HIV Immune Escape at an Immunodominant Epitope in HLA-B*27–Positive Individuals Predicts Viral Load Outcome

Palanee Ammaranond, David J. van Bockel, Kathy Petoumenos, Marylin McMurchie, Robert Finlayson, Melanie G. Middleton, Miles P. Davenport, Vanessa Venturi, Kazuo Suzuki, Linda Gelgor, John M. Kaldor, David A. Cooper and Anthony D. Kelleher
J Immunol January 1, 2011, 186 (1) 479-488; DOI: https://doi.org/10.4049/jimmunol.0903227

Abstract

The CTL response in HLA-B*27+ HIV-infected individuals is characterized by an immunodominant response to a conserved epitope in gag p24 (aa 263–272, KRWIILGLNK; KK10). Mutations resulting in substitution of the arginine (R264) at position 2 of this epitope have been identified as escape mutations. Nineteen HLA-B*27+ long-term nonprogressors were identified from an Australian cohort with an average follow-up of 16 y following infection. Viral and host genetic factors impacting on disease progression were determined at multiple time points. Twelve of 19 had wild-type sequences at codon 264 at all time points; 7 of 19 carried CTL escape variants. Median viral load and CD4+ T cell counts were not significantly different between these groups at enrollment. Viral load, as judged by levels at their last visit (1,700 and 21,000 RNA copies/ml, respectively; p = 0.01) or by time-weighted area under the curve was higher in the escape group (p = 0.02). Escape mutants at other HLA-B*27–restricted epitopes were uncommon. Moreover, host polymorphisms, such as CCR5Δ32, CCR2-64I, and SDF1-3′A, or breadth of TCR repertoire responding to KK10 did not segregate to wild-type or escape groups. Host and viral factors were examined for a relationship to viral load. The only factor to affect viral load was the presence of the R264 escape mutations at the immunodominant epitope. CTL escape at R264 in the KK10 epitope is a major determinant of subsequent viral load in these HLA-B*27+ individuals.

The phenomenon of HIV mutational escape from CTL pressure is well documented. Mutations within epitopes and in flanking regions resulting in reduced binding to the restricting MHC class I allele, altered T cell recognition, and altered processing have been described within most proteins of HIV-1 (1–12). Furthermore, there is evidence for the imprinting of mutations within viral genomes as evolution occurs within individuals and within populations (13–17). There are accumulating data suggesting that CD8+ T cell responses are closely associated with escape mutations occurring during acute or early infection, which ultimately impact upon disease progression (14, 18–22).

In models of macaque SIV infection, there is evidence for a relationship between generation of escape mutants and disease progression or vaccine failure (23–27). However, even here the numbers are small and are confounded by the fact that some of the most rapid progressors were those that never mounted a detectable CTL response (26). Therefore the relationship between disease progression and generation of escape mutations is still unclear.

One of the best-described CTL responses to HIV-1 is the immunodominant HLA-B*27–restricted response to a conserved epitope in Gag p24 (aa 263–272, KRWIILGLNK [KK10]) (28). Robust CTL responses directed toward this epitope are detectable from early in primary infection and are maintained throughout disease. T cell responses are usually detectable, despite the frequent generation of a common L268M variant at position 6, until well-characterized escape mutations occur at codon 264 (5, 6, 29, 30). These mutations result in conversion of the arginine (R) residue at position 2 of the epitope, which is critical for binding the B pocket of HLA-B*27 to lysine, glycine, threonine, or glutamine (5, 31, 32). Each of these substitutions results in an epitope with decreased binding affinity to the HLA-B*27 molecule, resulting in poorly presented epitopes that are weak stimulators of the CTL response. These mutations usually take years to arise. This delay, despite concerted immune pressure, seems to be due to the critical nature of the arginine at 264 for the formation and stability of the multimeric p24 capsid (33–35). The presence of a mutation at codon 264 in isolation has a substantial impact on viral fitness. The virus is intolerant of this nonsynonymous mutation and it is only rarely detected as an isolated variant in clinical samples. Viral fitness is restored by the presence of concomitant compensatory mutations, predominantly involving S173A and L268M, which restore the capacity for capsid unfolding (6, 35, 36). Within individuals, mutations seem to occur at the time of disease progression or vaccine failure (37).

