| Classical swine fever (CSF), caused by classical swine fever virus (CSFV), is a highly contagious disease of swine. CSFV is an enveloped RNA virus with its genome size of approximately 12.3 kb. Among the CSFV proteins, the structural proteins Erns, E2 and the nonstructural protein NS3 are immunogenic and capable of inducing antibodies. E2 plays a significant role in inducing neutralizing antibodies as well as entry into target cells and viral pathogenicity. Prevention of the disease in China relies on prophylactic immunization with C-strain vaccine. However, vaccinated animals could not be distinguished from pigs infected with field viruses by serological methods. Recent studies have revealed that field viruses have switched from genotypes of group 1 to those of group 2. CSFV infections have become endemic in some regions with atypical symptoms irrespective of vaccination. Therefore, development of novel molecular marker vaccines is still of great interests. The present study was aimed to:(1) explore the antigenic diversity of the E2 between group 1 and group 2 strains using specific monoclonal antibodies; (2) develop recombinant C-strain-based candidate vaccine strains containing the E2 gene from prevalent field isolates and evaluate their safety and immunogenicity; (3) examine the impact of Erns mutations on virus replication and formation of viral particles; and (4) develop a recombinant C-strain-based candidate vaccine strain containing Erns gene of bovine viral diarrhea virus with assessment of its safety and immunogenicity in pigs.1. Characterization of monoclonal antibodies against E2 of classical swine virus strainsMonoclonal antibodies to antigenic domains of glycoprotein E2-AD of strains SM and HZ 1-08 were prepared and used to examine the variable regions of antigenic diversity between C-strain and other strains, especially recent field isolates. Two hybridoma cell lines secreting mAb (2B10 and 4F4) were produced by fusing mouse myeloma cells (SP2/0) with spleen cells from BALB/c immunized with the purified recombinant E2 proteins. Western blot, IFA and ELISA analyses showed that the mAb 2B10 could only react with SM-strain, but not with C-strain or group 2 strains, while mAb 4F4 could react with group 2 strain, but not C-strain. It is known that Cys737 in involved in conformation of the E2 B/C domain, thus affecting recognition by specific antibodies. Cys737 mutation revealed that mAb 2B10 reacted with the mutant SM-E2s independent of the secondary structure while mAb 4F4 reacted with the conformational epitopes located in the B/C domain. Thus,2B10 was confirmed to recognize a linear epitopes, while 4F4 recognizes a conformational one. Site-directed mutagenesis by systematic alanine substitutions in SM-E2 protein showed that the epitope 707IXPXGXGG714 recognized by mAb 2B10 is present only on the E2 glycoprotein of strain Shimen, while Glu713 is the key residue for mAb 4F4 binding in group 2 strains. These two mAbs have diagnostic potentials.2. Characterization and immunogenicity of classical swine fever virus C-strain-based recombinant viruses containing the E2 gene of an field isolateSince the antigenic divergence is apparent between recent field isolates and the vaccine C-strain, vaccine efficacy could be compromised. Thus, we attempted to engineer recombinant CSF viruses based on the vaccine C-strain infectious clone pA-FL22 by exchanging the major antigenic region (870bp) of E2 and its hypervariable antigenic region 1 (90bp) with the equivalent regions of HZ 1-08. Two infectious recombinant CSF viruses, RecC-HZ-E2 and RecC-HARl, were generated, while the infectious clone with HAR1 deletion could not be rescued. The two recombinant viruses showed similar characteristics of in vitro replication to the recombinant parent C-strain (RecC). They could replicate in rabbits with characteristic profiles of fever and induced specific antibodies. Moreover, no clinical signs were observed post vaccination with recombinant CSFVs. In pigs there was slight viremia occurred at 14 day post-inoculation (dpi) with RecC-HZ-E2. Detectable antibody against E2 and Erns appeared at 7-10 dpi. The hyperimmune sera in pigs raised from RecC-HARl and RecC-HZ-E2 showed better neutralizing ability to QZ-14 than that with anti-C-strain sera. All pigs could be protected from challenges with strains SM and QZ-14, thus suggesting that the recombinant C-strain-based viruses could improve the protective efficacy to field isolates.3. Effects of amino acid substitution or deletion in the core antigenic region of Erns of classical swine fever virus on replicationAnti-Erns antibody could be detected in the sera of CSFV infected or vaccinated animals and its antigenic region 2 (AR2, aa84-160) is known to be immune-dominant. Single substitutions in aal 17-125 changed the reaction pattern of anti-CSFV sera in vitro, we tried to find the key domain or antigenic epitope related to antibody reactivity without having effects on virus replication for possible development of a ’negative marker’vaccine. Our data showed that single or continuous deletion mutation based on the backbone of C-strain failed to be rescued, while such changes including substitution of aa117-125 in strain SM did not alter its ability to yield progeny virus. Thus, we speculate that this region functions differently between strain SM and C-strain. We performed the same mutantions on the chimeric viruses C and G containing structural genes from C-strain and non-structural genes from strain SM or vice versa for the chimeric viruses D and E. All chimeric viruses could be rescued with deletion of aa117-118, but not with deletion of other residues. These findings suggest that the proteins other than Erns itself could also be involved in viral replication. Because the chimeric viruses were not able to replicate in rabbits and did not induce specific antibodies, their roles in differential diagnostic potentials could not be examined.4. Genetic rescuing and applying of recombinant CSFV vaccine strains containing the Erns gene of BVDVTo develop novel vaccines that can differentiate the infected from the vaccinated animals (DIVA strategy), a chimeric CSFV C-strain based virus carrying bovine viral diarrhea virus (BVDV) Erns (RecC-B-Erns) was constructed as a candidate marker vaccine. The complete Erns gene was replaced by the corresponding region of BVDV lb. The rescued virus RecC-B-Erns exhibited similar growth to RecC in ST and PK-15 cells. It was able to replicate in rabbits with characteristic profiles of fever and induced specific antibodies. Pigs vaccinated with the chimeric virus induced detectable antibodies at 7-10 dpi using the CSFV-specific E2 ELISA. There was 1-3-day delay of the anti-Erns antibodies as evaluated by BVDV-1b specific Erns ELISA. The immue sera collected at 28 dpi from pigs inoculated with RecC-B-Erns reacted with truncated Erns of BVDV (B-tErns-X), but had no cross-reaction with truncated Erns of C strain (C-tErns-Y). While sera induced by strain RecC and field strains reacted with C-tErns, but poorly with B-tErns. The pigs inoculated with RecC-B-Erns did not show any clinical signs and had specific antibody responses. We generated two mAbs 3E8 and 4D6 against Erns of C-strain and BVDV lb, respectively. They only recognized homologous Erns protein without any cross-reactivity. Thus, these epitopes could be used in developing a differentiating diagnostic system.In conclusion, this study identified an N-terminal region of glycoprotein E2 involved in antigenic divergence between CSFV group 1 and group 2 strains with HAR1 being closely related to generation of neutralizing antibodies. C-strain-based recombinant viruses RecC-HARl and RecC-HZ-E2 showed better neutralizing ability to field strains than that with anti-C-strain sera and provided protection against challenges with field strains. The chimeric virus RecC-B-Erns is safe to pigs and has good immunogenicity. These results would provide good foundation for development of a novel marker vaccine against classical swine fever. |
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