A genetic polymorphism of FREM1 is associated with resistance against HIV infection in the Pumwani sex worker cohort.

A subgroup of women enrolled in the Pumwani sex worker cohort remain seronegative and PCR negative for human immunodeficiency virus type 1 despite repeated exposure through high-risk sex work. Studies have shown that polymorphisms of genes involved in antigen presentation and viral restriction factors are associated with resistance to HIV infection. To discover other possible genetic factors underlying this HIV-resistant phenotype, we conducted an exploratory nonbiased, low-resolution, genome-wide single-nucleotide polymorphism (SNP) analysis comparing 60 HIV-resistant women to 48 HIV-infected controls. The SNP minor allele rs1552896, in an intron of FREM1, was significantly associated with the resistant phenotype (P = 1.68 × 10(-5); adjusted P = 2.37 × 10(-4); odds ratio [OR], 9.51; 95% confidence interval [CI], 2.82 to 32.05). We expanded the sample size by genotyping rs1552896 in the Pumwani cohort and comparing 114 HIV-resistant women to 609 HIV-infected controls and confirmed the association (P = 1.7 × 10(-4); OR, 2.67; 95% CI, 1.47 to 4.84). To validate the association in a second cohort, we genotyped 783 women enrolled in a mother-child health study and observed the minor allele of rs1552896 enriched in HIV-uninfected women (n = 488) compared to HIV-infected enrollees (n = 295) (P = 0.036; OR, 1.69; 95% CI, 0.98 to 2.93). Quantitative reverse transcription-PCR showed that FREM1 mRNA was highly expressed in tissues relevant for HIV-1 infection, and immunohistochemical analysis revealed that FREM1 protein is expressed in the ectocervical mucosa of HIV-resistant women. The significant association of rs1552896 with an HIV-resistant phenotype, together with the expression profile of FREM1 in tissues relevant to HIV infection, suggests that FREM1 is a potentially novel candidate gene for resistance to HIV infection.

Anti-HIV-1 activity of elafin is more potent than its precursor's, trappin-2, in genital epithelial cells.

Cervicovaginal lavage fluid (CVL) is a natural source of anti-HIV-1 factors; however, molecular characterization of the anti-HIV-1 activity of CVL remains elusive. In this study, we confirmed that CVLs from HIV-1-resistant (HIV-R) compared to HIV-1-susceptible (HIV-S) commercial sex workers (CSWs) contain significantly larger amounts of serine antiprotease trappin-2 (Tr) and its processed form, elafin (E). We assessed anti-HIV-1 activity of CVLs of CSWs and recombinant E and Tr on genital epithelial cells (ECs) that possess (TZM-bl) or lack (HEC-1A) canonical HIV-1 receptors. Our results showed that immunodepletion of 30% of Tr/E from CVL accounted for up to 60% of total anti-HIV-1 activity of CVL. Knockdown of endogenous Tr/E in HEC-1A cells resulted in significantly increased shedding of infectious R5 and X4 HIV-1. Pretreatment of R5, but not X4 HIV-1, with either Tr or E led to inhibition of HIV-1 infection of TZM-bl cells. Interestingly, when either HIV-1 or cells lacking canonical HIV-1 receptors were pretreated with Tr or E, HIV-1 attachment and transcytosis were significantly reduced, and decreased attachment was not associated with altered expression of syndecan-1 or CXCR4. Determination of 50% inhibitory concentrations (IC(50)) of Tr and E anti-HIV-1 activity indicated that E is ∼130 times more potent than its precursor, Tr, despite their equipotent antiprotease activities. This study provides the first experimental evidence that (i) Tr and E are among the principal anti-HIV-1 molecules of CVL; (ii) Tr and E affect cell attachment and transcytosis of HIV-1; (iii) E is more efficient than Tr regarding anti-HIV-1 activity; and (iv) the anti-HIV-1 effect of Tr and E is contextual.

