scholarly journals MVA Vector Vaccines Inhibit SARS CoV-2 Replication in Upper and Lower Respiratory Tracts of Transgenic Mice and Prevent Lethal Disease

2021 ◽  
Author(s):  
Ruikang Liu ◽  
Jeffrey L. Americo ◽  
Catherine A. Cotter ◽  
Patricia L. Earl ◽  
Noam Erez ◽  
...  
Keyword(s):  
Infectious Diseases ◽  
Vaccinia Virus ◽  
Neutralizing Antibodies ◽  
Severe Disease ◽  
Smallpox Vaccine ◽  
S Protein ◽  
Animal Protection ◽  
Vaccine Trials ◽  
Modified Vaccinia Virus Ankara ◽  
Initial Stage

AbstractReplication-restricted modified vaccinia virus Ankara (MVA) is a licensed smallpox vaccine and numerous clinical studies investigating recombinant MVAs (rMVAs) as vectors for prevention of other infectious diseases have been completed or are in progress. Two rMVA COVID-19 vaccine trials are at an initial stage, though no animal protection studies have been reported. Here, we characterize rMVAs expressing the S protein of CoV-2. Modifications of full length S individually or in combination included two proline substitutions, mutations of the furin recognition site and deletion of the endoplasmic retrieval signal. Another rMVA in which the receptor binding domain (RBD) flanked by the signal peptide and transmembrane domains of S was also constructed. Each modified S protein was displayed on the surface of rMVA-infected human cells and was recognized by anti-RBD antibody and by soluble hACE2 receptor. Intramuscular injection of mice with the rMVAs induced S-binding and pseudovirus-neutralizing antibodies. Boosting occurred following a second homologous rMVA but was higher with adjuvanted purified RBD protein. Weight loss and lethality following intranasal infection of transgenic hACE2 mice with CoV-2 was prevented by one or two immunizations with rMVAs or by passive transfer of serum from vaccinated mice. One or two rMVA vaccinations also prevented recovery of infectious CoV-2 from the lungs. A low amount of virus was detected in the nasal turbinates of only one of eight rMVA-vaccinated mice on day 2 and none later. Detection of subgenomic mRNA in turbinates on day 2 only indicated that replication was abortive in immunized animals.SignificanceVaccines are required to control COVID-19 during the pandemic and possibly afterwards. Recombinant nucleic acids, proteins and virus vectors that stimulate immune responses to the CoV-2 S protein have provided protection in experimental animal or human clinical trials, though questions remain regarding their ability to prevent spread and the duration of immunity. The present study focuses on replication-restricted modified vaccinia virus Ankara (MVA), which has been shown to be a safe, immunogenic and stable smallpox vaccine and a promising vaccine vector for other infectious diseases and cancer. In a transgenic mouse model, one or two injections of recombinant MVAs that express modified forms of S inhibited CoV-2 replication in the upper and lower respiratory tracts and prevented severe disease.

scholarly journals Neutralization Assay Using a Modified Vaccinia Virus Ankara Vector Expressing the Green Fluorescent Protein Is a High-Throughput Method To Monitor the Humoral Immune Response against Vaccinia Virus

2004 ◽  
Vol 11 (2) ◽  
pp. 406-410 ◽  
Author(s):  
Antonio Cosma ◽  
Silja Bühler ◽  
Rashmi Nagaraj ◽  
Caroline Staib ◽  
Anna-Lena Hammarin ◽  
...  
Keyword(s):  
Immune Response ◽  
Green Fluorescent Protein ◽  
Vaccinia Virus ◽  
High Throughput ◽  
Neutralizing Antibodies ◽  
Fluorescent Protein ◽  
Safety Concern ◽  
Neutralization Assay ◽  
Modified Vaccinia Virus Ankara ◽  
Green Fluorescent

