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Detection of secretory antibodies in the airway is highly desirable when evaluating mucosal protection by vaccines against a respiratory virus, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We show that intranasal delivery of an attenuated SARS-CoV-2 (Nsp1-K164A/H165A) induces both mucosal and systemic IgA and IgG in male Syrian hamsters. Interestingly, either direct intranasal immunization or airborne transmission-mediated delivery of Nsp1-K164A/H165A in Syrian hamsters offers protection against heterologous challenge with variants of concern (VOCs) including Delta, Omicron BA.1, BA.2.12.1 and BA.5. Vaccinated animals show significant reduction in both tissue viral loads and lung inflammation. Similarly attenuated viruses bearing BA.1 and BA.5 spike boost variant-specific neutralizing antibodies in male mice that were first vaccinated with modified vaccinia virus Ankara vectors (MVA) expressing full-length WA1/2020 Spike protein. Together, these results demonstrate that our attenuated virus may be a promising nasal vaccine candidate for boosting mucosal immunity against future SARS-CoV-2 VOCs., (© 2023. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Background: Highly pathogenic avian H5 influenza viruses have spread and diversified genetically and antigenically into multiple clades and subclades. Most isolates of currently circulating H5 viruses are in clade 2.3.2.1 or 2.3.4.4., Methods: Panels of murine monoclonal antibodies (mAbs) were generated to the influenza hemagglutinin (HA) of H5 viruses from the clade 2.3.2.1 H5N1 vaccine virus A/duck/Bangladesh/19097/2013 and the clade 2.3.4.4 H5N8 vaccine virus A/gyrfalcon/Washington/41088-6/2014. Antibodies were selected and characterized for binding, neutralization, epitope recognition, cross-reactivity with other H5 viruses, and the ability to provide protection in passive transfer experiments., Results: All mAbs bound homologous HA in an ELISA format; mAbs 5C2 and 6H6 were broadly binding for other H5 HAs. Potently neutralizing mAbs were identified in each panel, and all neutralizing mAbs provided protection in passive transfer experiments in mice challenged with a homologous clade influenza virus. Cross-reacting mAb 5C2 neutralized a wide variety of clade 2.3.2.1 viruses, as well as H5 viruses from other clades, and also provided protection against heterologous H5 clade influenza virus challenge. Epitope analysis indicated that the majority of mAbs recognized epitopes in the globular head of the HA. The mAb 5C2 appeared to recognize an epitope below the globular head but above the stalk region of HA., Conclusions: The results suggested that these H5 mAbs would be useful for virus and vaccine characterization. The results confirmed the functional cross-reactivity of mAb 5C2, which appears to bind a novel epitope, and suggest the therapeutic potential for H5 infections in humans with further development., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Broadly neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are sought to curb coronavirus disease 2019 (COVID-19) infections. Here we produced and characterized a set of mouse monoclonal antibodies (mAbs) specific for the ancestral SARS-CoV-2 receptor binding domain (RBD). Two of them, 17A7 and 17B10, were highly potent in microneutralization assay with 50% inhibitory concentration (IC 50 ) ≤135 ng/mL against infectious SARS-CoV-2 variants, including G614, Alpha, Beta, Gamma, Delta, Epsilon, Zeta, Kappa, Lambda, B.1.1.298, B.1.222, B.1.5, and R.1. Both mAbs (especially 17A7) also exhibited strong in vivo efficacy in protecting K18-hACE2 transgenic mice from the lethal infection with G614, Alpha, Beta, Gamma, and Delta viruses. Structural analysis indicated that 17A7 and 17B10 target the tip of the receptor binding motif in the RBD-up conformation. A third RBD-reactive mAb (3A6) although escaped by Beta and Gamma, was highly effective in cross-neutralizing Delta and Omicron BA.1 variants in vitro and in vivo. In competition experiments, antibodies targeting epitopes similar to these 3 mAbs were rarely enriched in human COVID-19 convalescent sera or postvaccination sera. These results are helpful to inform new antibody/vaccine design and these mAbs can be useful tools for characterizing SARS-CoV-2 variants and elicited antibody responses., (© 2023 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Few live attenuated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines are in pre-clinical or clinical development. We seek to attenuate SARS-CoV-2 (isolate WA1/2020) by removing the polybasic insert within the spike protein and the open reading frames (ORFs) 6-8, and by introducing mutations that abolish non-structural protein 1 (Nsp1)-mediated toxicity. The derived virus (WA1-ΔPRRA-ΔORF6-8-Nsp1 K164A/H165A ) replicates to 100- to 1000-fold-lower titers than the ancestral virus and induces little lung pathology in both K18-human ACE2 (hACE2) transgenic mice and Syrian hamsters. Immunofluorescence and transcriptomic analyses of infected hamsters confirm that three-pronged genetic modifications attenuate the proinflammatory pathways more than the removal of the polybasic cleavage site alone. Finally, intranasal administration of just 100 PFU of the WA1-ΔPRRA-ΔORF6-8-Nsp1 K164A/H165A elicits robust antibody responses in Syrian hamsters and protects against SARS-CoV-2-induced weight loss and pneumonia. As a proof-of-concept study, we demonstrate that live but sufficiently attenuated SARS-CoV-2 vaccines may be attainable by rational design., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Studies on Ebola virus disease (EVD) survivors and clinical studies on Ebola virus (EBOV) vaccine candidates have pinpointed the importance of a strong antibody response in protection and survival from EBOV infection. However, little is known about the T cell responses to EBOV or EBOV vaccines. We used HLA-A*02:01 (HLA-A2) transgenic mice to study HLA-A2-specific T cell responses elicited following vaccination with EBOV glycoprotein (EBOV-GP) presented with three different systems: (i) recombinant protein (rEBOV-GP), (ii) vesicular stomatitis replication-competent recombinant virus (VSV-EBOV-GP), and (iii) modified vaccinia Ankara virus recombinant (MVA-EBOV-GP). T cells from immunized animals were analyzed using peptide pools representing the entire GP region and individual peptides. Regardless of the vaccine formulation, we identified a minimal 9mer epitope containing an HLA-A2 motif (FLDPATTS), which was confirmed through HLA-A2 binding affinity and immunization studies. Using binding prediction software, we identified substitutions surrounding position 9 (S9V, P10V, and Q11V) that predicted enhanced binding to the HLA-A2 molecule. This enhanced binding was confirmed through in vitro binding studies and enhanced potency was shown with in vivo immunization studies using the enhanced sequences and the wild-type sequence. Of note, in silico studies predicted the enhanced 9mer epitope carrying the S9V substitution as the best overall HLA-A2 epitope for the full-length EBOV-GP. These results suggest that EBOV-GP-S9V and EBOV-GP-P10V represent more potent in vivo immunogens. Identification and enhancement of EBOV-specific human HLA epitopes could lead to the development of tools and reagents to induce more robust T cell responses in human subjects. IMPORTANCE Vaccine efficacy and immunity to viral infection are often measured by neutralizing antibody titers. T cells are specialized subsets of immune cells with antiviral activity, but this response is variable and difficult to track. We showed that the HLA-A2-specific T cell response to the Ebola virus glycoprotein can be enhanced significantly by a single residue substitution designed to improve an epitope binding affinity to one of the most frequent MHC alleles in the human population. This strategy could be applied to improve T cell responses to Ebola vaccines designed to elicit antibodies and adapted to target MHC alleles of populations in regions where endemic infections, like Ebola virus disease, are still causing outbreaks with concerning pandemic potential.

