Potential underestimation of HLA-C-restricted cytotoxic T-lymphocyte responses

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Rapid definition of five novel HLA-A*3002-Restricted human immunodeficiency virus-specific cytotoxic T-lymphocyte epitopes by elispot and intracellular cytokine staining assays

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) play a major role in control of viral replication. To understand the contribution of this antiviral response, an initial step is to fully define the specific epitopes targeted by CTL. These studies focused on CTL responses restricted by HLA-A*3002, one of the HLA-A molecules most prominent in African populations. To avoid the time-consuming effort and expense involved in culturing CTL prior to defining epitopes and restricting alleles, we developed a method combining Elispot assays with intracellular gamma interferon staining of peripheral blood mononuclear cells to first map the optimal epitopes targeted and then define the HLA restriction of novel epitopes. In two A*3002-positive subjects whose CTL responses were characterized in detail, the strongest response in both cases was to an epitope in p17 Gag, RSLYNTVATLY (residues 76 to 86). Using this method, CTL epitopes for which there were no motif predictions were optimized and the HLA restriction was established within 48 to 72 h of receipt of blood. This simple and convenient approach should prove useful especially in the characterization of CTL responses specific to HIV and other viruses, particularly in localities where performing cytotoxicity assays would be problematic

Substantial differences in specificity of HIV-specific cytotoxic T cells in acute and chronic HIV infection

Cytotoxic T lymphocytes (CTLs) play a vital part in controlling viral replication during human viral infections. Most studies in human infections have focused on CTL specificities in chronic infection and few data exist regarding the specificity of the initial CTL response induced in acute infection. In this study, HIV-1 infection in persons expressing human histocompatibility leukocyte antigen (HLA)-A*0201 was used as a means of addressing this issue. In chronic infection, the dominant HLA-A*0201–restricted CTL response is directed towards the epitope SLYNTVATL (“SL9”) in p17 Gag (residues 77–85). This epitope is targeted by 75% of HLA-A*0201–positive adults, and the magnitude of this A*0201-SL9 response shows a strong negative association with viral load in progressive infection. Despite using the highly sensitive peptide–major histocompatibility complex tetramer and intracellular cytokine assays, responses to the SL9 epitope were not detectable in any of 11 HLA-A*0201–positive subjects with acute HIV-1 infection (P = 2 × 10−6), even when assays were repeated using the SL9 peptide variant that was encoded by their autologous virus. In contrast, multiple responses (median 3) to other epitopes were evident in 7 of the 11 A*0201–positive subjects. Longitudinal study of two subjects confirmed that the A*0201-SL9 response emerged later than other CTL responses, and after viral set point had been reached. Together, these data show that the CTL responses that are present and that even may dominate in chronic infection may differ substantially from those that constitute the initial antiviral CTL response. This finding is an important consideration in vaccine design and in the evaluation of vaccine candidates

Selection, transmission, and reversion of an antigen-processing cytotoxic T-lymphocyte escape mutation in Human Immunodeficiency Virus Type 1 infection

Numerous studies now support that human immunodeficiency virus type 1 (HIV-1) evolution is influenced by immune selection pressure, with population studies showing an association between specific HLA alleles and mutations within defined cytotoxic T-lymphocyte epitopes. Here we combine sequence data and functional studies of CD8 T-cell responses to demonstrate that allele-specific immune pressures also select for mutations flanking CD8 epitopes that impair antigen processing. In persons expressing HLA-A3, we demonstrate consistent selection for a mutation in a C-terminal flanking residue of the normally immunodominant Gag KK9 epitope that prevents its processing and presentation, resulting in a rapid decline in the CD8 T-cell response. This single amino acid substitution also lies within a second HLA-A3-restricted epitope, with the mutation directly impairing recognition by CD8 T cells. Transmission of the mutation to subjects expressing HLA-A3 was shown to prevent the induction of normally immunodominant acute-phase responses to both epitopes. However, subsequent in vivo reversion of the mutation was coincident with delayed induction of new CD8 T-cell responses to both epitopes. These data demonstrate that mutations within the flanking region of an HIV-1 epitope can impair recognition by an established CD8 T-cell response and that transmission of these mutations alters the acute-phase CD8+ T-cell response. Moreover, reversion of these mutations in the absence of the original immune pressure reveals the potential plasticity of immunologically selected evolutionary changes

Mother-to-child transmission of HIV infection and CTL escape through HLA-A2-SLYNTVATL epitope sequence variation

Cytotoxic T lymphocytes (CTL) play a central role in containment of HIV infection. Evasion of the immune response by CTL escape is associated with progression to disease. It is therefore hypothesised that transmitted viruses encode escape mutations within epitopes that are required for successful control of viraemia. In order to test this hypothesis, escape through the dominant HLA-A2-restricted CTL epitope SLYNTVATL (p17 Gag residues 77–85 SL9) in the setting of mother-to-child-transmission (MTCT) was investigated. Initial data from two families in which the HIV-infected mother expressed HLA-A*0201 and had transmitted the virus to other family members were consistent with this hypothesis. In addition, analysis of the gag sequence phylogeny in one family demonstrated that CTL escape variants can be successfully transmitted both horizontally and vertically. To test the hypothesis further, a larger cohort of transmitting mothers (n=8) and non-transmitters (n=14) were studied. Variation within the SL9 epitope was associated with expression of HLA-A2 (P=0.04) but overall no clear link between variation from the SL9 consensus sequence and MTCT was established. However, the high level of background diversity within p17 Gag served to obscure any possible association between escape and MTCT. In conclusion, these studies highlighted the obstacles to demonstrating CTL escape arising at this particular epitope. Alternative strategies likely to be more definitive are discussed

