Human Leukocyte Antigen B58 Supertype and Human Immunodeficiency Virus Type 1 Infection in Native Africans
http://www.100md.com
《病菌学杂志》
Departments of Epidemiology Medicine Microbiology, University of Alabama at Birmingham, Birmingham, Alabama
National Blood Transfusion Service, Lusaka, Zambia
Project San Francisco, Kigali, Rwanda
Department of Global Health, Emory University, Atlanta, Georgia
ABSTRACT
Human leukocyte antigen (HLA) class I alleles can be grouped into supertypes according to their shared peptide binding properties. We examined alleles of the HLA-B58 supertype (B58s) in treatment-nave human immunodeficiency virus type 1 (HIV-1)-seropositive Africans (423 Zambians and 202 Rwandans). HLA-B and HLA-C alleles were resolved to four digits by a combination of molecular methods, and their respective associations with outcomes of HIV-1 infection were analyzed by statistical procedures appropriate for continuous or categorical data. The effects of the individual alleles on natural HIV-1 infection were heterogeneous. In HIV-1 subtype C-infected Zambians, the mean viral load (VL) was lower among B5703 (P = 0.01) or B5703-Cw18 (P < 0.001) haplotype carriers and higher among B5802 (P = 0.02) or B5802-Cw0602 (P = 0.03) carriers. The B5801-Cw03 haplotype showed an association with low VL (P = 0.05), whereas B5801 as a whole did not. Rwandans with HIV-1 subtype A infection showed associations of B5703 and B5802 with slow (P = 0.06) and rapid (P = 0.003) disease progression, respectively. In neither population were B1516-B1517 alleles associated with more favorable responses. Overall, B58s alleles, individually or as part of an HLA-B-HLA-C haplotype, appeared to have a distinctive impact on HIV-1 infection among native Africans. As presently defined, B58s alleles cannot be considered uniformly protective against HIV/AIDS in every population.
INTRODUCTION
The concept of human leukocyte antigen (HLA) supertypes, categories of alleles that share common peptide-binding motifs, offers a simplification of the complex HLA nomenclature by consolidating the huge spectrum of individual alleles into relatively few groups. Individual alleles assigned to each supertype have either proven or predicted ability to present antigenic peptides with similar anchoring residues at the second and C-terminal positions of peptide ligands. Initially proposed in the late 1990s, four HLA-A and five HLA-B supertype categories encompass the majority of known HLA class I alleles (24, 25). There have been subsequent efforts to improve supertype categorization (8, 22). However, independent of the supertype assignment method, the HLA epitopes for just a few of the major supertypes have been estimated to provide substantial population coverage for antigenic peptides. If conserved peptides that cross-react with representative HLA class I supertypes can be identified, a supertype-guided approach could simplify the development of human immunodeficiency virus type 1 (HIV-1) subunit vaccines. Furthermore, designing a multiepitope HIV-1 vaccine candidate to target a few supertypes would be considerably easier than tailoring one to highly population-specific allelic differences. However, the success of such a strategy would depend on numerous factors (13, 15), including the degree of uniformity in the nature and magnitude of the CD8+ cytotoxic T-lymphocyte (CTL) responses to viral peptides.
The associations of CTL responses and control of HIV-1 viremia appear to differ from one HLA class I supertype to another (20, 23, 31). However, shared epitope recognition by alleles within a single supertype would be expected to produce relatively uniform associations of immunologic or clinically relevant CTL responses. On the other hand, if functional differences among alleles within a single HLA supertype are substantial, then the knowledge of those differences would be crucial for predicting responses to vaccines designed on the basis of supertype. Since not all HLA class I supertypes have been uniform in their associations with HIV-1 outcomes (4, 31), the systematic assessment of alleles within the same supertype may identify important differences in their epitope-binding characteristics.
The HLA-B58 supertype (B58s) is associated with a favorable response in Caucasians infected with HIV-1 clade B and in Africans infected with clade C (20, 23, 31). Among B58s alleles (all B57 alleles, B5801-5802, and B1516-1517) (25). The HLA-B57 alleles have consistently been associated with a favorable disease course in populations with different HIV-1 viral subtypes and ethnic backgrounds (1, 6, 11, 28, 30). However, assessment of the other B58s alleles has been relatively sparse (4, 10, 14). We evaluated the degree of control of HIV-1 infection exerted by the different B58s alleles in two cohorts of infected native Africans. Our findings of contrasting associations for certain B58s alleles, along with apparent effect modification by their accompanying HLA-C alleles, highlight the distinctive contributions of individual B58s alleles and their local haplotypes.