Numerous vaccine candidates against SARS-CoV-2, the causative agent of the COVID-19 pandemic, are under development. The majority of vaccine candidates to date are designed to induce immune responses against the viral spike (S) protein, although different forms of S antigen have been incorporated. To evaluate the yield and immunogenicity of different forms of S, we constructed modified vaccinia virus Ankara (MVA) vectors expressing full-length S (MVA-S), the RBD, and soluble S ectodomain and tested their immunogenicity in dose-ranging studies in mice. All three MVA vectors induced spike-specific immunoglobulin G after one subcutaneous immunization and serum titers were boosted following a second immunization. The MVA-S and MVA-ssM elicited the strongest neutralizing antibody responses. In assessing protective efficacy, MVA-S-immunized adult Syrian hamsters were challenged with SARS-CoV-2 (USA/WA1/2020). MVA-S-vaccinated hamsters exhibited less severe manifestations of atypical pneumocyte hyperplasia, hemorrhage, vasculitis, and especially consolidation, compared to control animals. They also displayed significant reductions in gross pathology scores and weight loss, and a moderate reduction in virus shedding was observed post challenge in nasal washes. There was evidence of reduced viral replication by in situ hybridization, although the reduction in viral RNA levels in lungs and nasal turbinates did not reach significance. Taken together, the data indicate that immunization with two doses of an MVA vector expressing SARS-CoV-2 S provides protection against a stringent SARS-CoV-2 challenge of adult Syrian hamsters, reaffirm the utility of this animal model for evaluating candidate SARS-CoV-2 vaccines, and demonstrate the value of an MVA platform in facilitating vaccine development against SARS-CoV-2., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

