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Associations of human leukocyte antigen-G with resistance and susceptibility to HIV-1 infection in the Pumwani sex worker cohort

Turk, William J.R.a; Kimani, Joshuab,c; Bielawny, Tomasza; Wachihi, Charlesc; Ball, Terry Blakea,b,c; Plummer, Francis A.a,b; Luo, Maa,b

doi: 10.1097/QAD.0b013e32835ab1f2

Objective: To determine the association between human leukocyte antigens (HLA)-G genotypes and resistance or susceptibility to HIV-1.

Design: A group of sex workers in Pumwani, Kenya can be epidemiologically defined as resistant to HIV-1 infection despite frequent exposure and provide an example of natural protective immunity. HLA class I and II molecules have been shown to be associated with resistance/susceptibility to infection in this cohort. HLA-G is a nonclassical class I allele that is primarily involved in mucosal and inflammatory response, which is of interest in HIV-1 resistance.

Methods: In this study, we used a sequence-based typing method to genotype HLA-G for 667 women enrolled in this cohort and examined the influence of HLA-G genotypes on resistance or susceptibility to HIV-1 infection.

Results: The G*01 : 01:01 genotype was significantly enriched in the HIV-1-resistant women [P = 0.002, Odds ratio: 2.11, 95% confidence interval (CI): 0.259–0.976], whereas the G*01 : 04:04 genotype was significantly associated with susceptibility to HIV-1 infection (P = 0.039, OR:0.502, 95% CI:0.259–0.976). Kaplan-Meier survival analysis correlated with these results. G*01 : 01:01 genotype was associated with significantly lower rate of seroconversion (P = 0.001). Whereas, G*01 : 04:04 genotype was significantly associated with an increased rate of seroconversion (P = 0.013). The associations of these HLA-G alleles are independent of other HLA class I and II alleles identified in this population.

Conclusion: Our study showed that specific HLA-G alleles are associated with resistance or susceptibility to HIV-1 acquisition in this high-risk population. Further studies are needed to understand its functional significance in HIV-1 transmission.

aPublic Health Agency of Canada, National Microbiology Laboratory

bDepartment of Medical Microbiology and Infectious Disease, University of Manitoba, Winnipeg, Manitoba, Canada

cDepartment of Medical Microbiology, University of Nairobi, Nairobi, Kenya.

Correspondence to Dr Ma Luo, 1015 Arlington Street, Winnipeg, MB, R3E 3R2, Canada. Tel: +1 204 789 5072; fax: +1 204 784 4835; e-mail:

Received 7 June, 2012

Revised 20 September, 2012

Accepted 24 September, 2012

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HIV/AIDS is currently a global pandemic with an estimated 34 million people living with HIV/AIDS and 1.8 million AIDS-related deaths annually [1]. Currently, there are 22.5 million people living with HIV/AIDS in sub-Saharan Africa where the pandemic has hit the hardest. Although effective antiretroviral therapies have been developed to control disease progression, there is still no cure. One of the best hopes to stop the spread of HIV is developing effective vaccines and microbicides. Attempts have not yet been successful due to the rapid mutations of the HIV retrovirus and insufficient understanding of protective immunity against the virus.