Because carriage of HLA-B*27 has been associated with long-term nonprogression (38–40), it has been speculated that this advantage is specifically related to the negative impact of the escape mutation at codon 264 on viral fitness and the requirement for fitness-restoring compensatory mutations. However, possible confounders that could impact on disease progression, such as the carriage of chemokine receptor polymorphisms, have not been systematically addressed.

We chose to study the effect of the generation of the KK10 escape mutation on surrogate markers of disease progression, while taking into account known viral and host determinants that are associated with slower progression. This study was performed in the context of a cohort of long-term nonprogressors (LTNPs) prospectively recruited and followed since 1994 (41). During follow-up, more than half of this cohort has progressed in terms of reduced CD4+ T cell counts or being started on antiretroviral therapy (ART). We screened the cohort for individuals who carried HLA-B*27 and then examined these individuals for the presence of CTL escape mutants at R264 in KK10. We subsequently determined whether the presence of escape mutations at this single immunodominant epitope contributed to long-term outcome. The presence of escape mutations at R264 in the immunodominant HLA-B*27–restricted KK10 epitope impacts upon viral load outcomes in these individuals.

Materials and Methods

Study subjects

The Australian Long-Term Non-Progressor cohort was established in 1994, recruiting and following 94 patients. After gaining written informed consent, patients with asymptomatic HIV disease who had been infected with HIV for ≥8 y and had a CD4+ T cell count ≥500 cells/μl in the absence of any ART were enrolled in this study. Parameters, such as CD4+ and CD8+ T lymphocyte counts, viral load, treatment history, and clinical indicators of disease progression as defined by the revised World Health Organization clinical staging of HIV infection (42), were recorded and entered into a database. PBMCs and plasma samples were cryopreserved at up to yearly intervals. For this study, data were censored at the last available visit or at the visit at which ART was commenced.

HLA genotyping and detection of CCR5, CCR2, and SDF1 polymorphisms

Genomic DNA was extracted from 5 × 106 PBMCs (Qiagen, Valencia, CA). HLA class I and II DNA typing was performed by standard PCR sequence-specific priming through the Australian Red Cross Blood Service, Sydney. The identification of the CCR5Δ32, CCR2-64I, and SDF1-3′A polymorphisms was performed with minor variations of the previously described methodologies (43, 44).

Viral extraction, PCR, and sequencing reactions

Viral RNA was extracted and reverse transcribed from plasma, as previously described (31), from 21 HLA-B*27+ patients at multiple visits during up to 10 y of follow-up. Full-length gag p24 gene sequences were amplified by nested PCR and cloned, as previously described (31). A minimum of four clones were sequenced at each time point. Sequences were manually edited and assembled with Sequencher (Gene Codes, Ann Harbor, MI).

The same methodology was used to amplify sequences from p17 and nef using the following primer sets: p17, outer primers: P1A (5′-AGTGGCGCCCGAACAGG-3′) and P1B (5′-GAAGTGACATAGCAGGAACT-3′), inner primers: P2A (5′-TCTCGACGCAGGACTCG-3′) and P2B (5′-GAGGAAGCTGCAGAATGG-3′); nef, outer primers: nefof (5′-AGAGTTAGGCAGGGATATTCACC-3′) and nefor (5′-CAGCTGCTTATATGCAGGATC-3′), inner primers: neff (5′-ACCTCAGGTACCTTTAAGACCAAT-3′) and nefr (5′-GTCCCCAGCGGAAAGTC-3′). A 720-bp fragment of gp41 was amplified using primers gp412 (5′-AAGCCCTGTCTTATTCTTCTAGGTA-3′), gp413 (5′-ATACCTAAAATACCTAAAGGATCAACAGCTC-3′) and gp411 (5′-TGCTCTGGAAAACTCATTTG-3′). PCR products were analyzed on a 1.5% agarose gel before bulk sequencing. Sequencing reactions were carried out using a BigDye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA).

