Research On Modification, Expression And Immunogenicity Of HIV Vaccine Expressing Cellular Immune Antigen

Since the first reported AIDS case in 1981, AIDS has been spreading at an alarming rate in the world, seriously threatened human health. HIV vaccine has not been developed yet in the past decades, because of the specific biological characteristics and the lack of awareness of the mechanism on HIV infection and immune protection. The neutralizing antibody induction is important for the development of traditional vaccine, but for the HIV vaccine, besides the induction of cross-protective neutralizing antibody, wide spectrum cellular immuno response is also important for the high efficiency protection. It's getting a important strategy for HIV cellular immune vaccine to modify cellular immune antigen, use multi-antigens and combine immumization of "prime-boost "with different vaccines.In this research, five major cellular immune antigens of HIV-1 B/C subtype as target genes, which are gag, pol, rev, tat and nef, were selected to modify and optimize their gene sequences, codon bias and express structure. By comparison of the express level and immune response of antigens expressed pre and post optimization, it's expected to get more clear on the correlation of gene condon optimize and gene expression structure with the effective celluler immune response.To achieve the objection, four main tasks were carried out in this thesis:first, the selection and optimization of five selected HIV B/C subtype cellular immune antigens; second, the construction and identification of HIV DNA vaccine and rVV vaccine; third, the comparison of gene express levels between codon optimized and non-optimized genes and among various gene express structures; fourth, the comparison of celluler immune response induced by the different vaccine with genes optimized and non-optimized in gene condon or expression structure.First, to modify and optimize five selected HIV-1 B/C subtype cellular immune antigens. Based on eleven China mainland HIV-1 B/C subtype full genome of amino acids sequence, hgag, hpol,hRTN (fusing gene by rev,tat1,nef),hrev,htat,hnef were designed and synthesized according to the preferred codons by mammalian cell. All these six genes were identified correctly. In order to facilitate comparison, obtained cn54 subtype four wide type genes including gagpol,gag,pol,RTN(fusing gene by rev,tat1,nef)Second, constructed two HIV-1 vaccines with DNA (pVRC) and TianTan subtype replicating vaccinia virus (rVV) vector, which includes 6 genes optimized DNA vaccine pVRC-hgag, pVRC-hpol, pVRC-hRTN, pVRC-hrev, pVRC-htat, pVRC-hnef,6 genes optimized replicating vaccinia virus vector vaccine RVJ1175hgag, RVJ1175hpol, RVJ1175hRTN, RVJ1175hrev, RVJ1175htat, RVJ1175hnef. Meanwhile, in order to facilitate comparison, constructed four wild-type gene DNA vaccine pVRC-gagpol, pVRC-gag, pVRC-pol, pVRC-RTN,4 wild-type gene replicating vaccinia virus vector vaccine RVJ1175gagpol, RVJ1175gag, RVJ1175pol, RVJ1175RTN. All these vaccines were identified correctly.Third, the vaccines of codon optimized promoted the express level of gag, pol, rev, tat and nef gene, single express structure had higher express level than fusing genes. IF, WB and FCM methods were used to test the express level of optimized and non-optimized genes. Results shows that all optimized and non-optimized genes can express in pVRC and rVV, and genes express better in pVRC than rVV. Gag and pol had higher express level after gene optimization. The significant promote of express level is observed in pol gene, single pol gene had higher express level than gagpol fusing gene, but similar result was not observed in gag. As for rev, tat and nef, optimized single gene express slight higher level than optimized hRTN, and both optimized single rev, tat, nef gene and fusing hRTN had better express level than non-optimized RTN.Fourth, codon optimized DNA vaccine can significantly improve the Gag, Pol, Rev, Tat, Nef protein immune effects in mice, single structure Rev, Tat, Nef express better than fusion structure RTN. ELIspot and ICS methods were used to access the immune effect of optimized and non-optimized target genes in rVV and DNA vector. The results showed that after optimization both rVV and DNA vaccine can stimulate mice to produce cellular immune responses, the response of DNA vaccines are better than that of rVV, and no significant difference were found among optimized and non-optimized rVV vaccines. Among DNA vaccines, optimized hgag, hpol genes had better immune response than non-optimized gag and pol. For rev, tat, nef gene, except for opitimized hrev had higher immune response than optimized hRTN, no significant difference were found among other two single optimized gene and optimized hRTN gene, but they slightly higher than non-optimized RTN.Furthermore,this thesis reported two new Rev specific epitope peptide sequence, which are 6006:STYLGRPAEPVPLQL,6007:GRPAEPVPLQLPPLE. This thesis also verified that the specific cellular immune response of Gag and Pol were mediated by CB8+T cells, while the specific cellular immune response of Tat and Nef were medeiated by CD4+T cells.In summary, HIV-1 cellular immune antigen sequence, codon or structure modification and optimization will increase target gene express level and immune response. In the DNA vaccine, gene codon optimization and the gene separated structure can increase the gene expression levels and cellular immune effects, it can use this gene as a target antigen. In the recombinant vaccinia virus vaccine, because the compatibility of vaccinia virus on a wide codon, codon optimization had little effect on increasing cellular immunity, and fusion gene structure of the corresponding gene can induce similar cellular immune responses by separated structure genes, it may choose such genes as cellular immunity antigen.These founding provide clues for HIV cross-protective antigen and the immunities of different gene in DNA vaccine and rVV vector, and also provide an experimental basis for the combination of DNA vaccine and rVV.