The modified vaccinia virus Ankara (MVA), a severely attenuated strain of vaccinia virus, is a promising vector platform for viral-vectored vaccine development because of its attributes of efficient transgene expression and safety profile, among others. Thus, transgene stability in MVA is important to assure immunogenicity and efficacy. The global GC content of the MVA genome is 33%, and GC-rich sequences containing runs of C or G nucleotides have been reported to be less stable with passage of MVA vectors in cells. The production of recombinant MVA vaccines requires a number of expansion steps in cell culture, depending on production scale. We assessed the effect of extensive passage of four recombinant MVA vectors on the stability of the GC-rich herpes simplex type 2 (HSV-2) US6 gene encoding viral glycoprotein D (gD2) inserted at four different genomic sites, including the deletion (del) II and del III sites, the CP77 gene locus ( MVA_009-MVA_013 ) and the I8R - G1L intergenic region. Our data indicate that after 35 passages, there was a reduction in gD2 expression from del II, del III and CP77 sites. Sequencing analysis implicated US6 deletion and mutational events as responsible for the loss of gD2 expression. By contrast, 85.9% of recombinant plaques expressed gD2 from the I8R - G1L site, suggesting better accommodation of transgenes in this intergenic region. Thus, the I8R - G1L intergenic region may be more useful for transgene insertion for enhanced stability.

Vaccines that elicit broadly cross-neutralizing antibodies, including antibodies that target the conserved stem of hemagglutinin (HA), are being developed as a strategy for next-generation influenza vaccines that protect against influenza across multiple years. However, efficient induction of cross-neutralizing antibodies remains a challenge, and potential escape mutations have not been well characterized. Here we elicited cross-neutralizing antibodies by immunizing animals with the hemagglutinins from H5 and H9 subtype influenza A viruses that are sensitive to neutralization by stem antibodies. We further isolated and characterized an HA stem monoclonal antibody 4C2 that broadly neutralizes group 1 influenza viruses and identified HA mutations that reduced sensitivity to stem antibodies. Our results offer insights for next-generation influenza vaccine strategies for inducing cross-neutralizing antibodies., Competing Interests: The authors have declared that no competing interests exist.

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.

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.)

The robustness of immune responses to an antigen could be dictated by the route of vaccine inoculation. Traditional smallpox vaccines, essentially vaccinia virus strains, that were used in the eradication of smallpox were administered by percutaneous inoculation (skin scarification). The modified vaccinia virus Ankara is licensed as a smallpox vaccine in Europe and Canada and currently undergoing clinical development in the United States. MVA is also being investigated as a vector for the delivery of heterologous genes for prophylactic or therapeutic immunization. Since MVA is replication-deficient, MVA and MVA-vectored vaccines are often inoculated through the intramuscular, intradermal or subcutaneous routes. Vaccine inoculation via the intramuscular, intradermal or subcutaneous routes requires the use of injection needles, and an estimated 10 to 20% of the population of the United States has needle phobia. Following an observation in our laboratory that a replication-deficient recombinant vaccinia virus derived from the New York City Board of Health strain elicited protective immune responses in a mouse model upon inoculation by tail scarification, we investigated whether MVA and MVA recombinants can elicit protective responses following percutaneous administration in mouse models. Our data suggest that MVA administered by percutaneous inoculation, elicited vaccinia-specific antibody responses, and protected mice from lethal vaccinia virus challenge, at levels comparable to or better than subcutaneous or intramuscular inoculation. High titers of specific neutralizing antibodies were elicited in mice inoculated with a recombinant MVA expressing the herpes simplex type 2 glycoprotein D after scarification. Similarly, a recombinant MVA expressing the hemagglutinin of attenuated influenza virus rgA/Viet Nam/1203/2004 (H5N1) elicited protective immune responses when administered at low doses by scarification. Taken together, our data suggest that MVA and MVA-vectored vaccines inoculated by scarification can elicit protective immune responses that are comparable to subcutaneous vaccination, and may allow for antigen sparing when vaccine supply is limited.

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, 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., (Published by Elsevier Inc.)

