Animal experiments show impact of vaccination on reduction of SARS-CoV-2 virus circulation: A model for vaccine development?

BACKGROUND: In the pre-clinical phase, SARS-CoV-2 vaccines were tested in animal models, including exposure trials, to investigate protection against SARS-CoV-2. These studies paved the way for clinical development. The objective of our review was to provide an overview of published animal exposure results, focussing on the capacity of vaccines to reduce/prevent viral shedding. METHOD: Using Medline, we retrieved eighteen papers on eight different vaccine platforms in four animal models. Data were extracted on presence/absence of viral RNA in nose, throat, or lungs, and neutralizing antibody levels in the blood. RESULTS: All vaccines showed a tendency of reduced viral load after exposure. Particularly nasal swab results are likely to give an indication about the impact on virus excretion in the environment. Similarly, the reduction or prevention of viral replication in the bronchoalveolar environment might be related with disease prevention, explaining the high efficacy in clinical trials. DISCUSSION: Although it remains difficult to compare the results directly, the potential for a strong reduction of transmission was shown, indicating that the animal models predicted what is observed in the field after large scale human vaccination. This merits further attention for standardization of exposure experiments, with the intention to speed up future vaccine development.

Innate immune defenses induced by CpG do not promote vaccine-induced protection against foot-and-mouth disease virus in pigs.

Emergency vaccination as part of the control strategies against foot-and-mouth disease virus (FMDV) has the potential to limit virus spread and reduce large-scale culling. To reduce the time between vaccination and the onset of immunity, immunostimulatory CpG was tested for its capacity to promote early protection against FMDV challenge in pigs. To this end, CpG 2142, an efficient inducer of alpha interferon, was injected intramuscularly. Increased transcription of Mx1, OAS, and IRF-7 was identified as a sensitive measurement of CpG-induced innate immunity, with increased levels detectable to at least 4 days after injection of CpG formulated with Emulsigen. Despite this, CpG combined with an FMD vaccine did not promote protection. Pigs vaccinated 2 days before challenge had disease development, which was at least as acute as that of unvaccinated controls. All pigs vaccinated 7 days before challenge were protected without a noticeable effect of CpG. In summary, our results demonstrate the caution required when translating findings from mouse models to natural hosts of FMDV.

Animal genomics for animal health report: critical needs, problems to be solved, potential solutions, and a roadmap for moving forward.

The first International Symposium on Animal Genomics for Animal Health, held at the World Organisation for Animal Health (OIE) Headquarter, 23-25 October, 2007, Paris, France, assembled more than 250 participants representing research organizations from 26 countries. The symposium included a roundtable discussion on critical needs, challenges and opportunities, and a forward look at the potential applications of animal genomics in animal health research. The aim of the roundtable discussion was to foster a dialogue between scientists working at the cutting edge of animal genomics research and animal health scientists. In an effort to broaden the perspective of the roundtable discussion, the organizers set out four priority areas to advance the use of genome-enabled technologies in animal health research. Contributions were obtained through open discussions and a questionnaire distributed at the start of the symposium. This symposium report provides detailed summaries ofthe outcome ofthe roundtable discussion for each of the four priority areas. For each priority, the problems needing to be solved, according to the views of the participants, are identified, including potential solutions, recommendations, and lastly, concrete steps that could be taken to address these problems. This report serves as a roadmap to steer research priorities in animal genomics research.

A West Nile virus (WNV) recombinant canarypox virus vaccine elicits WNV-specific neutralizing antibodies and cell-mediated immune responses in the horse.

Successful vaccination against West Nile virus (WNV) requires induction of both neutralizing antibodies and cell-mediated immune responses. In this study, we have assessed the ability of a recombinant ALVAC-WNV vaccine (RECOMBITEK WNV) to elicit neutralizing antibodies and virus-specific cell-mediated immune responses in horses. In addition, we examined whether prior exposure to ALVAC-WNV vaccine would inhibit B and cell-mediated immune responses against the transgene product upon subsequent booster immunizations with the same vaccine. The results demonstrated that the recombinant ALVAC-WNV vaccine induced neutralizing antibodies and prM/E insert-specific IFN-gamma(+) producing cells against WNV in vaccinated horses. Prior exposure to ALVAC-WNV vaccine did not impair the ability of horses to respond to two subsequent booster injections with the same vaccine, although anti-vector-specific antibody and cell-mediated immune responses were induced in vaccinated horses. This report describes, for the first time, the induction of antigen-specific cell-mediated responses following vaccination with an ALVAC virus recombinant vaccine encoding WNV antigens. Moreover, we showed that both WNV-specific IFN-gamma producing cells and anti-WNV neutralizing antibody responses, are not inhibited by subsequent vaccinations with the same vector vaccine.

