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Functionally active HLA-G polymorphisms are associated with the risk of heterosexual HIV-1 infection in African women

Matte, Claudinea,b; Lajoie, Juliea,b; Lacaille, Juliea; Zijenah, Lynn Sc; Ward, Brian Jd; Roger, Michela,b

Basic Science: Concise Communications

Background: Heterosexual transmission of HIV-1 is the major route of infection worldwide. HLA-G molecules are involved in the inhibition of cell-mediated immune responses and could permit or even promote the propagation of infection in the female reproductive tract.

Objective: To examine whether HLA-G genetic variants are associated with the risk of heterosexual HIV-1 infection.

Methods: HLA-G polymorphism in DNA samples from 431 (228 HIV-positive and 203 HIV-negative) Zimbabwean women was determined by amplified-restriction fragment length polymorphism and DNA sequencing analyses.

Results: Six HLA-G alleles were identified in the study population. HLA-G*0105N, which does not encode functional HLA-G1 proteins, was significantly associated with protection from HIV-1 infection [odds ratio (OR), 0.51; 95% confidence interval (CI), 0.31–0.85; P = 0.0083). The HLA-G*010108 allele was associated with a 2.5-fold increased risk of HIV-1 infection (OR, 2.47; 95% CI, 1.32–4.64; P = 0.0038). In addition, two HLA-G*010108-containing genotypes were associated with elevated risk of HIV-1 infection: HLA-G*010108/010401 (OR, 23.6; 95% CI, 1.39–401.7; P = 0.0009) and G*010101/010108 (OR, 5.6; 95% CI, 1.24–25.3; P = 0.012).

Conclusion: This study demonstrates that functionally active HLA-G polymorphisms are associated with altered risk of HIV-1 infection in African women. This provides evidence to support the hypothesis that modulation of HLA-G expression by HIV-1 can contribute to the risk of infection. Targeted interventions to reduce or block HLA-G expression in genital tissues could lead to novel strategies for the prevention of heterosexual HIV-1 transmission.

From the aLaboratory of Immunogenetics, Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montreal General Hospital Research Institute and the bDepartment of Microbiology and Immunology, Montréal University, Montréal, Canada, the cDepartment of Immunology, University of Zimbabwe, Harare, Zimbabwe and the dResearch Institute of the McGill University Hospital Complex, Montréal, Canada.

Correspondence to Dr M. Roger, Département de microbiologie, Hôpital Notre-Dame du CHUM, 1560 Sherbrooke Est, Montréal, Québec, Canada H2L 4M1.

Received: 22 May 2003; revised: 7 July 2003; accepted: 15 July 2003.

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Although female-to-male transmission of HIV-1 can occur, the vast majority of cases involve transmission of the virus from males to females. It is well documented that HIV-1 can be isolated from the cervix and the vagina as cell-free and cell-associated virus [1–5]. HIV-1 can infect macrophage, dendritic and epithelial cells from female genital tissues [6,7], suggesting that these cells may be targets for initial infection by the virus. However, the precise molecular mechanism whereby virus-infected cells can spread across the female genital tissue remains largely unknown.

Viruses that infect humans have evolved a myriad of mechanisms for avoiding the immune system [8,9]. Some viral proteins regulate cell surface expression of HLA class I proteins to permit infected cells to resist cytotoxic T lymphocyte killing. However, the removal of HLA class I proteins can expose the infected cell to attack by natural killer (NK) cells, since these cells preferentially lyse targets that lack HLA class I expression. NK cells can be prevented from killing cells by interaction of specific NK inhibitory receptors with HLA-C, HLA-G and HLA-E [10,11]. HIV-1 Nef proteins selectively downregulate HLA-A and HLA-B, without affecting cell surface expression of HLA-E and HLA-C on lymphoid cells [12]. Similarly, infection of macrophage by human cytomegalovirus leads to upregulated cell surface expression of HLA-G while HLA-A and HLA-B are downregulated [13]. The selective regulation of HLA expression may permit HIV-infected cells to escape from cytotoxic T lymphocyte-mediated killing while avoiding NK cell-mediated lysis [14–16].

HLA-G is a non-classical major histocompatibility complex (MHC) class I antigen that is predominantly expressed on invasive trophoblastic cells [17] and is postulated to be an important mediator of maternal–fetal tolerance through inhibition of lysis by maternal NK cells [18]. HLA-G transcripts are also expressed at low level in a variety of normal human adult tissues, including thymic epithelial cells [19] and peripheral blood mononuclear cells [20]. Recent studies indicate that HLA-G expression is upregulated in inflammatory and neoplastic tissues [21–25]. HLA-G molecules are detected in activated macrophages and dentritic cells infiltrating lung carcinomas in infectious pulmonary diseases but not in normal tissue [22]. Macrophage surface expression of HLA-G is strongly induced in HIV-1-positive patients [26]. In addition to the actions of HIV-1 Nef proteins, the HIV-1 envelope protein gp41 upregulates the synthesis of interleukin-10 production in monocytes [27]. Interleukin-10 selectively enhances HLA-G expression in these cells while downregulating the expression of other HLA class I and II molecules [28]. Taken together, these observations suggest that HIV-1 uses both direct and indirect mechanisms to enhance the cell surface expression of HLA-G while downregulating the expression of other HLA class I proteins. This selective induction of HLA-G may permit HIV-1 to escape the mucosal immune response and facilitate the establishment of the infection in the female genital tract. The present study examines whether HLA-G is a good candidate gene for susceptibility to HIV-1 infection.