For protection from HIV-1 infection, more might not be better: a systematic analysis of HIV Gag epitopes of two alleles associated with different outcomes of HIV-1 infection.

A subset of women in the Pumwani Sex Worker Cohort, established in 1985 in Nairobi, Kenya, remains uninfected despite repeated high-risk exposure (HIV-exposed, seronegative [HESN]) through active sex work. This HESN phenotype is associated with several alleles of human leukocyte antigens (HLAs) and specific CD8(+) and CD4(+) T cell responses to HIV-1. The associations of HLA alleles with differential HIV-1 infection are most likely due to their different abilities to present antigen and the different immune responses they induce. The characteristics of epitopes of HLA alleles associated with different outcomes of HIV-1 infection might therefore point to a vital clue for developing an effective vaccine. In this study, we systematically analyzed HIV-1 clade A and D Gag CD8(+) T cell epitopes of two HLA class I alleles associated with different outcomes of HIV-1 infection. Binding affinity and off-rates of the identified epitopes were determined. Gamma interferon (IFN-γ) enzyme-linked immunospot (ELISpot) assays with patient peripheral blood mononuclear cells (PBMCs) validated the epitopes. Epitope-specific CD8(+) T cells were further phenotyped for memory markers with tetramer staining. Our study showed that the protective allele A*01:01 recognizes only three Gag epitopes. By contrast, B*07:02, the allele associated with susceptibility, binds 30 epitope variants. These two alleles differ most importantly in the spectrum of Gag epitopes they can present and not in affinity, off-rates, the location of the epitopes, or epitope-specific Tem/Tcm frequencies. The binding of more epitopes and strong IFN-gamma ELISpot responses are associated with susceptibility to HIV-1 infection, while more focused antigen recognition of multiple subtypes is protective. Rational vaccine design should take these observations into account.

The role of G protein gene GNB3 C825T polymorphism in HIV-1 acquisition, progression and immune activation.

BACKGROUND: The GNB3 C825T polymorphism is associated with increased G protein-mediated signal transduction, SDF-1α-mediated lymphocyte chemotaxis, accelerated HIV-1 progression, and altered responses to antiretroviral therapy among Caucasian subjects. The GNB3 825T allele is highly prevalent in African populations, and as such any impact on HIV-1 acquisition or progression rates could have a dramatic impact. This study examines the association of the 825T polymorphism with HIV-1 acquisition, disease progression and immune activation in two African cohorts. GNB3 825 genotyping was performed for enrolees in both a commercial sex worker cohort and a perinatal HIV transmission (PHT) cohort in Nairobi, Kenya. Ex vivo immune activation was quantified by flow cytometry, and plasma chemokine levels were assessed by cytokine bead array. RESULTS: GNB3 genotype was not associated with sexual or vertical HIV-1 acquisition within these cohorts. Within the Pumwani cohort, GNB3 genotype did not affect HIV-1 disease progression among seroconverters or among HIV-1-positive individuals after adjustment for baseline CD4 count. Maternal CD4 decline and viral load increase in the PHT cohort did not differ between genotypes. Multi-parametric flow cytometry assessment of T cell activation (CD69, HLA-DR, CD38) and Treg frequency (CD25(+)FOXP3(+)) found no differences between genotype groups. Plasma SDF-1α, MIP-1ß and TRAIL levels quantified by cytokine bead array were also similar between groups. CONCLUSIONS: In contrast to previous reports, we were unable to provide evidence to suggest that the GNB3 C825T polymorphism affects HIV-1 acquisition or disease progression within African populations. Ex vivo immune activation and plasma chemokine levels were similarly unaffected by GNB3 genotype in both HIV-1-negative and HIV-1-positive individuals. The paucity of studies investigating the impact of GNB3 polymorphism among African populations and the lack of mechanistic studies make it difficult to assess the true biological significance of this polymorphism in HIV-1 infection.

HIV-specific CD8+ T-cell proliferation is prospectively associated with delayed disease progression.