Effective priming of foals born to immune dams against influenza by a canarypox-vectored recombinant influenza H3N8 vaccine.

A classical limitation of early life immunization is the interference by maternally derived antibodies, which are known to inhibit the immune response to modified-live and killed vaccines. Several studies have convincingly shown that even minute amounts of maternally derived antibodies against equine influenza can strongly interfere with successful vaccination of foals born to immune mares. In this study we evaluated the response of foals born to vaccinated mares to immunization with a canarypox-vectored recombinant vaccine against equine influenza virus H3N8. The recombinant vaccine was able to efficiently prime foals in the presence of maternally derived immunity against influenza as was evidenced by a clear anamnestic antibody response when a secondary vaccination with the same vaccine was performed. The canarypox-vectored recombinant influenza vaccine therefore offers a unique opportunity to overcome the limitations of early life vaccination in the face of maternally derived immunity in foals.

A molecular approach to the identification of cytotoxic T-lymphocyte epitopes within equine herpesvirus 1.

Equine herpesvirus 1 (EHV-1) causes respiratory and neurological disease and abortion in horses. Animals with high frequencies of cytotoxic T lymphocytes (CTL) show reduced severity of respiratory disease and frequency of abortion, probably by CTL-mediated control of cell-associated viraemia. This study aimed to identify CTL epitopes restricted by selected major histocompatibility complex (MHC) class I alleles expressed in the equine leukocyte antigen (ELA) A3 haplotype. Effector CTL were induced from EHV-1-primed ponies and thoroughbreds with characterized MHC class I haplotypes and screened against P815 target cells transfected with selected EHV-1 genes and MHC class I genes. Targets that expressed EHV-1 gene 64 and the MHC B2 gene were lysed by effector CTL in a genetically restricted manner. There was no T-cell recognition of targets expressing either the MHC B2 gene and EHV-1 genes 2, 12, 14, 16, 35, 63 or 69, or the MHC C1 gene and EHV-1 genes 12, 14, 16 or 64. A vaccinia virus vector encoding gene 64 (NYVAC-64) was also investigated. Using lymphocytes from ELA-A3 horses, the recombinant NYVAC-64 virus induced effector CTL that lysed EHV-1-infected target cells; the recombinant virus also supplied a functional peptide that was expressed by target cells and recognized in an MHC-restricted fashion by CTL induced with EHV-1. This construct may therefore be used to determine the antigenicity of EHV-1 gene 64 for other MHC haplotypes. These techniques are broadly applicable to the identification of additional CTL target proteins and their presenting MHC alleles, not only for EHV-1, but for other equine viruses.

Antibody and IFN-gamma responses induced by a recombinant canarypox vaccine and challenge infection with equine influenza virus.

In horses, equine influenza virus (EIV) is a leading cause of respiratory disease. Conventional inactivated vaccines induce a short-lived immune response. By comparison, natural infection confers a long-term immunity to re-infection. An aim of new equine influenza vaccines is to more closely mimic natural infection in order to achieve a better quality of immunity. A new live recombinant vaccine derived from the canarypox virus vector and expressing haemagglutinin genes of EIV (subtype H3N8) has been developed. Stimulation of the immune system was studied after immunisation with this canarypox-based vaccine and challenge infection by exposure to a nebulised aerosol of EIV. The humoral immune response was evaluated by measuring serum antibody levels using the single radial haemolysis (SRH) assay. The cellular immune response was assessed by the measurement of interferon gamma (IFN-gamma) synthesis in peripheral blood mononuclear cells (PBMC). Clinical signs of the disease (temperature, coughing, nasal discharge, dyspnoea, depression and anorexia) and virus excretion were monitored after challenge infection. Clinical signs and virus shedding were significantly reduced in vaccinates compared with unvaccinated controls. EIV-specific immunity was stimulated by vaccination with a recombinant vaccine as serological responses were detected after immunisation. This study also provided the first evidence for increased IFN-gamma protein synthesis in vaccinated ponies following challenge infection with EIV compared with control ponies.

Use of DNA and recombinant canarypox viral (ALVAC) vectors for equine herpes virus vaccination.

