Heterogeneity in susceptibility to HIV-1 has been described in several cohorts [1–3]. This is exemplified in a subset of highly exposed but uninfected women enrolled in the Pumwani Female Sex Worker cohort , which was established in 1985 to prospectively study sexually transmitted infections, and continues to enroll members with biannual follow-up. In the cohort, overall seroprevalence is approximately 70% and most HIV-negative women seroconverted within 3 years of enrolment. However, a subgroup appears to be relatively resistant to HIV-1, they remain HIV seronegative and PCR negative for more than 3 years while continuing active sex work . Several factors have been shown to contribute this HIV-1 resistance [4,5], including class I and II human leucocyte antigens [6–9]. HLA class II DP (HLA-DP) has a heterodimeric binding cleft comprised of DP alpha 1 (DPA1) and DP beta 1 (DPB1) , and has a key role in presenting antigens to CD4+ T cells . The influence of HLA-DP has not been examined in the context of HIV-1 infection, despite its importance in other diseases [12–16]. To further understand the protective immune response in the HIV-resistant sex workers, we studied the associations of HLA-DP with resistance/susceptibility to HIV-1.
All resistant women in this study enrolled before 2000 (average follow-up 9.6 ± 4.3 years). HIV-positive women were considered susceptible. HIV-negative women with shorter follow-up were classified as negative and were not included in the comparisons between resistant and positive women. Owing to limited DNA, some women could not be typed for both DPA1 and DPB1. DPA1 was genotyped for 114 resistant, 157 HIV-negative and 703 HIV-positive individuals; DPB1 was genotyped for 111 resistant, 221 HIV-negative and 762 HIV-positive individuals; whereas both DPA1 and DPB1 were genotyped for 111 resistant, 152 HIV-negative and 681 HIV-positive individuals. Ethics committees at the University of Manitoba and University of Nairobi approved this study. Informed consent was obtained from all women enrolled.
Exon 2 of DPA1 and DPB1 was amplified using gene-specific primers [17,18]. DPA1 and DPB1 gene fragments were sequenced using the BigDye Cycle Sequencing Kit (Applied Biosystems, Foster City, California, USA) [19,20] and ABI 3100 Prism Genetic Analyzer (Applied Biosystems), and genotyped using Codon Express . Allele, genotype/haplotype frequencies, and Hardy–Weinberg calculations were estimated using Python for populations-32-0.6.0 (PyPop) . Genotyping results were analyzed with biological data using SPSS 15.0 (SPSS Inc., Chicago, Illinois, USA). Cross-sectional analysis was performed to identify associations of HLA-DP genotypes with HIV-1 susceptibility, using χ2, Fisher's exact test  and crosstabs analysis [odds ratio (OR), 95% confidence interval (95% CI)], were used to determine the relationship between binary outcomes and explanatory variables. Einot and Gabriel-adjusted P-values were calculated using modified syntax written by David Nichols of SPSS. Significant cross-sectional associations were included in binary logistic regression using a forward Wald method. To examine the role of HLA-DP in seroconversion, Kaplan–Meier and multivariate Cox regression analyses (backward Wald) were performed. Taking patient enrollment and samples being typed into account, a weighted parameter was generated using logistic regression, to adjust for crosstabs and binary logistic regression.
Several common HLA-DP alleles were identified. Out of the 17 HLA-DP alleles identified, four major alleles accounted for more than 90%: DPA1*010301 (38.04%), DPA1*0301 (20.07%), DPA1*020101 (17.66%) and DPA1*020202 (16.32%). Three alleles out of 51 identified accounted for more than 55% of DPB1 alleles: DPB1*010101 (24.36%), DPB1*0402 (19.70%) and DPB1*020102 (13.07%). Allele frequencies did not deviate significantly from Hardy–Weinberg equilibrium. DPA1*020202–DPB1*010101 (14.081%), DPA1*0301–DPB1*0402 (13.631%) and DPA1*010301–DPB1*020102 (11.841%), accounted for nearly 40% of the haplotypes. These frequencies were comparable with other populations around the world .
Cross-sectional analysis identified associations of HLA-DP genotypes with HIV-1 resistance. DPA1*010301 (P = 0.033, OR = 1.585, 95% CI = 1.036–2.425) and DPB1*3001 (P = 0.007, OR = 3.274, 95% CI = 1.451–7.384), were associated with HIV-1 resistance. DPA1*010301 homozygotes were enriched in the HIV-1-resistant group. Thirty DPA1–DPB1 haplotypes with five or more copies were examined. DPA1*010301–DPB1*3001 (P = 0.022, OR = 3.481, 95% CI = 1.260–9.611) and DPA1*0301–DPB1*5501 (P = 0.019, OR = 3.207, 95% CI = 1.265–8.132) were associated with resistance to HIV-1 infection. Low-frequency haplotypes with less than five copies in this population were grouped and analyzed together for rare haplotype associations, but no significant associations were found. All observed associations remained significant after adjustment for multiple comparisons. Further analysis using the weighted parameter also confirmed the observed associations.
