Construction And Experimental Immunization Of HIV Multi-Epitope Vaccines

Acquired Immunodeficiency Syndrome(AIDS) is a very serious disease which was occurred following the infection by Human Immunodeficiency Virus(HIV). In china, the major prevalent HIV-1 strains include subtype B, C and their recombinant virus. AIDS hazard mankind's health, it has the complicated immune suppression of various clinical symptom, including CD4+ T progressive decrease, disservice of cellular immune function. The existing therapeutic methods can not eliminate intracellular virus and lead to the opportunistic infection. Therefore, it is crucial to research and develop effective vaccines to prevent and therapy HIV infection.Recently, accompany the advancement of immunobiology and immunopathology about HIV, the concept of protective immunity deeply changed. Research indicated that HLA-I mediated cell immunity, especially the antigen specific CTL mediated cytotoxity playing the key contribution in control HIV infection, which help to eliminate intracellular virus. The effective protective immunity originated from a reasonable combination and mach of a group of epitopes. In design potent prophylactic or therapeutic vaccines, based on multi CTL epitopes can construct chimeric vaccine, thus may induce specific and wide immune responses which directly targeted multi CTL epitopes.So this research based on the immunology theory of epitope recognization and antigen presentation, exploitation molecular biology, molecular virology, bioinformatics methods, undertake the design of a novel HIV antigen molecule and construct two candidate DNA vaccines, then carry out the experiment immunization study. At the same time independently construct a novel recombinant fowlpox virus transfer vector, after that, progress the screen of rFPV candidate vaccine.Our laboratory according to the strategy of HIV-1 dominant antigen epitope vaccine design, choose HIV p24 protein as carrier molecule, have synthesized the novel multiple-epitope antigen MEGp24, which contain six T-cell epitopes. In line with the principal of balance immune responses, and emphasis to strengthen CTL induction, this research plan to overcome the deficiency about too much humoral immunity induction components of previously multiple-epitope antigen contained. Furthermore, there have more indication in recent research about HIV non-structure proteins in immune reaction and control virus replication. In acute HIV infection, the CD8+ CTL occurred within the primary weeks all targeted non-structure or structure protein, and these proliferative responses relate to the reduction of earlier viremia. Therefore this research advance in novel HIV-1 vaccine antigen epitope selection add 17 HIV CTL epitopes to design chimeric antigen, which emphasis the CTL epitopes belong to non-structure proteins.We have search the Los Alamos HIV Molecular Immunology Database, search for HIV-1 and HIV-2 CTL or CD8 T cell epitopes,from pol, Rt, It, Vpr, Tat, p17, gp160, nef, etc. choose 17 T cell epitopes,inserted into MEGp24 that construct novel multiple-epitope antigen MEGNp24. Its homology molecular modeling indicate that MEGNp24'spacial conformation maintain acinose structure, some conformational epitopes disposition the surface of whole protein, correspond the desire. Protease cutting analysis showed that some specific epitopes can be cutting correctly. The gene was then cloned into vetor pVAX1, recombinant plasmid pVAX-MEGNp24 containing the chemiric gene MEGNp24 was obtained. Vaccine delivery approaches suitable for use with large numbers of CTL epitopes include DNA plasmid and viral vector formats. Suicide DNA vaccine that derived from Semliki forest rivus RNA replicon can induce strong immune response. In this research, based on pSFV1, through molecular cloning, inserted SFV non-structure gene into pIRESneo plasmid downstream of CMV promoter and upstream Poly A, successfully constructed pCS that contained SFV RNA replicon. The study of mammalian cell transfection indicated that pCS-EGFP expressed green fluorescent protein successfully, confirmed the practical use of this expression system. Then constructed pCS-MEGNp24 and transfected into BHK-21, IFA confirmed that MEGNp24 also can be expressed in mammalian cells, indicated pCS may be used as DNA vaccine vector to construct multi epitope vaccines.The constructed HIV DNA vaccines were amplified in Jm109 strain of Escherichia coli., then pVAX-MEGNp24 and pCS-MEGNp24 was immunized into BALB/c mice. The two DNA vaccines were comparative study plus different adjuvant. Detection of cellular and humoral immunity level by CTL detect kit and cytokine detect kit. HIV-1 peptide-specific target cells lysis was detect, pVAX-MEGNp24 may achieve 80% while pCS-MEGNp24 may achieve 70%. The results demonstrated that specific humoral and cellular immune responses could be elicited by the recombinant vaccines. Moreover, it showed that pVAX-MEGNp24 may be activate higher immune responses. This research design five primes into PCR reaction and amplified TK gene of fowlpox virus, constructed rFPV transfer vector which recruit TK gene as framework and contain screen gene of gpt, LacZ gene and EGFP gene as report gene. Use synthetic poxvirus'early and late promoter PE/L and P7.5 as cascade promoter. This constructed rFPV transfer vector that possess independence rights. Transfer the vector into CEF showed that this new type transfer vector can express foreign protein as EGFP effectively, the transfer and expression efficency surpass the first generation vector. Then ligated MEGNp24 into rFPV transfer vector, got the recombinant fowlpox virus candidate vaccine, provide the foundation to further study of combined immunization.On the whole, this research according new route, investigated HIV recombinant DNA vaccine and rFPV vaccine which are based epitope, approach the methods and theory about novel antigen molecule design that cotained multi epitope, established foundation for developing potent HIV recombinant vaccines and its pre-clinical study.