Selection and accumulation of an HIV-1 escape mutant by three types of HIV-1-specific cytotoxic T lymphocytes recognizing wild-type and/or escape mutant epitopes

Abstract

It is known that cytotoxic T lymphocytes (CTLs) recognizing HIV-1 escape mutants are elicited in HIV-1-infected individuals, but their role in the control of HIV-1 replication remains unclear. We investigated the antiviral ability of CTLs recognizing the HLA-A*24:02-restricted Gag28 -36 (KYKLKHIVW) epitope and/or its escape mutant (KYRLKHIVW) elicited in the early and chronic phases of the infection. Wild-type (WT)-epitope-specific CTLs, as well as cross-reactive CTLs recognizing both WT and K30R (3R) epitopes, which were predominantly elicited at early and/or chronic phases in HLA-A*24:02(+) individuals infected with the WT virus, suppressed the replication of the WT virus but failed to suppress that of the 3R virus, indicating that the 3R virus was selected by these 2 types of CTLs. On the other hand, cross-reactive and 3R-specific CTLs, which were elicited in those infected with the 3R virus, did not suppress the replication of either WT or 3R virus, indicating that these CTLs did not contribute to the control of 3R virus replication. High accumulation of the 3R mutation was found in a Japanese population recently recruited. The selection and accumulation of this 3R mutation resulted from the antiviral ability of these Gag28-specific CTLs and high prevalence of HLA-A*24:02 in a Japanese population. The present study highlighted the mechanisms for the roles of cross-reactive and mutant-epitope-specific CTLs, as well as high accumulation of escape mutants, in an HIV-1-infected population.

Fig 1

Gag28-specific CD8⁺ T cells from individuals infected with WT virus at early and chronic phases. Gag28-specific CD8⁺ T cells were induced by stimulating PBMCs from early and chronic phases in 4 WT-virus-infected HLA-A*24:02⁺ individuals with WT or 3R peptide. The responses of these bulk-cultured cells to C1R-A2402 cells prepulsed with WT or 3R peptide at concentrations of 0.1 to 1,000 nM were analyzed by using the ICC assay.

Fig 2

Antiviral activity of Gag28-specific CTL clones generated from early-phase PBMCs from patient KI-092, infected with WT virus. Gag28-specific CTL clones were generated from early-phase PBMCs from KI-092 by stimulating them with WT peptide. The activities of 3 CTL clones (n = 3) were analyzed. (A) Cytotoxic activity toward 721.221-CD4-A2402 cells prepulsed with the WT or 3R peptide at concentrations of 1 to 1,000 nM. The cytotoxic activity was measured at an E:T ratio of 1:1. (B) Binding affinity to WT and 3R tetramers at concentrations of 1 to 100 nM. The MFI values of the T cell clones are shown. (C) Cytotoxic activity against 721.221-CD4-A2402 cells infected with NL-432gag^SF2 (WT virus) or NL-432gag^SF2-3R (3R virus). WT-virus-infected (49.1% of total cells were p24 Ag⁺) and 3R virus-infected (48.6% of total cells were p24 Ag⁺) cells were used as target cells. The cytotoxic activity was measured at E:T ratios of 0.5:1, 1:1, and 2:1. (D) Abilities of the clones to suppress the replication of WT or 3R viruses. The ability was tested at different E:T ratios. The error bars indicate standard deviations.

Fig 3

Antiviral activities of Gag28-specific CTL clones generated from PBMCs of patient KI-161, infected with WT virus. Gag28-specific CTL clones were generated from early-phase and chronic-phase PBMCs isolated from KI-161 after stimulating them with the WT and 3R peptides, respectively. Three types of Gag28-36-specific CTL clones, i.e., WT specific (left), cross-reactive (middle), and WT dominant (right), were generated from the early-phase PBMCs. (A) Cytotoxic activity against 721.221-CD4-A2402 cells prepulsed with the WT or 3R peptide at concentrations of 1 to 1,000 nM. The cytotoxic activity was measured at an E:T ratio of 1:1. (B) Binding affinity toward WT and 3R tetramers at concentrations of 1 to 100 nM. The MFIs of the T cell clones are shown. (C) Cytotoxic activity against 721.221-CD4-A2402 cells infected with WT virus or 3R virus. WT-virus-infected (49.0% of total cells were p24 Ag⁺) and 3R-virus-infected (50.0% of total cells were p24 Ag⁺) cells were used as target cells. The cytotoxic activity was measured at E:T ratios of 0.5:1, 1:1, and 2:1. (D) Abilities of the clones to suppress the replication of WT or 3R virus. The ability was tested at different E:T ratios. n, number of clones tested. The error bars indicate standard deviations.

Fig 4

Antiviral activities of cross-reactive and 3R-specific CTL clones generated from patients KI-091 and KI-163 infected with 3R virus. Gag28-specific CTL clones were generated from chronic-phase PBMCs isolated from patients KI-091 and KI-163 after their stimulation with 3R peptide. The following activities of these CTL clones were analyzed. (A) Cytotoxic activity against 721.221-CD4-A2402 cells prepulsed with the WT or 3R peptide at concentrations of 1 to 1,000 nM. The cytotoxic activity was measured at an E:T ratio of 1:1. (B) Binding affinity toward WT and 3R tetramers at concentrations of 1 to 100 nM. The MFIs of the T cell clones are shown. (C) Cytotoxic activity against 721.221-CD4-A2402 cells infected with WT virus or 3R virus. WT-virus-infected and 3R virus-infected cells were used as target cells. The frequency of p24 Ag⁺ cells among the HIV-1-infected cells was as follows: WT-virus-infected cells, 49.1% and 43.1% for CTL clones from KI-091 and KI-163, respectively, and 3R-virus-infected cells, 48.6% and 45.6% for CTL clones from KI-091 and KI-163, respectively. The cytotoxic activity was measured at E:T ratios of 0.5:1, 1:1, and 2:1. (D) Abilities of the clones to suppress the replication of WT or 3R virus. The abilities were tested at different E:T ratios. n, number of clones tested. The error bars indicate standard deviations.

Fig 5

Frequencies of the 3R mutation in a Japanese hemophiliac cohort and nonhemophiliac cohorts recruited from 1996 to 2002 and from 2008 to 2011. The frequencies of mutations at position 3 of the Gag28 epitope in chronically HIV-1-infected HLA-A*24:02⁺ or HLA-A*24:02⁻ hemophiliac individuals and nonhemophiliac individuals recruited from 1996 to 2002 or from 2008 to 2011 are shown. The consensus sequence of this epitope in HIV-1 subtype B is KYKLKHIVW. The frequency of the 3R mutation between HLA-A*24:02⁺ and HLA-A*24:02⁻ subjects in each cohort or that in HLA-A*24:02⁺ or HLA-A*24:02⁻ subjects among the 3 cohorts was statistically analyzed by using Fisher's exact test.

Fig 6

Replication kinetics of WT and 3R viruses in CD4⁺ T cells. CD4⁺ T cells (2 × 10⁵) isolated from PBMCs from a healthy donor were infected with WT or 3R virus in triplicate at a blue-cell-forming unit of 500 (in MAGIC-5 cells) in a total volume of 0.2 ml and then incubated at 37°C for 2 h. The infected cells were washed twice with R10 and then cultured in 1 ml of complete medium plus rIL-2 at 37°C. A 0.1-ml volume of the culture supernatants was collected at days 2 to 10 postinfection. The concentration of p24 Ag was measured by using ELISA.