Enumeration of CD8+ T cell responses by ELISPOT and MHC class I tetrameric complexes

IFN-γ ELISPOT and peptide-MHC (p-MHC) class I tetrameric complexes were used to enumerate responses to CD8+ T cell-specific epitopes on thawed PBMCs, as previously described (31, 45). Calculation of Ag-specific (tetramer+) CD8+ T cell population frequency and median fluorescence index (MFI) was performed using BD FACSDiva software (v 4.0; BD Biosciences, San Jose, CA).

Clonotypic analysis of TCR repertoire responding to KK10 epitope

PBMCs were stained with HLA-B*2705 tetrameric complexes carrying either of the two major variants (L or M268) of the KK10 epitope sequenced from plasma. HLA-A*0201 tetrameric complexes carrying the NV9 epitope (NLVPMVATV) from the pp65 matrix protein of CMV or HLA-B*2705 complexes carrying the RL9 epitope (RRIYDLIEL) from the EBN3AC nuclear Ag were used as comparator immunodominant populations. Cells were fixed with formaldehyde and sorted into tetramer+ populations, and RNA was extracted as previously described (45). Unbiased amplification of TCR β-chain first-strand cDNA template was achieved using a nonnested anchored template-switch RT-PCR with a singular 3′ TCR β-chain C region primer (5′-TTCTGATGGCTCAAACACAGCGAC-3′), as described previously (45, 46). Purified product was ligated into a TA-cloning vector (Invitrogen, Carlsbad, CA) and transformed into chemically competent Escherichia coli. A minimum of 23 clones were selected and vector product amplified using generic M13 primers and then sequenced using a BigDye v3.1 sequencing kit and an ABI 3730xl capillary sequencer (Applied Biosystems). Sequence data were aligned using Sequencher (Gene Codes), and clonotype identity was confirmed using the Immunogenetics online sequence analysis algorithm (http://imgt.cines.fr).

Statistical analysis

The Fisher exact test was used to compare categorical variables, and the Mann–Whitney U test was used to analyze continuous variables derived from the HLA-B*27+ individuals falling into wild-type (WT) and escape groups. Plasma viral load and CD4+ T cell covariates were measured as medians at censored time point (last available time point or start of treatment), as well as mean time-weighted area under the curve (AUC) for viral load (copies/ml) and for CD4+ T cell count. Time-weighted AUC was calculated as AUC (using the trapezoidal rule) divided by the patient’s total duration of follow-up (47). These tests were performed using SAS software (SAS Institute, Cary, NC).

Comparison of clonotype distribution was standardized for multiple samples using the Simpson diversity index (DS), which ranges in value from 0 (minimal diversity) to 1 (maximal diversity), accounting for the variety of TCR clonotypes and their clonal dominance (48). It is used to quantitatively assess differences in diversity between TCR repertoires. A randomization procedure was used to estimate the value of these indices using 23 clonotypic TCR sequences per time point to account for differences in the number of clonal sequences obtained per TCR repertoire. The calculation of diversity measures and the randomization technique were performed using Matlab (The Mathworks, Natick, MA).

Results

Demographic characteristics of HLA-B*27+ individuals

Twenty-two of the 94 patients enrolled in the LTNP cohort were HLA-B*2705+. On chart review, one subject was found to be on combined ART at cohort entry and was excluded from this study.

All 21 patients included were men, with a median age of 45 y (range: 29–64 y). Reported risk factors for HIV infection were men who have sex with men (47.6%) and were unspecified in the remainder. At study entry, median viral load was 4,000 copies/ml (range: 50–73,000 copies/ml), and median CD4+ T cell count was 651 cells/μl (range: 360–1190; Table I). All were infected with subtype B virus. Subject LT13 had a CD4+ T cell count <500 cells/μl at study entry; however, because he had not received any ART and had been diagnosed as HIV+ 12 y before inclusion in the study, he was included in this analysis. Two subjects were excluded from the analysis because they consistently had low-level plasma viral load (<200 copies/ml), and the gag p24 region could not be amplified from plasma or PBMC samples, despite repeated attempts with a range of primer pairs.

