Comprehensive screening for human immunodeficiency virus type 1 subtype-specific CD8 cytotoxic T lymphocytes and definition of degenerate epitopes restricted by HLA-A0207 and -C(W)0304 alleles

Abstract

For this report, the rapid identification and characterization of human immunodeficiency virus type 1 (HIV-1)-derived broadly cross-subtype-reactive CD8 cytotoxic T lymphocyte (CTL) epitopes were performed. Using a gamma interferon (IFN-gamma) Elispot assay-based approach and a panel of recombinant vaccinia viruses expressing gag, env, pol, and nef genes representing the seven most predominant subtypes and one circulating recombinant form of HIV-1, the subtype specificity and cross-subtype reactivity of a CD8 response were directly measured from circulating peripheral blood mononuclear cells (PBMC). Enhanced sensitivity of detection of CD8 responses from cryopreserved PBMC was achieved using autologous vaccinia virus-infected B-lymphoblastoid cell lines as supplemental antigen-presenting cells. Of eleven subjects studied, six exhibited broadly cross-subtype-reactive CD8-mediated IFN-gamma production (at least seven of eight subtypes recognized) to at least one major gene product from HIV-1. Screening of subjects showing broadly cross-subtype-specific responses in the vaccinia virus-based enzyme-linked immunospot (Elispot) assay using a panel of overlapping peptides resulted in the identification of cross-subtype responses down to the 20-mer peptide level in less than 3 days. Three subjects showed broad cross-subtype reactivity in both the IFN-gamma Elispot assay and the standard chromium release cytotoxicity assay. Fine mapping and HLA restriction analysis of the response from three subjects demonstrated that this technique can be used to define epitopes restricted by HLA-A, -B, and -C alleles. In addition, the ability of all three epitopes to be processed from multiple subtypes of their parent proteins and presented in the context of HLA class I molecules following de novo synthesis is shown. While all three minimal epitopes mapped here had previously been defined as HIV-1 epitopes, two are shown to have novel HLA restriction alleles and therefore exhibit degenerate HLA binding capacity. These findings provide biological validation of HLA supertypes in HIV-1 CTL recognition and support earlier studies of cross-subtype CTL responses during HIV-1 infection.

FIG. 1.

Determination of optimal ratio of PBMC to supplemental recombinant vaccinia virus-infected autologous BLCL for maximizing the sensitivity of IFN-γ-producing cell detection. Vertical bars represent the SFU/10⁶ PBMC for control wells (vP1170, vaccinia virus WR; stippled bars) and test wells (vP1288, expressing the IIIB pol gene; solid bars) performed in duplicate. (A) Cryopreserved PBMC thawed and rested overnight (as outlined in Results) prior to addition to assay. (B) Cryopreserved PBMC added directly to assay postthaw.

FIG. 2.

Comparison of sensitivity of HIV-1-specific IFN-γ-producing CD8 T-cell detection from cryopreserved PBMC using either supplemental vaccinia virus-infected BLCL (A and C) or direct addition of vaccinia virus to cryopreserved PBMC (B and D) for two subjects. Results are presented as total numbers of IFN-γ-producing cells (mean of triplicate wells) for either control depletion (solid bars) or CD8 depletion (stippled bars). Recombinant vaccinia viruses representing subtype A (gag and pol) and CRF01_AE (env and nef) were used.

FIG. 3.

Subtype and cross-subtype IFN-γ Elispot analysis for three subjects, AIHP-6 (A and B), VAIP-4 (C and D), and US101 (E and F). Cutoff for positivity (dotted line) is the 99% confidence interval for the negative control vSC8 (WR vaccinia virus, expressing β-galactosidase) performed in quadruplicate. Vertical bars represent the mean IFN-γ SFU/10⁶ PBMC of duplicate wells. Dependence on CD8 T cells is shown by immunomagnetic bead depletion in the lower panel of each pair. Subjects VAIP-4 and AIHP-6 were infected with HIV-1 subtype CRF01_AE, and subject US101 was infected with HIV-1 subtype B.

FIG. 4.

Cross-subtype IFN-γ Elispot analysis for eight subjects. Subjects US102 and US103 were from the United States, and the remaining six were Thai. Cutoff for positivity (dotted line) is the 99% confidence interval for the negative control vSC8, performed in quadruplicate. Vertical bars represent the mean IFN-γ SFU/10⁶ PBMC of duplicate wells. Subjects AIHP-8 and AIHP-9 were infected with HIV-1 subtype CRF01_AE, and subjects US102 and US103 were infected with HIV-1 subtype B, while the subtype infecting AIHP-3, AIHP-4, AIHP-5, and AIHP-200 was not identified.

FIG. 5.

Identification of immunodominant epitopes from three subjects showing broadly cross-subtype-reactive CD8 T-cell responses. Vertical bars represent the mean IFN-γ SFU/10⁶ PBMC of duplicate wells for both control depletion (solid bars) and CD8 depletion (stippled bars). Subject US101 was screened with 16 pools of five peptides representing the env gene from subtype B isolate MN (A), and the positive pool was subsequently broken down to individual peptides (B). Subjects AIHP-6 (C) and VAIP-4 (E) were screened with a 7 by 7 matrix of overlapping 20-mer peptides representing the gag gene from subtype A isolate 92UG037. Panel D shows the determination of peptide 27 (shaded, in pools 4A and 6B) as the immunodominant peptide for AIHP-6, and panel F shows peptide 30 (shaded, in pools 5A and 2B) as the immunodominant peptide for VAIP-4.