The vaccinia virus complement control protein (VCP) is a secreted viral protein that binds the C3b and C4b complement components and inhibits the classic and alternative complement pathways. Previously, we reported that an attenuated smallpox vaccine, LC16m8, which was derived from the Lister strain of vaccinia virus (VV-Lister), expressed a glycosylated form of VCP, whereas published sequence data at that time indicated that the VV-Lister VCP has no motif for N-linked glycosylation. We were interested in determining whether the glycosylation of VCP impairs its biological activity, possibly contributing to the attenuation of LC16m8, and the likely origin of the glycosylated VCP. Expression analysis indicated that VV-Lister contains substrains expressing glycosylated VCP and substrains expressing nonglycosylated VCP. Other strains of smallpox vaccine, as well as laboratory strains of vaccinia virus, all expressed nonglycosylated VCP. Individual Lister virus clones expressing either the glycosylated VCP or the nonglycosylated species were isolated, and partially purified VCP from the isolates were found to be functional equivalents in binding human C3b and C4b complement proteins and inhibiting hemolysis and in immunogenicity. Recombinant vaccinia viruses expressing FLAG-tagged glycosylated VCP (FLAG-VCPg) and nonglycosylated VCP (FLAG-VCP) were constructed based on the Western Reserve strain. Purified FLAG-VCP and FLAG-VCPg bind human C3b and C4b and blocked complement-mediated hemolysis. Our data suggest that glycosylation did not affect the biological activity of VCP and thus may not have contributed to the attenuation of LC16m8. In addition, the LC16m8 virus likely originated from a substrain of VV-Lister that expresses glycosylated VCP., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

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.

Whole-body bioimaging was employed to study the effects of passive immunotherapies on lethality and viral dissemination in BALB/c mice challenged with recombinant vaccinia viruses expressing luciferase. WRvFire and IHD-J-Luc vaccinia viruses induced lethality with similar times to death following intranasal infection, but WRvFire replicated at higher levels than IHD-J-Luc in the upper and lower respiratory tracts. Three types of therapies were tested: licensed human anti-vaccinia virus immunoglobulin intravenous (VIGIV); recombinant anti-vaccinia virus immunoglobulin (rVIG; Symphogen, Denmark), an investigational product containing a mixture of 26 human monoclonal antibodies (HuMAbs) against mature virion (MV) and enveloped virion (EV); and HuMAb compositions targeting subsets of MV or EV proteins. Bioluminescence recorded daily showed that pretreatment with VIGIV (30 mg) or with rVIG (100 μg) on day -2 protected mice from death but did not prevent viral replication at the site of inoculation and dissemination to internal organs. Compositions containing HuMAbs against MV or EV proteins were protective in both infection models at 100 μg per animal, but at 30 μg, only anti-EV antibodies conferred protection. Importantly, the t statistic of the mean total fluxes revealed that viral loads in surviving mice were significantly reduced in at least 3 sites for 3 consecutive days (days 3 to 5) postchallenge, while significant reduction for 1 or 2 days in any individual site did not confer protection. Our data suggest that reduction of viral replication at multiple sites, including respiratory tract, spleen, and liver, as monitored by whole-body bioluminescence can be used to predict the effectiveness of passive immunotherapies in mouse models.

Smallpox, a disease caused by variola virus, is estimated to have killed hundreds of millions to billions of people before it was certified as eradicated in 1980. However, there has been renewed interest in smallpox vaccine development due in part to zoonotic poxvirus infections and the possibility of a re-emergence of smallpox, as well as the fact that first-generation smallpox vaccines are associated with relatively rare, but severe, adverse reactions in some vaccinees. An understanding of the immune mechanisms of vaccine protection and the use of suitable animal models for vaccine efficacy assessment are paramount to the development of safer and effective smallpox vaccines. This article focuses on studies aimed at understanding the immune responses elicited by vaccinia virus and the various animal models that can be used to evaluate smallpox vaccine efficacy. Harnessing this information is necessary to assess the effectiveness and potential usefulness of new-generation smallpox vaccines.

Highly attenuated modified vaccinia virus Ankara (MVA) is being considered as a safer alternative to conventional smallpox vaccines such as Dryvax or ACAM 2000, but it requires higher doses or more-frequent boosting than replication-competent Dryvax. Previously, we found that passive transfer of A27 antibodies can enhance protection afforded by vaccinia immune globulin (VIG), which is derived from Dryvax immunized subjects. Here we investigated whether protective immunity elicited by MVA could be augmented by prime-boost or combination immunizations with a recombinant A27 (rA27) protein. We found that a prime/boost immunization regimen with rA27 protein and MVA, in either sequence order, conferred protection to mice challenged with a lethal dose of vaccinia virus strain Western Reserve (VV-WR), compared to no protection after immunizations with a similar dose of either MVA or rA27 alone. Moreover, protection was achieved in mice primed simultaneously with combination of both MVA and rA27 in different vaccination routes, without any boost, even though MVA or rA27 alone at the same dose gave no protection. These findings show that rA27 can synergize with MVA to elicit robust protection that has a dose-sparing effect on MVA and can accelerate protection by eliminating the need for a booster dose.