ABSTRACT Vaccination against smallpox is again considered in order to face a possible bioterrorist threat, but the nature and the level of the immune response needed to protect a person from smallpox after vaccination are not totally understood. Therefore, simple, rapid, and accurate assays to evaluate the immune response to vaccinia virus need to be developed. Neutralization assays are usually considered good predictors of vaccine efficacy and more informative with regard to protection than binding assays. Currently, the presence of neutralizing antibodies to vaccinia virus is measured using a plaque reduction neutralization test, but this method is time-consuming and labor-intensive and has a subjective readout. Here, we describe an innovative neutralization assay based on a modified vaccinia virus Ankara (MVA) vector expressing the green fluorescent protein (MVA-gfp). This MVA-gfp neutralization assay is rapid and sensitive and has a high-throughput potential. Thus, it is suitable to monitor the immune response and eventually the efficacy of a large campaign of vaccination against smallpox and to study the vector-specific immune response in clinical trials that use genetically engineered vaccinia viruses. Most importantly, application of the highly attenuated MVA eliminates the safety concern in using the replication-competent vaccinia virus in the standard clinical laboratory.

scholarly journals In contrast to TH2-biased approaches, TH1 COVID-19 vaccines protect Syrian hamsters from severe disease in the absence of dexamethasone-treatable vaccine-associated enhanced respiratory pathology

2021 ◽  
Author(s):  
Aileen Ebenig ◽  
Samada Muraleedharan ◽  
Julia Kazmierski ◽  
Daniel Todt ◽  
Arne Auste ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Viral Vectors ◽  
Severe Disease ◽  
Lymphoid Cells ◽  
Protein Vaccine ◽  
S Protein ◽  
Vaccine Candidates ◽  
Syrian Hamsters ◽  
Unvaccinated Control ◽  
Low Passage

Since December 2019, the novel human coronavirus SARS-CoV-2 has spread globally, causing millions of deaths. Unprecedented efforts have enabled development and authorization of a range of vaccines, which reduce transmission rates and confer protection against the associated disease COVID-19. These vaccines are conceptually diverse, including e.g. classical adjuvanted whole-inactivated virus, viral vectors, and mRNA vaccines. We have analysed two prototypic model vaccines, the strongly TH1-biased measles vaccine-derived candidate MeVvac2-SARS2-S(H) and a TH2-biased Alum-adjuvanted, non-stabilized Spike (S) protein side-by-side, for their ability to protect Syrian hamsters upon challenge with a low-passage SARS-CoV-2 patient isolate. As expected, the MeVvac2-SARS2-S(H) vaccine protected the hamsters safely from severe disease. In contrast, the protein vaccine induced vaccine-associated enhanced respiratory disease (VAERD) with massive infiltration of eosinophils into the lungs. Global RNA-Seq analysis of hamster lungs revealed reduced viral RNA and less host dysregulation in MeVvac2-SARS2-S(H) vaccinated animals, while S protein vaccination triggered enhanced host gene dysregulation compared to unvaccinated control animals. Of note, mRNAs encoding the major eosinophil attractant CCL-11, the TH2 response-driving cytokine IL-19, as well as TH2-cytokines IL-4, IL-5, and IL-13 were exclusively up-regulated in the lungs of S protein vaccinated animals, consistent with previously described VAERD induced by RSV vaccine candidates. IL-4, IL-5, and IL-13 were also up-regulated in S-specific splenocytes after protein vaccination. Using scRNA-Seq, T cells and innate lymphoid cells were identified as the source of these cytokines, while Ccl11 and Il19 mRNAs were expressed in lung macrophages displaying an activated phenotype. Interestingly, the amount of viral reads in this macrophage population correlated with the abundance of Fc-receptor reads. These findings suggest that VAERD is triggered by induction of TH2-type helper cells secreting IL-4, IL-5, and IL-13, together with stimulation of macrophage subsets dependent on non-neutralizing antibodies. Via this mechanism, uncontrolled eosinophil recruitment to the infected tissue occurs, a hallmark of VAERD immunopathogenesis. These effects could effectively be treated using dexamethasone and were not observed in animals vaccinated with MeVvac2-SARS2-S(H). Taken together, our data validate the potential of TH2-biased COVID-19 vaccines and identify the transcriptional mediators that underlie VAERD, but confirm safety of TH1-biased vaccine concepts such as vector-based or mRNA vaccines. Dexamethasone, which is already in use for treatment of severe COVID-19, may alleviate such VAERD, but in-depth scrutiny of any next-generation protein-based vaccine candidates is required, prior and after their regulatory approval.

