Large-scale screening of HIV-1 T-cell epitopes restricted by 12 prevalent HLA-A allotypes in Northeast Asia and universal detection of HIV-1-specific CD8+ T cells

Abstract

Background: Although the immune response of host T cells to human immunodeficiency virus (HIV) significantly influences the progression of the infection, the development of T-cell-based vaccines and therapies, as well as the clinical evaluation of specific T-cell functions, is currently markedly hindered by the absence of broad-spectrum, functionally validated HIV T-cell epitopes that account for the polymorphisms of the human leukocyte antigen (HLA) within an indicated geographic population. This study aimed to identify T-cell epitopes derived from the GP160, GAG, and POL proteins of the HIV-1 strain, specifically linked to 12 prevalent HLA-A allotypes, that collectively represent approximately 91% of the total gene frequency in Northeast Asian populations.

Methods: A total of 134 epitopes were predicted in silico and selected as potential candidates for further validation. Subsequently, peripheral blood mononuclear cells (PBMCs) were collected from 96 individuals with HIV-1 and cocultured ex vivo with each epitope candidate peptide, followed by the detection of activated CD8⁺ T cells. Peripheral blood mononuclear cells (PBMCs) were collected from 96 individuals with HIV-1 and cocultured ex vivo with each candidate peptide epitope, followed by the detection of activated CD8+ T cells. A total of 69 epitopes were validated as real-world HIV T-cell epitopes presented by 12 dominant HLA-A allotypes. Furthermore, the HLA-A cross-restriction for each epitope candidate was identified through peptide competitive binding assays using 12 transfected HMy2.CIR cell lines.

Results: A total of 45 epitopes demonstrated high affinity, while 31 epitopes displayed intermediate affinity. A broad-spectrum CD8⁺ T-cell epitope library containing 141 validated epitope peptides was used to universally detect HIV-1-specific CD8⁺ T cells via peptide-PBMC ex vivo coculture and intracellular IFN-γ staining. In 52 people with HIV-1, the number of reactive HIV-1 specific CD8⁺ T cells was significantly higher in the CD4⁺ T-cell-high patient group compared to the CD4⁺ T-cell-low patient group, and it correlated with the CD4⁺ T-cell-low patient group (<200/μL).

Conclusion: This study provides a broad-spectrum CD8⁺ T-cell epitope library aimed at developing a T-cell-directed HIV vaccine that offers high population coverage in Northeast Asia. In addition, it establishes a universal detection method for the clinical assessment of HIV-1-specific CD8⁺ T-cell responses.

Keywords: CD8+ T-cell; HIV-1; HLA-A; T-cell epitope; antigen-specific T-cell detection.

Figure 1

Sixty-nine epitope candidates were validated as real-world T-cell epitopes by peptide-PBMC ex vivo coculture experiments and intracellular IFN-γ staining. The PBMCs from 96 people with HIV-1 were cocultured for 20 h with the corresponding epitope candidate peptides, which could be represented by the patient’s HLA-A allotypes as virtually predicted. A total of 46 PBMCs from the peptide-PBMC cocultures presented positive T-cell responses. The frequencies of IFN-γ⁺ cells in the CD3⁺/CD8⁺ populations of each validated epitope and its negative control well in each PBMC sample are presented. NC, negative control well without peptide.

Figure 2

Binding affinity analysis of 112 HIV epitope candidates to the corresponding HLA-A molecules using competitive peptide binding assay. A series of unlabeled peptides and a fluorescent reference peptide were co-incubated with HMy2.CIR cell lines expressing the indicated HLA-A molecules. The competition binding inhibition (%) of each standby peptide at 5 and 15 μM was then calculated by determining the percentage of fluorescence-positive HMy2.CIR. The blue and violet lines are peak plots of cellular fluorescence intensity at 5 and 15 μM of the peptide to be detected, respectively. The gray filled line represents the maximum cellular fluorescence (cells using only the fluorescent reference peptide); the red line in the NC plot represents the background fluorescence of cells using only the RPMI 1640 culture medium as a fluorescent negative control.

Figure 3

Clinical detection of HIV-1 specific CD8⁺ T cells using 141 validated epitope peptides. The epitope peptides were grouped into three pools according to the derived protein (GP160, GAG, and POL) and ex vivo co-incubated for 15–20 h with PBMCs from 52 people with HIV-1 followed by IFN-γ ICS. (A) The histogram plots show the count of INF-γ⁺ CD8 T cells (negative control values were excepted) in the PBMCs of 52 people with HIV-1 after stimulation with three peptide pools. (B) The comparison of the count of IFN-γ⁺ CD8 T cells in the negative control, three peptide pools, the sum of three peptide pools, and positive control.