Variability in susceptibility to infectious disease has long been observed in human history either within a population or among different populations. When exposed to an infectious pathogen some individuals do not appear to become infected or responded with mild symptoms and recovered very quickly after being infected, whereas others succumbed to the infection. Similarly, heterogeneity in susceptibility to HIV-1 has been observed in several cohort studies [2–7]. Most individuals are susceptible to HIV-1 infection either through sexual transmission, blood transfusion, or other high-risk exposure. Some rare individuals, about 5% in the population, appear to be resistant to HIV-1 infection despite repeated exposure through high-risk sexual exposure or blood transfusion. Through two decades of research it has become clear that multiple factors are involved in the heterogeneous responses to HIV-1 infection and the genetic factors underlying the phenomenon are complex and diverse [6–23]. Polymorphisms of host genetic factors involved in host immune responses and biological process of HIV-1 infection, establishment and spread appear to play an important role in the heterogeneity in susceptibility to HIV-1 infection [18,24–53]. Several genetic factors, such as CCR5 δ-32 mutation [12,54–65], Trim5α polymorphisms [33,53,66–68], Interferon gamma regulation factor-1 polymorphisms [11,69] and specific human leukocyte antigens (HLA) class I and class II alleles [70–73] have been reported to influence a person's response to HIV-1 infection. Among the reported host genetic factors CCR5 δ-32 mutations is the only genetic defect that is shown to be resistant to HIV-1 infection [8,9]. However, this mutation is not present in African populations. Although it is well known that HLA class I and class II antigens present antigens to CD8+ and CD4+ T cells to initiate immune responses to infectious pathogens, the specific mechanisms of the associations of HLA class I and class II alleles with resistance or susceptibility to HIV-1 infection need to be carefully investigated.

The Pumwani Sex Worker Cohort was established in 1985 in Nairobi, Kenya to study risk factors in sexually transmitted infections. A sub-population of women in this cohort has remained polymerase chain reaction (PCR) and seronegative for HIV-1 despite repeated exposures through high-risk sex work [2]. These individuals have been defined as relatively resistant to HIV-1 infection. This phenomenon was not due to altered sex practices or behavioural differences or condom usage. The resistant phenotype also clustered in families, as relatives of those HIV-1-resistant women were less likely to be infected by HIV-1 (T. Blake Ball, personal communications).

Previous studies showed that this resistance to HIV-1 infection is associated with qualitatively different CD8+ and CD4+ T-cell responses [74–78]. More recently, lower baseline levels of pro-inflammatory cytokines were detected in these HIV-1-resistant individuals when compared to HIV-1-negative controls [79]. Microarray analysis has shown that HIV-resistant women have down regulation of genes involved in T-cell receptor signalling and natural killer (NK) cell cytotoxicity [80]. Host genetics factors play an important role in this natural resistance to HIV-1. Previous studies have shown that both HLA class I and class II alleles have been associated with resistance/susceptibility to HIV-1 infection in this cohort [70–73]. Thus, it is likely that both adaptive and innate immunity are contributed to this natural resistance.

HLA-G is a member of the nonclassical class I antigens [81] along with HLA-E and HLA-F. It is characterized by having low gene polymorphism, limited spatial distribution, low expression levels and seven different isoforms [25,82] due to the alternatively spliced mRNA transcripts that lack one or more exons. HLA-G has been implicated in fetal–maternal tolerance during pregnancy [83], inflammatory disease, autoimmune disease, infectious disease and tissue transplantation [84]. It is known to directly inhibit the cytolytic function of NK cells, the antigen-specific cytolytic function of CD8+ T cells, induce tolerogenic dendritic cells and affect the allo-proliferative responses of CD4+ T cells [85]. Due to its immunosuppressive properties and expression in mucosal and inflammatory tissue, HLA-G has been of great interest in relation to HIV infection. Soluble HLA-G (sHLA-G) has been detected in plasma of HIV-infected individuals [86,87]. Specific HLA-G polymorphisms have been found to be associated with mother-to-child transmission of HIV [88]. HLA-G polymorphisms have been reported to be associated with heterosexual transmission of HIV [89–91]. Because of the importance of HLA-G in modulating immune responses and its expression in mucosal tissue, we carry out this study to investigate the possible association of genetic polymorphisms of HLA-G with resistance or susceptibility to HIV-1 infection in the Pumwani cohort.

