Preparation Of Monoclonal Antibodies Against VP2and VP5Proteins Of Bluetongue Virus16and Identification Of B-cell Epiotpes

Bluetongue (BT) is an infectious, non-contagious, arthropod-borne viral disease caused by thebluetongue virus (BTV), which is an arbovirus belonging to the Orbivirus genus of the familyReoviridae. Sheep and goat are the principal victims and are most susceptible to BTV. There isconsiderable genetic and antigenic variation within the BTV serogroup, with26distinct serotypes andconsiderable strain variation within each serotype. As a result, there is little or no immune crossprotection among different BTV serotypes and it has been difficult to develop a useful multivalentvaccine. The development of serotype-specific differential diagnostic approaches is currently needed tofacilitate the control of BTV.The VP2protein of BTV is an important structural protein and is the principal antigen responsiblefor BTV serotype specificity. In this study, recombinant VP2protein was prepared using the BaculovirusExpression System, and the recombinant BTV16VP2protein was used to immunize mice. Wegenerated and mapped the reactivity of two BTV16-specific monoclonal antibodies (MAbs) andidentified two novel serotype-specific linear B cell epitopes on the BTV16VP2protein. To identify theB cell epitopes, we synthesized87pairs of complementary oligonucleotides encoding each16aminoacid long peptide sequence, covering the whole amino acid sequence of the BTV16VP2protein.Oligonucleotides were designed so that the adjacent peptides in the series had five amino acid residuesin common to maintain the linear B cell epitopes on BTV16VP2protein to a large extent.87MBP-fused polypeptides were produced in prokaryote expression systems. The indirect ELISA wasperformed using the expressed MBP-fused polypeptides as coating antigen to identify the epitope region.We next sought to identify the minimal linear peptide epitopes in the BTV16VP2protein recognized byMAb3G10and2B4. A panel of truncated polypeptides were synthesized in which amino acids wereprogressively deleted from the amino and/or the carboxy terminus of the16a.a. peptides recognized by3G10and2B4, defining34EWSGHDVTEIPNRRMF49and543DPYVKRTVK551as the minimal linearepitopes recognized by3G10and2B4respectively. The amino acid sequences corresponding to theregion encompassing these two BTV16VP2minimal linear epitopes from a number of BTV16strainswere identified and aligned. Both linear epitopes were highly conserved among the different BTV16strains. Amino acid alignments were also performed among different BTV serotypes and prototypemembers of the genus Orbivirus in the family Reoviridae. The MAb3G10and2B4epitopes were verypoorly conserved among other BTV serotype viruses and prototype members of the genus Orbivirus inthe family Reoviridae. The MAbs and their defined linear epitopes may support the development ofserotype-specific differential diagnostic tools and marker vaccines.Although the majority of neutralizing antibodies generated by the infected host are directed againstVP2, VP5also influences BTV serotype, possibly by influencing the conformation of the VP2molecules in the outer capsid layer. Recent findings revealed that VP2alone, while able to induce aneutralizing antibody response, cannot efficiently protect against BTV infection. The degree of protection, however, was much higher when VP2was used together with the minor outer capsid proteinVP5, no matter immunized with recombinant proteins or recombinant virus. But the mechanism bywhich VP5influences the generation of protective immune responses in collaboration with VP2isincompletely understood.To date, very little is known about the B-cell epitopes on the BTV VP5protein recognized byhumoral immune responses. In this study, we generated five BTV16VP5protein-specific MAbs, anddefined the linear epitopes recognized by MAbs using a series of peptides expressed as MBP-fusionpolypeptides, which covering the whole amino acid sepuence of BTV16VP5protein. To further refineour definition of the epitopes, a panel of truncated polypeptides were synthesized in which amino acidswere progressively deleted from the motifs identified in the initial screen, defining three new B cellepitopes on VP5protein:310ITANTREIQHIKEE323,188STMVKEYRQKIDALKA203and265LSGID269.Amino acid sequence alignment indicated that three epitopes were totally conserved among differentBTV16strains. The MAbs generated along with identified epitopes will be useful for examining VP5protein function and may contribute to our understanding of the fuction of VP5protein during immuneresponses.