Abstract
Particular HLA alleles are associated with reduced human immunodeficiency virus replication. It has been difficult, however, to characterize the immune correlates of viral control. An analysis of the influence of major histocompatibility complex class I alleles on viral control in 181 simian immunodeficiency virus SIVmac239-infected rhesus macaques revealed that Mamu-B*17 was associated with a 26-fold reduction in plasma virus concentrations (P < 0.001). Mamu-B*17 was also enriched 1,000-fold in a group of animals that controlled viral replication. Even after accounting for this group, Mamu-B*17 was associated with an eightfold reduction in plasma virus concentrations (P < 0.001). Mamu-B*17-positive macaques could, therefore, facilitate our understanding of the correlates of viral control.
Major histocompatibility complex (MHC) class I alleles have been associated with the control of human immunodeficiency virus (HIV) replication (1, 2, 3, 5, 13). The protective benefit of the MHC class I alleles HLA-B*27 and HLA-B*57 is well established, and cellular immune responses restricted by these alleles have been characterized (7, 13). Unfortunately, correlating particular responses with the control of viral replication has been difficult. The dual obstacles of HIV variability and allelic diversity of the human MHC class I loci confound interpretation of immune correlates in the rare patients who control the virus. An animal model for understanding the basis of this HLA class I-associated control of viral replication would facilitate an investigation of the underlying immune correlates of control.
The MHC-defined Indian rhesus macaque infected with simian immunodeficiency virus (SIV) provides a tractable model system to discern the influence of MHC class I alleles on the replication of a pathogenic AIDS virus. Previous studies have reported associations between rhesus macaque MHC class I alleles and control of virus replication (9, 10, 11, 12). Here we investigate the effect of high-frequency MHC class I alleles in the Indian rhesus macaque on the control of viral replication after challenge with a clonal virus isolate.
High-frequency MHC class I allele associations with chronic-phase viral control.
To investigate whether particular high-frequency MHC class I alleles are linked to the control of pathogenic SIVmac239 replication, we measured postchallenge plasma virus concentrations in 181 Indian rhesus macaques from two independent cohorts. Multiple chronic-phase plasma virus time points were measured for each animal, from 10 weeks postchallenge through the time of sacrifice (range, 10 to 320 weeks postchallenge). All animals were infected with the clonal isolate SIVmac239.
Remarkably, 13 animals maintained chronic-phase plasma virus concentrations that were 1,000-fold lower than those of the cohort geometric mean. This group was significantly enriched for four high-frequency MHC class I alleles: the closely linked Mamu-B*17 and Mamu-B*29 (P = 0.003), Mamu-A*02 (P = 0.03), and Mamu-A*11 (P = 0.03) (Table 1). Conversely, one allele, Mamu-B*01, was significantly underrepresented in this group. Although Mamu-B*01 was present in 60 of the 181 animals in the infected cohort, it was not present in any of the 13 “elite controllers” (P = 0.02) (Table 1). While 85% of the 181 animals had chronic-phase plasma virus concentrations within 2 log units of the cohort chronic-phase geometric mean of 457,000 viral RNA (vRNA) copies/ml, this group of elite controllers maintained plasma virus concentrations that were more than 3 log units lower (362 vRNA copies/ml) than the cohort chronic-phase geometric mean. In fact, the 13 elite controllers have maintained these plasma virus concentrations from week 10 postchallenge to the present (52 to 310 weeks postinfection). Interestingly, the percentage (7%) of macaques that controlled SIVmac239 replication to the lowest levels in this model system is very similar to the percentage (8%) of HIV-infected humans found to suppress their virus to comparable levels (6).
TABLE 1.