FIG. 5.

Identification of immunodominant epitopes from three subjects showing broadly cross-subtype-reactive CD8 T-cell responses. Vertical bars represent the mean IFN-γ SFU/10⁶ PBMC of duplicate wells for both control depletion (solid bars) and CD8 depletion (stippled bars). Subject US101 was screened with 16 pools of five peptides representing the env gene from subtype B isolate MN (A), and the positive pool was subsequently broken down to individual peptides (B). Subjects AIHP-6 (C) and VAIP-4 (E) were screened with a 7 by 7 matrix of overlapping 20-mer peptides representing the gag gene from subtype A isolate 92UG037. Panel D shows the determination of peptide 27 (shaded, in pools 4A and 6B) as the immunodominant peptide for AIHP-6, and panel F shows peptide 30 (shaded, in pools 5A and 2B) as the immunodominant peptide for VAIP-4.

FIG. 6.

Cytotoxic activity of peptide-stimulated cultures of cryopreserved PBMC. The identified immunodominant 20-mer peptides were used to generate effector cells from subjects AIHP-6 (A), VAIP-4 (B), and US101 (C). Vaccinia virus-infected autologous BLCL served as target cells. Percent specific killing is shown for target cells infected with the gag constructs for AIHP-6 and VAIP-4 and the env constructs for US101 at E:T ratios of 50:1 to 2:1. Autologous BLCL infected with vSC8 served as the negative controls.

FIG. 7.

Minimal epitope mapping of the gag-specific cross-subtype CD8 response of subject AIHP-6. (A) Overlapping 15-mer peptides (Pep) (representing HXB2 gag) were used to minimally map the epitope directly from cryopreserved PBMC. Only one 15-mer peptide (peptide 66) contained the 10-mer peptide KRWIILGLNK (gag 263-272) (underlined). (B) Percent specific killing of the parent 20-mer peptide (peptide 27 from 92UG037 gag, solid squares) and the minimal 10-mer peptide (solid circles) representing the immunodominant HLA-B27 epitope (predicted from the subject's HLA type) are compared to non-peptide-pulsed autologous BLCL (open diamonds). Effector cells were generated by 20-mer peptide stimulation of cryopreserved PBMC, and target cells (autologous BLCL) were pulsed overnight with each peptide at 10 μg/ml. (C) Alignment of the amino acid sequences for each of the gag constructs used in the study covering the minimal epitope. A critical arginine (R) to lysine (K) substitution is present at the P2 HLA-B27 binding site of the CRF01_AE (vT142) construct sequence.

FIG. 8.

Minimal epitope mapping and HLA restriction of the env-specific cross-subtype CD8 response of subject US101. (A) Overlapping 15-mer peptides (Pep) (representing TH023 env) were used to minimally map the epitope directly from cryopreserved PBMC. Two 15-mer peptides (peptides 137 and 138) contained the 9-mer peptide RAIEAQQHL (gag 557-565) (underlined). (B) Percent specific killing of the predicted minimal epitope RAIEAQQHL based on the HLA-C_W0304 motif. Effector cells were generated by 20-mer peptide stimulation (peptide 52 from MN env) of cryopreserved PBMC and target cells (autologous BLCL, allogeneic BLCL, and HLA-C_W0304-only matched BLCL) were pulsed overnight with each peptide at 10 μg/ml. (C) Alignment of the amino acid sequences for each of the env constructs used in the study covering the minimal epitope. Two critical central amino acid substitutions are present in the subtype H (vT239R) construct sequence.

FIG. 9.

Minimal epitope mapping of the gag-specific cross-subtype CD8 response of subject VAIP-4. (A) Overlapping 15-mer peptides (Pep) (representing HXB2 gag) were used to minimally map the epitope directly from cryopreserved PBMC. Only one 15-mer (peptide 74) contains the 9-mer peptide YVDRFYKTL (gag 296-304) (underlined). (B) Percent specific killing of the parent 15-mer peptide (peptide 74, solid squares) and the minimal 9-mer peptide (solid circles). Effector cells were generated by 20-mer peptide stimulation (peptide 30 from 92UG037 gag) of cryopreserved PBMC, and target cells (autologous BLCL) were pulsed overnight with each peptide at 10 μg/ml. (C) Alignment of the amino acid sequences for each of the gag constructs used in the study covering the minimal epitope.

FIG. 10.

HLA restriction analysis of the gag-specific cross-subtype CD8 response of subject VAIP-4 mapped to peptide YVDRFYKTL (gag 296-304). Effector cells were generated by 20-mer peptide stimulation (peptide 30 from 92UG037 gag) of cryopreserved PBMC, and target cells were pulsed overnight with peptide at 10 μg/ml. Percent specific killing of autologous and partially HLA class I-matched BLCL is shown at E:T ratios of 50:1 (solid bars), 25:1 (Z-hatched bars), 10:1 (S-hatched bars), and 2:1 (stippled bars). The HLA class I type of each target cell is shown along the x axis.