In this study, experimental canarypox virus (ALVAC) and plasmid DNA recombinant vaccines expressing the gB, gC and gD glycoproteins of EHV-1 were assessed for their ability to protect conventional ponies against a respiratory challenge with EHV-1. In addition, potential means of enhancing serological responses in horses to ALVAC and DNA vaccination were explored. These included co-administration of the antigen with conventional adjuvants, complexation with DMRIE-DOPE and co-expression of the antigen along with equine GM-CSF. Groups of EHV primed ponies were vaccinated twice intra-muscularly with one dose of the appropriate test vaccine at an interval of 5 weeks. Two to 3 weeks after the second vaccination, ponies were infected intra-nasally with the virulent Ab4 strain of EHV-1 after which they were observed clinically and sampled for virological investigations. The results demonstrated that DNA and ALVAC vaccination markedly reduced virus excretion after challenge in terms of duration and magnitude, but failed to protect against cell-associated viremia. Noteworthy was the almost complete absence of virus excretion in the group of ponies vaccinated with ALVAC-EHV in the presence of Carbopol adjuvant or DNA plasmid formulated with aluminium phosphate. The administration of the DNA vaccine in the presence of GM-CSF and formulated in DMRIE-DOPE and of the ALVAC vaccine in the presence of Carbopol adjuvant significantly improved virus neutralising antibody responses to EHV-1. These findings indicate that DNA and ALVAC vaccination is a promising approach for the immunological control of EHV-1 infection, but that more research is needed to identify the immunodominant protective antigens of EHV-1 and their interaction with the equine immune system.

Characterisation of CTL and IFN-gamma synthesis in ponies following vaccination with a NYVAC-based construct coding for EHV-1 immediate early gene, followed by challenge infection.

Equine herpesvirus-1 (EHV-1) is a ubiquitous pathogen of horses, which continues to cause respiratory and neurological disease and abortion, despite the widespread use of vaccines. Cell mediated immunity (CMI) is thought to play a major role in protection against infection with EHV-1. The aim of this study was to characterise the virus-specific CMI response in ponies vaccinated with vP1014, a vaccinia-based construct (NYVAC) coding for the immediate early gene (gene 64) of EHV-1. This gene product is a CTL target protein for an equine MHC class I allele expressed on the A3 haplotype. EHV-primed yearling ponies expressing this haplotype were vaccinated once (n = 1), three (n = 1), or four times (n = 2), and one pony was kept as an unvaccinated control. Cytotoxic T lymphocyte (CTL) activity and interferon gamma (IFN-gamma) synthesis were measured before and after vaccination and challenge infection with EHV-1. Multiple immunisations with vP1014 resulted in increased CTL activity and IFN-gamma synthesis specific for EHV-1 compared with unvaccinated or singly vaccinated ponies. The phenotype of EHV-1 specific T-cells synthesising IFN-gamma was also modified by immunisation. In the unvaccinated pony, the predominant population synthesising IFN-gamma after EHV-1 stimulation was CD8alpha+. In contrast, multiply vaccinated ponies demonstrated an increased proportion of CD8alpha- T-cells synthesising IFN-gamma. The results demonstrated that vaccination with a NYVAC-based construct coding for gene 64 stimulated CMI. This immune response alone did not protect against challenge infection. However, the study does illustrate that vaccinia-based vaccines can stimulate CMI in the horse and may therefore contribute to protection against disease caused by EHV-1.

Recombinant canarypox vectored West Nile virus (WNV) vaccine protects dogs and cats against a mosquito WNV challenge.

The safety and efficacy of a canarypox vector expressing PrM and E genes of West Nile virus (WNV) (ALVAC-WNV) was evaluated in dogs and cats. One group of 17 dogs (vaccinated with 10(5.6) TCID(50)) and two groups of cats (groups 1 [n=14] vaccinated with 10(7.5) TCID(50) and 2 [n=8] 10(5.6) TCID(50)) were vaccinated twice at 28-day intervals. Fifteen dogs and eleven cats served as negative controls. The cats and dogs were challenged 120 and 135 days after the second immunization, respectively via the bites of Aedes albopictus mosquitoes infected with WNV. The first dose of vaccine induced a detectable antibody response in four dogs and five cats (one immunized with low and four with high doses). After the second dose, all the vaccinated dogs and all of the cats, immunized with high dose had detectable antibody titers, whereas only four of eight cats in the low dose group were seropositive. None of the vaccinated dogs and one vaccinated cat developed viremia following the WNV mosquito-challenge. In contrast, 14 of the 15 control dogs and 9 of the 11 control cats developed viremia. The experimental vaccine described in this study may be of value in the prevention of WNV infection in dogs and cats.

