Difference Of T Cell And B Cell Activation In Two Proteins With Similar Antigenicity But Great Distinct Immunogenicity

An E.coli expressed recombinant antigen NE2(HEV ORF2 aa394-606) was able to aggregate into homo-oligomer,and one of it's N terminal extension mutant which was expressed in E.coli and named HEV 239(HEV ORF2 aa368-606) was found to aggregate into particle.The antigenicity of HEV 239 is virtually identical to E2,which is not particulate but soluble.However,HEV 239 is over 200 times more immunogenic than NE2.Some researches found that the ability to be effectively captured by the antigen presenting cells is the reason why the particle antigen possessed great immunogenicity.But someone inclined to deem the particle antigen's strong immunogenicity as a result of it's ability to activate naive B cells. The research based on why HEV 239 and NE2 possessed similar antigenicity but different immunogenicity will contribute to a better understanding of the mechanisms involved in the significant high immunogenicity of particulate antigen and may provide knowledge for the rational design and development of future vaccines:At first we compared the different ability of protein 239 and NE2 to elicit strong t cell response.BALB/c mice were immunized s.c.with 20μg of the HEV 239,E2,mixed with alum adjuvant,respectively.Spleen cells were harvested 2 weeks later and HEV 239-,or E2-specific T cells were enumerated by IFN-γ-ELISPOT assay andIL-5-ELISPOT.The results showed that HEV 239 vaccination evoked much stronger antigen-specific Th1 and Th2 response than E2 vaccination,and maybe it is one of the reasons why the.NE2 possessed weaker immunogenecity.Secondly,Forty-six overlapping 15-mer peptides that span the entire length of HEV 239 were used as stimulator of IFN-γ-ELISPOT assay to identify T cell epitope of HEV 239.Results showed that peptide P34 (sequence:HSKTFFVLPLRGKLS)was strong reactive,peptide P35(sequence: FVLPLRGKLSFWEAG) was moderately reactive,the other peptides were not reactive.Further study was performed to characterize the T cell epitopes contained in peptide P34 and P35.It was found that peptide P34 contains a Th epitope when peptide 35 contains a Tc epitope.Thirdly,different dose of NE2 and 239 was used as stimulator in IFN-γ-ELISPOT assay to activate the spleen cells came from the 50μg P34 CFA immunized mouse and the P34 specific T cells were enumerated.The results showed that high concentration of 239 and NE2 were able to activate antigen specific T cell response,but when stimulated with low concentration of antigen,protein 239 can also elicit T cell response while NE2 can not induce detectable T cell response.It suggested that only high dose NE2 not low dose can evoke T ceil response.In order to verify if the disability of NE2 to elicit strong Th response has affected it's immunogenicity.Groups of five mice each were immunized s.c.with P34,P35 or a non-reactive control peptide P18.The animals were boosted 4 weeks later with 5,10 or 20μg of E2.Blood samples were collected immediately before boosting and then weekly in following 3 weeks.None of the animals primed with P18 showed detectable antibody response to HEV even after high dose E2 boosting,whereas antibody responses can be induced in all of the P34-primed mice and in some of the P35-primed mice after 10 or 20μg E2 boosting.The priming effect of P34 is significantly better than P35,and both are significantly stronger than that of control (P18).It is suggested that the low efficiency of T cell activation be one of the reasons for the poor intrinsic immunogenicity of NE2.The fourth part of this paper we compared the B cell activation by HEV 239 and E2,groups of three athymic BALB/c mice each were immunized intraperitoneally with 0.2,2,20,100μg HEV 239 or E2,mixed with alum adjuvant.Antibodies against HEV were tested on day 7 and day 14.Results showed that HEV 239 could induce T cell independent antibody production at as low as 0.2μg dose,whereas E2 could not induce antibody production even when 100μg E2 was immunized.It suggested that the disability for NE2 to activate naive B cell may contributes to it's weak immunogenicity. At last,the H-2~d restricted Th epitope acquired by former experiment was used to help a B epitope to produce it's monoclonal antibody.The B epitope came from a Avian Influenza virus antigen which was can not to be expressed independently.Several mousse were inoculated with the peptide which contains a B cell epitope of the Avian Influenza virus antigen and the H-2~d restricted Th epitope acquired by former experiments. And we aquired a monoclonal antibody from the immunize mouse.In conclusion,we identified one H-2~d restricted Th and Tc epitope in HEV 239,primarily clarified the reason why 239 and NE2 possessed similar antigenicity but different immunogenicity and provided some knowledge for the rational design and development of future vaccines.In addition,The Th epitope had been used to help a b epitope to produced it's specific monoclonal antibody.