View this table:
Table I. Characteristics of HLA-B*27+ individuals at enrollment and end point visits

Characterization of immunodominant HLA-B*27–restricted epitope KK10

The gag p24 region was successfully sequenced from 19 individuals. At a minimum, each of these patients had clonal sequences derived from plasma collected at enrollment or the censoring time point (Fig. 1, Table I). Twelve of 19 carried R264 in all sequences at each sampled time point (median: 14.5 y; range, 8–19 y). Among these, four carried the WT sequence (KRWIILGLNK) and eight carried the common variant L268M sequence (KRWIIMGLNK).

FIGURE 1.
FIGURE 1.

Compiled individual time course for CD4 counts (A), plasma viral load (B), and plasma virus sequence variation (C) in KK10 HLA-B*27+ patients included in this study. The x-axis represents time from enrollment in the study (years of follow-up). Patient data for CD4+ T cell count and viral load (legend for A and B) are represented by viral sequence analysis; WT (solid line) and immune escape (dashed line). C, Viral immune escape is shown in parallel represented by common nonsynonymous mutations at codons 264 and 268.

Seven carried variant strains with mutations at aa 264, consistent with previously described CTL escape mutants: KKWIIMGLNK (n = 2), KKWIVIGLNK (n = 1) (5, 6, 29, 32), KGWIILGLNK (n = 3) (6, 49), or KQWIIMGLNK (n = 1) (31) (Table I). In five of seven (LT4, LT6, LT13, LT14, and LT19), the virus carried one of these mutations at the initial and last visits. One of seven (LT3) had WT virus at aa 264 at entry but developed an escape mutation (R264G) 2 y later, which then increased to fixation. One of seven (LT2) had a WT virus at the baseline visit; 9 mo later, a mixture of viral sequences coding for R264, R264K, and R264G was observed (Fig. 1).

Enumeration of immunological response to KK10 epitope variants

Staining of PBMC populations with HLA-B*27 tetrameric complexes bearing the appropriate cognate epitope KRWIILGLNK or KRWIIMGLNK revealed substantial proportions of CD8+ T cells specific for this immunodominant epitope at time points when WT or the L268M variant sequence was present. For patient groups maintaining p24 Gag WT or viral immune escape sequence, there was no clear difference in the frequency of KK10-specific CD8+ T cells (median: 4.1 versus 3.6%; range, 1.6–8.8%; Fig. 2).

FIGURE 2.
FIGURE 2.

Features of KK10-specific CD8+ T cells response at study entry as measured by frequency (%) and avidity (MFI) of cells staining with HLA-B*27 tetramer carrying cognate peptide in patients maintaining WT virus (LT1, LT5, LT8, LT17) and those developing an escape mutant (LT2, LT3) during the period of observation.

Similarly, substantial IFN-γ responses were measured by ELISPOT from PBMCs stimulated with WT or L268M variant of the epitope if measured at pre-escape time points. Responses were not seen to peptides representing escape variants KKWIIMGLNK, KGWIILGLNK, or KQWIIMGLNK at pre- or postescape time points. Following generation of these escape variants at KK10, responses to the subdominant p17 epitope IRLRPGGKK were detected in patients LT4 and LT14 subsequent to development of escape mutations at codon 264. Consistent with the published data, these responses were not detected in LT3 and LT9, who had not developed an escape mutation at codon 264 at the study entry time point examined (Supplemental Fig. 1) (32).