Efficacy of a recombinant equine influenza vaccine against challenge with an American lineage H3N8 influenza virus responsible for the 2003 outbreak in the United Kingdom.

Fifteen influenza-naive Welsh mountain ponies were randomly assigned to three groups of five. A single dose of a recombinant ALVAC vaccine was administered intramuscularly to five of the ponies, two doses, administered five weeks apart, were administered to five, and the other five served as unvaccinated, challenge controls. Two weeks after the completion of the vaccination programme, the ponies were all challenged by exposure to an aerosol of influenza virus A/eq/Newmarket/5/03. Their clinical signs were scored daily for 14 days according to a standardised scoring protocol, and nasal swabs were taken daily for 10 days to monitor the excretion of virus. The challenge produced severe clinical signs of influenza (fever, coughing, nasal discharge and dyspnoea) in all five control ponies, but the vaccinated ponies developed only mild disease, consisting of a serous nasal discharge lasting for only one day. The excretion of virus was almost completely suppressed in the vaccinated ponies, but the control ponies shed the virus for up to seven days after the challenge.

Recombinant canarypoxvirus vaccine carrying the prM/E genes of West Nile virus protects horses against a West Nile virus-mosquito challenge.

An ALVAC (canarypoxvirus)-based recombinant (vCP2017) expressing the prM and E genes derived from a 1999 New York isolate of West Nile virus (WNV) was constructed and assessed for its protective efficacy in horses in two different experiments. In the first trial, a dose titration study was conducted to evaluate both serum neutralising antibody responses to WNV and duration of immunity. In the second trial the onset of protection was determined. Twenty-eight adult horses received two doses of vCP2017 administered intramuscularly at 5-week intervals and sixteen horses comprised age-matched non-vaccinated controls. Individual sera were taken periodically and tested for neutralising antibodies against WNV. Horses were challenged by allowing WNV-infected Aedes albopictus mosquitoes to feed on them two weeks (second trial) or one year (first trial) after the second vaccination. After challenge, horses were monitored for clinical signs of disease, and blood samples were collected for detection of WNV viremia and antibody. In both trials, all vaccinated horses developed neutralising antibodies against WNV. None of the vaccinated or control horses developed clinical signs of WNV disease upon challenge. None of the nine horses challenged 2 weeks after primary vaccination and only one of the ten vaccinated horses challenged 1 year after vaccination developed detectable viremia after challenge, whereas more than 80% of the controls became infected. Results from these studies demonstrated that a primary course of two doses of vCP2017 provides both antibody response and an early immunity in horses against WNV viremia.

Efficacy of a canarypox virus-vectored vaccine against feline leukaemia.

Canarypox virus recombinant vaccines have a unique efficacy and safety profile for the vaccinated host because the canarypox virus is non-replicative in mammalian hosts. After the vaccination of a mammalian species, recombinant canarypox viruses express the inserted genes but cannot multiply in the host. They stimulate a strong immune response in the absence of any virus amplification in the host or any viral spread into the environment. A new canarypox-based recombinant vaccine is the canarypox-feline leukaemia virus (FeLV) vaccine (EURIFEL FeLV; Merial) that expresses the FeLV env and gag protective genes. This paper describes experiments which demonstrate that it is effective against any oronasal FeLV challenge. The protection was shown to be solid against an oronasal challenge one year after the initial vaccination, and was effective against a very severe 'in-contact' challenge. Furthermore, the canarypox virus-FeLV vaccine was effective without an adjuvant.

Quantitative analysis of the antigen-specific IFNgamma+ T cell-mediated immune response in conventional outbred pigs: kinetics and duration of the DNA-induced IFNgamma+ CD8+ T cell response.