scholarly journals Antibody Profiling by Proteome Microarray Reveals the Immunogenicity of the Attenuated Smallpox Vaccine Modified Vaccinia Virus Ankara Is Comparable to That of Dryvax

2007 ◽  
Vol 82 (2) ◽  
pp. 652-663 ◽  
Author(s):  
D. Huw Davies ◽  
Linda S. Wyatt ◽  
Frances K. Newman ◽  
Patricia L. Earl ◽  
Sookhee Chun ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Cross Reactivity ◽  
Smallpox Vaccine ◽  
Nonstructural Proteins ◽  
Modified Vaccinia Virus Ankara ◽  
Core Proteins ◽  
Other Information ◽  
Work Supports ◽  
Virus Strains ◽  
Chicken Embryo Fibroblasts

ABSTRACT Modified vaccinia virus Ankara (MVA) is a highly attenuated vaccinia virus that is under consideration as an alternative to the conventional smallpox vaccine Dryvax. MVA was attenuated by extensive passage of vaccinia virus Ankara in chicken embryo fibroblasts. Several immunomodulatory genes and genes that influence host range are deleted or mutated, and replication is aborted in the late stage of infection in most nonavian cells. The effect of these mutations on immunogenicity is not well understood. Since the structural genes appear to be intact in MVA, it is hypothesized that critical targets for antibody neutralization have been retained. To test this, we probed microarrays of the Western Reserve (WR) proteome with sera from humans and macaques after MVA and Dryvax vaccination. As most protein sequences of MVA are 97 to 99% identical to those of other vaccinia virus strains, extensive binding cross-reactivity is expected, except for those deleted or truncated. Despite different hosts and immunization regimens, the MVA and Dryvax antibody profiles were broadly similar, with antibodies against membrane and core proteins being the best conserved. The responses to nonstructural proteins were less well conserved, although these are not expected to influence virus neutralization. The broadest antibody response was obtained for hyperimmune rabbits with WR, which is pathogenic in rabbits. These data indicate that, despite the mutations and deletions in MVA, its overall immunogenicity is broadly comparable to that of Dryvax, particularly at the level of antibodies to membrane proteins. The work supports other information suggesting that MVA may be a useful alternative to Dryvax.

scholarly journals Different Patterns of Immune Responses but Similar Control of a Simian-Human Immunodeficiency Virus 89.6P Mucosal Challenge by Modified Vaccinia Virus Ankara (MVA) and DNA/MVA Vaccines

2002 ◽  
Vol 76 (15) ◽  
pp. 7625-7631 ◽  
Author(s):  
Rama Rao Amara ◽  
Francois Villinger ◽  
Silvija I. Staprans ◽  
John D. Altman ◽  
David C. Montefiori ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
T Cells ◽  
Vaccinia Virus ◽  
Neutralizing Antibodies ◽  
Vaccine Trial ◽  
Good Control ◽  
Aids Vaccine ◽  
Modified Vaccinia Virus Ankara ◽  
Immunodeficiency Virus ◽  
Group A

ABSTRACT Recently we demonstrated the control of a mucosal challenge with a pathogenic chimera of simian and human immunodeficiency virus (SHIV-89.6P) by priming with a Gag-Pol-Env-expressing DNA and boosting with a Gag-Pol-Env-expressing recombinant modified vaccinia virus Ankara (DNA/MVA) vaccine. Here we evaluate the ability of the MVA component of this vaccine to serve as both a prime and a boost for an AIDS vaccine. The same immunization schedule, MVA dose, and challenge conditions were used as in the prior DNA/MVA vaccine trial. Compared to the DNA/MVA vaccine, the MVA-only vaccine raised less than 1/10 the number of vaccine-specific T cells but 10-fold-higher titers of binding antibody for Env. Postchallenge, the animals vaccinated with MVA alone increased their CD8 cell numbers to levels that were similar to those seen in DNA/MVA-vaccinated animals. However, they underwent a slower emergence and contraction of antiviral CD8 T cells and were slower to generate neutralizing antibodies than the DNA/MVA-vaccinated animals. Despite this, by 5 weeks postchallenge, the MVA-only-vaccinated animals had achieved as good control of the viral infection as the DNA/MVA group, a situation that has held up to the present time in the trial (48 weeks postchallenge). Thus, MVA vaccines, as well as DNA/MVA vaccines, merit further evaluation for their ability to control the current AIDS pandemic.

