Viral suppression of multiple escape mutants by de novo CD8(+) T cell responses in a human immunodeficiency virus-1 infected elite suppressor

Abstract

Elite suppressors or controllers (ES) are HIV-1 infected patients who maintain undetectable viral loads without treatment. While HLA-B*57-positive ES are usually infected with virus that is unmutated at CTL epitopes, a single, dominant variant containing CTL escape mutations is typically seen in plasma during chronic infection. We describe an ES who developed seven distinct and rare escape variants at an HLA-B*57-restricted Gag epitope over a five year period. Interestingly, he developed proliferative, de novo CTL responses that suppressed replication of each of these variants. These responses, in combination with low viral fitness of each variant, may contribute to sustained elite control in this ES.

Figure 1

Proviral and plasma virus variants from ES8 spanning five years. Clones were amplified using nested, limiting dilution PCR and RT-PCR. Sequence logos were created utilizing the WebLogo program from University of California at Berkeley http://weblogo.berkeley.edu/. For clones amplified at each time point, Logos show the proportion of an amino acid residue and substitutions at a given site. The date the sequence was obtained, the source of the sequence, and the number of clones for each time point are shown, as well as the sequence of the HLA-B*57-restricted IW9 and TW10 epitopes.

Figure 2

Mutations present in the TW10 epitope of plasma viral variants dramatically impact viral fitness. (A) Schematic for the development of NL43nGFP based viruses to test the impact of plasma TW10 variants on viral fitness. Gag from ES8-1a virus which contains the TSTLAEQVAW TW10 mutant was mutated using site-directed mutagenesis to produce gag with the six other variants present in the plasma, as well as wild type TW10 and T242N on this ES8-1a backbone. Site-directed mutagenesis was also utilized to insert T242N into the NL43 gag, and gag from ES8-17 was amplified. Each of these gag mutants were then inserted into NL43eGFP backbone. All viruses derived from ES8-1a, the viral variant representing plasma viral variants, also had the I147M mutation, as indicated. (B) Fitness of viral variants was measured on day 6 post infection. Data shown are infection of two uninfected donors infected with two separate virus preparations in duplicate. Percent infection was normalized to transfection efficiency, and to the percent infection of WT NL43 which was on average 42%. (C) Replication of viral variants ES8-17 and ES8-1a. ES8-17 is wild type at the TW10 epitope and was cultured from resting CD4⁺ T cells from ES8. ES8-1a has mutation at TW10 and was cultured from activated CD4⁺ T cells. Results are the average of triplicate samples of p24 supernatant of activated CD4⁺ T cells infected with each virus. P-values shown above each time point compare the p24 of ES8-17 and ES8-1a using a one-tailed Student's T-test.

Figure 3

CD8+ T cells from ES8 mount a proliferative response to autologous TW10 variants. (A) CD8⁺ T cells from ES8 mount a strong response to wild type TW10 and autologous variants, but not to TW10 with the T242N mutation. IFN-γ ELISPOT measuring the response of CD8+ T cells from ES8 to wild type TW10, autologous variants, and the T242N mutant. Data is from 2 hours (left), 4 hours (center), and 6 hours (right) of stimulation with the peptides. (B) IFN-γ ⁺ CD8⁺ lymphocytes that have been stimulated with wild type TW10 and autologous variants are undergoing proliferation. Representative dot plot and histogram shown for stimulation with one of the autologous mutants, TSTLTEQVAW (top). Gating is on the population of CD8⁺ T cells that are IFN-γ ⁺ (bottom). (C) HLA-B*57 tetramer⁺ cells are producing IFN-γ only in response to wild type TW10 peptide, but not to autologous variants. Representative dot plot and histogram shown for stimulation with one of the autologous mutants, TSTLTEQVAW (top left) and wild type TW10 (top right). Gating is on CD8⁺ T cells (bottom). Total of 500,000 events were collected for panels B and C, and the peptide with which cells were stimulated indicated in the legend.

Figure 4

Unstimulated CD8⁺ T cells from ES8 block infection by autologous and wild type virus (described in Figure 2a) as effectively as cells stimulated with autologous or wild type TW10 peptides. CD4⁺ T cells from the patient were infected via spinoculation with wild type NL43 and NL43-based viruses expressing autologous TW10 epitopes, as described in Figure 2a. Cells were then co-cultured with CD8⁺ T cells from ES8 which were either unstimulated or had been stimulated with the TVA, TIA, or AVA peptides for four days. Co-culture with infected cells was for seven days. Results shown are averages of triplicate experiments with error bars indicating standard deviation.