For protection from HIV-1 infection, more might not be better: a systematic analysis of HIV Gag epitopes of two alleles associated with different outcomes of HIV-1 infection.

A subset of women in the Pumwani Sex Worker Cohort, established in 1985 in Nairobi, Kenya, remains uninfected despite repeated high-risk exposure (HIV-exposed, seronegative [HESN]) through active sex work. This HESN phenotype is associated with several alleles of human leukocyte antigens (HLAs) and specific CD8(+) and CD4(+) T cell responses to HIV-1. The associations of HLA alleles with differential HIV-1 infection are most likely due to their different abilities to present antigen and the different immune responses they induce. The characteristics of epitopes of HLA alleles associated with different outcomes of HIV-1 infection might therefore point to a vital clue for developing an effective vaccine. In this study, we systematically analyzed HIV-1 clade A and D Gag CD8(+) T cell epitopes of two HLA class I alleles associated with different outcomes of HIV-1 infection. Binding affinity and off-rates of the identified epitopes were determined. Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assays with patient peripheral blood mononuclear cells (PBMCs) validated the epitopes. Epitope-specific CD8(+) T cells were further phenotyped for memory markers with tetramer staining. Our study showed that the protective allele A*01:01 recognizes only three Gag epitopes. By contrast, B*07:02, the allele associated with susceptibility, binds 30 epitope variants. These two alleles differ most importantly in the spectrum of Gag epitopes they can present and not in affinity, off-rates, the location of the epitopes, or epitope-specific Tem/Tcm frequencies. The binding of more epitopes and strong IFN-gamma ELISpot responses are associated with susceptibility to HIV-1 infection, while more focused antigen recognition of multiple subtypes is protective. Rational vaccine design should take these observations into account.

An integrative bioinformatic approach for studying escape mutations in human immunodeficiency virus type 1 gag in the Pumwani Sex Worker Cohort.

Human immunodeficiency virus type 1 (HIV-1) is able to evade the host cytotoxic T-lymphocyte (CTL) response through a variety of escape avenues. Epitopes that are presented to CTLs are first processed in the presenting cell in several steps, including proteasomal cleavage, transport to the endoplasmic reticulum, binding by the HLA molecule, and finally presentation to the T-cell receptor. An understanding of the potential of the virus to escape CTL responses can aid in designing an effective vaccine. To investigate such a potential, we analyzed HIV-1 gag from 468 HIV-1-positive Kenyan women by using several bioinformatic approaches that allowed the identification of positively selected amino acids in the HIV-1 gag region and study of the effects that these mutations could have on the various stages of antigen processing. Correlations between positively selected residues and mean CD4 counts also allowed study of the effect of mutation on HIV disease progression. A number of mutations that could create or destroy proteasomal cleavage sites or reduce binding affinity of the transport antigen processing protein, effectively hindering epitope presentation, were identified. Many mutations correlated with the presence of specific HLA alleles and with lower or higher CD4 counts. For instance, the mutation V190I in subtype A1-infected individuals is associated with HLA-B*5802 (P = 4.73 x 10(-4)), a rapid-progression allele according to other studies, and also to a decreased mean CD4 count (P = 0.019). Thus, V190I is a possible HLA escape mutant. This method classifies many positively selected mutations across the entire gag region according to their potential for immune escape and their effect on disease progression.

Identification and characterization of HLA-A*0301 epitopes in HIV-1 gag proteins using a novel approach.

Identification of CTL epitopes correlated to immune protection is important for the development of vaccines that enhance T-cell mediated immune responses. The correlation of positively selected amino acids (PS) of HIV-1 with host HLA alleles can identify regions containing potential T-cell epitopes. However, the specific epitopes have to be identified and characterized using overlapping peptides through T-cell functional assays. In this study we used a new approach to identify and characterize potential epitopes in the gag region containing PS mutations that significantly correlated with HLA-A*0301. The iTopia Epitope Discovery System was used to rapidly screen a panel of peptides overlapping the regions containing PS mutations and the peptides identified were assessed for relative affinity and complex stability. The potential epitopes were then validated by interferon gamma (IFN-gamma) ELISpot assays with patient PBMCs. Using this approach we identified/confirmed the predicted HLA-A*0301 epitopes in two regions of gag containing PS mutations V7I and K403R, one previously reported and the other novel. Five of the seven peptides that bound to A*0301 contained the K403R mutation and corresponded to the documented LARNCRAPRK-A3 supertype epitope. Two epitope variants, RASVLSGGK and RASILSGGK containing the V7I mutation, were identified using the iTopia Epitope Discovery System, however only the consensus variant (RAK9C) was confirmed using the ELISpot assay and it represents a novel A*0301 epitope.