The effects of CTL escape at KK10 on surrogate markers of disease progression

We next investigated the effect of carrying an escape mutation at codon 264 of Gag on surrogate markers of disease progression by dividing the group into those that carried WT or escape mutations at codon 264. The time courses of plasma viral RNA levels, CD4+ T cell counts, and proportions of molecular clones carrying WT or variant sequence in individual patients are summarized in Fig. 1.

At study entry there was no difference in CD4+ T cell count (median cells/μl: WT = 703; escape = 651; p = 0.19) or viral load (median copies/ml: WT = 1850; escape = 8800; p = 0.06) between these two groups. These groups were compared at the last follow-up time point available or on the day that ART was commenced. The median follow-up to this time point from diagnosis was 14.5 and 13.0 y for the WT and escape groups, respectively. Median CD4+ T cell counts at the follow-up time point were 634 and 396 cells/μl in WT and escape groups, respectively (p = 0.09). However, viral load in the WT group was significantly lower than that of the escape group (median copies/ml: 1,700 and 21,000, respectively; p = 0.01; Table II). Time-weighted AUC for CD4+ T cell count and viral load variables were compared, to allow for correction of different lengths of follow-up between subjects. Viral load was significantly lower in those who maintained R264 in KK10 compared with those who did not (copies/ml: WT = 1,784; escape = 21,000; p = 0.02). Using this analysis, there was also a statistical difference in this measure of change in CD4+ T cell numbers between these two groups (cells/μl: WT = 745; escape = 518; p = 0.02; Table II).

View this table:
Table II. Distribution of virological and immunological parameters among WT and R264 escape mutation groups in HLA-B*27+ individuals

Finally, we considered the commencement of ART as another clinical marker of disease progression. Four of 7 in the escape group but only 1 of 12 in the WT group had commenced therapy during the period of observation (p = 0.04, Fisher exact test). This series of univariate analyses suggests that development of an escape mutation at KK10 results in higher levels of viral load and perhaps lower levels of CD4+ T cells. Further to this observation, we subsequently investigated whether there were other differences between these two groups, apart from the generation of the escape mutation at codon 264, which might explain the higher viral loads observed. Particular attention was paid to host and viral factors that are associated with altered disease progression.

Characterization of host chemokine receptor polymorphisms and TCR repertoire

The presence of CCR5Δ32, CCR2-64I, and SDF1-3′A polymorphisms was assessed in all 19 LTNPs (Table III). Thirteen (68.4%) were homozygous for WT CCR5. Six (31.6%) were heterozygous for the Δ32 mutation, and none was homozygous for this polymorphism. Fifteen were homozygous for WT CCR2 (78.9%), four were heterozygous (21.0%), and none was homozygous for the CCR2-64I polymorphism. Nine (47.4%) were homozygous for WT SDF1, nine (47.4%) were heterozygous, and one (5.3%) was homozygous for the SDF1-3′A mutation. None of the subjects who were homozygous or heterozygous for the CCR5Δ32 and CCR2-64I polymorphisms carried both. Three subjects were heterozygous for CCR5Δ32 and SDF1-3′A, whereas only one individual was heterozygous for CCR2-64I and SDF1-3′A. The carriage of these host genetic factors was not different between the groups carrying WT or escape viral sequence at codon 264 (Table II).

View this table:
Table III. Distribution of polymorphisms in chemokine coreceptors CCR5 and CCR2 and chemokine SDF1

TCR repertoire at study entry for cells responding to cognate variant epitope (KK10) in four patients who did not develop an escape mutation in KK10 (LT1, LT5, LT8, and LT17) was compared with two patients who eventually developed an escape mutation in KK10 (LT2 and LT3). Clonal expansion or diversity to the KK10 and control herpesvirus epitopes was determined using DS, which standardizes the analysis of T cell repertoire data between patients. No substantial differences were found between WT or escape patients responding to the KK10 or control epitopes with regard to the number of clonotypes (range: WT = 2–6; escape = 2–4) or repertoire diversity (DS range: WT = 0.00–0.76; escape = 0.32–0.50; Table IV).