MATERIALS AND METHODS
Subjects. We analyzed data from participants in two cohorts whose available biologic materials permitted resolution of HLA B58s: one included HIV-1-infected Zambian sex partners, and the other included sexually active HIV-1-infected Rwandan women. The design and conduct of these studies have been described elsewhere (9, 18, 28). The selection of mostly HIV-1 subtype C-infected Zambians (n = 423) was based on the availability of HIV-1 RNA measurements. The selection of mostly HIV-1 subtype A-infected Rwandan women (n = 202) was based on the availability of clinical and hematological indicators of disease progression.
HIV-1 outcomes. HIV-1 RNA levels (viral loads [VLs]) were measured by a Roche Amplicor 1.0 assay (Roche Diagnostic Systems Inc., Branchburg, N.J.). Logarithmic (log10) transformation allowed modeling of VL as a continuous, normally distributed variable. Categorical VL analysis involved groups with <10,000, 10,000 to 100,000, and >100,000 copies/ml VL. Rwandan women were monitored from the date when infection was first documented until death or for 8 years and categorized as relatively slow (n = 101), intermediate (n = 86), or rapid (n = 15) progressors (27).
HLA class I typing and haplotype assignment. HLA class I alleles were detected by PCR with sequence-specific primers (Pel-Freez Clinical Systems, Brown Deer, Wis.) for both cohorts. B58s alleles were resolved to their four-digit specificities by automated reference-strand conformation analysis. HLA-B and HLA-C haplotypes in Zambians were manually assigned after linkage disequilibrium analysis (28).
Statistical analysis. For the Zambian cohort, the Mann-Whitney test was used to compare the median log10 VLs among subjects with various B58s alleles. For Zambian couples with a previously established correlation between VL levels in epidemiologically linked transmitters and seroconverters (29), we used generalized estimating equation (GEE) methodology (32) to assess the association between B58s alleles and VL. Because VLs in subjects with preexisting seropositivity at enrollment or seroconverters during follow-up could also reflect time-sensitive effects of HLA alleles, we stratified the subjects by serostatus to account for such differences in the analysis of the effects of B58s alleles. In separate stratification, we also aimed to dissect the effects of individual B58s alleles from those of their closely linked HLA-C alleles.
For the Rwandan cohort, the Jonckheere-Terpstra test was used to compare the proportions of subjects in the HIV-1 disease progression categories. Proportional odds regression analysis was used to measure the proportional odds ratios (POR) and 95% confidence intervals (CI) for the B58s alleles.
SAS version 9.01 (SAS Institute, Cary, N.C.) and GraphPad Prism version 4.0 were used for all statistical analyses and graphs.
RESULTS
B58s alleles and HIV-1 outcomes in Zambians. Of those Zambians whose specimens were typed at the HLA-B locus, 127 (30%) carried at least one B58s allele. Compared with Caucasians, Zambians were more likely to carry B58s alleles, including B5802. Frequencies of individual B58s alleles in this population (2N = 846) ranged from 1.7% for B1516-1517 to 5.8% for B5703 (Fig. 1). The mean log10 VL for all seropositive individuals was 4.67 (47,000 copies/ml). Overall, subjects with B58s alleles showed a slightly lower log10 VL than those without it (log10 VL = –0.12, P = 0.18). However, VLs differed markedly across groups carrying certain individual B58s alleles. HLA-B5703 was associated with low VLs in both linear (mean VL = 4.36, log10 VL = –0.34, P = 0.01) and categorical (P = 0.004) analyses (Fig. 2a). Conversely, B5802 was associated with higher VLs in both linear (mean VL = 4.97, log10 VL = +0.33, P = 0.02) and categorical (P = 0.007) analyses. For subjects with B5801, the median log10 VL (4.61) was not appreciably different from that found for subjects with B5703 (4.49) (P = 0.54) and not significantly lower than the median for subjects with non-B58s alleles (P = 0.23). The small number of individuals with B1516-1517 had a nonsignificantly higher mean VL (mean VL = 4.77, log10 VL = +0.12, P = 0.53) than those without any B58s allele.