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Materials and methods

Study population

The study was conducted among 667 individuals enrolled before 2002 in the Pumwani sex worker cohort, an open prospective cohort established in 1985 in the heart of Pumwani slum in Nairobi, Kenya to study risk factors in sexually transmitted diseases. The majority of the women were from South-Central and South–West Kenya, and regions of Tanzania and Uganda around Lake Victoria. More than 95% of them are Bantus and 5% of them are Nilotes. The sexual transmitted disease status was not the requirement for the enrolment. The criteria for enrolment, sample collection and sexually transmitted infection testing have been described elsewhere [2]. The patients enrolled in the cohort have been followed biannually since the cohort establishment. In addition to research, it provides services related to sexually transmitted infections and HIV prevention and care, including consultation, provision of free condoms and treatment of other infections. Despite effective intervention programmes, the annual incidence of HIV-1 infection among initially seronegative women is currently four per 100 person years: a dramatic decrease from the initial annual incidence of 45%. The women classified as resistant in this study were all enrolled in the cohort before 1999 with an average follow-up time of 9.6±4.3 years. The control group for the genetic association studies are all HIV positive. Ethics committees at the University of Manitoba and the University of Nairobi have approved this study. Informed consent was obtained from all women enrolled in the study.

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DNA isolation

Genomic DNA was extracted from whole blood, buffy coat, B cells, peripheral blood mononuclear cells and peripheral blood lymphocytes using either the QIAamp DNA Mini Kit (Qiagen Inc., Mississauga, Ontario, Canada) or the BioRobot EZ1 (Qiagen Inc.) following manufacturer's instructions. DNA concentration and optical density (A260/A280) were determined using the Nano Drop (Nano Drop Technologies Inc., Wilmington, Delaware, USA) and NanoDrop 3.0.1 software (Coleman Technologies Inc., Glen Mills, Pennsylvania, USA).

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PCR and sequencing primers

PCR and sequencing primers were designed on the basis of HLA-G genomic DNA sequences obtained from NCBI's Genome Biology website (http://, Homo sapiens genome build 35.1) using PrimerSelect (Lasergene; DNASTAR, Madison, Wisconsin, USA) and Sequencher 4.5 (Gene Codes). The PCR primer set HLAGPCRF and HLAGPCRR designed to amplify a 994 bp fragment which covers partial intron 1, exon 2, intron 2, exon 3, and partial intron 3 of the HLA-G gene. The primer set HLAGEX4PCRF and HLAGEX4PCRR produce a 463 bp fragment, which covers partial intron 3, exon 4, and partial intron 4. Relevant information for PCR primer sequences, annealing temperatures and fragment sizes can be found in Table 1.

Table 1

Table 1

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PCR reactions

The 50 μl final PCR reaction mixture consists of 60 mmol/l Tris-HCl (pH 9.0), 1.5 mmol/l MgCl2, 15 mmol/l (NH4)2SO4, 25 μmol/l of each dNTP, 0.1% gelatin, 6.25 pmol of each primer, 1.25 Unit of Taq DNA polymerase (Gibco/ BRL, Life Technologies, Burlington, Ontario, Canada) and 50–100 ng DNA. The cycle parameters used in the PTC-200 Peltier Thermal Cycler (MJ Research, Inc., Watertown, Massachusetts, USA) was 45 cycles of 1 min at 96°C, 1 min at 63°C (exon 2 and 3) or 60°C (exon 4), and 2 min at 72°C. This was followed by a 10-min incubation at 72°C. To confirm the successful amplification of the gene, 1% agarose gel electrophoresis was used to examine the correctly sized PCR products. The PCR products were then purified using Multiwell Filter Plates (Pall Corp., Ann Arbor, Michigan, USA).

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Sequencing and human leukocyte antigens-G genotyping

The purified PCR products reaction was sequenced using BigDye v3.1 from the ABI Prism BigDye Cycle sequencing kits (Applied Biosystems, Foster City, California, USA). Sequencing primers were developed on the basis of genomic sequences to sequence each particular exon. The specific primers sequences as well as optimal annealing temperatures are listed in Table 1. The sequencing products were analyzed on an ABI 3130xl Genetic Analyzer (Applied Biosystems). The HLA-G alleles were typed using computer software CodonExpress (University of Manitoba, Winnipeg, MB, Canada) developed based on a taxonomy-based sequence analysis method [92,93]. The HLA-G database was downloaded from IMTG/HLA Database (