View this table:
Table IV. Diversity of TCR repertoire at study entry for HIV (KK10) and control herpesvirus CMV and EBV epitope-specific CD8+ T cell populations

MFI of staining with p-MHC class I tetramers was analyzed as a surrogate marker of Ag-specific CD8+ T cell avidity for Ag. The calculated MFI of patients maintaining WT virus at codon 264 of KK10 was lower than those who developed escape (median MFI of KK10-specific CD8+ T cells: WT = 5,396; escape = 19,138; Fig. 2). However, this analysis was limited because only two of the escape group had WT sequence at entry to the study.

Viral variation at subdominant HLA-B*27–restricted epitopes

The HLA-B*2705–restricted subdominant epitope in Gag p17, aa 19–27, IRLRPGGKK, which may become dominant after escape at the p24 (aa 263–272) epitope (5, 32), was bulk sequenced at the last available time point. All patients carried the WT sequence at this epitope (Table V).

View this table:
Table V. Summary of sequences found at subdominant HLA-B*27–restricted epitopes in p17, gp41, and nef (derived from bulk sequence data)

In 18 of 19 patients, the HLA-B*27–restricted epitope in gp41 (aa 786–795), GRRGWEALKY, was successfully sequenced at the last assessed time point (Table V). Fifteen carried the WT sequence; 3 of 18 carried the possible escape mutations R787Q (n = 1) or R787H (n = 2). Ten carried mutations at position 792 (A792V/T/I/P/L). This is not an anchor residue for binding to HLA-B*27.

As previously documented, none of these individuals carried a nef-deleted virus (50). The region of nef containing the HLA-B*27–restricted epitope RRQDILDLWI (aa 105–114) was successfully sequenced from plasma virus of 16 patients. Two of 16 carried mutations at the anchor residue R106G (n = 1) and R106E (n = 1), which represent possible escape mutations. Four of 16 had mutations at position 108 (D108E), and 11 had amino acid substitutions of V to I at position 114 (Table V).

There was no difference between the WT and escape groups with regard to the carriage of mutations of the anchor residue, which represent candidate escape mutations, within any of these three subdominant HLA-B*2705–restricted epitopes (Table II). Variation was not noted in either group within the Gag p17 (aa 19–27) epitope. Two of those in the WT group and one in the escape group had a mutation at position 2 in the gp41 epitope (aa 786–795; p > 0.99). Two subjects in the WT group and none in the escape group had mutated sequences at position 2 of the nef epitope (aa 105–114; p = 0.51; Table II).

Distribution of possible confounding host and viral factors associated with viral load between WT and escape groups

Because polymorphisms within the genes coding for the SDF1 chemokine and CC chemokine receptors have been associated with delayed disease progression, we looked for confounding associations between carriage of these mutations and viral load. No difference was observed in the median viral load when patients were divided by carriage of CCR2-64I or CCR5Δ32 polymorphisms (p = 0.22 and p = 0.19, respectively; Table VI). It was not possible to statistically interpret differences in viral load for the SDF1-3′A polymorphism, because only 1 of the 19 patients studied was homozygous for this mutation. Further, there was no impact on viral load when patients were divided by carriage of viral immune escape variants in subdominant HLA-B*27–restricted epitopes in gp41 (p = 0.95) or Nef (p = 0.87; Table VI). Moreover, when determining confounding immunological parameters, there was no segregation of TCR diversity in those who had diversity of TCR repertoire determined with viral load (Table IV).

View this table:
Table VI. The effect of potential confounders on viral load in HLA-B*27+ individuals

HLA class I and II genotyping was assessed to determine whether other HLA alleles might influence disease progression following immune escape (Table VII) (51). HLA-A and -B alleles were determined for all (n = 19) patients, and HLA-DRB1 alleles were determined for 17 patients. HLA types were examined for featured haplotypes, homozygosity, and alleles previously associated with control (52, 53), in the absence of immunodominant influence (54) or with escape (38, 55, 56). Equal distribution of HLA-B*27 homozygosity observed in WT and escape groups (n = 2 in both), combined with a lack of a haplotype or another HLA-allele associated with the escape group, suggests that other HLA-A, -B, and -DR alleles had no considerable impact on viral load, although the sample size precluded any formal analyses. Together, these analyses are consistent with there being no significant impact of these cofactors on viral load variation in our cohort. Taken together, the univariate analyses performed suggest that the presence of an escape mutation at codon 264 in p24 was the only variable measure that was associated with higher viral load outcome (p = 0.01).