It is now well established that antigen-specific CD8(+) T cells play a major role in vaccine-induced immunity against intracellular pathogens and tumor cells. The detection of these immune cells in outbred animals has been hampered mainly by the need to generate individual autologous antigen-presenting cells (APCs) due to the high degree of polymorphism of the major histocompatibility complex (MHC) Class I loci. We used individually derived immature porcine dendritic cells infected with a pox-based recombinant viral vector to ex vivo stimulate PBMCs from vaccinated conventional pigs. The frequencies of antigen-specific T cells was determined by the number of IFNgamma-secreting cells in a quantitative enzyme-linked immune spot (ELISPOT) assay. Using this approach we were able to rank different pseudorabies virus (PRV) vaccines strategies for their ability to prime viral-specific IFNgamma(+) T cells. Plasmid DNA has recently emerged as a promising tool with multiple applications in the field of infectious diseases, allergy and cancer. We showed for the first time in this study that DNA immunization induced a long-lived antigen-specific IFNgamma(+) T cells response in conventional pigs. Additional studies allowed us to show that these virus-specific IFNgamma(+) responding cells detected in this ELISPOT assay were MHC-restricted and comprised in the CD8alpha(bright) pig T cell subset. These new data confirm the usefulness of DNA vaccines to control diseases requiring cellular immunity in pigs.

Functional and phenotypic characterization of distinct porcine dendritic cells derived from peripheral blood monocytes.

Dendritic cells (DCs) are bone marrow-derived antigen-presenting cells that have an exquisite capacity to interact with T cells and modulate their responses. Little is known about porcine DCs despite the fact that they represent an important target in strategies that are aimed at modulating resistance to infection in pigs and may be of major importance in transplantation biology. We generated immature monocyte-derived porcine dendritic cells (MoDCs) directly from adherent peripheral blood cells treated with porcine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The cells were observed via electron microscopy and their phenotype was characterized using monoclonal antibodies. The functionality of the porcine MoDCs was demonstrated showing that the cells were capable of different specialized functions relevant to antigen capture and were potent stimulators in a primary allo-mixed leucocyte reaction. Treatment of the MoDCs with porcine cell line-derived necrotic factors resulted in the phenotypic and functional maturation of MoDCs. We confirmed also that monocyte-derived DCs were differentially regulated by cytokines, showing that transforming growth factor-beta1 (TGF-beta1) is able to redirect monocytic precursors into the differentiation pathway of Langerhans' cells presenting typical Birbeck granules. Interestingly, and in contrast to the human and murine model, we showed that the monocyte-derived porcine Langerhans'-type cells (MoLCs) were much more potent activators of allogeneic T cells than MoDCs obtained without TGF-beta1.

Enhanced protective response and immuno-adjuvant effects of porcine GM-CSF on DNA vaccination of pigs against Aujeszky's disease virus.

This study was conducted to investigate whether the co-delivery of DNA encoding porcine cytokines would enhance a protective immune response in pigs to a Pseudorabies virus (PRV; or Aujeszky's disease virus) DNA vaccine. Aujeszky's disease in pigs results in respiratory and nervous symptoms with important economic losses. To evaluate cytokine effects, eukaryotic expression vectors were constructed for porcine GM-CSF, IL-2 and IFN-gamma. cDNA for each of these cytokines was inserted under the control of a CMV promoter in the pcDNA3 plasmid and cytokine expression was confirmed after DNA transfection in various mammalian cell cultures by bioassays (GM-CSF and IL2) and ELISA (IFN-gamma). Pigs were vaccinated by single intramuscular injection with plasmid DNA encoding PRV gB and gD along with various combinations of cytokine plasmid constructs. Pig serum was tested for the production of antibody by isotype specific anti-PRV ELISA. Pigs were then challenged with the highly virulent PRV strain NIA3 on day 21 after vaccination. The survival and growth rate of pigs were monitored for seven days after the viral challenge. The co-administration of GM-CSF plasmid increased the immune response induced by gB and gD PRV DNA vaccine. This immune response was characterized by an earlier appearance of anti-PRV IgG2, a significantly enhanced anti-PRV IgG1 and IgG2 antibody response, a significantly decreased and shortened viral excretion in nasal swabs and an improved protection to the viral challenge. In contrast, the co-administration of porcine IL-2 or IFN-gamma had no adjuvant effects. Our results thus demonstrate for the first time that the application of porcine GM-CSF gene in a DNA vaccine formulation can exert immuno-adjuvant and protective effects with single vaccination in the natural host pig against Aujeszky's disease.

Non-essential loci in the BamHI-I and -F fragments of the HVT FC126 genome.

