Introduction The majority of individuals infected with HIV-1 develop an acute viral syndrome within 7–21 days of infection, characterized primarily by fever, lymphadenopathy, and cutaneous rash in the presence of very high levels of HIV-1 replication [1,2]. Both innate and adaptive immune responses, including natural killer cell responses, HIV-1-specific CD4+ and CD8+ T cell responses, and neutralizing antibodies have been associated with the subsequent resolution of the clinical symptoms and the decline of HIV-1 RNA levels to so-called viral set point levels. In particular, the first appearance of HIV-1-specific CD8+ T cells in the peripheral blood has been shown to be temporally associated with the initial decline of HIV-1 viremia during primary infection [3,4], suggesting a crucial role of these early virus-specific T cells in the control of viral replication. This is further supported by the lack of decline in viral replication in simian immunodeficiency virus-infected macaques depleted of CD8+ lymphocytes [5–7] and the selection of viral strains containing sequence variations within targeted CD8+ T cell epitopes during this early phase of infection [8–11], indicative of HLA class I-restricted immune selection pressure on the virus. Despite this presumed immune-mediated decline in acute viremia, HIV-1-specific CD8+ T cell responses in primary infection are of lower magnitude and more narrowly directed against a limited number of epitopes than are HIV-1-specific CD8+ T cell responses detected in chronic infection [12–15], indicating that the quality and specificity, rather than the quantity, of virus-specific CD8+ T cell responses may be associated with the initial control of viral replication [16–20]. However, very little is known about the individual epitopes targeted during primary HIV-1 infection and their immunodominance pattern, as these studies are complicated by the high HLA class I diversity in humans, requiring large numbers of participants with primary infection to draw meaningful conclusions. To date, studies of the specificity of HIV-1-specific CD8+ T cell responses during primary HIV-1 infection have been limited in size, focused on individuals expressing specific HLA alleles of interest, or assessed the protein specificity of these CD8+ T cells without determining the individual targeted epitopes in the context of the restricting HLA class I molecules [12–15,21–25]. Here, we describe the characterization of HIV-1-specific CD8+ T cell responses on the single-epitope level in a cohort of 104 individuals identified during primary HIV-1 infection. The aim of the study was to identify the immunodominant CD8+ T cell epitopes within HIV-1 that are targeted during primary HIV-1 infection, and to assess the contribution of CD8+ T cell responses restricted by the individual HLA class I molecules to the total virus-specific CD8+ T cell response early in infection. Methods Study Participants A total of 104 HIV-1-infected individuals were enrolled in this study at the Massachusetts General Hospital in Boston, Massachusetts, United States, and a private medical clinic (Jessen-Praxis) in Berlin, Germany. Of this group, 69 (66%) individuals were identified during acute HIV-1 infection, as defined by either a negative HIV-1 p24 ELISA or an evolving HIV-1 Western blot (fewer than three bands), and the remaining 35 (34%) were identified within the first 6 mo of HIV-1 infection, as defined by a negative HIV-1 p24 ELISA during the past 6 mo or a negative detuned HIV-1 ELISA at the time of enrollment. The majority of study participants were men who have sex with men (98 [94%]) and individuals of Northern European descent (83 [80%]). The average viral load at presentation was 3,527,188 HIV-1 RNA copies/ml (range 50–84,200,000 copies) and the average CD4+ T cell count was 520 cells/μl (range 42–1,334). The majority of individuals (88 [85%]) initiated HAART during primary infection. The assessment of HIV-1-specific CD8+ T cell responses was performed on frozen peripheral blood mononuclear cell (PBMC) samples collected 8 wk (± 10 d) following initial presentation. This time point was chosen because previous studies had demonstrated that a substantial subset of individuals with acute HIV-1 infection have no detectable or only very weakly detectable HIV-1-specific CD8+ T cell responses at presentation, and that the HAART-induced decline in virus-specific T cell responses occurs after 8 wk of treatment in individuals treated with HAART during acute or early HIV-1 infection [12]. The study was approved by the respective institutional review boards and was conducted in accordance with human experimentation guidelines of the Massachusetts General Hospital, and all study participants provided informed consent prior to enrollment in the study. HLA Typing High- and intermediate-resolution HLA class I typing was performed by sequence-specific PCR according to standard procedures. DNA was extracted from PBMCs using the Puregene DNA Isolation Kit for blood (Gentra