View this table:
Table VII. Summary table of HLA class I and II DNA typing determined from genomic DNA

Discussion

Although escape mutations have been repeatedly demonstrated in HIV-infected individuals, the association of escape variants and disease progression is variable, depending upon the escape mutation and the restricting HLA element (14, 18). In particular, the association of mutations at R264 inducing reduced affinity to HLA-B*27 and progression has not been definitively demonstrated (5, 6, 57, 58). To address this question, we chose a candidate epitope (KK10) that is known to be uniformly immunodominant (28, 30, 59) and that is restricted by an HLA allele associated with reduced rates of disease progression (38–40). In this study, we considered the effect of a range of other viral and host factors that might impact on disease progression, in addition to considering the effect of viral sequence variation resulting in known escape mutations within the conserved immunodominant KK10 epitope in Gag p24. These factors included: 1) host genetic factors described as impacting upon disease progression: polymorphisms in chemokine coreceptors (CCR5, CCR2) and the natural ligand of CXCR4 (SDF1); 2) diversity of TCR repertoire responding to the KK10 epitope; and 3) sequence variation at the known subdominant HLA-B*27–restricted epitopes in gag, env, and nef genes (6, 28, 30, 57, 60, 61), as well as the presence of deletions within the nef gene (62).

We found that, even among this group selected for their slow disease progression, those with escape mutations at codon 264 in KK10 developed higher viral loads relative to those that maintained WT virus. This was true whether the measure of viral load was taken at the last available time point or was measured by time-weighted AUC analysis, which compensates for the different periods of follow-up among individuals. The magnitude of the difference in viral load of 1.1 log10 copies/ml is relatively large compared with that attributed to escape mutations occurring in chronic infection; a recent analysis suggested that the presence of a single escape mutation is associated with a <10% increase in viral load (63). The size of this effect is likely to reflect the importance of this immunodominant CTL response to viral control in these individuals prior to escape and the extent of viral fitness postescape when compensatory mutations are present (35, 64). This finding suggests that the presence of escape mutations at an immunodominant epitope is associated with a higher viral load. In contrast, CD4+ T lymphocyte numbers were not consistently statistically different between those carrying WT and escape polymorphisms at codon 264 of KK10. This may be due to treatment guidelines at the time of commencing ART in the majority of these patients (late 1990s, early 2000s). Those guidelines, based on the contemporaneous Department of Health and Human Services guidelines, encouraged early administration of therapy, prior to significant immune depletion and at relatively high CD4+ T cell counts (65). This would prevent the CD4+ T cell count depletion that usually follows increased viral loads. Therefore, the failure to consistently demonstrate a deleterious effect of escape on CD4+ T cell count may reflect treatment of high or increasing viral load prior to any substantial decrease in CD4+ T cell count. This argument is strengthened by the observation that those with escape mutations were more likely to begin ART than were those who maintained WT sequence at codon 264 of gag.

The distribution of other possible determinants of plasma viral load was not different between those developing escape mutations and those maintaining WT sequences at codon 264. This indicated that generation of escape mutations was not influenced by these factors. Further, if the effect of these other possible confounding factors on viral load were considered separately, there was no significant influence on viral load outcome within this group. The data presented in this article are consistent with previously published data, in that the CD8+ T cell responses to KK10 prior to escape were robust (5, 57, 66, 67). Further, as expected, the generation of KK10 escape mutants with low affinity for HLA-B*27 was associated with loss of this immune response, providing circumstantial evidence that loss of this immunodominant response results in reduced control of the virus replication.

