Identification Of Conservative Cytotoxic T-Lymphocyte Epitopes Of Human Immunodeficiency Virus Type 1 Clade B Proteins Gag, Nef, Pol And Env In China

Acquired immunodeficiency syndrome(AIDS)is a worldwide infectious disease, which is mainly caused by Human Immunodeficiency Virus type I (HIV-1).It is estimated by UNAIDS and WHO in the report of AIDS epidemic update: special report on HIV/AIDS that since the beginning of the epidemic in 1981 a total of 39.5 million people were living with HIV and about 4,300,000 people were newly infected HIV in 2006. About 11,000 people are infected with HIV every day globally, at the same time 8000 people are died of HIV infection. Efforts to control disease progression associated with HIV-1 infection have led to the use of a combination of antiviral drugs, referred to as highly active antiretroviral therapy (HAART). Treatment with HAART has dramatically decreased the morbidity and mortality caused by HIV-1. HAART effectively lowers viral load and allows for a partial reconstruction of immunological function in a majority of HIV-1-infected patients. Despite the initial success of HAART in controlling HIV-1 replication, this therapeutic approach also has its limitations, which due to the serious adverse effects of the drugs, emergence of drug-resistant HIV-1 mutants and poor adherence. Thus, developing a vaccine preventing HIV-1 infection is urgently needed. Virus-specific cytotoxic T-lymphocyte (CTL) responses play a critical role in the control of HIV-1 infection and may play an important part in therapeutic and prophylactic HIV-1 vaccines. The identification of virus-specific epitopes with optimal immunogenicity that are efficiently recognized by CTL is the first step in the development of future vaccines. The specificity of HIV-1-reactive cellular immune responses depends largely on the genetic background of a population, as CD8+ T cell epitopes are presented to the T cell receptor by HLA class I on their respective cell surfaces. Sequence heterogenicity of HIV-1 are frequently associated with particular host HLA class I alleles. Therefore it is very important to study virus-specific CTL responses in different HIV epidemic regions and ethnic populations. Recent data indicate that China is one of the regions in the world in which the HIV-1 epidemic is expanding most rapidly, about 650,000 individuals are estimated to have been infected in 2006 since the first case of AIDS was identified in China in 1985. These figures regarding the HIV-1 epidemic in China emphasize that a safe, effective, and affordable vaccine is urgently needed. The common alleles of Chinese population is different from these of Caucasian populations and the mainly circulating viral strain is also different from that in other regions, which determine a different profile of CTL response from that in Caucasian and Africa populations. Therefore identification of HIV-1-specific CTL epitopes restricted by common alleles in Chinese population is of critical importance in planning further vaccine trials in China. The most severe HIV-1 epidemic is occurring in China. HIV-1 subtype B (HIV-1B) is the main circulating viral clade in this region and HIV-1B specific CTL responses is cross-reactive to other viral strains. Therefore in this project, we study CTL responses in the individuals infected with HIV-1B. CTLs were shown to be an important component of the immune response to control HIV-1 infection. The definition of conservative and dominant epitopes across the HIV-1B genome that are targeted by CTL is critical for vaccine design. Therefore we present HLA class I typing and the analysis of cumulative CTL responses in China firstly. The characteristics of the predominant virus that causes the HIV-1 epidemic in a certain geographic area and also the genetic background of the population, through the distribution of common HLA class I alleles, might impact dominant CTL responses in the vaccinee and in the general population. The enzyme-linked immunospot (ELISpot) gamma interferon assay has been shown to be a reliable tool to map optimal CTL epitopes, correlating well with other methods, such as intracellular staining, tetramer staining, and the classical chromium release assay. Using ELISpot with overlapping synthetic peptides spanned HIV-1B proteins, we analyzed HIV-1B-specific CTL responses of HIV-1-infected blood donors. Profiles of cumulative ELISpot-based CTL responses combined with sequence diversity analysis provide additional information of immunodominant regions across the HIV-1B genome. Results of previous studies suggest that the construction of a poly-epitope HIV-1 vaccine that includes multiple copies of immunodominant CTL epitopes across the viral genome, derived from predominant HIV-1 strains, might be a logical approach to the design of a vaccine against AIDS.In this study, the HIV-1-specific T-cell responses were measured in HIV-1-infected blood donors by using overlapping peptidespanning the entire HIV-1 clade B consensus sequences in the ELISpot assay. The major contents and results of this study are as following:1. Using nested multiplex PCR, fourteen individuals infected with HIV-1B have been identified. Combining with previous result of viral subtyping, sixty-six individuals in all with HIV-1B infection have enrolled in this study. Eight HLA class I specificities present in over 10% of the study population:A*02,A*24,A*11, B*13, Cw*03, Cw*07, Cw*01, Cw*06, which is corresponding with previous findings. Two HLA class I specificities B*15 and B*40 is slight less than 10%, the findings indicated that the genetic background of study population is match that of the Chinese population well.2. Virus-specific CTL responses in patients with HIV-1 clade B infection were analyzed with the gamma interferon (IFN-γ)-enzyme-linked immunospot assay by using synthetic overlapping peptides corresponding to the HIV-1B consensus sequence. Fourteen immunodominant regions were identified. These peptides were located in Gag (six peptides), Nef (four peptides), Pol (one peptide) and Env (two peptides) all study subjects recognized at least one overlapping peptide. Gag, Nef, Pol and Env were the most frequently targeted proteins, which is consistent with previous studies in infected Caucasians and Africans. However, within these HIV-1 proteins, T cell responses clustered in different regions as different overlapping peptides were targeted by virus-specific T cells in the different study populations.3. Combining with the HIV-1 B sequences from China in Los Alamos HIV database, the variability of fourteen immunodominant regions were analysis, nucleotide sequences in immunodominant regions of HIV-1 B genome but Pol were relative conserved verus corresponding viral genome. Amino acid diversity in immunodominant regions of HIV-1 B proteins but Pol were less than that of the corresponding entire proteins. These findings indicated most immunodominant regions were conservative.4. Twenty-one epitopes were idenfified from these immunodominant regions restricted by common alleles in Chinese population as the following: Eleven epitop(VKVVEEKAF,EKAFSPEV,FSPEVIPMF,SPEVIPMF,VIPMFSAL,TPQDLNTM, EGATPQDL, GQMREPRGSDI, YVDRFYKTL, RDYVDRFF KT, TLRAEQATQD)located in protein Gag, six epitopes(QVPLRPMTYK, WIYHTQGYF,TQGYFPDWQNY,GYFPDWQNY, YFPDWQNYT, NYTP GPGVRY)located in protein Nef, four epitopes(LWVTVYYGV, TVYY GVPVWK,VYYGVPVWKEA, RAIEAQQHL ) located in protein Env. Epitopes in protein Nef proved elicitd adverse CTL responses in previous studies should be omitted from the vaccine construct. Epitopes in proteins Gag and Env that elicit protective CTL responses may be important for future vaccine development in China.In summary, the CTL response profiles of HIV-1 B infected Chinese were different from these in Caucasians and Africans. Fourteen immunodominant regions and twenty-one epitopes restricted by common alleles in Chinese population were idnetified in this study. These epitopes may elicit strong CTL responses and be important for future vaccine development in China.