Study On Mechanisms Of Immune Protection Against HIV-1 Infection

Objective:Up to date,Acquired Immune Deficiency Syndrome,also known as AIDS,has claimed the lives of more than 25 million people worldwide.It has become one of the four biggest killers that threaten human life.In recent years,the most conventional clinical treatment for AIDS is highly active antiretroviral therapy(HAART).However,there are big unmet goals due to its high cost,poor compliance from patients,serious side effects and incapability to eradicate virus.HIV is divided into:HIV-1 type and HIV-2 type.The main epidemic currently is HIV-1.Therefore,the development of safe and effective anti-HIV-1 vaccines is particularly urgent.Researches have found that CD8~+C ytotoxic T Lymphocyte(CTL)plays an important role in control of the replication of HIV-1.Thus,development of CTL-based HIV-1vaccines has become a hot topic.This current study is to define the mechanisms of immune protection in HIV-1 infection in aspect of CTL,neutralizing antibody and NK cells.We will study its antiviral mechanisms from four aspects:First,In vitro proliferation assays were used to study the factors affecting the proliferative response of HIV-1 specific C TL;Second,different antigen peptides were used to directly stimulate HIV-1 specific CTL.The cells were cloned into cell lines to test their antiviral function.Third,the CTL clones that recognize the same epitope were tested in antiviral functions,TCR clonotypes,expression of exhaustion and activation molecules and secretion of cytokines.Last,since single factor is not sufficient to protect against HIV-1 infection,combination of CTL,NK cells and monoclonal neutralizing antibodies are evaluated for their relative contribution and cooperation.Method:(1)Three different antigen peptide stimulation methods were used to stimulate the proliferation of HIV-1 specific C TL in vitro.The effect of exogenous cell growth factor IL-2 on the proliferation of HIV-1 specific CTL was evaluated in the culture system.(2)Twelve CTL clone cells generated from limiting dilution were tested in a variety of in vitro functional experiments,including IFN-?ELISpot assay,killing activity experiments,degranulation function experiments.(3)Antiviral functional assays,including IFN-?ELISpot assay,cytotoxicity assay and virus inhibition assay on X4 and R5 virus wild-type strains and antigenic epitope mutant strains,were performed using 9 CTL clone cells that recognize HIV-1 epitope B*27KK10;Exhaustion and activation molecules and cytokines associated with antiviral function were evaluated by PCR and flow cytometry respectively.(4)The relative contribution and synergistic effect of neutralizing antibodies,CTL,and NK cells against viruses were tested using antibody neutralization assays and virus inhibition assays,respectively.Result:(1)The ability to stimulate CTL proliferation was comparable using antigenic peptides directly or antigenic peptides pre-incubated for 2 hours.Peptide-pulsed mixed APCs(CTL-depleted PBMCs)stimulated CTL proliferation superiorly to the previous two stimulators.(2)Different HLA-restricted epitope-specific CTL clone cells have different abilities to kill target cells and the killing activity is related to the degranulation function.(3)B*27-KK10 specific C TL can effectively inhibit HIV-1 replication and different TCR-specific CTL clones that recognize the B*27-KK10 epitope have different viral inhibitory abilities based on TCR usage;CTL clone highly secreting MIP-1?is superior in inhibition of HIV-1virus replication;V?15 TCR clonotype E501 can simultaneously inhibit X4 and R5virus replication and cross-recognize wild-type and mutant strains of the virus.(4)Neutralizing antibody b12 neutralized HIV-1 infection of CD4~+T cells but did not inhibit HIV-1 replication in CD4~+T cells and C TL are superior in inhibition of HIV-1replication in CD4~+T cells;neutralizing antibody b12 did not synergize with CTL to inhibit HIV-1 replication in CD4~+T cells but synergized with NK cells to inhibit HIV-1 replication in CD4~+T cells.Conclusion:(1)In the culture system without the addition of IL-2,peptide-pulsed mixed APCs stimulate proliferation of HIV-1 specific CTL more effectively.(2)There is a common regulatory mechanism among several functional responses of HIV-1 specific C TL clone cells against viruses.(3)The ability of the same HLA-restricted epitope-specific C TL clones to resist viruses is not the same,which is determined by the TCR clonotype.(4)C TL alone is not enough to prevent HIV-1 infection and control virus replication.It also requires the participation of NK cells and neutralizing antibodies.