Human immunodeficiency virus (HIV)-specific CD8(+) T-cell proliferation is consistently correlated with enhanced host HIV immune control, but whether proliferative responses are a cause or consequence of immune protection is unclear. We measured Env-specific CD8(+) T-cell proliferation and interferon (IFN)-γ secretion in HIV-infected participants with CD4 counts >200, who then completed 121 person-years of prospective follow-up to monitor HIV disease progression. In all, 13 of 31 participants (42%) reached end point during longitudinal follow-up. Strong Env-specific CD8(+) T-cell proliferation (>10% of CD8(+) T cells) was observed in 14/31 participants at baseline, and this was associated with a longer time to HIV disease progression end point, stratified baseline CD4 count (P=0.016). No associations were observed for IFN-γ ELISPOT responses and progression (P>0.2). Strong proliferation remained significant in multivariate Cox regression analyses (P=0.044) as an independent predictor of delayed HIV disease progression, along with baseline CD4 count (P=0.04). Duration of HIV infection was associated with more rapid progression in univariate, but not multivariate, analysis (P=0.112). Age and baseline viral load were not predictive of progression. HIV-specific CD8(+) T-cell proliferation was a correlate of protective immunity in this prospective study; such responses may be important for HIV vaccine protection.

Epitope mapping of HIV-specific CD8+ T cell responses by multiple immunological readouts reveals distinct specificities defined by function.

The limited success of HIV vaccine candidates to date highlights our need to better characterize protective cell-mediated immunity (CMI). While HIV-specific CD8(+) T cell responses have been defined largely by measuring gamma interferon (IFN-γ), these responses are not always protective, and it is unclear whether the same epitopes would predominate if other functional parameters were examined. Here, we assessed the epitope specificity of HIV-specific CD8(+) T cell responses by multiparametric flow cytometry, measuring five CD8(+) T cell functions (IFN-γ, macrophage inflammatory protein 1ß [MIP-1ß], tumor necrosis factor alpha [TNF-α], interleukin-2 [IL-2], and proliferative capacity) in 24 chronically HIV-infected individuals. Sixty-nine epitope-specific responses to 50 epitopes within p24 were measured. Surprisingly, most epitope-specific responses were IFN-γ negative (50/69 responses). Many responses had polyfunctional (33%) and proliferative (19%) components. An inverse association between IL-2 and proliferation responses was also observed, contrary to what was described previously. We confirm that long-term nonprogressors (LTNP) have more polyfunctional responses and also have higher-magnitude and broader p24-specific proliferation and higher levels of IL-2 and TNF-α production than do progressing controls. Together, these data suggest that the specificity of CD8(+) T cell responses differs depending on the immunological readout, with a 3.5-fold increase in breadth detected by including multiple parameters. Furthermore, the identification of epitopes that elicit polyfunctional responses reinforces the need for the comprehensive evaluation of HIV vaccine candidates, and these epitopes may represent novel targets for CMI-based vaccines.

Molecular definition of vaginal microbiota in East African commercial sex workers.

Resistance to HIV infection in a cohort of commercial sex workers living in Nairobi, Kenya, is linked to mucosal and antiinflammatory factors that may be influenced by the vaginal microbiota. Since bacterial vaginosis (BV), a polymicrobial dysbiosis characterized by low levels of protective Lactobacillus organisms, is an established risk factor for HIV infection, we investigated whether vaginal microbiology was associated with HIV-exposed seronegative (HESN) or HIV-seropositive (HIV(+)) status in this cohort. A subset of 44 individuals was selected for deep-sequencing analysis based on the chaperonin 60 (cpn60) universal target (UT), including HESN individuals (n = 16), other HIV-seronegative controls (HIV-N, n = 16), and HIV(+) individuals (n = 12). Our findings indicate exceptionally high phylogenetic resolution of the cpn60 UT using reads as short as 200 bp, with 54 species in 29 genera detected in this group. Contrary to our initial hypothesis, few differences between HESN and HIV-N women were observed. Several HIV(+) women had distinct profiles dominated by Escherichia coli. The deep-sequencing phylogenetic profile of the vaginal microbiota corresponds closely to BV(+) and BV(-) diagnoses by microscopy, elucidating BV at the molecular level. A cluster of samples with intermediate abundance of Lactobacillus and dominant Gardnerella was identified, defining a distinct BV phenotype that may represent a transitional stage between BV(+) and BV(-). Several alpha- and betaproteobacteria, including the recently described species Variovorax paradoxus, were found to correlate positively with increased Lactobacillus levels that define the BV(-) ("normal") phenotype. We conclude that cpn60 UT is ideally suited to next-generation sequencing technologies for further investigation of microbial community dynamics and mucosal immunity underlying HIV resistance in this cohort.