Vaccination with Dryvax elicits a broad humoral response against many viral proteins. Human vaccinia immune globulin was used to screen the secreted proteins from cells infected with Dryvax or the candidate smallpox vaccine LC16m8 to determine whether the protective humoral response included antibodies against secreted viral proteins. Many proteins were detected, with the primary band corresponding to a band of 28 or 30 kDa in cells infected with Dryvax or LC16m8, respectively. This was identified as the vaccinia virus complement protein (VCP), which migrated more slowly in LC16m8-infected cells due to post-translational glycosylation. Vaccinia virus deleted in VCP, vVCPko, protected mice from a lethal intranasal challenge of vaccinia Western Reserve strain. Mice vaccinated with purified VCP demonstrated a strong humoral response, but were not protected against a moderate lethal challenge of vaccinia virus, suggesting that the humoral response against VCP is not critical for protection.

The immune response elicited by LC16m8, a candidate smallpox vaccine that was developed in Japan by cold selection during serial passage of the Lister vaccine virus in primary rabbit kidney cells, was compared to Dryvax in a mouse model. LC16m8 carries a mutation resulting in the truncation of the B5 protein, an important neutralizing target of the extracellular envelope form of vaccinia virus (EV). LC16m8 elicited a broad-spectrum immunoglobulin G (IgG) response that neutralized both EV and the intracellular mature form of vaccinia virus and provoked cell-mediated immune responses, including the activation of CD4+ and CD8+ cells, similarly to Dryvax. Mice inoculated with LC16m8 had detectable but low levels of anti-B5 IgG compared to Dryvax, but both Dryvax and LC16m8 sera neutralized vaccinia virus EV in vitro. A truncated B5 protein (approximately 8 kDa) was expressed abundantly in LC16m8-infected cells, and both murine immune sera and human vaccinia virus immunoglobulin recognized the truncated recombinant B5 protein in antigen-specific enzyme-linked immunosorbent assays. At a high-dose intranasal challenge (100 or 250 50% lethal doses), LC16m8 and Dryvax conferred similar levels of protection against vaccinia virus strain WR postvaccination. Taken together, the results extend our current understanding of the protective immune responses elicited by LC16m8 and indicate that the relative efficacy in a mouse model rivals that of previously licensed smallpox vaccines.

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.

The licensed smallpox vaccine Dryvax is used as the standard in comparative immunogenicity and protection studies of new smallpox vaccine candidates. Although the correlates of protection against smallpox are unknown, recent studies have shown that a humoral response against the intracellular mature virion and extracellular enveloped virion (EV) forms of vaccinia virus is crucial for protection. Using a recombinant Semliki Forest virus (rSFV) vector system, we expressed a set of full-length EV proteins for the development of EV antigen-specific enzyme-linked immunosorbent assays (ELISAs) and the production of monospecific antisera. The EV-specific ELISAs were used to evaluate the EV humoral response elicited by Dryvax and the nonreplicating modified vaccinia virus Ankara (MVA) in mouse vaccination experiments comparing doses and routes of vaccination. Quantitatively similar titers of antibodies against EV antigens A33R, A56R, and B5R were measured in mice vaccinated with Dryvax and MVA when MVA was administered at a dose of 10(8) plaque-forming units. Further, a substantial increase in the EV-specific antibody response was induced in mice inoculated with MVA by using a prime-boost schedule. Finally, we investigated the abilities of the EV-expressing rSFV vectors to elicit the production of polyclonal monospecific antisera against the corresponding EV proteins in mice. The monospecific serum antibody levels against A33R, A56R, and B5R were measurably higher than the antibody levels induced by Dryvax. The resulting polyclonal antisera were used in Western blot analysis and immunofluorescence assays, indicating that rSFV particles are useful vectors for generating monospecific antisera.