To assess the effect of duration of HIV-1 infection on our findings, we compared transmitting partners who were HIV-1 positive at study entry (i.e., seroprevalent subjects) with partners who seroconverted after entry (i.e., seroconverters). The contrasting effects of B5703 and B5802 on VL were consistently apparent following stratification (Table 1), although the magnitude and significance of the B5802 disadvantage were diminished among seroconverters. The protective effect for B5801 was suggested among seroprevalent subjects (mean VL = 4.5, log10 VL = –0.28, P = 0.06) and particularly among those with the B5801-Cw03 haplotype (mean VL = 4.19, log10 VL = –0.65, P = 0.02) but absent in seroconverters. However, low numbers of seroconverters with B58s alleles may have accounted for the observed differences in the magnitude of the effects. The association with VL could not be meaningfully assessed in B1516-1517 seroconverters (n = 4).
There were 143 haplotype combinations consisting of a B58s allele and its imputed linked HLA-C allele identified among all Zambians studied, each with two presumed haplotypes (2N = 844). Notably, (i) the association of B5703 with lower VLs was strong in the presence of Cw18 but entirely lost in its absence; (ii) B5801 commonly formed haplotypes with both Cw03 and Cw07, but only the B5801-Cw03 haplotype was associated with appreciably lower VLs (Table 2) ; and (iii) the association of the B5802-Cw06 haplotype and that of B5802 overall with higher VLs were strong and indistinguishable due to the very strong disequilibrium between these B and Cw alleles. Because the carriage of Cw18 appeared statistically to modify the effect of B5703 in the Zambian population, we explored the role of Cw18 further. Its carriage was associated with lower VLs among all subjects (mean VL = 4.3, log10 VL = –0.43, P < 0.0001) as well as in the subsets of those with and without B5703 (mean VL = 4.25, log10 VL = –0.72, P < 0.01, and mean VL = 4.39, log10 VL = –0.34, P = 0.03, respectively). However, because Cw18 also shows linkage disequilibrium with B8101, another allele associated with lower VLs in Zulu/Xhosa populations (14) and in our Zambian population (mean VL = 4.34, log10 VL = –0.35, P = 0.03), the independent Cw18 effect could be assessed only in the absence of both B5703 and B8101. Even in that small subgroup (n = 6), Cw18 showed an effect (log10 VL = –0.33), albeit statistically insignificant (P = 0.3), of a magnitude similar to that seen with B5703 and B8101 carriers.
B58s alleles and HIV disease progression in Rwandans. Of 202 seropositive women whose specimens were typed at the HLA-B locus, 73 (36.1%) carried at least one B58s allele. Frequencies of individual B58s alleles in this population (2N = 404) ranged from 2% for B1516-1517 to 3.7% for B5801, 7.4% for B5703, and 8.2% for B5802. The proportions of each HIV-1 disease progression group who carried B58s alleles did not differ (P = 0.81). B5703 was associated with a relatively favorable disease course (POR = 0.47; 95% CI = 0.21 to 1.07; P = 0.06), whereas B5802 was strongly associated with accelerated HIV-1 progression (POR = 3.46; 95% CI = 1.6 to 7.7; P = 0.003) (Fig. 2b). No trends were apparent for B5801 (P = 0.46) or B1516-1517 (P = 0.25).
DISCUSSION
Our observations for two populations of Africans infected with different HIV-1 subtypes demonstrate functional heterogeneity for individual alleles within the B58 supertype. We found no appreciable advantage of the B58 supertype as a whole on HIV/AIDS, in contrast to several previous studies (3, 16, 23, 31). Different HIV-1 subtypes or HLA class I supertype frequencies could have accounted for the observed population-specific effects of supertypes on viral control and immune escape (31). The more likely reason, however, is that contributions of the individual component alleles of the B58s were not examined (3, 16) or that their analysis was limited by the rarity of certain B58s alleles (23, 31). In particular, the very low frequency of B5802 among Caucasians (5) precluded assessment of its contribution to the protection by B58s seen in the Multicenter AIDS Cohort Study.
Our results confirm the favorable effect of B5703 and unfavorable effect of B5802 on VL previously reported for HIV-1 subtype C in South Africa (14). Additionally, our findings extend the evidence to include the acceleration by B5802 of the disease progression among Rwandans with the HIV-1 clade A. An earlier report on the failure of B5802 to prsent immunodominant HIV-1 subtype C Gag peptides in subjects from Botswana (19) accords with poor control of viremia in Zambians and disease progression in Rwandans with B5802. The available experimental and epidemiologic data point to structural and functional features of B5802 that set it apart from the other members of the B58 supertype with regard to its capacity to respond to HIV-1 subtype C peptides. Specifically, it has been suggested that changes in amino acid side chains (94IT, 95IL, and 97RW) in the -2 helix of HLA class I molecules affect the key structures of the antigen binding groove such as the tyrosine bed and the F-pocket (2, 12, 21, 26), thereby impairing the presentation of immunodominant HIV-1 peptides.