HIV­exposed seronegative commercial sex workers show a quiescent phenotype in the CD4+ T cell compartment and reduced expression of HIV­dependent host factors.

Studies of human immunodeficiency virus (HIV)-exposed seronegative individuals are crucial to inform vaccine design. In the present study we demonstrated that HIV-exposed seronegative commercial sex workers produce lower levels of proinflammatory cytokines at baseline than HIV-negative control subjects. We also showed that CD4+ T cells of HIV-exposed seronegative commercial sex workers have a characteristically lower level of gene expression that can be seen in differentially expressed genes and systems crucial for HIV replication, such as the T cell receptor pathway and previously identified HIV dependency factors. This apparent lowered activation results in a phenomenon we term "immune quiescence," which may contribute to host resistance to HIV.

Clade-specific evolution mediated by HLA-B*57/5801 in human immunodeficiency virus type 1 clade A1 p24.

HLA-B*57-mediated selection pressure leads to a typical escape pathway in human immunodeficiency virus type 1 (HIV-1) CD8 epitopes such as TW10. Whether this T242N pathway is shared by all clades remains unknown. We therefore assessed the nature of HLA-B*57 selection in a large, observational Kenyan cohort where clades A1 and D predominate. While T242N was ubiquitous in clade D HLA-B*57(+) subjects, this mutation was rare (15%) in clade A1. Instead, P243T and I247L were selected by clade A1-infected HLA-B*57 subjects but not by HLA-B*5801(+) subjects. Our data suggest that clade A1 consensus proline at Gag residue 243 might represent an inherent block to T242N escape in clade A1. We confirmed immunologically that P243T and I247L likely represent escape mutations. HLA-B*57 evolution also differed between clades in the KF11 and IW9 epitopes. A better understanding of clade-specific evolution is important for the development of HIV vaccines in regions with multiple clades.

CD26/dipeptidyl peptidase IV (CD26/DPPIV) is highly expressed in peripheral blood of HIV-1 exposed uninfected female sex workers.

BACKGROUND: Design of effective vaccines against the human immunodeficiency virus (HIV-1) continues to present formidable challenges. However, individuals who are exposed HIV-1 but do not get infected may reveal correlates of protection that may inform on effective vaccine design. A preliminary gene expression analysis of HIV resistant female sex workers (HIV-R) suggested a high expression CD26/DPPIV gene. Previous studies have indicated an anti-HIV effect of high CD26/DPPIV expressing cells in vitro. Similarly, high CD26/DPPIV protein levels in vivo have been shown to be a risk factor for type 2 diabetes. We carried out a study to confirm if the high CD26/DPPIV gene expression among the HIV-R were concordant with high blood protein levels and its correlation with clinical type 2 diabetes and other perturbations in the insulin signaling pathway. RESULTS: A quantitative CD26/DPPIV plasma analysis from 100 HIV-R, 100 HIV infected (HIV +) and 100 HIV negative controls (HIV Neg) showed a significantly elevated CD26/DPPIV concentration among the HIV-R group (mean 1315 ng/ml) than the HIV Neg (910 ng/ml) and HIV + (870 ng/ml, p < 0.001). Similarly a FACs analysis of cell associated DPPIV (CD26) revealed a higher CD26/DPPIV expression on CD4+ T-cells derived from HIV-R than from the HIV+ (90.30% vs 80.90 p = 0.002) and HIV Neg controls (90.30% vs 82.30 p < 0.001) respectively. A further comparison of the mean fluorescent intensity (MFI) of CD26/DPPIV expression showed a higher DPP4 MFI on HIV-R CD4+ T cells (median 118 vs 91 for HIV-Neg, p = 0.0003). An evaluation for hyperglycemia, did not confirm Type 2 diabetes but an impaired fasting glucose condition (5.775 mmol/L). A follow-up quantitative PCR analysis of the insulin signaling pathway genes showed a down expression of NFκB, a central mediator of the immune response and activator of HIV-1 transcription. CONCLUSION: HIV resistant sex workers have a high expression of CD26/DPPIV in tandem with lowered immune activation markers. This may suggest a novel role for CD26/DPPIV in protection against HIV infection in vivo.