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Statistical analysis

Analyses were performed with SPSS13.0 for Windows statistical analysis software package. Python for Populations (PyPop)-32-0.6.0 [94], a software program for population genetics, was used to calculate allele frequencies and to test for deviations from Hardy–Weinberg equilibrium. HLA-G allele frequencies were calculated by direct counting. Phenotypic frequencies were expressed as the percentage of individuals bearing the corresponding allele specificity. Chi-square testing and logistical regression was used to determine whether any significant associations exist between HLA-G and resistance or susceptibility to HIV. Kaplan–Meier survival analysis was conducted to examine the effect of HLA-G on seroconversion. Only the alleles with frequencies above 5% in the population were analyzed for their association with resistance or susceptibility to HIV infection. For binary logistic regression analysis, all alleles significantly associated with resistance or susceptibility to HIV-1 infection were included in the model. The previously reported HLA class I and class II alleles associated with either resistance or susceptibility to HIV-1 infection were included in the multivariate analysis to verify the independent associations of HLA-G with differential susceptibility to HIV-1 infection identified in this study.

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Allele frequency distribution of human leukocyte antigens-G in the Pumwani sex worker cohort

We genotyped exon 2, 3 and 4 of HLA-G of 667 patients enrolled in the Pumwani sex worker cohort in Nairobi, Kenya. These exons encode the α 1, 2 and 3 domains of HLA-G. 17 HLA-G alleles were identified in this population (Table 2). The 17 HLA-G alleles encode six different proteins (G*01 : 01, G*01 : 03, G*01 : 04, G*01 : 06, G*01 : 10 and G*01 : 11) and one null allele (G*01 : 05N). Five HLA-G alleles were detected at the frequencies above 5% in the population with G*01 : 01:01 (38.5%) and G*01 : 01:02 (19.8%) as the most frequently observed alleles. Together with G*01 : 03 (9.28%), G*01 : 04:04 (9.87%) and G*01 : 05N (6.44%), the five HLA-G alleles are accounted for 83.9% of all HLA-G alleles in the population.

Table 2

Table 2

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Association of human leukocyte antigens-G genotypes with resistance and susceptibility to HIV infection

The frequencies of HLA-G*01 : 01:01 heterozygotes and homozygotes were significantly higher in the HIV-resistant women than in the HIV-positive women (48.42 versus 41.15%, 25.26 versus 15.9%, P = 0.005). The G*01 : 01:01 phenotype was significantly associated with resistance to HIV-1 infection (P = 0.002) (Table 3). The women with this genotype are more than two times less likely to be HIV infected [odds ratio (OR) = 2.11, 95% confidence interval (CI): 0.259–0.976).

Table 3

Table 3

The HLA-G*01 : 04:04 phenotype was significantly enriched in the HIV-positive group (P = 0.039) (Table 3). Women with this allele have a two-fold of risk of being infected by HIV-1 than those do not have this allele (OR = 0.502, 95% CI: 0.259–0.976).

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Association of human leukocyte antigen-G genotypes with reduced or increased risk of HIV seroconversion

Kaplan–Meier survival analysis was conducted among 289 women who entered the cohort seronegative to examine whether G*01 : 01:01 was associated with slower rate of seroconversion (Fig. 1a). The results showed that women with G*01 : 01:01 are significantly less likely to seroconvert than women without this allele (Log rank: 10.368, P = 0.001). Whereas, G*01 : 04:04 was significantly associated with an increased rate of seroconversion (Fig. 1b) (Log rank: 6.148, P = 0.013). These findings were consistent with the results from the cross-tab analysis, which provides further confirmation.