Particular high-frequency MHC class I alleles are significantly overrepresented among elite controllersa
| Animal or parameter | Presence of MHC class I allele or value for parameter
|
||||||
|---|---|---|---|---|---|---|---|
| A*01 | A*02 | A*08 | A*11 | B*01 | B*17 | B*29 | |
| aj11 | − | + | − | + | − | + | + |
| 00078 | − | − | + | − | − | − | + |
| 01064 | − | + | − | − | − | − | − |
| 95061b | + | + | − | − | − | + | + |
| 95071 | − | + | − | − | − | + | + |
| 95096c | + | − | − | + | − | + | + |
| 96112d | − | − | + | − | − | + | + |
| 98014e | − | + | − | + | − | + | + |
| 98016 | − | + | − | − | − | + | + |
| 98057 | − | − | − | + | − | − | − |
| 99C051f | − | + | − | − | − | + | + |
| 99x032 | − | + | + | − | − | − | − |
| 96C114g | + | − | − | − | − | + | + |
| Frequency in elite controllers (%) (n = 13) | 23 | 62h | 23 | 31h | 0 | 69i | 77i |
| Frequency in whole cohort (%) (n = 181) | 39 | 28 | 28 | 8 | 33 | 19 | 19 |
Animals that maintained chronic phase plasma virus concentrations below 1,000 vRNA copies/ml are shown. We genotyped all animals by sequence-specific PCR for seven high-frequency MHC class I alleles (4) (W. M. Rehraurer et al., unpublished data) and infected all of them with cloned SIVmac239. Significant enrichments of the elite controller group for a particular genotype are indicated in bold. Enrichment of this elite controller group for MHC class I alleles was determined with logistic regression in R 2.2.0 (R Foundation for Statistical Computing, Vienna, Austria). Animals were vaccine naive, except where indicated. We performed independent statistical analyses for vaccinated and nonvaccinated animals for all alleles and found no significant differences between plasma virus concentrations of vaccinated and nonvaccinated animals of each genotype (data not shown). +, presence of indicated allele; −, absence of indicated allele.
DNA/MVA GagCM9 epitope only.
Lipopeptide GagCM9 epitope only.
CpG in incomplete Freund’s adjuvant.
Intramuscular injections of 107 irradiated peripheral blood mononuclear cells from animal 2055.
SIVgagDNA5mgs/FL-ad5SIVgag.
Microvesicles and inactivated SIV protein.
P = 0.03.
P = 0.003.
We performed independent statistical analyses for vaccinated and nonvaccinated animals for all alleles and found no significant differences between plasma virus concentrations of vaccinated and nonvaccinated animals of every genotype (data not shown). While 97/181 (54%) of the overall cohort received an experimental vaccine, 6/13 (46%) of the elite controllers received an experimental vaccine.
The most dramatic enrichment of the elite controller group was for animals positive for both Mamu-B*17 (Mamu-B*17+) and Mamu-B*29 (Mamu-B*29+). While 19% of the overall cohort was Mamu-B*17+/Mamu-B*29+, 69% of the elite controllers were positive for both of these alleles (P = 0.003). While Mamu-B*17 binds at least 16 SIVmac239-derived peptides and thus restricts various SIV-specific cellular immune responses (8), there is no evidence to date that the closely linked (linkage R square value, 0.87) Mamu-B*29 allele encodes a gene product that binds any SIVmac239-derived peptides.
Mamu-B*17+/Mamu-B*29+ animals exhibited a 26-fold decrease in geometric mean chronic-phase plasma virus concentrations, compared to the rest of the cohort (P < 0.001). The Mamu-B*17+/Mamu-B*29+ animals consisted of two discrete subgroups: the nine Mamu-B*17+/Mamu-B*29+ animals that controlled their chronic-phase plasma virus concentrations to below 1,000 vRNA copies/ml formed a separate distribution from the 25 Mamu-B*17+/Mamu-B*29+ animals with more typical chronic-phase plasma virus concentrations. We therefore analyzed the Mamu-B*17+/Mamu-B*29+ animals, taking into account the influence of the Mamu-B*17+/Mamu-B*29+ elite controller group (Fig. 1). When we accounted for these elite controller Mamu-B*17+/Mamu-B*29+ animals, we discovered that these two alleles were still associated with an eightfold reduction in geometric mean chronic-phase plasma virus concentrations (P < 0.001).
FIG. 1.
Contribution of high-frequency MHC class I alleles to variation in chronic-phase plasma virus concentrations in 181 Indian rhesus macaques. Changes in geometric mean (dots) chronic-phase (week 10 to present) plasma virus concentrations relative to the whole cohort (represented by the red line) are shown with 95% confidence intervals (bars). Purple, P value of <0.001; green, P value of <0.05 and >0.001. Determinations of allelic effects on chronic-phase plasma virus concentration are given with mixed effects analyses, taking into account elite controller status, MHC class I genotype, and vaccination. All statistics were performed in R 2.2.0 with the lme4 package (R Foundation for Statistical Computing, Vienna, Austria). A total of 128 animals were infected at the WNPRC, and 53 animals were infected at the National Cancer Institute.