scholarly journals Modified Vaccinia Virus Ankara as a Viral Vector for Vaccine Candidates against Chikungunya Virus

Biomedicines ◽  
2021 ◽  
Vol 9 (9) ◽  
pp. 1122
Author(s):  
Juan García-Arriaza ◽  
Mariano Esteban ◽  
Daniel López
Keyword(s):  
Vaccinia Virus ◽  
Chikungunya Virus ◽  
Vaccine Development ◽  
Viral Vector ◽  
Severe Disease ◽  
Febrile Illness ◽  
Structural Proteins ◽  
Effective Vaccine ◽  
Vaccine Candidates ◽  
Modified Vaccinia Virus Ankara

There is a need to develop a highly effective vaccine against the emerging chikungunya virus (CHIKV), a mosquito-borne Alphavirus that causes severe disease in humans consisting of acute febrile illness, followed by chronic debilitating polyarthralgia and polyarthritis. In this review, we provide a brief history of the development of the first poxvirus vaccines that led to smallpox eradication and its implications for further vaccine development. As an example, we summarize the development of vaccine candidates based on the modified vaccinia virus Ankara (MVA) vector expressing different CHIKV structural proteins, paying special attention to MVA-CHIKV expressing all of the CHIKV structural proteins: C, E3, E2, 6K and E1. We review the characterization of innate and adaptive immune responses induced in mice and nonhuman primates by the MVA-CHIKV vaccine candidate and examine its efficacy in animal models, with promising preclinical findings needed prior to the approval of human clinical trials.

scholarly journals Recombinant Modified Vaccinia Virus Ankara Expressing a Soluble Form of Glycoprotein B Causes Durable Immunity and Neutralizing Antibodies against Multiple Strains of Human Cytomegalovirus

2004 ◽  
Vol 78 (8) ◽  
pp. 3965-3976 ◽  
Author(s):  
Zhongde Wang ◽  
Corinna La Rosa ◽  
Rebecca Maas ◽  
Heang Ly ◽  
John Brewer ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Human Cytomegalovirus ◽  
Neutralizing Antibodies ◽  
Natural Infection ◽  
Glycoprotein B ◽  
Soluble Form ◽  
Effective Vaccine ◽  
Viral Pathogen ◽  
Modified Vaccinia Virus Ankara ◽  
Viral Vaccine

ABSTRACT Human cytomegalovirus (CMV) is a viral pathogen that infects both genders, who remain asymptomatic unless they receive immunosuppressive drugs or acquire infections that cause reactivation of latent virus. CMV infection also causes serious birth defects following primary maternal infection during gestation. A safe and effective vaccine to limit disease in this population continues to be elusive. A well-studied antigen is glycoprotein B (gB), which is the principal target of neutralizing antibodies (NAb) towards CMV in humans and has been implicated as the viral partner in the receptor-mediated infection by CMV in a variety of cell types. Antibody-mediated virus neutralization has been proposed as a mechanism by which host immunity could modify primary infection. Towards this goal, an attenuated poxvirus, modified vaccinia virus Ankara (MVA), has been constructed to express soluble CMV gB (gB680-MVA) to induce CMV NAb. Very high levels of gB-specific CMV NAb were produced after two doses of the viral vaccine. NAb were durable within a twofold range for up to 6 months. Neutralization titers developed in immunized mice are equivalent to titers found clinically after natural infection. This viral vaccine, expressing gB derived from CMV strain AD169, induced antibodies that neutralized CMV strains of three different genotypes. Remarkably, preexisting MVA and vaccinia virus (poxvirus) immunity did not interfere with subsequent immunizations of gB680-MVA. The safety characteristics of MVA, combined with the robust immune response to CMV gB, suggest that this approach could be rapidly translated into the clinic.