The sequence of BamHI-I fragment of the herpesvirus of turkeys (HVT) FC126 strain DNA was analyzed for the presence of potential open reading frames (ORFs). Four complete (ORFs 2 to 5) and 2 partial ORFs (ORFs 1 and 6) were detected. ORFs 2 and 3 were homologous to the HSV-1 UL55 and the EHV-1 gene 3, respectively. The ORF 6 was already partially sequenced by Smith et al. (Virology 207, 205-216, 1995), and was homologous to a Marek's disease virus (MDV) ORF located in a similar position (ORF 21; Ross et al., Virus Genes 7, 33-51, 1993a). No significant homology was found for the other ORFs. ORF 4 was antisense to ORF 3. Two HVT recombinants having an expression cassette inserted into two intergenic sites were generated and tested for viremia in chickens. Results demonstrated that these 2 intergenic loci are non-essential for in vitro and in vivo HVT replication. A 650 bp deletion in the repeat region flanking UL (TRL and IRL (BamHI-F)) has been identified in some DNA molecules of HVT FC126 strain. This deletion covers the entire truncated pp38 homologous ORF and the N-terminus of a small ORF which has no detectable homology with any known gene. Our results indicate that (1) this genomic region including the HVT pp38 homologue was not essential for in vitro and in vivo growth of HVT, and (2) this deletion had no apparent effect on Marek's disease (MD) protection induced by HVT.

Herpesvirus of turkey recombinant viruses expressing infectious bursal disease virus (IBDV) VP2 immunogen induce protection against an IBDV virulent challenge in chickens.

Two recombinant herpesviruses of turkey (HVT) expressing the VP2 protein of infectious bursal disease virus (IBDV or Gumboro disease virus) have been constructed: vHVT001 and vHVT002. The VP2 open reading frame was inserted at the locus of the small subunit of ribonucleotide reductase gene (HSV-1 UL40 homolog) without any exogenous promoter in vHVT001 and at the locus of gl gene (HSV-1 US7 homolog) under the control of the human cytomegalovirus immediate-early promoter in vHVT002. The isolation of these recombinant viruses indicated that the deleted genes were not required for replication of HVT in chicken embryo fibroblasts. Efficacy of these recombinant viruses against IBDV strain 52/70 and Marek's disease virus (MDV strain RB1B) virulent challenges was evaluated in chickens vaccinated at 1 day of age. In the IBDV challenge, a good protection against mortality and bursal gross lesion was observed in vHVT002-vaccinated chickens: 100% with 10(5) PFU dose and 60% with 10(4) PFU dose; in contrast, only a weak level of protection was achieved after vaccination with vHVT001. Protection levels against MDV challenge obtained with vHVT001 and vHVT002 were low (around 10%) compared to that induced by the parental HVT (84%). In spite of the low protection level against MDV, this is the first report which describes induction of full protection against IBDV with a single inoculation of a recombinant virus.

Localization of antigenic sites of the S glycoprotein of feline infectious peritonitis virus involved in neutralization and antibody-dependent enhancement.

The S glycoprotein of feline infectious peritonitis virus (FIPV) has been shown to contain the antigenic sites responsible for eliciting both neutralization and antibody-dependent enhancement. To determine the region of S responsible, overlapping DNA fragments spanning the entire S gene were cloned and expressed as fusion proteins by in vitro transcription and translation. Fusion proteins containing relevant epitopes were identified by radioimmunoprecipitation with neutralizing and enhancing FIPV-specific monoclonal antibodies (MAbs). A region spanning residues 509 to 673 reacted with most MAbs tested. Translation in the presence of microsomal membranes did not enhance reactivity, suggesting that glycosylation is not essential for recognition by the MAbs. To localize the antigenic sites further, several MAb-resistant (mar) mutants of FIPV were cloned and sequenced. Amino acid residues that contribute to the neutralizing and enhancing epitopes were localized to two regions, designated A1 and A2, which show partial overlap with the homologous antigenic site A of transmissible gastroenteritis virus. Site A1 contains residues 568 and 591 and is homologous with part of subsite Aa of transmissible gastroenteritis virus. Site A2 contains residues 643, 649, and 656. Double mutations in sites A1 and A2 were found in mar mutants derived from neutralizing and enhancing MAbs 23F4.5 and 18A7.4, while a single mutation in site A2 was found in a mar mutant derived from MAb 24H5.4, which is neutralizing but not enhancing. The data suggest that site A2, which includes residues 643 to 656, is a dominant neutralizing site of FIPV and that sites A1 and A2 may act in concert to induce antibody-dependent enhancement.