Comprehensive immunological evaluation reveals surprisingly few differences between elite controller and progressor Mamu-B*17-positive simian immunodeficiency virus-infected rhesus macaques

Abstract

The association between particular major histocompatibility complex class I (MHC-I) alleles and control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication implies that certain CD8(+) T-lymphocyte (CD8-TL) responses are better able than others to control viral replication in vivo. However, possession of favorable alleles does not guarantee improved prognosis or viral control. In rhesus macaques, the MHC-I allele Mamu-B*17 is correlated with reduced viremia and is overrepresented in macaques that control SIVmac239, termed elite controllers (ECs). However, there is so far no mechanistic explanation for this phenomenon. Here we show that the chronic-phase Mamu-B*17-restricted repertoire is focused primarily against just five epitopes-VifHW8, EnvFW9, NefIW9, NefMW9, and env(ARF)cRW9-in both ECs and progressors. Interestingly, Mamu-B*17-restricted CD8-TL do not target epitopes in Gag. CD8-TL escape variation occurred in all targeted Mamu-B*17-restricted epitopes. However, recognition of escape variant peptides was commonly observed in both ECs and progressors. Wild-type sequences in the VifHW8 epitope tended to be conserved in ECs, but there was no evidence that this enhances viral control. In fact, no consistent differences were detected between ECs and progressors in any measured parameter. Our data suggest that the narrowly focused Mamu-B*17-restricted repertoire suppresses virus replication and drives viral evolution. It is, however, insufficient in the majority of individuals that express the "protective" Mamu-B*17 molecule. Most importantly, our data indicate that the important differences between Mamu-B*17-positive ECs and progressors are not readily discernible using standard assays to measure immune responses.

FIG. 1.

Mamu-B*17-positive animals exhibit a range of chronic virus loads. The mean chronic-phase (>10 weeks postinfection) virus loads of 185 SIV-infected Indian rhesus macaques were divided into Mamu-B*17-positive and -negative groups. Animals that express Mamu-B*08 were removed from analysis because they are highly disposed to control SIVmac239 replication (36). Animal r98016, which expresses both Mamu-B*17 and -B*08, is included here. The horizontal lines through each grouping represent the mean virus loads of that group. The horizontal line at 1 × 10³ vRNA cEq/ml plasma is the cutoff for EC status, as previously reported (36).

FIG. 2.

The Mamu-B*17-restricted response is focused primarily on four epitopes. Mamu-B*17-positive macaques were tested in the chronic phase of SIV infection for responses to 35 previously defined Mamu-B*17-restricted epitopes (47) using IFN-γ ELISPOT. WPI, weeks postinfection. The y axis represents the number of animals making a response (as defined below) to the given epitope. Only epitopes that showed at least one positive response in chronic infection are shown. All ELISPOTS were performed in duplicate. ELISPOT responses were measured as spot-forming cells (SFC) per million PBMCs. The mean number of spots in unstimulated (no peptide) wells was subtracted from each well. ELISPOT responses were considered positive if the number of spots (per million PBMCs) in replicate wells exceeded background plus two times the standard deviation and was >50. An asterisk indicates that data for the cRW9 epitope were previously published (41) and represent data from 15 progressors and 5 ECs.

FIG. 3.

Amino acid variation was observed in three Mamu-B*17-restricted epitopes. Most of the coding regions for Vif and Nef were sequenced at the time of euthanasia or late chronic SIV infection in 31 Mamu-B*17-positive and 31 Mamu-B*17-negative animals. Of 31 Mamu-B*17-positive animals, four were ECs. An asterisk indicates that viral sequences from ECs represent the recrudescent virus after experimental CD8 cell depletion in four Mamu-B*17-positive ECs (19). We could not obtain sequences from other ECs due to their extremely low viral loads. Double asterisks indicate that macaque r95003 (Mamu-B*17 negative) made an HW8-specific response, as well as a cRW9-specific response, and harbored SIV with escape mutations in both epitopes (41). Triple asterisks indicate that this variant confers escape from an overlapping Mamu-A*02-restricted epitope, Nef_159-167YY9 (58).

FIG. 4.

Mamu-B*17-positive animals recognize common escape variants of Mamu-B*17-restricted epitopes. IFN-γ ELISPOT responses to titrated peptides representing wild-type and common escape variants of the four primary Mamu-B*17-restricted epitopes, Vif HW8 (left panels), Nef MW9 (second from left), Nef IW9 (second from right), and Env FW9 (far-right panels). The autologous (aut) viral sequences (the epitope sequences at the time of ELISPOT testing) are in insets in each panel, aligned to wild-type (wt) SIVmac239. Five chronically infected animals were tested: progressors r02039 (a) and r90092 (b), former EC r96112 (c), and ECs r95071 (d) and r98016 (e). (f) Animal r95071 harbored SIV with an M-to-I variant of the MW9 epitope at position 1 after experimental in vivo CD8 cell depletion (experiment done in reference 19), but currently harbors an M-to-T variant. The M1I-specific response greatly expanded upon return of CD8⁺ cells, while wild-type-specific cells did not (g). All ELISPOTS (in panels a to e and g) were performed in duplicate. Responses were measured as spot-forming cells (SFC) per million PBMCs. The mean number of spots in unstimulated (no peptide) wells was subtracted from each well. ELISPOT responses were considered positive if the number of spots (per million PBMCs) in replicate wells exceeded the background plus two times the standard deviation and was >50. Error bars represent the mean ± standard error for each measurement. The horizontal line in panels a thru e is the cutoff for what is considered positive.