The mechanisms by which elite suppressors (ES) control viral replication remain unclear. Although several studies [1,2] have shown that some may be infected with a defective virus, we recently demonstrated that some ES are infected with a fully replication-competent virus . This strongly suggests that host factors play a role in the control of viral replication. The HLA-B*57 and HLA-B*27 [4–7] alleles are over-represented among ES, which has been interpreted as evidence for a role of CD8+ T cells in the control of viremia. Support for this hypothesis came from multiple studies showing that HIV-1-specific CD8+ T cells from ES were qualitatively superior to those from patients with progressive disease [8–13].
However, a recent genome-wide screen has identified single nucleotide polymorphisms (SNP) in two genes that are associated with low viral loads in untreated patients . The protective SNP in one of the genes, HLA complex P5 (HCP5), is in strong linkage disequilibrium with the HLA-B*5701 allele . It is thus possible that the HCP5 polymorphism and not HLA-B*5701 plays a direct role in the control of viremia, and that the qualitatively superior HLA-B*57-restricted CD8+ T cell functions observed in ES are a result, rather than a cause, of the control of viral replication. On the other hand, the other protective polymorphism, associated with the HLA-C gene , may control expression levels of HLA-C proteins and thus enhance presentation of HIV-1-specific antigens. To determine the role of these polymorphisms in the control of HIV-1 replication in ES, we performed genotypic analysis on peripheral blood mononuclear cells from 19 patients.
The target SNPs were amplified from genomic DNA by polymerase chain reaction (PCR) reactions using locus-specific primers. The primers used for detecting the HCP SNP were HCP5-F1: TAC CCT CAT TGT GTG ACA GCA, and HCP5-R1: GTC GTG GGA TTT TGC ACT TC. The primers used for detecting the HLA-C associated SNP were HLA-C-F2: AGG GTG GTG CCA AGT ATG AG, and HLA-C-R2: CTT CTA GAG CCC CGT GGA G. The PCR conditions used included denaturation at 94 °C for 3 min, followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 68 °C for 30 s. Polymorphisms were detected by sequence analysis of gel-purified PCR products (Qiagen, Valencia, California, USA). All heterozygous SNPs were confirmed by cloning.
As shown in the Table 1, nine ES were positive for the HLA-B*5703 allele and one was HLA-B*5702-positive. Both alleles are closely related to HLA-B*5701, which was found to be in strong linkage disequilibrium with the protective HCP5 SNP. It is thus surprising that none of the HLA-B*5702/5703 ES were positive for the protective HCP5 allele. By contrast, the protective allele was present in an HLA-B*5701 patient with a low but detectable viral load and an HLA-B*5701 patient with progressive disease. One of 16 ES was homozygous and three others were heterozygous for the protective HLA-C-associated SNP. This allele has a relatively high frequency in Sub-Saharan Africans and thus would be expected to be relatively common in our African–American cohort of patients. A chi-square analysis showed that the observed frequency in our patients of African descent who are homozygous (0.07) and heterozygous (0.143) for the protective SNP is not significantly different from that expected based on the frequencies observed in a cohort of Sub-Saharan African patients (0.07 and 0.368, respectively) in the NCBI database (q = 0.48). This is not a perfect analysis because African–American patients may have significant genetic differences from Sub-Saharan Africans and, although these data need to be confirmed in a larger cohort of ES, the preliminary data suggest that the protective allele cannot explain the control of viremia in the majority of our patients.
It is interesting that 12 of the 16 patients controlled viral replication without having either of the two protective SNPs with the highest impact on viral load in untreated patients. In addition, three of four patients (ES4, ES8 and ES10) from whom we have isolated replication-competent virus in the past  were negative for both protective alleles. Thus, there are clearly some patients who are able to control pathogenic HIV-1 without HCP5 or the HLA-C associated polymorphisms.
It is unclear why none of our HLA-B*5703-positive ES expressed the protective HCP5 allele. The frequency of this allele in the NCBI database is very low in Sub-Saharan Africans and thus would also be expected to be also low in our cohort, which is predominantly comprised of patients of African descent. Of note, one African-American HLA-B*5701+ patient with progressive disease was positive for this HCP5 protective allele. Further analysis is warranted to determine whether the lack of the protective HCP5 allele in our cohort is due to the predominance of African–American patients or the lack of HLA-B*5701 patients. There may also be other genes in linkage disequilibrium with HLA-B*5703 that play a major role in the control of viral replication in these patients. It will thus be important to perform genome-wide analyses in cohorts of patients of African descent.
The clinical history of Progressor # 2 (P2) has features that argue both for and against a role for genetic factors other than HLA-B*57 in the control of viral replication. This HLA-B*5703 patient, who has neither the HCP5- or HLA-C-associated protective SNPs, completely controlled a pathogenic CXCR4-tropic HIV-1 isolate shortly after primary infection . This control may have occurred before an adaptive cellular immune response could have developed. Furthermore, the low frequency of latently infected cells observed shortly after seroconversion suggests that he never had high levels of viremia . This is consistent with a strong initial innate immune response. However, he experienced a virologic breakthrough 12 months after seroconversion. Full viral genome sequencing before and after viremia revealed only changes in two HLA-B*57-restricted CD8+ T cell epitopes, reversions of the M184V drug resistance mutation and a novel Vpu mutation. The M184V reversion was not accompanied by a major increase in viral fitness, so it is unclear how any of these mutations would affect an innate response. On the other hand, the temporal relationship between the development of the escape mutations and virologic breakthrough strongly suggests a role of the adaptive immune response in the control of viremia. Further studies will be needed to determine how much of a role other genetic factors play in the control of viral replication in HLA-B*5703-positive ES.
Funded by NIH Grants K08 AI51191-04 and R56 AI73185-01A1 and the Howard Hughes Medical Institute. We thank Dr Rafael Irizarry for helpful discussions.
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