The data are also consistent with the published literature in that in the majority of individuals who carry HLA-B*27, these escape mutations take many years to arise. This is likely due to the need for the accumulation of compensatory mutations that overcome the negative effects on viral fitness induced by escape mutations at codon 264 in KK10 if present alone (35, 64). However, the fact that escape seems to arise while individuals have relatively intact immune systems seems to be inconsistent with the observation from Gao et al. (68), in that the major benefit of carriage of HLA-B*27 occurs late in the disease process in those with <200 CD4+ T cells/μl. These late protective effects can likely be attributed to the long-term maintenance of WT KK10 sequence in a subset of HLA-B*27+ individuals (35). Intervention with ART limits our ability to explore this hypothesis in the context of this data set.

Interestingly, there was no statistical difference between the WT and the escape groups with regard to the presence of escape mutations at HLA-B*27–restricted epitopes in Gag p17, Nef, and gp41, indicating that there is no rapid accumulation of escape mutants at subdominant epitopes following the generation of escape in KK10. Where present, variations in these subdominant epitopes did not show any statistically significant impact on CD4+ T cell counts or viral load.

Unlike a previous study in vaccinated macaques challenged with SIV (20), we were unable to demonstrate, using a standardized quantitative measurement, a relationship between the breadth of TCR repertoire and the propensity for control of virus during chronic infection. This may be an artifact of sample size, because estimations from this data set make experiments to formally test this hypothesis unfeasibly large. Although this study attempted to address the impact on a range of host factors, as well as viral escape on viral control, it does not address the impact of qualitative differences in the T cell response to the KK10 epitope. Clonal turnover and senescence may impact outcome, as identified by a previous study (66).

To the best of our knowledge, this is the first study of an HLA-B*27 population to show an exclusive association between the presence of escape mutations and increases in plasma viral load. Although escape mutations in the HLA-B*27 population were reported in other studies, ours is the largest study of HLA-B*27+ individuals to date. Although our sample size is small, it is more than double the number of HLA-B*27+ subjects included in any other published work (5, 6, 29, 49). An additional caveat is the possibility that the escape mutations present at study entry had been present since transmission (32, 37, 69) or at least arose very early in the disease course (6, 30), and the observed loss of viral control is related to unmeasured parameters. The frequency of these escape mutations within databases and the literature suggest that this is an unlikely scenario but is one that cannot be completely excluded. Significant challenges remain for the identification of correlates of protection in nonprogressors. In conclusion, we found that the plasma RNA level was significantly higher following the development of escape compared with those that maintain WT virus (p = 0.01). These results suggest that escape mutation in HLA-B*27+ individuals is a major determinate of disease control, as determined by plasma concentrations of HIV-1 RNA.

Acknowledgments

We thank Drs. Ben Anderson (St Leonard's Medical Centre, St Leonard’s, New South Wales, Australia), Nicholas C. Doong (Burwood Road Medical Centre, Burwood, New South Wales, Australia), Mark Kelly (AIDS Medical Centre, Brisbane, Queensland, Australia), Ross Price (Taylor Square Private Clinic), and all patients who contributed to this study.

Disclosures The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by the Australian Government Department of Health and Ageing, the National Health and Medical Research Council, and the Australian Research Council.

  • The views expressed in this publication do not necessarily represent the position of the Australian Government. The National Centre in HIV Epidemiology and Clinical Research is affiliated with the Faculty of Medicine, University of New South Wales.

  • The online version of this article contains supplemental material.

  • Abbreviations used in this paper:

    ART
    antiretroviral therapy
    AUC
    area under the curve
    DS
    Simpson diversity index
    KK10
    KRWIILGLNK
    MFI
    median fluorescence index
    N/A
    not able to amplify
    ND
    not done
    NS
    not able to be sequenced using the traditional sequencing
    PID
    patient identifier
    p-MHC
    peptide-MHC
    WT
    wild-type.

  • Received October 5, 2009.
  • Accepted October 13, 2010.

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