Fig. 1

Fig. 1

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Multivariate analysis

As HLA class I and II genes are closely located on chromosome 6, the associations may be attributed to linkage disequilibrium between different HLA alleles. We conducted binary logistic regression analysis to determine whether either of the allele associations was dependent or independent of previously reported HLA class I and class II alleles that are significantly associated with either resistance to susceptibility to HIV-1 infection. Since DPA1*01 : 03:01, DRB1*01 and A*01 have been reported to be associated with resistance to HIV-1 infection in this cohort [70–72,95], we included these alleles in the binary logistic regression analysis with G*01 : 01:01. The results showed that the association of G*01 : 01:01 was independent from DPA1*01 : 03:01, DRB1*01 and A*01(Table 4a). Because DRB1*15 : 03, DPB1*04 : 02 and A*23 : 01 have been reported to be associated with susceptibility to HIV-1 infection in this cohort [70–72,95], we included these alleles in the logistic regression analysis with G*01 : 04:04. The analysis showed that the association of G*01 : 04:04 with susceptibility to HIV-1 infection was independent of DRB1*15 : 03, DPB1*04 : 02 and A*23 : 01 (Table 4b).

Table 4

Table 4

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The primary route of HIV infection is through mucosal heterosexual transmission, with women more at risk of infection than men [1]. HLA-G is of particular interest in HIV-1 infection due to its expression at mucosal surfaces and its influence in immunotolerance and inflammatory responses. Identification of two specific functional HLA-G alleles, G*01 : 01:01 and G*01 : 04:04 associated with resistance or susceptibility to HIV-1 acquisition in the sex workers of the Pumwani sex worker cohort is novel and could have important implications.

G*01 : 01:01 is the most common allele in the Pumwani cohort at 38.5% (Table 2). The fact that it is the most frequent allele in almost all populations [96] suggests that perhaps it has had a selective advantage over the course of human evolution that extends beyond HIV-1 infection. G*01 : 04:04 differs from G*01 : 01:01 by a mutation at codon 110 (CTC → ATC) resulting in an amino acid change of Leucine to iso-Leucine. This mutation is located in exon 3 and could potentially change the protein configuration of the antigen-binding pocket or affect interactions between HLA-G and its killer cell receptor (KIR) ligand. This is one potential explanation of the differences seen but needs to be followed up with functional studies.

Our findings are different from the previously reported associations in a West African (Benin) population [89] and an Italian Caucasian population [90]. In the West African population a significant association was found between G*01 : 05N and protection from HIV-1 infection and an association with susceptibility for two haplotypes, G*01 : 01:08/01 : 04:01 and G*01 : 01:01/01 : 01:08. In the Italian population G*01 : 05N was associated with an increased risk for HIV-1 infection. In our study the G*01 : 05N null allele was not found to be significantly associated with either resistance or susceptibility to HIV-1 infection, even though it had a frequency of 6.43% in the population.

There are several possible explanations for the differences. It is possible that the differences could be due to ethnicity. Africa is a genetically diverse place and thus the West African population could be different from the East African population. There were also significant differences in the HLA-G genotyping methods. For one, the other studies did not use a sequence-based typing method and did not include exon 4, which is necessary to determine all the functional HLA-G alleles. It is possible that the association in the West African study with susceptibility to the haplotype G*01 : 01:08/01 : 04:01 is correlated with G*01 : 04:04 in our study, as the difference between G*01 : 04:01 and G*01 : 04:04 is at codon 267 of exon 4 CCG → CCA. However without typing of exon 4, this remains speculative. Another major difference between our study and the others is the study populations. The HIV-1-resistant women in the Pumwani sex worker cohort have been very well defined and have been followed for more than 25 years [2], whereas the other cohort were HIV negative, but not necessarily resistant and with only cross-sectional analysis. Thus, it is more difficult to ascertain the patient phenotype. Lastly, our study population is larger with better power. The Italian study had 374 individuals enrolled; whereas the West African study had 431 individuals, compared with our 598 used in association analysis. Although these differences by no means discredit their results they are important factors to consider.