Patterns of Immunodominance in HIV-1–specific Cytotoxic T Lymphocyte Responses in Two Human Histocompatibility Leukocyte Antigens (HLA)-identical Siblings with HLA-A*0201 Are Influenced by Epitope Mutation

Primary human immunodeficiency virus (HIV) infection is controlled principally by HIV-specific cytotoxic T lymphocytes (CTL) to a steady-state level of virus load, which strongly influences the ultimate rate of progression to disease. Epitope selection by CTL may be an important determinant of the degree of immune control over the virus. This report describes the CTL responses of two HLA-identical hemophiliac brothers who were exposed to identical batches of Factor VIII and became seropositive within 10 wk of one another. Both have HLA-A*0201. The CTL responses of the two siblings were very dissimilar, one donor making strong responses to two epitopes within p17 Gag (HLA-A*0201–restricted SLYNTVATL and HLA-A3–restricted RLRPGGKKK). The sibling responded to neither epitope, but made strong responses to two epitopes presented by HLA-B7. This was not the result of differences in presentation of the epitopes. However, mutations in both immunodominant epitopes of the p17 Gag responder were seen in proviral sequences of the nonresponder. We then documented the CTL responses to two HLA-A*0201–restricted epitopes, in Gag (SLYNTVATL) and Pol (ILKEPVHGV) in 22 other HIV-infected donors with HLA-A*0201. The majority (71%) generated responses to the Gag epitope. In the 29% of donors failing to respond to the Gag epitope in standard assays, there was evidence of low frequency memory CTL responses using peptide stimulation of PBMC, and most of these donors also showed mutations in or around the Gag epitope. We concluded that HLA class I genotype determines epitope selection initially but that mutation in immunodominant epitopes can profoundly alter the pattern of CTL response

Passive Sexual Transmission of Human Immunodeficiency Virus Type 1 Variants and Adaptation in New Hosts

Human immunodeficiency virus type 1 (HIV-1) genetic diversity is a major obstacle for the design of a successful vaccine. Certain viral polymorphisms encode human leukocyte antigen (HLA)-associated immune escape, potentially overcoming limited vaccine protection. Although transmission of immune escape variants has been reported, the overall extent to which this phenomenon occurs in populations and the degree to which it contributes to HIV-1 viral evolution are unknown. Selection on the HIV-1 env gene at transmission favors neutralization-sensitive variants, but it is not known to what degree selection acts on the internal HIV-1 proteins to restrict or enhance the transmission of immune escape variants. Studies have suggested that HLA class I may determine susceptibility to HIV-1 infection, but a definitive role for HLA at transmission remains unproven. Comparing populations of acute seroconverters and chronically infected patients, we found no evidence of selection acting to restrict transmission of HIV-1 variants. We found that statistical associations previously reported in chronic infection between viral polymorphisms and HLA class I alleles are not present in acute infection, suggesting that the majority of viral polymorphisms in these patients are the result of transmission rather than de novo adaptation. Using four episodes of HIV-1 transmission in which the donors and recipients were both sampled very close to the time of infection we found that, despite a transmission bottleneck, genetic variants of HIV-1 infection are transmitted in a frequency-dependent manner. As HIV-1 infections are seeded by unique donor-adapted viral variants, each episode is a highly individual antigenic challenge. Host-specific, idiosyncratic HIV-1 antigenic diversity will seriously tax the efficacy of immunization based on consensus sequences

Evolution and transmission of stable CTL escape mutations in HIV infection

Increasing evidence indicates that potent anti-HIV-1 activity is mediated by cytotoxic T lymphocytes (CTLs); however, the effects of this immune pressure on viral transmission and evolution have not been determined. Here we investigate mother–child transmission in the setting of human leukocyte antigen (HLA)-B27 expression, selected for analysis because it is associated with prolonged immune containment in adult infection. In adults, mutations in a dominant and highly conserved B27-restricted Gag CTL epitope lead to loss of recognition and disease progression In mothers expressing HLA-B27 who transmit HIV-1 perinatally, we document transmission of viruses encoding CTL escape variants in this dominant Gag epitope that no longer bind to B27. Their infected infants target an otherwise subdominant B27-restricted epitope and fail to contain HIV replication. These CTL escape variants remain stable without reversion in the absence of the evolutionary pressure that originally selected the mutation. These data suggest that CTL escape mutations in epitopes associated with suppression of viraemia will accumulate as the epidemic progresses, and therefore have important implications for vaccine design