While B5802 has structurally been predicted to function inadequately, B5801 would be expected to resemble B57 alleles in controlling HIV-1 infection. However, we did not detect a uniform advantage for all Zambian B5801 carriers. The protective effect of B5801 was particularly apparent among seroprevalent subjects in conjunction with a closely linked Cw03 allele. The absence of a B5801 benefit among seroconverters may imply a later effect of B5801 as distinct from the early protection well established for B57 in a study of recent seroconverters (1) and in our own. Thus, despite their similarity in both predicted and reported binding motifs, B5801 differed somewhat from B5703 in its associations with HIV-1 outcomes in our study. Together, our data from Africans suggest important functional differences between B58 and B57 in the context of HIV/AIDS.
Although the preeminence of HLA-B alleles in HIV/AIDS has been demonstrated experimentally and epidemiologically (7, 14), in our Zambian cohort certain HLA-C alleles in linkage disequilibrium with their corresponding HLA-B alleles appeared to be contributing to their effect on VL. In particular, Cw18 showed significant associations with low VL both in the presence and in the absence of B5703, with which it is in linkage disequilibrium; conversely, VL was higher in subjects carrying B5703 who lacked Cw18. Compared with the effect of B5703 overall, the magnitude of the VL association with the B5703-Cw18 haplotype was greater. In Rwandan subjects, neither the number of patients with the B5703-Cw18 haplotype nor the strength of linkage disequilibrium between the two alleles of the haplotype was sufficient to assess the effects of the two alleles with appropriate stratification.
The advantage of B5801 likewise appeared dependent on the presence of the Cw03 allele in Zambians. For B5802 and Cw0602, the linkage disequilibrium was so uniform that an independent deleterious effect of B5802 could not be established. For reasons that are unclear, in Zambians these HLA-B-HLA-C haplotype combinations may have exerted particularly strong joint effects on the usual class I-mediated CTL pathway. It is also possible that HLA-C-restricted CTL responses may influence the epitopes targeted by relevant HLA-B alleles. A third possibility is that HLA-C alleles are involved through their additional role as ligands for killer immunoglobulin-like receptors (KIRs). However, HLA-KIR interactions are complex; they can drive both activating and inhibiting KIR effects (16, 17). Similar contributions of HLA-C alleles were not observed in Rwandans, probably due to differences in HLA-B-HLA-C haplotype frequencies and the prevalent HIV-1 subtype.
Our study had several strengths and limitations. We were able to examine the effects of B58s alleles in two African populations with distinct circulating viral subtypes. The capability of assessing the effects of B58s alleles among both seroprevalent and recently seroconverted individuals was also advantageous. Our longitudinal study design enabled us to detect the protective effect of B5801 among the seroprevalent but not the recently seroconverted Zambians. This effect had been predicted in seroprevalent South Africans (14) but may not be generalizable to subjects in earlier stages of infection. The relatively high prevalence of several B58s alleles among Africans provided sufficient power to evaluate the associations of HIV-1 outcomes with individual B58s alleles, except for B1516-1517, which has also been shown to be protective (10). The assessment of this pair of alleles was limited by small numbers in both cohorts. An absence of virologic data for Rwandans further limited our assessment, which was confined to categorical analysis of clinical outcomes.
In summary, B58s alleles or their haplotypes exert effects distinct enough from each other that the properties of all alleles of the B58 supertype should not be considered the same. Because HLA alleles interact with products of other genes inside and outside of the major histocompatibility complex as well as products of the virus itself, it is rather unlikely that their supertype classification based solely on CTL function can entirely capture their pluripotential effects. Further systematic investigation of individual alleles within other known HLA supertypes could prove equally informative for studies of infection and immunity.
ACKNOWLEDGMENTS
This work was supported by several grants (AI40591, AI42454, AI41530, and AI41951) from the National Institute of Allergy and Infectious Diseases (NIAID), with additional funding from the Center for AIDS Research at the University of Alabama at Birmingham.