Adverse events associated with the use of live smallpox vaccines have led to the development of a new generation of attenuated smallpox vaccines that are prepared in cultured cells as alternatives. The inability to conduct direct clinical evaluation of their efficacy in humans demands that licensure be based on animal studies and exhaustive evaluation of their in vitro properties. One of the most important characteristics of live viral vaccines is their genetic stability, including reversion of the vaccine strain to more virulent forms, recombination with other viral sequences to produce potentially pathogenic viruses, and genetic drift that can result in decrease of immunogenicity and efficacy. To study genetic stability of an immunoessential vaccinia virus gene in a new generation smallpox vaccine, an advanced oligonucleotide microchip was developed and used to assay for mutations that could emerge in B5R gene, a vaccinia virus gene encoding for a protein that contains very important neutralizing epitopes. This microarray contained overlapping oligonucleotides covering the B5R gene of modified vaccinia virus Ankara (MVA), a well-studied candidate smallpox vaccine. The microarray assay was shown to be able to detect even a single point mutation, and to differentiate between vaccinia strains. At the same time, it could detect newly emerged mutations in clones of vaccinia strains. In the work described here, it was shown that MVA B5R gene was stable after 34 passages in Vero and MRC-5 cells that were proposed for use as cell substrates for vaccine manufacture. Potentially, the proposed method could be used as an identity test and could be extended for the entire viral genome and used to monitor consistency of vaccine production., ((c) 2007 Wiley-Liss, Inc.)

A previous report described a prime-boost immunization strategy using plasmid and modified vaccinia virus Ankara (MVA) vectors expressing herpes simplex virus 2 glycoprotein D (gD). Enhanced humoral and cellular immune responses were elicited by the prime-boost combination compared to plasmid DNA immunization alone. Surprisingly, a more diverse antibody isotype response, and a greater antibody and cellular immune response, was obtained if the gD MVA vector was used as the priming immunization rather than the gD plasmid vector. The present report evaluates the use of a needle-free delivery platform (Biojector) for delivery of plasmid and MVA gD-expressing vectors in a prime-boost immunization strategy. Needle-free delivery of both plasmid and MVA gD expression vectors was efficient, reproducible, and elicited a strong immune response in immunized mice. Biojector delivery of plasmid DNA was able to evoke a broader isotype response and cellular immune response than that obtained by gene gun delivered plasmid DNA. Further, DNA priming by Biojector delivery as part of a prime-boost procedure with MVA-gD2 resulted in a diverse antibody isotype distribution and enhanced cellular immune responses, similar to the responses obtained when MVA-gD2 was used as the priming immunization. Thus, needle-free delivery of plasmid DNA may provide additional flexibility and options for effective prime-boost vaccination.

Using vaccinia immune globulin (VIG), a high-titer antibody preparation from immunized subjects, we demonstrate that the humoral immune response in humans is directed against numerous antigens in the Dryvax vaccine strain. Western blot and immunoprecipitation analyses revealed highly antigenic proteins associated with both the extracellular enveloped virus and intracellular mature virus forms. The modified vaccinia virus Ankara (MVA), a new generation smallpox vaccine that is attenuated for replication in humans, expresses most, but not all, of the major vaccinia antigens recognized by antibodies in VIG, lacking the highly antigenic protein corresponding to the A-type inclusion body protein. Since new-generation smallpox vaccines such as MVA will require extensive comparison to traditional smallpox vaccines in animal models of immunogenicity and protection, we compared the vaccinia virus antigens recognized by VIG to those recognized by sera from Dryvax and MVA immunized mice. The humoral immune response in immunized mice is qualitatively similar to that in humans.

Vaccinia virus encodes an enzyme with DNA modifying activity that cleaves and inefficiently cross-links cruciformic DNA. This enzyme is contained within the virion, expressed at late times postinfection, and processes DNA in an energy-independent, Mg2+ ion-independent manner. Viral nuclease activity was measured in extracts from cells infected with well-defined viral mutants. Since some viral extracts lacked nuclease activity, the gene encoding the activity was postulated to be one of the open reading frames absent in the viruses lacking activity. Inducible expression of each candidate open reading frame revealed that only the gene VACWR035, or K4L, was required for nuclease activity. A recombinant virus missing only the open reading frame for K4L lacked nuclease activity. Extracts from a recombinant virus expressing K4L linked to a FLAG polypeptide were able to cleave and cross-link cruciformic DNA. There were no significant differences between the virus lacking K4L and wild-type vaccinia virus WR with respect to infectivity, growth characteristics, or processing of viral replicative intermediate DNA, including both telomeric and cross-linked forms. Purification of the K4L FLAG polypeptide expressed in bacteria yielded protein containing nicking-joining activity, implying that K4L is the only vaccinia virus protein required for the nicking-joining enzymatic activity.

