In the current study we demonstrated that HIV-1-specific CD8+
T-cell responses as measured by the IFN-γ ELISPOT assay correlated inversely with vRNA. Moreover, this correlation was largely due to responses directed against Gag and specifically the p24 protein. Responses to Gag also correlated directly with absolute CD4+
T-cell counts, as did responses directed against the Pol precursor and IN. Our data demonstrate two separate and independent immune functions that correlate with vRNA: response to the p24 whole protein (likely due to CD4+
T cells) and response to 20-mer peptide pools representing Gag (due to CD8+
T cells, as demonstrated above). In view of the known significant correlations of vRNA and CD4+
T-cell counts with HIV-1 disease progression (2
), these immune responses to Gag (as detected by the ELISPOT assay) would likely correlate with progression to AIDS as well.
Our study is the first to demonstrate correlations of HIV-1-specific T cells with both vRNA and absolute CD4+
T cells. It is also the first to see correlations of markers of disease progression with more than one protein (p24 and IN). Although at least two groups have correlated the frequency of HIV-1-specific cells with vRNA using the tetramer assay (34
), the results have not been duplicated by other groups (4a
). The reasons that our study demonstrated correlations between immune responses and markers of HIV-1 disease progression could be due to a number of factors. In this study, we used a total of 250 peptides spanning all of the Gag, Pol, Env, and Nef proteins, thereby studying a much greater number of potential epitopes. We purposely quantified the IFN-γ responses, vRNA measurements, and absolute CD4+
T-cell counts from the same blood specimen for each subject. Additionally, the majority of patients were off medications for at least 6 months prior to sampling or had detectable vRNA despite ART. Furthermore, our cohort included patients in all stages of chronic HIV-1 infection, as evidenced by the broad range in their vRNA and absolute CD4+
T-cell counts (Table ). Finally, assays used to measure the HIV-1-specific CD8+
T-cell responses differ in their methods of optimization. As an example, the intracellular cytokine staining technique utilizes costimulatory molecules (4a
) that are not routinely used in the ELISPOT assay, and it is possible that certain assay optimization techniques may negate in vivo immune response differences that may exist among HIV-1-infected individuals.
Our ability to correlate responses to p24 with markers of disease progression is novel, and it is likely that these responses are due to CD8+ rather than CD4+ T-cell responses. We demonstrated that CD8+ T cells were being measured by using our ELISPOT technique in eight of eight subjects tested. The responses were due exclusively to CD8+ T cells in seven of these subjects and were mapped to HLA class I-restricted epitopes in all six of the individuals tested. Additionally, responses to the p24 whole protein did not correlate with p24 peptide responses, indicating that they measured separate effects. Therefore, HIV-1-specific CD8+ T cells were responsible for the majority of peptide-stimulated IFN-γ ELISPOT responses and correlated with markers of disease progression independently of CD4+ T-cell responses.
The reasons for the observed correlations seen with p24 peptide responses are unclear, but may be due to a number of factors. The p24 protein was one of the most frequently recognized subunit proteins, and the greatest magnitude of responses were directed to this protein when adjusted for the AA score (Fig. ). The reasons for this high frequency and magnitude of response are likely due to AA sequence conservation. Indeed, the two regions demonstrating the strongest correlations with markers of disease progression (p24 and IN) are also the most highly conserved proteins among the clade B viruses (36
The inability to correlate Env-specific responses with risk factors for HIV-1 disease progression is likely due to the low magnitude of these responses in our cohort (median, 88 SFU/106
PBMC). Musey et al. were able to demonstrate a correlation between freshly isolated Env-specific CTL and vRNA (42
). However, unlike our study, this group looked at HIV-1-specific CD8+
T cells in early infection where responses to Env were immunodominant; being present in up to 80% of their subjects. The inability to correlate vRNA with Nef-specific responses cannot be explained by the low magnitude of reactivity directed towards this protein, as the median response was 388 SFU/106
PBMC (Fig. ). In fact, Nef-specific responses were immunodominant (i.e., had the greatest magnitude) in 6 of the 27 subjects.
The relative lack of genetic sequence conservation in the Env and Nef regions in clade B viruses could explain the inability to correlate IFN-γ responses specific to these proteins with markers of disease progression. Among the different proteins of clade B viruses (36
), Env and Nef are the most variable of the proteins tested in this study. A possible consequence of this genetic variability is that the measured immune responses to the Env and Nef proteins no longer serve to control viral replication due to the presence of CTL escape mutants (5
). This hypothesis may be particularly pertinent to the Nef-specific responses, as several studies have demonstrated the presence of CTL escape mutants to this protein (13
One important limitation of this study is the use of peptides derived from HIV clones that may not actually represent autologous virus. It is therefore safe to assume that the T-cell responses seen in our study underrepresent the true response. Use of autologous viral peptides not only would increase the frequency and magnitude of responses, but may also change some of the correlations seen in our study. Clearly sequencing autologous virus in order to design individual immune reagents should be performed in the future to more accurately measure HIV-1-specific CD8+ T-cell responses.
The goal of many of the newer candidate HIV-1 vaccines is to elicit strong CD8+ T-cell responses in an attempt to prevent HIV-1 disease in both infected and uninfected individuals. The current study presents further evidence that the HIV-1 Gag protein, particularly its p24 capsid subunit, should be included in any vaccine designed to elicit cell-mediated immune responses. Studies currently in progress, which aim to refine the specificity of CD8+ T-cell responses to HIV-1 and examine these responses longitudinally, will likely elucidate other proteins to be included in an optimal CTL-based vaccine.