Figure 1.
CD4+ T cell responses targeting HIV Gag are associated with immunological control.
Persons with a CD4+ T cell response against a pool of Gag peptides have significantly lower viral loads and higher CD4 counts than those with no response against the Gag peptide pool. A total of 373 subjects were screened and statistics were calculated using the Mann Whitney test.
Figure 2.
The majority of the HIV-1 C clade CD4+ epitopes cluster in the Gag region.
ELISPOT screening was conducted on 32 randomly selected subjects. (A) 33 CD4+ restricted epitopes were identified, with 27 epitopes located in the Gag region, 3 in the Polymerase region, 1 in the Vpr region and 2 in the Nef region. There were no CD4+ epitopes present in the Vif, Vpu, Rev, Tat, or Envelope proteins. (B) The peptides identified by the ELISPOT assay utilizing CD8+ depleted PBMCs were confirmed by flow cytometry using whole PBMCs to be CD4+ restricted. The dot plot above represents data from the negative control on the left and a CD4+ IFN-γ response to the p24 Gag peptide YVDRFFKTLRAEQATADV.
Table 1.
Most frequently recognized clade C CD4+ T cell epitopes.
Figure 3.
The total HIV-1–specific CD4+ T cell response is dominated by Gag.
Using IFN-γ ELISPOT assay to identify HIV-1 specific CD4+ T cell epitopes in 32 subjects, we found that irrespective of the viral load, the Gag protein is the most commonly targeted and therefore makes the greatest contribution to the overall CD4+ T cell response. Subjects 1–16 had viral loads below the median, while subjects 18–32 had viremia greater than the median (36 550 copies/ml plasma). Acc (accessory) denotes the Vpr, Vpu and Vif proteins pooled together. Subjects 24 and 25 both targeted a single Vpr protein.
Figure 4.
Correlation of total breadth and magnitude of CD4+ T cell response to viral load.
(A) The total breadth of the HIV-1-specific CD4+ T cell response was obtained by summing the number of peptides identified by the ELISPOT assay and was found to correlate inversely with viral load. (B) The total magnitude of the response was calculated by adding the number of spot forming cells for each peptide response. Here we found no association between the magnitude of the response and viral load. More specifically the breadth (C) and magnitude (D) of Gag-specific CD4+ T cell responses, correlated inversely with viral load. Excluding the outlier with 17 peptide responses, these correlations continued to be significant for both breadth (E) and magnitude (F).
Figure 5.
Dual cytokine staining to pools of overlapping peptides.
Dual IFN-γ/IL-2 ICS was conducted on 10 subjects. (A) The dot plot on the left shows the unstimulated control while the one on the right shows the simultaneous detection of IFN-γ and IL-2 in response to stimulation with a pool of Gag peptides. The cells were gated on CD4+ T cells. (B) The cytokine response to all HIV-1 protein pools were added to obtain the total HIV-1 cytokine specific response. The percentage of HIV-1-specific CD4+ T cells producing only IFN-γ was significantly higher than the percentage of HIV-1-specific CD4+ T cells producing both IFN-γ and IL-2.
Figure 6.
Cross clade recognition of the p24 Gag peptide YVDRFFKTLRAEQATQDV.
In a subset of 28 subjects, we found that the C and B clade versions of peptide YVDRFFKTLRAEQATQDV induced the same magnitude of IFN-γ response in 7 patients, as detected by the ELISPOT assay.