Significant adverse events are associated with vaccination with the currently licensed smallpox vaccine. Candidate new-generation smallpox vaccines such as the replication-defective modified vaccinia virus Ankara (MVA) produce very few adverse events in experimental animals and in limited human clinical trials conducted near the end of the smallpox eradication campaign. Efficacy evaluation of such new-generation vaccines will be extraordinarily complex, however, since the eradication of smallpox precludes a clinical efficacy trial and the correlates of protection against smallpox are unknown. A combination of relevant animal efficacy studies along with thorough comparative immunogenicity studies between traditional and new-generation smallpox vaccines will be necessary for vaccine licensure. In the present study, a variety of immune responses elicited by MVA and the licensed smallpox vaccine Dryvax in a murine model were compared, with a focus on mimicking conditions and strategies likely to be employed in human vaccine trials. Immunization of mice with MVA, using several relevant vaccination routes including needle-free delivery, elicited humoral and cellular immune responses qualitatively similar to those elicited by vaccination with Dryvax. Similar levels of vaccinia-specific IgG and neutralizing antibody were elicited by Dryvax and MVA when higher doses (approximately 1 log) of MVA were used for immunization. Antibody levels peaked at about 6 weeks post-immunization and remained stable for at least 15 weeks. A booster immunization of either MVA or Dryvax following an initial priming immunization with MVA resulted in an enhanced IgG titer and neutralizing antibody response. In addition, both Dryvax and various MVA vaccination protocols elicited antibody responses to the extracellular enveloped form of the virus and afforded protection against a lethal intranasal challenge with vaccinia virus WR.

Construction of a herpes simplex virus 2 (HSV-2) bacterial artificial chromosome (BAC) is described. BAC vector sequences were inserted into the thymidine kinase gene of HSV-2 by homologous recombination. DNA from cells infected with the resulting recombinant virus was transformed into E. coli, and colonies containing the HSV-2 BAC (HSV2-BAC) were isolated and analyzed for the expected genotype. HSV2-BAC DNA was infectious when transfected back into mammalian cells and the resulting virus was thymidine kinase negative. When used to immunize mice, the HSV2-BAC DNA elicited a strong HSV-2 specific antibody response that was equal to or greater than live virus immunization. Further, HSV2-BAC immunization was protective when animals were challenged with a lethal dose of virus. The utility of the HSV2-BAC for construction of recombinant virus genomes was demonstrated by elimination of the HSV-2 glycoprotein D (gD) gene. A recombinant HSV-2 BAC with the gD gene deleted was isolated and shown to be incapable of producing infectious virus following transfection unless an HSV gD gene was expressed in a complementing cell line. Immunization of mice with the HSV2 gD-BAC also elicited an HSV-2 specific antibody response and was protective. The results demonstrate the feasibility of DNA immunization with HSV-2 bacterial artificial chromosomes for replicating and nonreplicating candidate HSV-2 vaccines, as well as the utility of BAC technology for construction and maintenance of novel HSV-2 vaccines. The results further suggest that such technology will be a powerful tool for dissecting the immune response to HSV-2.

The study of immunodominance within microbe-specific CD8 T cell responses has been challenging. We used a previously undescribed approach to create unbiased panels of CD8 cytotoxic T lymphocyte clones specific for herpes simplex virus type 2, a pathogen with a complex genome encoding at least 85 polypeptides. Circulating herpes simplex virus type 2-specific cells were enriched and cloned after sorting for expression of the skin homing-associated receptor, cutaneous lymphocyte-associated antigen, bypassing restimulation with antigen. The specificity of the resultant cytotoxic clones was determined. Clonal frequencies were compared with each other and with the total number of cytotoxic clones. For each subject within the homing receptor-positive compartment, the CD8 cytotoxic response was dominated by T cells specific for only a few peptides. Previously undescribed antigens and epitopes in viral tegument, capsid, or scaffold proteins were immunodominant in some subjects. Clone enumeration analyses were confirmed in some subjects with dominance studies by using herpes simplex mutants, vaccinia recombinants, and/or enzyme-linked immune spots. We conclude that among circulating cells expressing a homing-associated receptor, during chronic herpes type 2 infection, the CD8 T cell response becomes quite focused despite the presence of many potential antigenic peptides.

Several reports have indicated that prime-boost strategies of vaccination can enhance the level of specific immunity induced by nucleic acid vaccines. The present report describes such a strategy with herpes simplex virus (HSV)-2 glycoprotein D (gD), using combinations of plasmid vector that expresses gD (pgD2) and a recombinant modified vaccinia virus Ankara vector that expresses gD (MVA-gD2). The IgG antibody response to gD and the HSV-2 neutralizing antibody response were greatest when the MVA-gD2 vector was used as the priming immunization and then was boosted with either pgD2 or MVA-gD2. Determination of the isotype profile of MVA-gD2-primed mice revealed a much broader distribution of isotypes than that seen after DNA vaccination. In addition, antigen-stimulated spleen cells from mice primed with MVA-gD2 and boosted with either MVA-gD2 or pgD2 produced higher levels of interleukin-2 and interferon-gamma than did those from pgD2-primed mice, indicating that a prime-boost immunization strategy that uses the MVA and plasmid DNA vector dramatically enhances and diversifies the humoral and cellular immune response to HSV-2 gD.