Systems, Minneapolis, Minnesota, United States). IFN-γ Enzyme-Linked Immunosorbent Spot Assay HIV-1-specific CD8+ T cell responses were quantified by IFN-γ enzyme-linked immunosorbent spot (ELISPOT) assay, using a panel of 173 peptides corresponding to described optimal clade B cytotoxic T lymphocyte epitopes [26]. PBMCs were plated at 100,000 cells per well with peptides at a final concentration of 10−5 molar in 96-well plates and processed as described [12]. PBMCs were incubated with media alone (negative control) or PHA (positive control). The number of specific IFN-γ secreting T-cells were counted using an automated ELISPOT reader (AID, Strassberg, Germany), calculated by subtracting the average negative control value and expressed as spot-forming cells (SFCs)/106 input cells. Negative controls were always 30 SFCs/106 input cells or fewer. A response was considered positive at 50 SFCs/106 input cells or more and when the total number of spots were at least three times greater than the mean number of spots in the negative control wells. Hazard Ratios for Disease Outcomes The hazard ratios (HRs) for the various HLA alleles were determined previously using Cox model analyses [27–30]. Briefly, 1,217 HIV-1-infected individuals for whom the dates of seroconversion were known were derived from four cohorts: the Multicenter AIDS Cohort Study (MACS, n = 522), the Multicenter Hemophilia Cohort Study (MHCS, n = 322), the San Francisco City Clinic Cohort (SFCCC, n = 87), and the AIDS Linked to Intravenous Experience (ALIVE, n = 286) study. Survival analyses were performed on seroconverters from all the cohorts combined and included all patients without regard to racial group. Four AIDS-related outcomes were considered end points of survival analysis: a CD4+ T lymphocyte count of less than 200/mm3 (hereafter termed “CD4 50% of study participants) by HLA-B alleles (9/96 HLA-B epitopes tested versus 0/73 HLA-A epitopes; p 0.1 for all comparisons, unpublished data). Figure 4 Immunodomination of HLA-B57- and HLA-B27-Restricted HIV-1-Specific CD8+ T Cell Responses The percent contribution (left graphs) and the absolute magnitude (right graphs, given as SFCs per million input PBMCs [SFC/Mill PBMC]) of HLA-A1-, -A2-, -A3-, and -A24-restricted HIV-1-specific CD8+ T cell responses in individuals expressing these HLA class I alleles alone, or in conjunction with HLA-B57 or HLA-B27, are shown. Each dot represents data for one individual. The contribution, as well as the absolute magnitude, of HIV-1-specific CD8+ T cell responses directed against HLA-A1-, -A2-, and -A24-restricted CD8+ T cell epitopes was significantly lower in participants that also coexpressed HLA-B57 or HLA-B27. The same trend was observed for HLA-A3, but did not reach statistical significance. HLA-A1-, -A2-, -A3-, and -A24-restricted HIV-1-specific CD8+ T cell response did not differ between individuals expressing other frequent HLA class B alleles, such as HLA-B7, -B8, -B35, or -B44 (unpublished data). HLA Class I Allele-Specific Relative Hazard for Disease Progression Correlates Inversely with the Contribution to the HIV-1-Specific CD8+ T Cell Response The above data for HLA-B57 and HLA-B27 demonstrate that these two alleles, which have been strongly associated with slower HIV-1 disease progression, contribute substantially to the total HIV-1-specific CD8+ T cell response during primary HIV-1 infection. We next tested whether the contribution of an individual HLA class I allele to the total HIV-1-specific CD8+ T cell response in primary infection is associated with the HR for that allele in progression to four HIV-1 disease outcomes (CD4 <200, AIDS 1987, AIDS 1993, and death), using data from four HIV-1 cohorts (MACS, MHCS, SFCCC, and ALIVE) [27–30]. For all four outcomes, the contribution of an HLA allele to the HIV-1-specific CD8+ T cell response during primary infection showed a consistent inverse correlation with the respective HR (Figure 5), and this inverse correlation trended toward significance for the outcomes AIDS 1987 (p = 0.06), AIDS 1993 (p = 0.07), and reached significance for death (p = 0.045). While the correlation was strongly driven by HLA-B27 and HLA-B57, this analysis, which compared two discrete groups of patients, suggests that the ability of an HLA class I allele to dominate the HIV-1-specific T cell responses during primary infection might have an important impact on its protective role during the subsequent course of infection. Figure 5 Correlation between the Contribution of Individual HLA Class I Alleles to the Total HIV-1-Specific CD8+ T Cell Response during Primary Infection and the HR for HIV-1 Infection Outcome The percent contribution of individual HLA class I alleles to the total HIV-1-specific CD8+ T cell response during primary infection was correlated to the HR for four different HIV-1 infection outcomes (time to CD4 <200, time to AIDS 1987, time to AIDS 1993, and time to death) for the respective HLA alleles. Discussion Several studies suggest that innate