We are grateful to investigators, staff, and participants of the Zambia-Emory HIV-1 Research Project (ZEHRP) and Project San Francisco for their valuable contributions to this work. We also thank I. Brill, G. Cloud, and A. Moore for their help in data management.
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National Blood Transfusion Service, Lusaka, Zambia
Project San Francisco, Kigali, Rwanda
Department of Global Health, Emory University, Atlanta, Georgia
ABSTRACT
Human leukocyte antigen (HLA) class I alleles can be grouped into supertypes according to their shared peptide binding properties. We examined alleles of the HLA-B58 supertype (B58s) in treatment-nave human immunodeficiency virus type 1 (HIV-1)-seropositive Africans (423 Zambians and 202 Rwandans). HLA-B and HLA-C alleles were resolved to four digits by a combination of molecular methods, and their respective associations with outcomes of HIV-1 infection were analyzed by statistical procedures appropriate for continuous or categorical data. The effects of the individual alleles on natural HIV-1 infection were heterogeneous. In HIV-1 subtype C-infected Zambians, the mean viral load (VL) was lower among B5703 (P = 0.01) or B5703-Cw18 (P < 0.001) haplotype carriers and higher among B5802 (P = 0.02) or B5802-Cw0602 (P = 0.03) carriers. The B5801-Cw03 haplotype showed an association with low VL (P = 0.05), whereas B5801 as a whole did not. Rwandans with HIV-1 subtype A infection showed associations of B5703 and B5802 with slow (P = 0.06) and rapid (P = 0.003) disease progression, respectively. In neither population were B1516-B1517 alleles associated with more favorable responses. Overall, B58s alleles, individually or as part of an HLA-B-HLA-C haplotype, appeared to have a distinctive impact on HIV-1 infection among native Africans. As presently defined, B58s alleles cannot be considered uniformly protective against HIV/AIDS in every population.
INTRODUCTION
The concept of human leukocyte antigen (HLA) supertypes, categories of alleles that share common peptide-binding motifs, offers a simplification of the complex HLA nomenclature by consolidating the huge spectrum of individual alleles into relatively few groups. Individual alleles assigned to each supertype have either proven or predicted ability to present antigenic peptides with similar anchoring residues at the second and C-terminal positions of peptide ligands. Initially proposed in the late 1990s, four HLA-A and five HLA-B supertype categories encompass the majority of known HLA class I alleles (24, 25). There have been subsequent efforts to improve supertype categorization (8, 22). However, independent of the supertype assignment method, the HLA epitopes for just a few of the major supertypes have been estimated to provide substantial population coverage for antigenic peptides. If conserved peptides that cross-react with representative HLA class I supertypes can be identified, a supertype-guided approach could simplify the development of human immunodeficiency virus type 1 (HIV-1) subunit vaccines. Furthermore, designing a multiepitope HIV-1 vaccine candidate to target a few supertypes would be considerably easier than tailoring one to highly population-specific allelic differences. However, the success of such a strategy would depend on numerous factors (13, 15), including the degree of uniformity in the nature and magnitude of the CD8+ cytotoxic T-lymphocyte (CTL) responses to viral peptides.
The associations of CTL responses and control of HIV-1 viremia appear to differ from one HLA class I supertype to another (20, 23, 31). However, shared epitope recognition by alleles within a single supertype would be expected to produce relatively uniform associations of immunologic or clinically relevant CTL responses. On the other hand, if functional differences among alleles within a single HLA supertype are substantial, then the knowledge of those differences would be crucial for predicting responses to vaccines designed on the basis of supertype. Since not all HLA class I supertypes have been uniform in their associations with HIV-1 outcomes (4, 31), the systematic assessment of alleles within the same supertype may identify important differences in their epitope-binding characteristics.
The HLA-B58 supertype (B58s) is associated with a favorable response in Caucasians infected with HIV-1 clade B and in Africans infected with clade C (20, 23, 31). Among B58s alleles (all B57 alleles, B5801-5802, and B1516-1517) (25). The HLA-B57 alleles have consistently been associated with a favorable disease course in populations with different HIV-1 viral subtypes and ethnic backgrounds (1, 6, 11, 28, 30). However, assessment of the other B58s alleles has been relatively sparse (4, 10, 14). We evaluated the degree of control of HIV-1 infection exerted by the different B58s alleles in two cohorts of infected native Africans. Our findings of contrasting associations for certain B58s alleles, along with apparent effect modification by their accompanying HLA-C alleles, highlight the distinctive contributions of individual B58s alleles and their local haplotypes.
