| Since human immunodeficiency virus (HIV) was identified as the pathogen of acquire immune deficiency syndrome (AIDS) in the 1980's, great efforts have been made to fight against this virus rampant on the earth. An effective vaccine is in urgent need for preventing or treating HIV infections worldwide. Throughout the research and development of HIV vaccines, researchers have focused on promoting cellular and humoral immune response, and maintaining the memory immune response. At our lab, IL-15 has been demonstrated to promote antitumor immune response by enhancing the CTL and NK cell activities. Then IL-15 plasmid was shown to enhance the HIV gag DNA vaccine induced cellular and humoral immune response, promote differentiation of memory CD8+T cells precursors into central memory T cells, and maintain the long-term memory immune response in mice. In this study, the effects of recombinant IL-15 for HIV vaccine both in vaccinia virus Tiantan strain vectors in mice, and the effects of plasmid IL-15 for multivalent HIV DNA vaccine in primates were further evaluated.In the study using vaccinia virus Tiantan strain as the vector, human IL-15 cDNA was inserted into the replicative vaccinia virus vector, and then the expression of IL-15 and the bioactivity of IL-15 in supernatant of in vitro culture were investigated. Mice were co-immunized with recombinant IL-15 vaccinia virus and recombinant HIV gag vaccinia virus, and the HIV gag specific cellular immune response was detected by IFN-y secreting ELISPOT analysis. The ELISPOT analysis indicated that IL-15 did not promote the frequency of HIV gag specific IFN-y secreting CD8+T cells in mice neither in high dose group nor in low dose group, but rather made the frequency of HIV specific CD8+T cells lower. Also, recombinant IL-15 vaccinia virus coadministration did not bring any benefit to the humoral immune response.In the study in primates, rhesus monkeys were co-immunized with IL-15 plasmid and HIV multivalent DNA vaccine; then recombinant HIV vaccinia virus vaccine was used for boosting immunization. After three times of DNA immunization, there was no significant difference between the group of co-immunized with IL-15/HIV DNA vaccine and the group immunized with HIV DNA vaccine alone in terms of HIV specific cellular and humoral immune response. After boosting with the recombinant HIV Tiantan strain vaccinia virus (rTV), the number of IFN-y spot forming cells per million PBMC was higher in IL-15 coadministrated monkeys than that of HIV vaccine immunized alone monkeys; furthermore, the titer of anti-HIV Env antibody was significantly higher in IL-15 co-immunized group than that in the group without IL-15. Also, flow cytometric analysis indicated that the percentage of CD8+memory T cells in peripheral blood was significantly higher in IL-15 co-immunization group than that in HIV vaccine immunized group at almost all the time points detected, which suggested that IL-15 could promote the turnover of effector T cells into memory T cells.Taken together, recombinant IL-15 Tiantan strain vaccinia virus did not augment the immune response induced by recombinant HIV gag vaccinia virus, but made it weaker, suggesting that IL-15 might not be an ideal adjuvant for the Tiantan strain vaccinia virus system. However, in rhesus monkeys, IL-15 plasmid coadministration increased the percentage of memory T cells in the peripheral blood, and augmented the long-lasting humoral immune response, which suggested that IL-15 could enhance the long-time immune response of HIV vaccine.The second part of the study is about screening and identification of the CDR3 of TCR V81 in HIV-infected patients along with corresponding antigen epitope peptides.γδT cells, only 1-10% of T lymphocytes in peripheral blood, are the linkage of the innate immunity and the adaptive immunity, and play an important role in anti-tumor and anti-infection immunity. It has been reported that the absolute number ofγδT cells in the peripheral blood of HIV infected patients were 4-5 fold higher than that in healthy controls, and most of theγδT cells in HIV patients were 81 in subtype, which is only 5-10% ofγδT cells in healthy people. On the contrary, Vy9/82γδT cells as the major subtype ofγδT cells in normal were decreased in number and generally anergic during anti-HIV immune response. The mechanism of these changes has not been clarified so far. Our lab focuses on the study ofγδT cells, and has proved that the CDR3 in the TCRδchain is the vital region during the antigen recognition ofγδTCR. Based on the above-mentioned studies, my study included: First, PBMCs from 9 HIV infected patients were collected and the cDNA of Vδ1γδTCR were amplified by RT-PCR, and then the cDNA of Vδ1 was connected into pGEM-T vector and sequenced. Two dominant sequences of CDR3δ, CALGVTTALIQ WGFVYTDKLIF(named HP1) and CALGEPPIYFNWGIRVTDKPIF(named HP2) were obtained.Secondly, two CDR38 peptides, HP1 and HP2, were used as probes to screen the Ph.D.-12TM Phage Display Peptide Library. Three dominant 12-mers peptides with a common motif of WHW:WHWQWTPWSIQP, WHWNAWNWSSQQ and WHWSWIQNAAPN, were obtained after non-specific elution and specific elution. Then the three 12-mers peptides were synthesized and their biological functions were observed in vitro. The binding of synthesized peptides andγδTCR was identified by ELISA (enzyme-linked immunosorbent assay) and flow cytometry at molecular and cellular levels. Furthermore, the synthesized peptides could stimulate the proliferation ofγδT cells in solid-phase expansion assay by flow cytometry and MTT anaylsis.In brief, two dominant CDR3 sequences of TCR81 in peripheral blood of HIV infected patients, and theγδ1 T cell recognized candidate epitope peptides by screening the Ph.D.-12TM Phage Display Peptide Library using the dominant CDR3δas probes were obtained, which support a new research model for the effect of Vδ1 T cells in HIV infections.
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