and adaptive immunological events occurring during primary HIV-1 infections play a crucial role in determining the level of viral replication during the subsequent disease process. The characterization of the individual epitopes frequently and consistently targeted by these early and potent virus-specific CD8+ T cells is therefore of major interest both for the identification of highly immunogenic targets for HIV-1 vaccines and for studies of the mechanisms underlying immunodominance in HIV-1 infection. Here we demonstrate in a cohort of 104 individuals with primary HIV-1 infection that only a subset of known CD8+ T cell epitopes is frequently and consistently targeted in the initial stages of HIV-1 infection, when viral load drops on average more than 1,000-fold. We found, furthermore, that the HLA class I genotype determines the level of contribution of CD8+ T cells responses restricted by individual alleles to the total HIV-1-specific T cell response. HLA alleles that contribute strongly to the initial virus-specific CD8+ T cell response represented those alleles associated with slower HIV-1 disease progression. In the present study we used peptides corresponding to optimal HIV-1-specific CD8+ T cell epitopes to assess their frequency of recognition during primary HIV-1 infection. While the use of described optimal cytotoxic CD8+ T lymphocyte epitopes within HIV-1 may have resulted in an underestimation of total HIV-1-specific T cell responses, this approach allowed us to identify a subset of immunodominant epitopes that are very frequently targeted during this early phase of HIV-1 infection. In contrast, other well-characterized epitopes restricted by the same HLA class I alleles and located within the same HIV-1 proteins were only rarely recognized, demonstrating consistent immunodominance patterns of HIV-1-specific CD8+ T cell responses during primary HIV-1 infection. Furthermore, CD8+ T cell responses in primary infection were directed against a narrow repertoire of HIV-1 epitopes, suggesting that a limited number of CD8+ T cell responses are sufficient to control HIV-1 replication during the initial phase of infection. While these data do not allow for direct conclusions regarding the epitope-specific CD8+ T cell responses that need to be induced by vaccines in order to mediate protective immunity, they suggest that the presence of broadly directed and strong HIV-1 CD8+ T cell responses is not an absolute requirement for protective immunity. Little is known about the mechanisms that determine immunodominant T cell responses in HIV-1 infection, but several factors—including the intracellular expression levels of the respective HIV-1 protein, the kinetics by which an epitope is processed, its binding affinity to the HLA class I molecule, and the binding affinity of the peptide–HLA class I complex to the CD8+ T cell receptor and its repertoire—have been implicated in the shaping of immunodominance patterns [38,39]. The current study did not allow us to address the individual contributions of these “intrinsic” factors to the observed hierarchies in epitope-specific CD8+ T cell responses. However, we demonstrate here that the sequence variability within HIV-1 is not a major factor in determining immunodominance pattern during primary HIV-1 infection, as frequently targeted epitopes did not significantly differ in their sequence variability within the HIV-1 population from rarely targeted epitopes restricted by the same HLA allele within this study cohort. Taken together, these studies of HIV-1-specific CD8+ T cell responses during primary HIV-1 infection on the single-epitope level resulted in the identification of several immunodominant epitopes within HIV-1 that are frequently targeted in infected individuals of Northern European descent. HLA class I molecules are codominantly expressed on antigen-presenting cells, such that all six HLA class I allotypes (if heterozygous for HLA-A, -B, and -C) can present viral epitopes and theoretically prime virus-specific CD8+ T cell responses. However, epitopic peptides compete for presentation by HLA molecules, and different HLA class I molecules may differ in their ability to present the respective viral epitopes on the cell surface. Here we demonstrate that selected HLA class I molecules contribute significantly more than other HLA class I molecules to the total HIV-1-specific CD8+ T cell response detected during primary infection. Interestingly, the presence of HLA-B57 and HLA-B27, two alleles that contributed more than 65% to the total antiviral CD8+ T cell response among individuals carrying these alleles, reduced the relative contribution of HIV-1-specific CD8+ T cell responses restricted by other HLA molecules expressed in the same individual, as well as the absolute virus-specific CD8+ T cell response restricted by these other alleles. The large number of persons examined in this study allowed us to assess the impact of HLA-B27 and -B57 on the HIV-1-specific T cell responses restricted by other