MATERIALS AND METHODS
Subjects. We analyzed data from participants in two cohorts whose available biologic materials permitted resolution of HLA B58s: one included HIV-1-infected Zambian sex partners, and the other included sexually active HIV-1-infected Rwandan women. The design and conduct of these studies have been described elsewhere (9, 18, 28). The selection of mostly HIV-1 subtype C-infected Zambians (n = 423) was based on the availability of HIV-1 RNA measurements. The selection of mostly HIV-1 subtype A-infected Rwandan women (n = 202) was based on the availability of clinical and hematological indicators of disease progression.
HIV-1 outcomes. HIV-1 RNA levels (viral loads [VLs]) were measured by a Roche Amplicor 1.0 assay (Roche Diagnostic Systems Inc., Branchburg, N.J.). Logarithmic (log10) transformation allowed modeling of VL as a continuous, normally distributed variable. Categorical VL analysis involved groups with <10,000, 10,000 to 100,000, and >100,000 copies/ml VL. Rwandan women were monitored from the date when infection was first documented until death or for 8 years and categorized as relatively slow (n = 101), intermediate (n = 86), or rapid (n = 15) progressors (27).
HLA class I typing and haplotype assignment. HLA class I alleles were detected by PCR with sequence-specific primers (Pel-Freez Clinical Systems, Brown Deer, Wis.) for both cohorts. B58s alleles were resolved to their four-digit specificities by automated reference-strand conformation analysis. HLA-B and HLA-C haplotypes in Zambians were manually assigned after linkage disequilibrium analysis (28).
Statistical analysis. For the Zambian cohort, the Mann-Whitney test was used to compare the median log10 VLs among subjects with various B58s alleles. For Zambian couples with a previously established correlation between VL levels in epidemiologically linked transmitters and seroconverters (29), we used generalized estimating equation (GEE) methodology (32) to assess the association between B58s alleles and VL. Because VLs in subjects with preexisting seropositivity at enrollment or seroconverters during follow-up could also reflect time-sensitive effects of HLA alleles, we stratified the subjects by serostatus to account for such differences in the analysis of the effects of B58s alleles. In separate stratification, we also aimed to dissect the effects of individual B58s alleles from those of their closely linked HLA-C alleles.
For the Rwandan cohort, the Jonckheere-Terpstra test was used to compare the proportions of subjects in the HIV-1 disease progression categories. Proportional odds regression analysis was used to measure the proportional odds ratios (POR) and 95% confidence intervals (CI) for the B58s alleles.
SAS version 9.01 (SAS Institute, Cary, N.C.) and GraphPad Prism version 4.0 were used for all statistical analyses and graphs.
RESULTS
B58s alleles and HIV-1 outcomes in Zambians. Of those Zambians whose specimens were typed at the HLA-B locus, 127 (30%) carried at least one B58s allele. Compared with Caucasians, Zambians were more likely to carry B58s alleles, including B5802. Frequencies of individual B58s alleles in this population (2N = 846) ranged from 1.7% for B1516-1517 to 5.8% for B5703 (Fig. 1). The mean log10 VL for all seropositive individuals was 4.67 (47,000 copies/ml). Overall, subjects with B58s alleles showed a slightly lower log10 VL than those without it (log10 VL = –0.12, P = 0.18). However, VLs differed markedly across groups carrying certain individual B58s alleles. HLA-B5703 was associated with low VLs in both linear (mean VL = 4.36, log10 VL = –0.34, P = 0.01) and categorical (P = 0.004) analyses (Fig. 2a). Conversely, B5802 was associated with higher VLs in both linear (mean VL = 4.97, log10 VL = +0.33, P = 0.02) and categorical (P = 0.007) analyses. For subjects with B5801, the median log10 VL (4.61) was not appreciably different from that found for subjects with B5703 (4.49) (P = 0.54) and not significantly lower than the median for subjects with non-B58s alleles (P = 0.23). The small number of individuals with B1516-1517 had a nonsignificantly higher mean VL (mean VL = 4.77, log10 VL = +0.12, P = 0.53) than those without any B58s allele.
