Amino Acid Substitutions In Hypervariable Region Of Epi- Demic Classical Swine Fever Virus E2 On Adaptive Im- Mune Responses Induced By Vaccine C Strain

Classical swine fever is a highly contagious diease of pigs, causing severe economic loss to the swine industry. Its causative agent is classical swine fever virus (CSFV) that belongs to the genus Pestivirus within the Flaviviridae family. CSFV is a small envel-oped RNA virus with a single positive-sense strand of about 12.3-kb. Its genome is flanked by 5’and 3’ untranslated regions and contains a single ORF encoding a poly-protein of 3898 amino acids. Cellular and viral proteases process the polyprotein into 12 mature viral proteins:Npro, C, Ems, El, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B. Of the four strunctural proteins, E2 is the major immunogenic-protein in-volved in production of neutral antibodies. E2-gene based subtyping has shown the transition of CSFV from genotype Ⅰ to genotype Ⅱ in the field isolates, suggesting ge-netic divergence of this major antigenic protein. Recent studies have indicated that that mutations with certain single amino acids might affect its immunogenecity, thus affect-ing its reactivity to antibodies induced by the genotype Ⅰ vaccine C-strain. Majority of the amino acids involved in antigenic reaction with the neutral antibodies are located in the B/C region. However, there is lack of information regarding the antigenic diver-gence of E2 and its impact on the protective efficacy of the C-strain vaccine. This study was aimed (1) to produce the C-strain E2-based mutant proteins containing AR3 regions or amino acid substituions therein from genotype Ⅱ isolates by prokaryotic and bacu-lovirus-based expression systems, and (2) to examine impacts of such replacement or substitution on their reactivity to polyclonal antibodies produced by C-strain vaccina-tion and on the activation of peripheral blood mononuclear cell (PBMC) from C-strain vaccinated pigs in response to stimulation by the recombinant mutant proteins.Recombinant E2 proteins containing the E2 antigenic regions AD and BC from genotype Ⅱ strains QZ-07 and HZ 1-08 expressed in E. coli showed significantly lower reactivity to the C-strain hyperimmune sera than the C-strain E2 counterpart P< 0.05). Comparison of the genotypes Ⅰ and Ⅱ E2 sequences revealed three hypervariable anti-genic regions (AR1, AR2 and AR3). We generated truncated E2 proteins (without the transmembrane region) from C-strain and the other genotype Ⅱ field isolates as well as C-strain E2-based mutant proteins containing AR1, AR2 and AR3 regions form the field isolates in a modified baculovirus expression system. ELISAand Western blotting indicated that the recombinant E2 containing AR3 from field isolates showed lowest reactivity to C-strain hyperimmune sera (P< 0.05) among the E2 proteins tested. In order to examine if antigenic variations on the E2 region affect cell-mediated immunity, the recombinant E2 proteins were used to stimulate the PBMCs from C-strain vac-cinated pigs. By ELISPOT and flowcytometry, we did not observe any significant dif-ference in antibody producing and IFN-y-producing cells in response to antigenic stim-ulation. These results suggest that E2 is mainly involved in humoral immune response and genetic variations of E2 in the antigenic regions, particularly the AR3 region, affect their reactivity to C-strain hyperimmune sera.To further examine the relationship between E2-AR3 mutations and their reactivity to C-strain induced antibodies, we chose 10 amino acids of significant difference among different genotype strains for baculovirus expression. This was accomplished by sub-stitution of the C-strain E2 with single amino acids based on the field isolates. Western blotting revealed no significant difference of immunoreactivity among the mutant pro-teins. ELISA showed that all mutant proteins had reduced reactivity to the C-strain an-tibodoes with the E37N and V45Q (ranked based on the AR3 region) being most sig-nificant (P< 0.05), suggesting that these two amino acids might be related to confor-mational changes of E2.To conclude, our work has provided clear evidence that genetic variations of E2 antigenic regions of genotype Ⅱ field isolates affect the reactivity to C-strain hyperim-mune sera and the AR3 region plays a major role. Within the AR3 region, the two resi-dues E37N and V45Q are the major contributor to such changes, suggesting that these two residues might contribute to conformational changes of E2. Our findings may offer some insights in further research on improved vaccine development, either live attenu-ated or E2 subunit vaccines.