Research On HLA-mediated Immune Escape Pathways In HIV-1 Subtype B’Nef Protein

BackgroundIn vivo/vitro observations and animal experiments indicated that cytotoxic T-lymphocyte (CTL)responses play a critical role in the control of HIV-1 replication, CD8+CTLs could destroy infected cells through recognizing the peptide-HLA complexes displayed on the cell surface. In order to deal with the immune pressure exerted by HLA-I molecules, however, HIV-1 evolves a series of strategies to evade the recognition by cytotoxic T-lymphocytes. In theroy, escape variants should have an advantage of replication, while recent researches indicated that HLA-mediated escape could reduce the replicative capabilities of the virus, and this is due in part to the escaped variants impaired the virus fitness. Recently, with the improvement of the analytical methods, researchers found that the pattern of CTL escape mutation is broadly predictable based on the host HLA profile at the population level. At present, a great number of HLA-associated polymorphisms (HLA-APs) in the HIV-1 Gag/Pol/Nef proteins have been identified, however, limited study was conducted in Asia, so this study will take HIV-1 subtype B’ Nef protein for an example, to analyze the distribution and characteristics of HLA-APs and to investigate their influence on CD4+T cell counts and plasma viral loads in HIV-1 subtype B’ infected individuals.Objective(1) To identify HLA-mediated immune escape of Nef protein in HIV-1 subtype B’ chronically infected and treatment-naive individuals through the Phylogenetic corrected Logistic regression model;(2) To analyze the distribution and variable characteristics of HLA-mediated immune escape in HIV-1 subtype B’ Nef and to investigate their correlations with the CD4 T cell counts and plasma viral loads in HIV-1 subtype B’infected individuals.MethodsStudy subjects:Our study subjects composed of 163 chronically HIV-1 infected antiretroviral naive individuals who were infected through former paid plasma donation in central China. All samples were collected from the study subjects after the informed consent. And the ethical approval for this study was granted by the Ethics committee of National Center for AIDS/STD Control and Prevention, China CDC.Methods:Whole blood of the study subjects was used for CD4+T cell counts testing and HLA genotyping (HLA genotyping was conductd by using PCR-SSP). HIV-1 RNA were extracted from plasma, Prime 5 was used to design the special primer forNef gene amplification. Nested-PCR was used to amplify Nef gene, and the sequences were assembled by the Sequencher, then adjusted manually in the software of bioedit, and finally, all sequences were uploaded to the HIV sequence database for alignment. Then, Maximum likelihood tree was constructed through the online PHYML project. The entropy score in each amino acid of Nef protein was calculated by the online tool in HIV sequence database. Next, the frequencies and distributions of HLA class I and HLA haplotypes with linkage disequilibrium of the study subjects were determined. And the HLA-mediated inmune escapes were identified using the phylogenetically corrected logistic regression model. Results could be divided into two categories:positive correlations mean that the amino acids significantly enriched in the presence of the certain HLA-I allele. And negative correlations indicate that the amino acids significantly depleted in the presence of the certain HLA-I allele, and vice versa. Positive correlation was also called "adapted form" or "escape form"; and negative correlations also called "nonadapted form" or "reversion form". The distribution and variable characteristics of HLA-mediated immune escape in HIV-1 subtype B’ Nef and their influence on HIV-1 disease status were analyzed statistically.Results(1) In the two digit resolution, we totally identified fifty-two HLAclass-I alleles(A/B/Cw), thirteen two-focus HLA haplotypes and two three-locus HLA haplotypes with linkage disequilibrium in the study subjects. The high frequently HLA class-I alleles were HLA-A*02/A*11/A*24/A*30; HLA-B*13/B*40/B*51; HLA-Cw*03/Cw*06/Cw*08; the high frequently two-focus HLA haplotypes with linkage disequilibrium were HLA-A*30/B*13, HLA-A*30/Cw*06 and HLA-B*30/Cw*06; and the high frequently three-locus HLA haplotype with linkage disequilibrium was HLA-A*30/B*13/Cw*06.(2) At the significance level of p<0.023 and q<0.05, we totally identified 137 HLA-mediated polymorphisms (HLA-APs). These HLA-APs were distributed in 62 amino acid residues of the Nef protein accounted for forty-five percentages of Nef codons. The numbers of HLA-APs adapted were almost the same with the number of that nonadapted. The frequency of HLA-APs occurred in the known CTL epitoes were also nearly the same with those outside the epitopes. The amount of HLA-APs in the three region of Nef were N-teriminal>central core region>C-terminal. The entropy of the amino acid sites with HLA-APs in thenef three sub-regions were higher than that of the sites with no HLA-APs.(3) Three broad categories of differential patterns of immune escape in Nef were observed. First, the majority of cases where different HLA alleles restricting the same epitope select for different escape mutation; Second, two HLA alleles selected for the same escape mutation but to a varying degree; Finally, the "escape" amino acid for one allele represented the "escape" form, and for another allele represented the "reversion" form, a phenomenon termed "tug-of-war" escape. Compared with IHAC chorts,16% HLA-APs found in our study were also reported in the IHAC cohort, but the strength of HLA immune pressure were different;and 35% HLA-APs were not reported in the IHAC chorts and they were associted with the common Chinese HLA alleles.(4)For the 9-peptide epitopes, positions 2 and 9 represented the most frequently escape sites, while N-terminal flanking residue+2 was less frequent; For 8,10,11-peptide epitopes, positions 2 represented the most frequently escape sites, while C-terminal residue was less frequent, the strength of anchor residues was higher than flanking region; in addition, the anchor residue escape in 9-mers epitopes was predicted to reduce peptide-HLA binding by a median of 5.03-fold and 2.17-fold for 8,10,11-mers epitopes, respectively.(5) The number of HLA-APs identified as adapted form was correlated with higher CD4+ T cell counts (p=0.033,r=0.195) and lower viral load(p=0.079,r=-0.164); we also observed three escape mutations (45S/163S/196R) associated with higher CD4+T cell counts(5S:p=0.0152; 163S:p=0.04; 196R:p=0.0298) and tower viral load.(45S:p=-0.0002;163S;p=-0.0005;196R: p<0.0001). And those above 3 mutations were HLA-B*13 resticted.Conclusion137 HLA-APs were identified in 62 AA sites in HIV-1 subtype B’Nef protein, the number of adapted form and nonadapted form HLA-APs was nearly the same. The entropy of the sites with HLA-APs was statistically higher than that of the sites with no HLA-APs, indicating that HLA-mediated immune pressure shape HIV-1 sequence diversity. Three different type of immune escape were identified, people from different ethinic groups infected with the same clade virus displayed distinct pattern of immune escape, this difference in part due to the different profiles of HLA alleles. In addition, the HLA-APs tend to occurr in the anchor residue of epitope, and the anchor residue escape was predicted to reduce peptide binding than nonanchor residues more signifcantly. Escape mutation, especially those HLA-B*13 associated 45S,163S and 196R mutations were correlated with higher CD4+T cell counts and lower viral load, which imply that these mutations could impair virus fitness.