Identification of a novel HLA-A24-restricted cytotoxic T-lymphocyte epitope derived from HIV-1 Gag protein (1998)

Exposure to HIV elicits a strong response of cytotoxic T lymphocytes (CTL) in HIV-infected individuals [1,2]. CTL are considered to play a crucial role in elimination of virus during the asymptomatic phase from HIV-infected individuals and in individuals who have a high chance of exposure to the virus but remain uninfected [3]. Therefore, identification of strong and common CTL epitopes will stimulate understanding of the more precise role of CTL in HIV-infected individuals and the development of an HIV vaccine. HLA-A24 (A*2402) is shared by approximately 70% of Japanese and is also a common allele worldwide [4,5]. To date, 12 HLA-A24-restricted HIV-1-specific CTL epitopes have been identified [6–8]. Amongst them, strong epitopes are derived from Env and Nef proteins, whereas three Gag-derived epitopes and one Pol-derived epitope are weak. Here we report the identification of a new strong and relatively common HLA-A24-restricted CTL epitope derived from HIV-1 Gag protein. In our previous study [7], we identified 11 HLA-A24-restricted CTL epitopes by reverse immunogenetics. The peptides used carry Tyr at position 2 (P2) and Leu, Ile or Phe at the carboxyl-terminus, which have been reported as HLA-A24 anchor residues [9]. We showed that Trp at the carboxyl-terminus is an HLA-A*2402 anchor residue [10], and hence we tried to identify additional HLA-A24-restricted CTL epitopes that have Trp at the carboxyl-terminus. Seventeen octamer to 11-mer peptides carrying Tyr at P2 and Trp at the carboxyl-terminus were synthesized and their binding affinity to HLA-A*2402 molecules was tested. Sixteen peptides bound to HLA-A*2402 molecules [10]. These peptides were then used for induction of the specific CTL. Peripheral blood lymphocytes (PBL) were obtained from three HIV-1-seropositive individuals carrying the HLA-A24 molecule (YY, HLA-A24/-,B51/59, Cw1/-; YK, HLA-A24/-,B46/52, Cw1/-; SG, HLA-A24/27,B35/51,Cw3/-) and cultured in the presence of the mixture of four to eight peptides at 1 μM each. After 7 days of culture, induction of CTL activity against Epstein-Barr virus (EBV)-transformed B-cell line Tm (Tm-EBV; HLA-A*1101/A*2402,B*5201/B*5201, Cw7/C*1202) was assessed by standard 51Cr release method [7]. Six peptides induced peptide-specific CTL activity (data not shown). These peptide-specific bulk cultures were restimulated with phytohemagglutinin-stimulated autologous cell lines pulsed with the corresponding mixture of peptide and then cultured for another week. Their cytolytic activity was evaluated using C1R cells transfected with the HLA-A*2402 gene (C1R-A*2402) pulsed with individual peptide at an effector-to-target ratio of 20 : 1. Bulk CTL cultures showed specific cytotoxicity for six peptides. In particular, strong CTL activity against C1R-A*2402 pulsed with nonamer peptide KYKLKHIVW (HIV-1SF2 position at amino acids 28–36, SF2-Gag28–9) was observed in two out of three individuals (YY, 83.0%; SG, 18.2% of relative specific lysis, which is the percentage of specific lysis of Tm-EBV pulsed with the peptide at 1 μM minus the percentage of specific lysis of Tm-EBV without peptide). For confirmation, CTL clones specific for SF2-Gag28–9 were generated from the bulk culture of YY by a limiting dilution method. The clones obtained killed C1R-A*2402 cells pulsed with the peptide in a peptide concentration-dependent manner (Fig. 1A, B). The minimum peptide concentration recognized by these CTL clones was very low (0.1–1.0 nM; Fig. 1B). They also killed C1R-A*2402 cells infected with Gag-expressing recombinant vaccinia virus, although they did not kill the control HLA-A, B-negative C1R cells (Fig. 1A). These results indicated that SF2-Gag28–9 is a naturally processed peptide epitope.Fig. 1: . Recognition of SF2-Gag28–9 by HLA-A24-restricted cytotoxic T lymphocytes (CTL). (A) Cytotoxicity of two CTL closes against C1R and C1R-A*2402 cells pulsed with the peptide (open bars and hatched bars, respectively) and those infected with Gag-expressing recombinant vaccinia virus (dotted bars and closed bars, respectively). Data are shown as relative specific lysis (percentage of specific lysis of the target cells pulsed with the peptide minus percentage of specific lysis of those without the peptide, or percentage specific lysis of the target cells infected with Gag-recombinant vaccinia virus minus percentage of specific lysis of those infected with wild-type vaccinia virus). (B) Cytotoxicity of the CTL clone 1 (•, ○) and 2 (▪, □) for C1R-A*2402 cells (•, ▪) and C1R cells (○, □) pulsed with titrated peptide. Cytotoxicity of the CTL clone 1 and 2 for the target cells was tested at an effector-to-target cell (E : T) ratio of 2 : 1. (C) Cytotoxicity in the cultured cells from 12 HIV-1-seropositive individuals carrying HLA-A24 after the first in vitro stimulation with the peptide. Data are shown as percentage of relative specific lysis against Tm-EBV at an E : T ratio of 50 : 1. More than 10% relative specific lysis was evaluated as positive response because relative specific lysis for control peptide was 2.2 ± 3.1%.To investigate whether the epitope was strong and common amongst HIV-1-infected individuals carrying HLA-A24 molecules, PBL from 12 HIV-1-seropositive individuals carrying HLA-A24 were stimulated with peptide SF2-Gag28–9, and at day 8, cytotoxic activity against Tm-EBV pulsed with the corresponding peptide was measured. Specific CTL activity (more than 10% relative specific lysis) was observed in bulk culture from individuals YY, KM, SG and ICH (Fig. 1C). Taken together, these results indicate that the newly identified epitope SF2-Gag28–9 is a strong and relatively common epitope. Thus, it was demonstrated that peptides carrying Trp at the carboxyl-terminus are presented to CTL by HLA-A*2402 molecules. There are nine variant sequences at the corresponding position to SF2-Gag amino acids 28–36 amongst HIV-1 clade B strains, and 68.7% (22 out of the 32 strains) possess variant sequences (Theoretical Biology and Biophysics, Los Alamos National Laboratory, Los Alamos, New Mexico, USA). SF2-Gag28–9 induced specific CTL activity in the bulk culture from four out of 12 HIV-1-seropositive individuals by a single stimulation. It is possible that the specific CTL was not induced by in vitro stimulation with SF2-Gag28–9 in the other eight individuals because they were infected with viruses carrying different amino-acid sequences at this position. Analysis of mutations in SF2-Gag28–9 and the CTL response to these mutant epitopes in the clinical course of HIV-1-seropositive individuals carrying HLA-A24 are now under investigation.