To assess the effect of duration of HIV-1 infection on our findings, we compared transmitting partners who were HIV-1 positive at study entry (i.e., seroprevalent subjects) with partners who seroconverted after entry (i.e., seroconverters). The contrasting effects of B5703 and B5802 on VL were consistently apparent following stratification (Table 1), although the magnitude and significance of the B5802 disadvantage were diminished among seroconverters. The protective effect for B5801 was suggested among seroprevalent subjects (mean VL = 4.5, log10 VL = –0.28, P = 0.06) and particularly among those with the B5801-Cw03 haplotype (mean VL = 4.19, log10 VL = –0.65, P = 0.02) but absent in seroconverters. However, low numbers of seroconverters with B58s alleles may have accounted for the observed differences in the magnitude of the effects. The association with VL could not be meaningfully assessed in B1516-1517 seroconverters (n = 4).
There were 143 haplotype combinations consisting of a B58s allele and its imputed linked HLA-C allele identified among all Zambians studied, each with two presumed haplotypes (2N = 844). Notably, (i) the association of B5703 with lower VLs was strong in the presence of Cw18 but entirely lost in its absence; (ii) B5801 commonly formed haplotypes with both Cw03 and Cw07, but only the B5801-Cw03 haplotype was associated with appreciably lower VLs (Table 2) ; and (iii) the association of the B5802-Cw06 haplotype and that of B5802 overall with higher VLs were strong and indistinguishable due to the very strong disequilibrium between these B and Cw alleles. Because the carriage of Cw18 appeared statistically to modify the effect of B5703 in the Zambian population, we explored the role of Cw18 further. Its carriage was associated with lower VLs among all subjects (mean VL = 4.3, log10 VL = –0.43, P < 0.0001) as well as in the subsets of those with and without B5703 (mean VL = 4.25, log10 VL = –0.72, P < 0.01, and mean VL = 4.39, log10 VL = –0.34, P = 0.03, respectively). However, because Cw18 also shows linkage disequilibrium with B8101, another allele associated with lower VLs in Zulu/Xhosa populations (14) and in our Zambian population (mean VL = 4.34, log10 VL = –0.35, P = 0.03), the independent Cw18 effect could be assessed only in the absence of both B5703 and B8101. Even in that small subgroup (n = 6), Cw18 showed an effect (log10 VL = –0.33), albeit statistically insignificant (P = 0.3), of a magnitude similar to that seen with B5703 and B8101 carriers.
B58s alleles and HIV disease progression in Rwandans. Of 202 seropositive women whose specimens were typed at the HLA-B locus, 73 (36.1%) carried at least one B58s allele. Frequencies of individual B58s alleles in this population (2N = 404) ranged from 2% for B1516-1517 to 3.7% for B5801, 7.4% for B5703, and 8.2% for B5802. The proportions of each HIV-1 disease progression group who carried B58s alleles did not differ (P = 0.81). B5703 was associated with a relatively favorable disease course (POR = 0.47; 95% CI = 0.21 to 1.07; P = 0.06), whereas B5802 was strongly associated with accelerated HIV-1 progression (POR = 3.46; 95% CI = 1.6 to 7.7; P = 0.003) (Fig. 2b). No trends were apparent for B5801 (P = 0.46) or B1516-1517 (P = 0.25).
DISCUSSION
Our observations for two populations of Africans infected with different HIV-1 subtypes demonstrate functional heterogeneity for individual alleles within the B58 supertype. We found no appreciable advantage of the B58 supertype as a whole on HIV/AIDS, in contrast to several previous studies (3, 16, 23, 31). Different HIV-1 subtypes or HLA class I supertype frequencies could have accounted for the observed population-specific effects of supertypes on viral control and immune escape (31). The more likely reason, however, is that contributions of the individual component alleles of the B58s were not examined (3, 16) or that their analysis was limited by the rarity of certain B58s alleles (23, 31). In particular, the very low frequency of B5802 among Caucasians (5) precluded assessment of its contribution to the protection by B58s seen in the Multicenter AIDS Cohort Study.
Our results confirm the favorable effect of B5703 and unfavorable effect of B5802 on VL previously reported for HIV-1 subtype C in South Africa (14). Additionally, our findings extend the evidence to include the acceleration by B5802 of the disease progression among Rwandans with the HIV-1 clade A. An earlier report on the failure of B5802 to prsent immunodominant HIV-1 subtype C Gag peptides in subjects from Botswana (19) accords with poor control of viremia in Zambians and disease progression in Rwandans with B5802. The available experimental and epidemiologic data point to structural and functional features of B5802 that set it apart from the other members of the B58 supertype with regard to its capacity to respond to HIV-1 subtype C peptides. Specifically, it has been suggested that changes in amino acid side chains (94IT, 95IL, and 97RW) in the -2 helix of HLA class I molecules affect the key structures of the antigen binding groove such as the tyrosine bed and the F-pocket (2, 12, 21, 26), thereby impairing the presentation of immunodominant HIV-1 peptides.