Kaplan–Meier analysis showed that individuals with DPA1*010301, the common genotype associated with HIV-1 resistance, were significantly less likely to seroconvert (P = 0.012, log rank = 8.837) (Fig. 1a), but DPA1*010301 homozygosity did not substantially reduce the risk of seroconversion. In addition, the genotypes and haplotypes associated with HIV-1 resistance had a trend towards slower seroconversion. Genotypes associated with increased rate of seroconversion included DPA1*0302 (P = 0.029, log rank = 4.754) (Fig. 1b), DPB1*0402 homozygotes (P = 0.007, log rank = 9.970) (Fig. 1c), DPA1*0301–DPB1*0402 (P = 0.008, log rank = 7.031) (Fig. 1d), DPB1*010101/1801 (P = 0.022, log rank = 5.211), and DPA1*020101 homozygotes (P = 0.015, log rank = 5.960). Their corresponding allele frequencies were also enriched in HIV-positive women.
Binary logistic regression showed that the associations of DPA1*010301 and DPB1*3001 genotypes with HIV-1 resistance were independent of HIV-1 resistant HLA genotypes previously described such as DQB1*050301, DQB1*0603, DQB1*0609, DRB*1102, DRB1*01 and the class I supertype A2/6802 [7–9]. The association of DPA1*010301 with slower seroconversion was also independent of DRB1*01 and DRB1*1102 genotypes  by Cox regression. Among the HLA-DP genotypes and haplotypes associated with increased rate of seroconversion, only the DPA1*020101 homozygote became nonsignificant when analyzed with DRB1*1503–DRB5*010101 haplotype, DRB1*030201 genotype , and A*2301 , suggesting that they could be linked.
Analysis of protein sequences encoded by exon 2 of DP alleles between genotypes of HIV-1 resistance/susceptibility revealed variability. At residue 11, DPA1*010301 encoded alanine, whereas DPA1*0302 encoded methionine. Variability was also observed at residues 8, 9, 11, 55, 56, 69 and 84–87 between DPB1*3001 and DPB1*0402. This suggests that this variability could have functional characteristics and future studies should determine their role in differential antigen presentation.
The DPA1*010301 genotype is possessed by 60.4% of individuals in the cohort. As the most common HLA-DPA1 genotype, its association with HIV-1 resistance and delayed seroconversion is striking. This suggests DP molecules play a role that is different from the rare allele advantage observed for HLA class I genes in HIV-1-disease progression . Vaccine strategies based on mechanisms of protection offered by a common HLA class II allele may have broader applications than those associated with class I antigens. Heterozygosity in the HLA class I region is thought to confer an advantage in HIV-1-disease progression, possibly owing to the greater ability to present a larger variety of viral epitopes [6,26]. It has been suggested that this advantage may also extend to HLA class II alleles . Indeed, homozygotes of susceptible alleles such as DPA1*020101 and DPB1*0402 rapidly seroconverted. Although DPA1*010301 was associated with HIV-1 resistance and slower seroconversion, homozygosity for DPA1*010301 did not provide additional protection for either HIV-1 resistance or seroconversion. The advantage of two protective alleles may have been neutralized by a disadvantage of homozygosity.
The present study is the first to show associations of HLA-DP molecules with resistance/susceptibility to HIV-1 infection. HLA-DP is clearly an important factor in the immune response to HIV-1, and additional study of the mechanism of protection is warranted. These findings provide further understanding of the mechanisms of underlying protective immunity to HIV-1, and might ultimately contribute to the design of an effective vaccine.
R. Hardie wrote and edited the final drafts of the manuscript, performed experiments and data analysis. E. Knight and B. Bruneau conducted experiments and data analysis, and contributed to writing and editing the paper. C. Semeniuk and K. Gill contributed to experiments and data analysis. N. Nagelkerke edited the paper and consulted on statistical analysis. J. Kimani and C. Wachihi helped with editing, maintaining the cohort and collected biological data. E. Ngugi helped with editing and contributed to establishment of the cohort. M. Luo designed the study, performed data analysis, and edited the paper. Francis A. Plummer established and maintained the cohort, designed the study, edited the paper, and secured funding.
The present study was supported by a grant from the Bill and Melinda Gates Foundation and the Canadian Institutes of Health Research through the Grand Challenges in Global Health Initiative. This study was also supported through a grant from the NIH (R01 AI56980). This work would not be possible without the participation of the women of the Pumwani Sex Worker cohort as well as the dedicated staff who work with the cohort. Thomas Bielawny provided technical assistance. Dr Francis A. Plummer is a Tier 1 CIHR Canada Research Chair.
There were no conflicts of interest.
Presented at International AIDS Conference, Toronto, Ontario, Canada, 13–18 August 2006. International Centre for Infectious Diseases Research and Innovation Retreat, Winnipeg, Manitoba, Canada, 28–29 October 2005, 23–25 October 2006. Public Health Agency of Canada Conference, Winnipeg, Manitoba, Canada, 20–21 March 2006, 12–13 March 2007. Canadian Association for HIV Research Conference, Toronto, Ontario, Canada, 26–29 April 2007
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