Viruses have evolved many mechanisms by which they can evade the host immune system. HIV-1 virus evading cytotoxic T-lymphocytes responses is a classical example. However, HLA class I expression inhibit the function of NK cells and only those cells that lack class I expression are targets for lysis by NK cells. The HIV-1 Nef protein has been shown to downregulate both HLA-A and HLA-B, a plausible means for avoiding host detection [97]. HIV-1 also induces upregulation of interleukin-10 (IL-10) which selectively upregulates HLA-G expression while downregulating HLA class I and II antigens. Importantly, HLA-G is known to interact and inhibit NK cells through specific KIR complexes. Thus, it had been postulated that the lack of expression of HLA-G protein due to the G*01 : 05N null allele might mean less inhibition of NK cells and thus the better elimination of HIV-1-infected cells [89]. Although our study did not correlate with this hypothesis for G*01 : 05N, it did add credence to the idea that HLA-G polymorphism may affect acquisition of HIV-1. A recent study of mucosal expression of sHLA-G found that high levels were associated with HIV-infected commercial sex workers (CSW) when compared with uninfected CSW and uninfected non-CSW [98]. These high mucosal levels contrast to a previous study of theirs that found sHLA-G in plasma had lower expression in HIV-positive individuals [98]. Another study has found that plasma sHLA-G levels vary considerably based on HLA-G polymorphism [99]. Therefore it seems that sHLA-G expression is different depending on which compartment one is investigating, mucosal or blood, and depending on HLA-G polymorphism. Of particular interest is that sHLA-G in the mucosal tract for those who were either heterozygous or homozygous for G*01 : 04:04 was found to be high regardless of whether they were HIV-infected CSW, uninfected CSW or uninfected non-CSW [98]. As the G*01 : 04:04 was significantly associated with susceptibility in our population, it is not unreasonable to speculate that it is possibly due to higher levels of baseline sHLA-G expression in the mucosal tract. However sHLA-G needs to be directly measured in mucosa and functional studies need to be carried out to elucidate the mechanism by which sHLA-G in HIV-1 replication in future studies.

The highly exposed seronegative (HESN) individuals who are resistant to HIV-1 infection have shown that HIV infection can be prevented in nature. Understanding of this natural resistance to HIV-1 infection can help to develop novel preventions to HIV-1. Our study contributes an important piece of the puzzle to further understanding this phenomenon. Functional studies need to be carried out for the identified associations of HLA-G*01 : 01:01 with resistance to HIV-1 infection and HLA-G*01 : 04:04 with susceptible to HIV-1 infection in the Pumwani sex worker cohort. Given the size of the population and the well defined phenotype, we will investigate the function of the identified association in future studies to assess whether HLA-G polymorphisms could be targeted to develop prevention methods to protect against HIV infection.

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This work was supported by Public Health Agency of Canada, and partially supported by the Canadian Institutes of Health Research and the Bill and Melinda Gates Foundation. We thank Tony Kariri for maintaining the databases of Pumwani cohorts at the University of Nairobi and Bing-hua Liang for managing the database at the University of Manitoba. We also thank the staff of the Majengo clinic, Jane Njoki, Jane Makene, Elazabeth Bwibo, Edith Amatiwa for their dedication, and the women of the Pumwani Sex Worker Cohort for their continued participation. Dr Francis A. Plummer is a Canadian Institutes of Health Research Senior Investigator and is currently a Tier I CIHR Canada Research Chair.

W.J.R.T. conducted the HLA-G genotyping typing, data analysis and drafting the article. J.K. and C.W. contributed to cohort management and biological data acquisition, sample collection and review the article. T.B. contributed to HLA-G genotyping typing and review the article. T.B.B. contributed to cohort maintenances and biological data acquisition, sample collection and review the article. F.A.P. contributed to cohort establishment and maintenances, biological data acquisition, sample collection and review the article. M.L. designed the study for HLA-G, contributed to HLA-G genotyping, data analysis, and drafting the article.

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Conflicts of interest

All the authors declare no conflict of interest.

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disease association; highly exposed seronegative; HIV-1; human leukocyte antigens-G; sequence-based typing

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