While B5802 has structurally been predicted to function inadequately, B5801 would be expected to resemble B57 alleles in controlling HIV-1 infection. However, we did not detect a uniform advantage for all Zambian B5801 carriers. The protective effect of B5801 was particularly apparent among seroprevalent subjects in conjunction with a closely linked Cw03 allele. The absence of a B5801 benefit among seroconverters may imply a later effect of B5801 as distinct from the early protection well established for B57 in a study of recent seroconverters (1) and in our own. Thus, despite their similarity in both predicted and reported binding motifs, B5801 differed somewhat from B5703 in its associations with HIV-1 outcomes in our study. Together, our data from Africans suggest important functional differences between B58 and B57 in the context of HIV/AIDS.
Although the preeminence of HLA-B alleles in HIV/AIDS has been demonstrated experimentally and epidemiologically (7, 14), in our Zambian cohort certain HLA-C alleles in linkage disequilibrium with their corresponding HLA-B alleles appeared to be contributing to their effect on VL. In particular, Cw18 showed significant associations with low VL both in the presence and in the absence of B5703, with which it is in linkage disequilibrium; conversely, VL was higher in subjects carrying B5703 who lacked Cw18. Compared with the effect of B5703 overall, the magnitude of the VL association with the B5703-Cw18 haplotype was greater. In Rwandan subjects, neither the number of patients with the B5703-Cw18 haplotype nor the strength of linkage disequilibrium between the two alleles of the haplotype was sufficient to assess the effects of the two alleles with appropriate stratification.
The advantage of B5801 likewise appeared dependent on the presence of the Cw03 allele in Zambians. For B5802 and Cw0602, the linkage disequilibrium was so uniform that an independent deleterious effect of B5802 could not be established. For reasons that are unclear, in Zambians these HLA-B-HLA-C haplotype combinations may have exerted particularly strong joint effects on the usual class I-mediated CTL pathway. It is also possible that HLA-C-restricted CTL responses may influence the epitopes targeted by relevant HLA-B alleles. A third possibility is that HLA-C alleles are involved through their additional role as ligands for killer immunoglobulin-like receptors (KIRs). However, HLA-KIR interactions are complex; they can drive both activating and inhibiting KIR effects (16, 17). Similar contributions of HLA-C alleles were not observed in Rwandans, probably due to differences in HLA-B-HLA-C haplotype frequencies and the prevalent HIV-1 subtype.
Our study had several strengths and limitations. We were able to examine the effects of B58s alleles in two African populations with distinct circulating viral subtypes. The capability of assessing the effects of B58s alleles among both seroprevalent and recently seroconverted individuals was also advantageous. Our longitudinal study design enabled us to detect the protective effect of B5801 among the seroprevalent but not the recently seroconverted Zambians. This effect had been predicted in seroprevalent South Africans (14) but may not be generalizable to subjects in earlier stages of infection. The relatively high prevalence of several B58s alleles among Africans provided sufficient power to evaluate the associations of HIV-1 outcomes with individual B58s alleles, except for B1516-1517, which has also been shown to be protective (10). The assessment of this pair of alleles was limited by small numbers in both cohorts. An absence of virologic data for Rwandans further limited our assessment, which was confined to categorical analysis of clinical outcomes.
In summary, B58s alleles or their haplotypes exert effects distinct enough from each other that the properties of all alleles of the B58 supertype should not be considered the same. Because HLA alleles interact with products of other genes inside and outside of the major histocompatibility complex as well as products of the virus itself, it is rather unlikely that their supertype classification based solely on CTL function can entirely capture their pluripotential effects. Further systematic investigation of individual alleles within other known HLA supertypes could prove equally informative for studies of infection and immunity.
ACKNOWLEDGMENTS
This work was supported by several grants (AI40591, AI42454, AI41530, and AI41951) from the National Institute of Allergy and Infectious Diseases (NIAID), with additional funding from the Center for AIDS Research at the University of Alabama at Birmingham.
We are grateful to investigators, staff, and participants of the Zambia-Emory HIV-1 Research Project (ZEHRP) and Project San Francisco for their valuable contributions to this work. We also thank I. Brill, G. Cloud, and A. Moore for their help in data management.
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