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DNA extracts were obtained from 431 unrelated women enrolled in the ZVITAMBO project in Zimbabwe, Africa. This is a randomized placebo-controlled clinical trial of 14 000 mother–child pairs with the main objective of investigating the impact of immediate postpartum vitamin A supplementation on vertical transmission of HIV-1. All eligible participants were recruited immediately postpartum from one of seven recruitment sites (maternity clinics and hospitals) in Greater Harare. Written informed consent was obtained from all participating mothers. Samples were drawn consecutively from two groups: 228 HIV-1-seropositive women and 203 HIV-1-seronegative women (as controls). The patients’ serological status was determined by enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blot. The majority (90%) of the subjects in the study are of the Shona ethnic group. HIV-1 infection was reported to be solely via heterosexual transmission.

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Genomic DNA was extracted from whole peripheral blood using Qiagen DNA extraction kit (Qiagen, Mississauga, Ontario, Canada). HLA-G polymorphism was defined by nucleotide sequence variations in exons 2 and 3 that encode α-1 and α-2 domains of the protein, which interact with bound peptides on T cell receptors [29]. Consequently, allelic designations are based on nucleotide sequence variants at codons 31 (ACG←TCG), 57 (CCG←CCA), 69 (GCC←GCT), 93 (CAC←CAT), 107 (GGA←GGT), 110 (CTC← ATC) and 130 (CTG←–TG). Of these variants, only three result in amino acid substitutions at the protein level: codon 31 (Thr←Ser), codon 110 (Leu←Ile) and codon 130 (Leu←Cys). Genotyping of these HLA-G exon 2 and 3 variants was performed on isolated genomic DNA by amplified-restriction fragment length polymorphism and DNA sequencing methods as previously described [30,31]. These methods allow the characterization of all possible HLA-G polymorphic sites. Significance of allelic and genotypic differences between groups was assessed by the chi-square test.

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Six different alleles were detected in HLA-G in 431 Zimbabwean women. In the overall study population, regardless of HIV status, HLA-G*010101 was the predominant allele, with a frequency of 42.3% followed by G*010401 (20.7%), G*010102 (18.8%), G*0105N (8.0%), G*010108 (5.9%) and G*0103 (4.3%). The HLA-G*010101 allele corresponds to the wild-type sequence originally described by Geraghty et al. [32]. HLA-G*010102 and G*010108 are two alleles from the G*010101 lineage that are defined by synonymous and conservative mutations at codons 57 and 93, respectively. HLA-G*0103 and G*010401 are defined by non-synonymous mutations at codons 31 and 110, respectively. HLA-G*0105N has a single base deletion at codon 130 that causes a shift in the reading frame, resulting in a truncated non-functional HLA-G1 protein [33]. Nevertheless, other HLA-G isoforms are still produced in HLA-G*0105N homozygous individuals [33].

The distribution of HLA-G alleles among HIV-positive and HIV-negative groups is shown in Table 1. There were no significant differences between the groups with respect to HLA-G* 010101, G*010102, G*0103, and G*010401. However, there were significant differences in the distribution of the HLA-G*0105N and G*010108 alleles between groups. HLA-G*0105N was observed less frequently in HIV-positive women (5.7%) than in HIV-negative women (10.6%; P = 0.0083). This observation strongly suggests that the presence of the G*0105N allele is a protection factor [odds ratio (OR), 0.51; 95% confidence interval (CI), 0.31–0.85) for HIV infection. By comparison, the HLA-G*010108 allele was observed more frequently in HIV-positive women (8.1%) than in HIV-negative women (3.5%) (P = 0.0038), representing a 2.5-fold increased risk of HIV infection (OR, 2.47; 95% CI, 1.32–4.64). The latter result is reflected in the analysis of the distribution of HLA-G genotypes between groups (data not shown) showing two HLA-G*010108-containing genotypes associated with elevated risk of HIV infection. HLA-G*010108/010401 and G*010101/010108 genotypes were observed more frequently in the HIV-positive women than in the HIV-negative women (P = 0.0009 and P = 0.012, respectively) and were associated with substantially increased risk of HIV infection (OR, 23.6; 95% CI, 1.39–401.7 and OR 5.6; 95% CI, 1.24–25.3, respectively).