alleles, but the high HLA diversity limited this assay to the most frequent alleles, including HLA-A1, -A2, -A3, and -A24. For all four HLA-A alleles studied, the magnitude of HIV-1-specific CD8+ T cell responses restricted during primary infection was dramatically lower in the presence of HLA-B27 and -B57, but not in the presence of other HLA-B alleles. These data suggest a selective disadvantage of CD8+ T cell responses restricted by these alleles in the presence of HLA-B27 and -B57, and is consistent with the concept of immunodomination by HLA-B27- and HLA-B57-restricted HIV-1-specific CD8+ T cell responses [38]. Immunodomination in the evolution of dominant virus-specific CD8+ T cell responses, first studied in mice, refers to the ability of CD8+ T cells specific for immunodominant epitopes to suppress the CD8+ T cell response to subdominant epitopes. In these mouse studies, it was shown that the elimination of an immunodominant epitope resulted in the development of strong CD8+ T cell responses to otherwise subdominant epitopes [40–45]. Subsequent studies in humans demonstrated that immunodominant Epstein-Barr virus- and cytomegalovirus-specific CD8+ T cell responses restricted by HLA-A2 were reduced in those who coexpressed HLA-B7 [46,47]. Furthermore, simian immunodeficiency virus-infected rhesus macaques expressing both Mamu-A*01 and -A*02 were recently shown to exhibit a significant delay in the development of a CD8+ T cell response to a Mamu-A*02-restricted epitope that represented the immunodominant response in Mamu-A*02+ macaques in the absence of Mamu-A*01 expression [48]. Similar to HLA-B27 and -B57, two HLA alleles associated with slow disease progression in HIV-1-infected humans [27–29,36,37], Mamu-A*01 has been associated with slower disease progression in simian immunodeficiency virus-infected macaques [49–53], indicating that immunodomination by these major histocompatibility complex alleles in macaques and humans may contribute to their superior ability to control viral replication. Furthermore, these three alleles, which are associated with slower disease progression in HIV-1 infection, all restrict a single highly immunodominant epitope-specific CD8+ T cell response during primary infection (TW10 for B57 [23], KK10 for B27, and TL8 for Mamu-A*01 [8,51]), suggesting that the combination of these specific epitopes in the context of the restricting major histocompatibility complex class I allele may be crucial for the described strong antiviral activity. Further studies will be needed to elucidate the mechanisms underlying the immunodominance of virus-specific CD8+ T cell responses restricted by these alleles. We tested directly for an association between the contribution of an individual HLA class I allele to the total virus-specific CD8+ T cell response in primary HIV-1 infection and its protective effect on disease progression. The average contribution of each studied HLA class I allele to the total HIV-1-specific T cell response detected using optimal CD8+ T cell epitopes was calculated in the 104 study participants, and correlated with the HR for the respective alleles for four different HIV-1 disease outcomes, as determined previously in the combined MHCS, ALIVE, MACS, and SFCCC cohort studies [27–30]. For each of the four outcomes considered (progression to CD4<200, AIDS 1987, AIDS 1992, and death), the antiviral contribution of an allele was negatively correlated with its HR, and this inverse correlation was borderline significant for the outcomes AIDS 1987 (p = 0.06), AIDS 1993 (p = 0.07), and death (p = 0.045). These data suggest that HLA class I alleles that contribute robustly to the initial CD8+ T cell response against HIV-1 delay progression to AIDS. It is notable that individuals in these different cohorts of HIV-1 infected participants were grouped according to low-resolution two-digit HLA class I typing, and this association was strongly driven by the associations for HLA-B57, HLA-B51, and HLA-B27. Future studies of larger cohorts that take high-resolution HLA class I subtypes into account will be needed to provide greater detail on the contribution of specific HLA class I subtypes to the total HIV-1-specific CD8+ T cell response and disease progression. Taken together, these studies in a large cohort of individuals with primary HIV-1 infection demonstrate consistent immunodominance patterns of HIV-1-specific CD8+ T cell responses during primary infection and provide a mechanistic link for the observed protective effect of specific HLA class I alleles on HIV-1 disease progression. Understanding the precise factors that allow epitope-specific CD8+ T cell responses restricted by individual HLA class I alleles to dominate over other epitopes restricted by the same allele, as well as over epitope-specific T cell responses restricted by other alleles, will require additional studies and be an important step in efforts to modulate antiviral T cell responses generated by therapeutic immunizations or vaccinations.
