rized to group 2A had a progressive disease (Figure 4C). The differential disease outcomes associated with groups 2A and 2B indicated that genotypes which contained a HCP5-G allele associated with disease acceleration despite containing two separate ‘protective’ alleles namely a HLA-B*57 and HLA-C59-C allele (group 2A). Thus, the genotype-phenotype relationship for group 2A (which contains both HLA-B*57 and HCP5G alleles) may explain why when examined in a multivariate model, possession of the HCP5-G allele was associated with both disease acceleration and higher viral loads (Table 2, model 6). The phenotype of subjects in group 3 was also very instructive (Figure 4B). These subjects were homozygous for HLA-C59-C and possessed B*57 and had among the slowest rates of disease progression (Figure 4B). This occurred despite the fact that most subjects in group 3 also possessed the HCP5-G allele. Thus, among those who possessed B*57, the disease-accelerating effects associated with the HCP5-G allele was evident in those with one (group 2A) but not two (group 3) HLA-C59-C alleles (Figure 4B). These observations underscore our previous thesis [34,41] that analyses conducted at the allele level obscure complex genotype- phenotype relationships that occur when the phenotypic effects of both haplotypes are accounted for. These survival curves also underscore two other points: First, a HLA-B*57-containing genotype does not require the presence of a ‘protective’ HLA-C allele to convey a protective effect on disease progression. This is reflected by the observation that subjects in group 1 who have a B*57 allele and are homozygous for the HLA- C59-T allele have a slow disease course. Second, the maximal ‘protective’ effects associated with possession of a HLA-C59-C allele occurs only when it is present in a HLA-B*57-containing genotype. This is reflected by the observation that subjects assigned to B*57- containing genotypic groups 2B or 3 have a much slower disease course than those categorized to B*57-lacking genotypic groups 5 and 6 (Figure 4, B and C, compare survival curves for groups 2B and 3 vs those for groups 5 and 6). B*57-containing genotypes and progressive disease There is clinical, virologic and immunologic data indicating a conundrum: some subjects despite possessing a ‘protective’ B*57 have a progressive disease course [20,22,42]. We surmised that the genotype-phenotype relationships shown in Figure 4B and Figure 4C might address this paradox as they showed that subjects with HLA-B*57-containing genotypes classify into two categories. The first group are those subjects with genetic features present in genotypic groups 1, 2B or 3 as these genotypic groups are associated with a non-progressive disease (NPD) course (Figure 4, B and C), and collectively these genotypes are designated here as B*57-NPD genotypes in Figure 4D. In the overall cohort, B*57- NPD genotypes conferred a nearly 83% slower rate of disease progression compared to genotypes that lack a B*57 allele (RH = 0.17, 95% CI = 0.07–0.41, P = 7.261025; Figure 4D, left panel). These associations detected at the level of the entire cohort (Figure 4D, left panel) remained consistent after adjustment for covariates known to influence disease progression in this cohort, including ethnicity [39,43]. The second group are those subjects categorized to group 2A and, compared to those with B*57-NPD genotypes, the clinical course of subjects in this group is characterized by a progressive disease (PD; designated as B*57-PD genotype in Figure 4D). Remarkably, the disease course of those with a B*57-PD genotype is similar to those who lack a B*57 allele (Figure 4D). Consistent genotype-phenotype relationships were also evident when similar analyses were conducted separately in HIV+ EAs and AAs before and after adjusting for covariates (Figure 4D; middle and right HLA, HCP5 and ZNRD1 in HIV panels) or when the analyses were restricted to subjects who did not receive HAART (Figure S4). In these HAART-free subjects, those assigned to the B*57-NPD genotypic group had a nearly 80% lower risk of progressing to AIDS than those who lacked a B*57 allele (RH = 0.21; 95% CI = 0.08–0.57; P = 0.002). Based on the aforementioned findings we surmised that the comparison of the immunologic (CD4+ T cell profiles and DTH skin test reactivity) and virologic (viral load) profiles would reveal a step-wise worsening in subjects categorized to B*57-NPD, B*57- PD and those lacking a B*57 allele. In general agreement with this, as a general rule, there was a step-wise decrease in the baseline, cumulative and nadir CD4+ T cell counts as well as DTH responses in subjects assigned to B*57-NPD, B*57-PD and those lacking B*57 (Figure 4E). Conversely, in subjects categorized to B*57-NPD, B*57-PD and those lacking B*57 there was a step-wise increase in the steady-state viral load (Figure 4E). The only exception to this general rule was that the baseline CD4+ T cell counts in subjects assigned to B*57-NPD and B*57-PD were similar, but they were higher than the cell counts found in those who lacked a B*57 allele (Figure 4E). To validate that B*57-containing NPD and PD genotypes convey contrasting phenotypic effects during HIV disease course we used a complementary approach and determined the distributions of these genotypes as a function of increasing AIDS- free status and steady-state viral loads with the intent of testing two premises. First, although increasing AIDS-free status is associated with a stepwise enrichment of subjects with a B*57-containing genotype (Figure 3D), the greatest enrichment should be for those with B*57-NPD genotypes. Second, the B*57-NPD and not B*57- PD genotypes should be enriched for subjects with a low steady- state viral load. The data shown in Figure 4F (left panel) provides evidence in support for the first premise. Nearly 12 percent of those HIV-positive individuals who were AIDS-free after 10 years of follow-up possessed a B*57-containing genotype, and strikingly ,90% of these subjects were those with a B*57-NPD genotype (Figure 4F, left panel). The data shown in Figure 4F (right panel) provides evidence in support of the second premise as among subjects with a steady-state viral load lower than 10,000 copies/ml, there was an overrepresentation of those with B*57-NPD genotypes. It was noteworthy that the proportion of subjects with a B*57-PD genotype was similar in those who had a steady-state viral load of ,10,000 and $55,000 copies/ml (Figure 4F). Thus, these distribution patterns (Figure 4F) provide additional validity for the categorization of B*57-containing genotypes into B*57- NPD and B*57-PD genotypes as a means to improve the accuracy of identifying those B*57-carrying subjects who may have a slower disease course or control of viral replication during the early phases of infection. Viral load-independent effects of B*57-NPD genotypes Based on the well-established, strong relationship between steady-state viral load and AIDS development [28,29], one interpretation of the aforementioned findings might be that the B*57-NPD genotypes convey disease-retardation by simply influencing the extent of early viral replication. However, in a recent study, we found that although the steady-state viral load is a strong predictor of AIDS risk, it accounted for only ,12% of the variability in AIDS progression rates [39]. The findings by Rodriguez et al also suggest that the contribution of viral load to the explained variability in the rate of decline in CD4+ T cell counts might not be very large [44,45]. These data indicated that parameters that are independent of viral load are also critical determinants of AIDS pathogenesis. We therefore used nested multivariate Cox proportional hazards models (Table 4) to assess PLoS ONE | www.plosone.org 12 November 2008 | Volume 3 | Issue 11 | e3636