scholarly journals Recombinant Modified Vaccinia Virus Ankara Expressing the Spike Glycoprotein of Severe Acute Respiratory Syndrome Coronavirus Induces Protective Neutralizing Antibodies Primarily Targeting the Receptor Binding Region

2005 ◽  
Vol 79 (5) ◽  
pp. 2678-2688 ◽  
Author(s):  
Zhiwei Chen ◽  
Linqi Zhang ◽  
Chuan Qin ◽  
Lei Ba ◽  
Christopher E. Yi ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Vaccinia Virus ◽  
Receptor Binding ◽  
Mammalian Cells ◽  
Neutralizing Antibodies ◽  
Binding Region ◽  
Major Mechanism ◽  
Modified Vaccinia Virus Ankara ◽  
Viral Vaccine ◽  
Receptor Binding Region

ABSTRACT Immunization with a killed or inactivated viral vaccine provides significant protection in animals against challenge with certain corresponding pathogenic coronaviruses (CoVs). However, the promise of this approach in humans is hampered by serious concerns over the risk of leaking live severe acute respiratory syndrome (SARS) viruses. In this study, we generated a SARS vaccine candidate by using the live-attenuated modified vaccinia virus Ankara (MVA) as a vector. The full-length SARS-CoV envelope Spike (S) glycoprotein gene was introduced into the deletion III region of the MVA genome. The newly generated recombinant MVA, ADS-MVA, is replication incompetent in mammalian cells and highly immunogenic in terms of inducing potent neutralizing antibodies in mice, rabbits, and monkeys. After two intramuscular vaccinations with ADS-MVA alone, the 50% inhibitory concentration in serum was achieved with reciprocal sera dilutions of more than 1,000- to 10,000-fold in these animals. Using fragmented S genes as immunogens, we also mapped a neutralizing epitope in the region of N-terminal 400 to 600 amino acids of the S glycoprotein (S400-600), which overlaps with the angiotensin-converting enzyme 2 (ACE2) receptor-binding region (RBR; S318-510). Moreover, using a recombinant soluble RBR-Fc protein, we were able to absorb and remove the majority of the neutralizing antibodies despite observing that the full S protein tends to induce a broader spectrum of neutralizing activities in comparison with fragmented S proteins. Our data suggest that a major mechanism for neutralizing SARS-CoV likely occurs through blocking the interaction between virus and the cellular receptor ACE2. In addition, ADS-MVA induced potent immune responses which very likely protected Chinese rhesus monkeys from pathogenic SARS-CoV challenge.

scholarly journals COVID-19 vaccine candidates based on modified vaccinia virus Ankara expressing the SARS-CoV-2 spike induce robust T- and B-cell immune responses and full efficacy in mice

2021 ◽  
Author(s):  
Juan García-Arriaza ◽  
Urtzi Garaigorta ◽  
Patricia Pérez ◽  
Adrián Lázaro-Frías ◽  
Carmen Zamora ◽  
...  
Keyword(s):  
T Cell ◽  
Animal Models ◽  
Vaccinia Virus ◽  
Virus Replication ◽  
T Cell Responses ◽  
Effector Memory ◽  
S Protein ◽  
Vaccine Candidates ◽  
Cell Responses ◽  
Modified Vaccinia Virus Ankara