Epstein-Barr virus (EBV) has been identified in a wide range of neoplastic and non-neoplastic disorders. The EBV open reading frame BHRF1 encodes a protein with partial sequence and functional homology to the anti-apoptotic onco-protein Bcl-2 and may therefore have a role in the proliferation of EBV positive cells. We have developed a rat monoclonal antibody against pBHRF1, which can detect BHRF1 in paraffin sections. While a number of mutant versions of BHRF1 were recognised, the monoclonal did not detect the BHRF1 homologue encoded by Herpesvirus papio or two mutants with deletions in the BH2 region. This novel rat monoclonal antibody (6A9) was used to examine tissue sections from 39 cases of non-keratinising undifferentiated nasopharyngeal carcinoma (NPC), 6 cases of metastatic NPC, 7 cases of EBV-positive NPC with squamous differentiation from Chinese patients, 15 cases of EBV-positive post-transplant lymphoproliferative disorder (PTLD), 6 EBV-containing lymphoblastoid cell lines, and 2 cases of oral hairy leukoplakia (OHL). In 11 cases of undifferentiated NPC, RT-PCR data were available for comparison with the immunohistochemistry. Both cases of OHL and two cases of LCL were positive for BHRF1 but none of the PTLD showed positive staining. All cases of undifferentiated NPC were positive for Bcl-2 but only one BHRF1 positive cell was identified in 1 of 39 cases of primary undifferentiated NPC. The 6A9 antibody produced less background staining and no nuclear positivity compared with the commercially available mouse monoclonal 5B11. It is concluded that BHRF1 can not be detected by immunohistochemistry in NPC and therefore it appears not to play a significant anti-apoptotic role in the progression of this EBV-associated tumour. The 6A9 monoclonal appears to be superior to 5B11 for the detection of pBHRF1 in tissue sections., (Copyright 2001 Wiley-Liss, Inc.)

The human tumour virus Epstein-Barr virus (EBV) encodes a 17 kDa protein, BHRF1, which is a member of the BCL:-2 family and has been shown to suppress apoptosis. The role of this gene in the life-cycle of EBV has not been fully elucidated. In order to identify motifs conserved in herpesviruses and possibly shed light on its function we isolated a BHRF1 homologue from herpesvirus papio (cercopithecine herpesvirus-12) a closely related gammaherpesvirus of baboons. The gene, hvpBHRF1, also encodes a 17 kDa protein which shares 64% identity and 79% similarity with EBV BHRF1 at the amino acid level. In biological assays, hvpBHRF1 and BHRF1 conferred similar levels of protection on human keratinocytes induced to apoptose with cis-platin.

BHRF 1, a component of the restricted early antigen (EA) complex of the Epstein-Barr virus (EBV) lytic cycle, encodes a 17 kDa putative transmembrane protein with both sequence and functional homology to the Bcl-2 proto-oncogene. To determine whether there was any sequence variation over the BHRF1 open reading frame (ORF), 15 EBV isolates from different geographical regions and from both healthy donors and patients with EBV-associated diseases were sequenced. A small number of base changes which resulted in amino acid substitutions in the BHRF1 protein were found relative to the prototype B95.8 EBV sequence and these were predominantly clustered near the amino terminus of the BHRF1 protein outside conserved domains identified in the Bcl-2 homologues. In transient transfection assays none of the mutations in the BHRF1 ORF from eight different EBV isolates had a significant effect on BHRF1 protein localization compared to the B95.8 BHRF1 protein. However, transient expression of the adenovirus 12 19K protein or Bcl-2 resulted in localization patterns distinct from that observed with BHRF1 protein. Whilst all eight EBV isolates and E1B-19K gave comparable levels of protection to the DNA-damaging agent cis-platin, Bcl-2 did not afford significant protection. Thus, despite several amino acid changes in the BHRF1 ORF of some of the EBV isolates studied, the ability of the protein to protect against cis-platin induced apoptosis is conserved. The highly conserved nature of BHRF1 amongst different EBV isolates at both the sequence and functional level supports the proposed important role of BHRF1 in delaying cell death, thereby maximizing the production of progeny virus and facilitating the establishment of virus persistence.