An Analysis On The Immunogenicity And Cross-reactivity Of Four CoxA16Strains

Coxsackievirus group A16virus (CA16) is a member of Picomaradae Enterovirus. It is one of the major causes of Hand, Foot and Mouth Disease (HFMD). Previous studies thought that CA16infection normally causes no clinical symptoms or few severe cases, therefore, not many studies have done on it. However, recent studies reported that there are more CA16caused severe HFMD cases, especially in some areas, severe cases caused by CA16has risen to more than20%. The data tells that fundamental studies on pathogenicity and immunological characterization of CA16is becoming important in order for vaccine development. We use different CA16strains which separated from different region of China to establish the phylogeny tree and genotyping, to obtain basic molecular genetic information on CA16epidemiology in China. We then chose4CA16strains which separated from different areas in Mainland China, these four CA16virus were inactivated by formalin and purified by AKTA purifier system, then use these sample to immunize mouse to compared amd analysis the immunogenicity and cross-reactivity of these four strains CA16.We hope that we can provide some data on CA16whole viral inactivated particle vaccine development.We have made phylogenetic tree for19CA16strains based on the VP1nucleotide sequence of CA16. The results show that the standard strain(G10) compared to the CA16strains which separated indecade has changed a lot on the genome. Meanwhile, epidemiological CA16strains in China changed from Bl genotype before2000to B2a and B2b in2000-2010as shown in the phylogenetic tree. In which, the CA16strains obtained in2008-2010were mainly B2b genotype. We also noticed the differences in nucleotide sequences and encoding protein amino acid sequences between different strains, which implies the phenotype of surface antigen might be different. Therefore, we chose four CA16strains (G20, MY08, KM208and KMM/08) that were separated from different areas in Mainland China to study their immunogenicity and cross-reactivity. These4strains were all separated in2008-2010, apart from KMM/08, which is B2a genotype, all the others are B2b genotype.The formalin inactivated CA16crude virus bulk was40-fold concentrated using a100-kD cut-off diafiration membrane in a trangential filter cassette, CA16was isolated using an AKTA purifier chromatography purification system equipped with Sepharose Fast Flow6gel. Fractions were collected and analyzed by ELISA. A group of six female BALB/c mice was immunized intraperitoneal with inactivated CA16immunogen 640EU/mice,and the animals were bootsed with the same dose at four-week intervals after priming. Western Blotting was then used to validate the binding of immune serum and CA16viral proteins. The results indicated that neutralizing antibody presented in the immune serum was able to binding of VP1and VP2.Subsequently, we compared the immunogenicity of these four CA16strains,we found that G20, KMM/08and KM208were significantly higher than MY08strain (P<0.05) on the immunogenicity. As we know that the major neutralizing antigen epitope to induce neutralizing antibody are mostly presented at VPl, then we analyzed the VP1amino acid sequences for the4strains. The results showed that G20, KMM/08and KM208have exactly same VP1amino acid sequences, while three different amino acid sites were found in MY08. Further analysis indicated that the mutation at site102of MY08VP1(N�'D) mutant neutral amino acid to acidic amino acid. This mutation is just within a conserved linear neutralizing antigen epitope site at VPl94-108peptides (TMPTTGTGTGTGNTDGYVN), which might be the cause of weaker immunogenicity of MY08strain.We also compared the cross-reactivity of these four CA16strains, and we found that these four CA16strains were elicited different levels cross-reactvity for other CA16strains, however, the cross-reactvity of G20、KM208、KMM/08strain for other CA16strains were higher than MY08strain (P>0.05),This result maybe cause of the MY08strain have a weaker immunogenicity. Moreover, the cross-reactvity of G20、KM208、 KMM/08strain for MY08were lower than the cross-reactvity of G20、KM208、KMM/08strain for each other, the possible explanation might be the mutation at VPl affect the recognition and bingding of MY08strain virus and neutralization antibody, then the neutralization antibody cannot efficiently combine CA16virus.