Vaccines against SARS-CoV-2, the causative agent of the COVID-19 pandemic, are urgently needed. We developed two COVID-19 vaccines based on modified vaccinia virus Ankara (MVA) vectors expressing the entire SARS-CoV-2 spike (S) protein (MVA-CoV2-S); their immunogenicity was evaluated in mice using DNA/MVA or MVA/MVA prime/boost immunizations. Both vaccines induced robust, broad and polyfunctional S-specific CD4+ (mainly Th1) and CD8+ T-cell responses, with a T effector memory phenotype. DNA/MVA immunizations elicited higher T-cell responses. All vaccine regimens triggered high titers of IgG antibodies specific for the S, as well as for the receptor-binding domain; the predominance of the IgG2c isotype was indicative of Th1 immunity. Notably, serum samples from vaccinated mice neutralized SARS-CoV-2 in cell cultures, and those from MVA/MVA immunizations showed a higher neutralizing capacity. Remarkably, one or two doses of MVA-CoV2-S protect humanized K18-hACE2 mice from a lethal dose of SARS-CoV-2. In addition, two doses of MVA-CoV2-S confer full inhibition of virus replication in the lungs. These results demonstrate the robust immunogenicity and full efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic. IMPORTANCE The continuous dissemination of the novel emerging SARS-CoV-2 virus, with more than 78 million infected cases worldwide and higher than 1,700,000 deaths as of December 23, 2020, highlights the urgent need for the development of novel vaccines against COVID-19. With this aim, we have developed novel vaccine candidates based on the poxvirus modified vaccinia virus Ankara (MVA) strain expressing the full-length SARS-CoV-2 spike (S) protein, and we have evaluated their immunogenicity in mice using DNA/MVA or MVA/MVA prime/boost immunization protocols. The results showed the induction of a potent S-specific T-cell response and high titers of neutralizing antibodies. Remarkably, humanized K18-hACE2 mice immunized with one or two doses of the MVA-based vaccine were 100% protected from SARS-CoV-2 lethality. Moreover, two doses of the vaccine prevented virus replication in lungs. Our findings prove the robust immunogenicity and efficacy of MVA-based COVID-19 vaccines in animal models and support its translation to the clinic.

scholarly journals Improved Survival in Rhesus Macaques Immunized with Modified Vaccinia Virus Ankara Recombinants Expressing Simian Immunodeficiency Virus Envelope Correlates with Reduction in Memory CD4+ T-Cell Loss and Higher Titers of Neutralizing Antibody

2009 ◽  
Vol 83 (11) ◽  
pp. 5388-5400 ◽  
Author(s):  
Ilnour Ourmanov ◽  
Takeo Kuwata ◽  
Robert Goeken ◽  
Simoy Goldstein ◽  
Ranjani Iyengar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Vaccinia Virus ◽  
Simian Immunodeficiency Virus ◽  
Neutralizing Antibodies ◽  
Cell Loss ◽  
Survival Advantage ◽  
Virus Envelope ◽  
Modified Vaccinia Virus Ankara ◽  
Immunodeficiency Virus

ABSTRACT Previous studies demonstrated that immunization of macaques with simian immunodeficiency virus (SIV) Gag-Pol and Env recombinants of the attenuated poxvirus modified vaccinia virus Ankara (MVA) provided protection from high viremia and AIDS following challenge with a pathogenic strain of SIV. Although all animals became infected, plasma viremia was significantly reduced in animals that received the MVA-SIV recombinant vaccines compared with animals that received nonrecombinant MVA. Most importantly, the reduction in viremia resulted in a significant increase in median and cumulative survival. Continued analysis of these animals over the subsequent 9 years has shown that they maintain a survival advantage, although all but two of the macaques have progressed to AIDS. Importantly, improved survival correlated with preservation of memory CD4+ T cells in the peripheral blood. The greatest survival advantage was observed in macaques immunized with regimens containing SIV Env, and the titer of neutralizing antibodies to the challenge virus prior to or shortly following challenge correlated with preservation of CD4+ T cells. These data are consistent with a role for neutralizing antibodies in nonsterilizing protection from high viremia and associated memory CD4+ T-cell loss.

scholarly journals Characterization and Use of Mammalian-Expressed Vaccinia Virus Extracellular Membrane Proteins for Quantification of the Humoral Immune Response to Smallpox Vaccines

2007 ◽  
Vol 14 (8) ◽  
pp. 1032-1044 ◽  
Author(s):  
Alonzo D. García ◽  
Clement A. Meseda ◽  
Anne E. Mayer ◽  
Arunima Kumar ◽  
Michael Merchlinsky ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Semliki Forest Virus ◽  
Serum Antibody ◽  
Humoral Response ◽  
Smallpox Vaccine ◽  
Vector System ◽  
Humoral Immune ◽  
Modified Vaccinia Virus Ankara ◽  
Monospecific Antisera ◽  
Antibody Levels

ABSTRACT 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 108 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.

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