Table 1

Table 1

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Recent studies have suggested that the selective induction of HLA-G molecules may be used by viruses and tumours to evade host immunosurveillance [13,21–23,26]. In this study, we have established that functionally active HLA-G polymorphisms are associated with altered risk of HIV-1 infection in African women. Although we did not have access to sera from sexual partners of the women in our study, the vast majority of Shona women are infected through heterosexual contacts. As a result, we believe that our findings of increased or decreased HIV-1 infection rates reflect facilitation or inhibition of heterosexual transmission. The validation of theses associations in a relatively large cohort provides strong evidence supporting the role of HLA-G molecules in such transmission. There was a highly significant correlation between HLA-G*0105N allele and protection from HIV-1 infection. This allele does not encode functional soluble or membrane-bound HLA-G1 proteins [33]. HLA-G1 is the major ligand by which HLA-G inhibits NK cell-mediated lysis [34–36]. We propose, therefore, that the absence or reduced expression of HLA-G1 in individuals carrying HLA-G*0105N would allow NK cells to destroy HIV-infected cells, leading to protection from infection.

Our results also showed that HLA-G*010108 is significantly associated with increased risk of HIV-1 infection. This allele is defined by a synonymous substitution (Pro) at codon 57. Although HLA-G codon 57 variant discordance between a mother and her child has been reported to reduce the risk of perinatal HIV-1 transmission [37], we were unable to confirm this finding in our African population [38]. Because the polymorphism at codon 57 does not change the amino acid composition, it can be presumed that it does not affect the protein's function. It is, therefore, difficult to envisage the mechanism by which such a silent mutation could have a direct influence on transmission. However, it is conceivable that the variant at codon 57 is closely linked to a functional polymorphism elsewhere in the gene. In fact, the codon 57 variant is present on the HLA-G*010401 allele together with a non-synonymous substitution at codon 110 (Leu←Ile). Our analysis of the genotypic distribution between groups revealed that the HLA-G*010108/010401 genotype is associated with a substantial increased risk of HIV infection (OR, 23.6; 95% CI, 1.39–401.7; P = 0.0009). The mean soluble HLA-G plasma levels in individuals carrying the HLA-G*0101401 allele are significantly higher than in those with the HLA-G*010101 wild-type allele [39]. Therefore, it is tempting to speculate that the relatively high level of HLA-G expression in subjects with the HLA-G*010108/010401 genotype may permit HIV-infected cells to inhibit NK-mediated cytolysis and promote viral transmission. Although the HLA-G*0101401 allele exhibits an amino acid variation at codon 110 (Leu←Ile), this replacement would not be predicted to have any drastic effect on the structure or function of HLA-G, because both amino acids are small hydrophobic residues. Interestingly, all of the individuals with the HLA-G*010108/01041 genotype are homozygous for the variant at codon 57. The mechanism by which the polymorphism at codon 57 in association with codon 110 variant would affect HLA-G production remains to be identified.

In conclusion, our results show that HLA-G polymorphisms are associated with heterosexual HIV-1 infection. Importantly, our findings not only provide convincing support for a plausible mechanism by which HLA-G might help HIV-1 to evade cell-mediated immune response but also may be utilized in designing novel preventive interventions. Further studies aimed at investigating the impact of downregulation or inhibition of HLA-G expression in genital tissues on prevention of HIV-1 transmission in high-risk populations may provide useful information. This approach is not limited to HIV-1 infection but may be relevant for the prevention or therapy of other viral pathogens and cancers.

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The authors would like to thank all the participants in this study, co-principal investigators K. Nathoo and J. Humphrey and the ZVITAMBO Study Group: H. Chidawanyika, P. Iliff, A. Mahomva, F. Majo, L. Malaba, M. Mbizvo, L. Moulton, K. Mutasa, J. Mutsambi, M. Ndhlovu, L. Propper, A. Ruff, N. Tavengwa, C. Zunguza, P. Zvandasara.

Sponsorship: This work was supported in part by grant PG-50917 from the Elizabeth Glaser Pediatric AIDS Foundation. M. Roger is supported by career award from Fonds de la Recherche en Santé du Québec (FRSQ). The ZVITAMBO project is supported by the Canadian International Development Agency (R/C Project 690/M3688), Cooperative Agreement DAN 0045-A-005094-00 between the US Agency for International Development and the Johns Hopkins School of Hygiene and Public Health, and the Rockefeller Foundation. Note: The ZVITAMBO project is a collaborative project of the University of Zimbabwe, the Harare City Health Department, the Johns Hopkins School of Hygiene and Public Health, and the Montreal General Hospital Research Institute, McGill University.

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Africa; HIV-1; HLA-G; heterosexual transmission; virus-cell interaction

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