Previous reports have indicated an association between the expression of Mamu-A*01 and a reduction in chronic-phase plasma virus concentrations in animals infected with SIVmac239 (9) and SIVmac251 (10, 12) and lower lymph node-associated virus concentrations in SHIV89.6P infection (14). Mamu-A*01 was associated with only slightly decreased chronic-phase plasma virus concentrations in an expanded SIVmac239-infected cohort (11). Decreased chronic-phase plasma virus concentrations, however, were not observed in Mamu-A*01+ cohorts infected with SIVsmE660, SHIV89.6P, or SHIVKU2 (12). In our further expanded cohorts, this allele was significantly associated with a threefold reduction in geometric mean plasma virus concentrations after we took into account vaccination and elite controller status (P = 0.006) (Fig. 1).
It is possible that genes closely linked to Mamu-B*17 and Mamu-A*01 might influence the ability of the virus to replicate in vivo. There were, however, no significant differences in acute-phase peak plasma virus concentrations in animals of all genotypes before the onset of cellular immune responses (Fig. 2). This suggests that the accentuated decline from acute-phase peak to chronic-phase plasma virus concentrations in Mamu-B*17+ and Mamu-A*01+ animals was not due to a differential intrinsic susceptibility of their CD4+ T cells to infection with pathogenic SIVmac239 or to the enhanced effect of some non-MHC class I host restriction factor.
FIG. 2.
The presence of particular high-frequency MHC class I alleles does not affect acute-phase peak plasma virus concentrations. Changes in geometric mean acute-phase peak (single highest value from weeks 1 to 3 for each animal) plasma virus concentrations relative to the whole cohort (represented by the red line) with 95% confidence intervals were estimated using linear regression. No significant associations between acute-phase peak plasma virus concentrations and MHC class I genotype were detected.
These Mamu-B*17+ animals promise to provide useful and clear-cut models for the understanding of the influence of particular MHC class I alleles on viral control. Because a comprehensive SIVmac239 epitope mapping study has been performed for Mamu-B*17 (8), Mamu-B*17+ animals provide a unique opportunity to analyze particular CD8+ T-cell specificities linked to successful control of viral replication. For example, we can ablate the SIV-specific CD8+ T-cell response in these Mamu-B*17+ elite controllers with depleting antibodies. Additionally, since SIVmac239 has proven recalcitrant to effective neutralization by antibody responses, we can examine the effect of particular MHC class I-restricted cellular immune responses without the potentially confounding effects of neutralizing antibody.
A substantial proportion of captive Indian rhesus macaques express Mamu B*17. Of the Indian rhesus macaques at the Wisconsin National Primate Research Center (WNPRC), approximately 16% are Mamu-B*17+. Given the influence of this allele on the outcome of SIV infection, Mamu-B*17+ animals should be removed from vaccine cohorts to avoid the attribution of Mamu-B*17-associated control of viral replication to possible effects of vaccination. Simply balancing control and vaccine groups with these macaques is insufficient, because spontaneous control occurs in a sizeable subset of Mamu-B*17+ animals. Until the precise determinants of this control are defined, the use of Mamu-B*17+ animals should be avoided except for analysis of the causes underlying their exceptional viral control.
Acknowledgments
We gratefully acknowledge Eva Rakasz, Kirsten Bomblies, and Matt Reynolds for helpful discussions, Jess Maxwell, Crystal Glidden, and Tim Jacoby for MHC typing, and Ron Desrosiers for SIVmac239 inocula.
This work was supported by NIH grants R01 AI052056-03 and R24 RR016038-05; NIH contract HHSN26620040008C/N 01-AI-40088; the WNPRC base grant 5P51 RR 000167; federal funds from the National Cancer Institute, National Institutes of Health, under contract no. NO1-CO-12400; and by the Intramural Research Program of the NIH, National NO1-CO-12400, Cancer Institute, Center for Cancer Research.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the U.S. Government.
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