of Cross-reactive T Cells*

T cell cross-reactivity describes the phenomenon whereby a single T cell can recognize two or more different peptide antigens presented in complex with MHC proteins. Cross-reactive T cells have previously been characterized at the population level by cytokine secretion and MHC tetramer staining assays, but single-cell analysis is difficult or impossible using these methods. In this study, we describe development of a novel peptide-MHC heterodimer specific for cross-reactive T cells. MHC-peptide monomers were independently conjugated to hydrazide or aldehyde-containing cross-linkers using thiol-maleimide coupling at cysteine residues introduced into recombinant MHC heavy chain proteins. Hydrazone formation provided bi-specific MHC heterodimers carrying two different peptides. Using this approach we prepared heterodimers of the murine class I MHC protein H-2K(b) carrying peptides from lymphocytic choriomeningitis virus and vaccinia virus, and used these to identify cross-reactive CD8+ T cells recognizing both lymphocytic choriomeningitis virus and vaccinia virus antigens. A similar strategy could be used to develop reagents to analyze cross-reactive T cell responses in humans.

A Recombinant Flagellin-Poxvirus Fusion Protein Vaccine Elicits Complement-Dependent Protection Against Respiratory Challenge with Vaccinia Virus in Mice

Bacterial flagellin is a potent adjuvant that enhances adaptive immune responses to a variety of protein antigens. The vaccinia virus antigens L1R and B5R are highly immunogenic in the context of the parent virus, but recombinant forms of the proteins are only weakly immunogenic. Therefore we evaluated the humoral response to these antigens in mice when flagellin was used as an adjuvant. Flagellin-L1R and flagellin-B5R fusion proteins were more potent than flagellin, L1R, and B5R as separate proteins. At least three immunizations with flagellin-L1R and flagellin-B5R fusion proteins were required to confer protection in mice against challenge with vaccinia virus. Immune mice exhibited only limited signs of disease following challenge. Additionally, virus neutralization titers correlated with protection. Depletion of complement using cobra venom factor resulted in a marked decrease in the survival of immunized mice after challenge with vaccinia virus. Our results are consistent with the conclusion that flagellin-L1R and flagellin-B5R fusion proteins are effective in eliciting protective immunity against vaccinia virus that is dependent, in large part, on complement.

Complement-bound human antibodies to vaccinia virus B5 antigen protect mice from virus challenge

Evaluation of: Benhnia MR, McCausland MM, Laudenslager J et al. Heavily isotype dependent protective activities of human antibodies against the vaccinia virus extracellular virion antigen B5. J. Virol. 83, 12355–12367 (2009).The mechanism of protection afforded by vaccinia virus (VACV) – the smallpox vaccine – is a key issue for the development of modern vaccines and countermeasures. Antibodies to VACV antigens of the extracellular virion (EV) form play a central role in protection against poxvirus diseases in animal models, and contribute to the protection of immunized humans against poxviruses. B5, a viral EV protein, is conserved among different orthopoxviruses and antibodies to B5 that protect mice against VACV challenge. Antibodies to B5 are primarily responsible for neutralization of vaccinia EVs, yet the mechanism of EV neutralization by antibodies to B5 is not fully understood. The paper under evaluation demonstrates that most of the neutralization in vitro and protection in vivo in a mouse model, by monoclonal human anti-B5 IgGs, is heavily dependent on the ability of the IgGs to bind complement (C3 and C1q). Similarly, IgGs capable of complement binding control complement-dependent cytotoxicity of VACV-infected cells. Human polyclonal antibodies induced by the smallpox vaccine were similarly dependent on complement for EV neutralization and the complement-dependent destruction of infected cells. These findings not only contribute to a better understanding of the mechanism of protection by antibodies, but might also help in the development and evaluation of newly-developed therapeutic and prophylactic antibody-based products against virulent orthopoxviruses, and for the prevention or treatment of smallpox vaccine-related post-vaccinal adverse effects.

Uncovering the Interplay Between CD8, CD4 and Antibody Responses to Complex Pathogens

Vaccinia virus (VACV) was used as the vaccine strain to eradicate smallpox. VACV is still administered to healthcare workers or researchers who are at risk of contracting the virus, and to military personnel. Thus, VACV represents a weapon against outbreaks, both natural (e.g., monkeypox) or man-made (bioterror). This virus is also used as a vector for experimental vaccine development (cancer/infectious disease). As a prototypic poxvirus, VACV is a model system for studying host-pathogen interactions. Until recently, little was known about the targets of host immune responses, which was likely owing to VACVs large genome (>200 open reading frames). However, the last few years have witnessed an explosion of data, and VACV has quickly become a useful model to study adaptive immune responses. This review summarizes and highlights key findings based on identification of VACV antigens targeted by the immune system (CD4, CD8 and antibodies) and the complex interplay between responses.

Protection against Lethal Vaccinia Virus Challenge by Using an Attenuated Matrix Protein Mutant Vesicular Stomatitis Virus Vaccine Vector Expressing Poxvirus Antigens

Recombinant vesicular stomatitis viruses (VSV) are excellent candidate vectors for vaccination against human diseases. The neurovirulence of VSV in animal models requires the attenuation of the virus for use in humans. Previous efforts have focused on attenuating virus replication. Studies presented here test an alternative approach for attenuation that uses a matrix (M) protein mutant (rM51R) VSV as a vaccine vector against respiratory infection. This mutant is attenuated for viral virulence by its inability to suppress the innate immune response. The ability of rM51R VSV vectors to protect against lethal respiratory challenge was tested using a vaccinia virus intranasal challenge model. Mice immunized intranasally with rM51R vectors expressing vaccinia virus antigens B5R and L1R were protected against lethal vaccinia virus challenge. A single immunization with the vectors provided protection against vaccinia virus-induced mortality; however, a prime-boost strategy reduced the severity of the vaccinia virus-induced disease progression. Antibody titers measured after the prime and boost were low despite complete protection against lethal challenge. However, immunized animals had higher antibody titers during the challenge, suggesting that memory B-cell responses may be important for the protection. Depletion experiments demonstrated that B cells but not CD8 T cells were involved in the protection mediated by rM51R vaccine vectors that express B5R and L1R. These results demonstrate the potential of M protein mutant VSVs as candidate vaccine vectors against human diseases.