Human immunodeficiency virus (HIV) type 1 proviral hypermutation correlates with CD4 count in HIV-infected women from Kenya.

APOBEC3G is an important innate immune molecule that causes human immunodeficiency virus type 1 (HIV-1) hypermutation, which can result in detrimental viral genome mutations. The Vif protein of wild-type HIV-1 counteracts APOBEC3G activity by targeting it for degradation and inhibiting its incorporation into viral particles. Additional APOBEC cytidine deaminases have been identified, such as APOBEC3F, which has a similar mode of action but different sequence specificity. A relationship between APOBEC3F/G and HIV disease progression has been proposed. During HIV-1 sequence analysis of the vpu/env region of 240 HIV-infected subjects from Nairobi, Kenya, 13 drastically hypermutated proviral sequences were identified. Sequences derived from plasma virus, however, lacked hypermutation, as did proviral vif. When correlates of disease progression were examined, subjects with hypermutated provirus were found to have significantly higher CD4 counts than the other subjects. Furthermore, hypermutation as estimated by elevated adenine content positively correlated with CD4 count for all 240 study subjects. The sequence context of the observed hypermutation was statistically associated with APOBEC3F/G activity. In contrast to previous studies, this study demonstrates that higher CD4 counts correlate with increased hypermutation in the absence of obvious mutations in the APOBEC inhibiting Vif protein. This strongly suggests that host factors, such as APOBEC3F/G, are playing a protective role in these patients, modulating viral hypermutation and host disease progression. These findings support the potential of targeting APOBEC3F/G for therapeutic purposes.

An integrative bioinformatic approach for studying escape mutations in human immunodeficiency virus type 1 gag in the Pumwani Sex Worker Cohort.

Human immunodeficiency virus type 1 (HIV-1) is able to evade the host cytotoxic T-lymphocyte (CTL) response through a variety of escape avenues. Epitopes that are presented to CTLs are first processed in the presenting cell in several steps, including proteasomal cleavage, transport to the endoplasmic reticulum, binding by the HLA molecule, and finally presentation to the T-cell receptor. An understanding of the potential of the virus to escape CTL responses can aid in designing an effective vaccine. To investigate such a potential, we analyzed HIV-1 gag from 468 HIV-1-positive Kenyan women by using several bioinformatic approaches that allowed the identification of positively selected amino acids in the HIV-1 gag region and study of the effects that these mutations could have on the various stages of antigen processing. Correlations between positively selected residues and mean CD4 counts also allowed study of the effect of mutation on HIV disease progression. A number of mutations that could create or destroy proteasomal cleavage sites or reduce binding affinity of the transport antigen processing protein, effectively hindering epitope presentation, were identified. Many mutations correlated with the presence of specific HLA alleles and with lower or higher CD4 counts. For instance, the mutation V190I in subtype A1-infected individuals is associated with HLA-B*5802 (P = 4.73 x 10(-4)), a rapid-progression allele according to other studies, and also to a decreased mean CD4 count (P = 0.019). Thus, V190I is a possible HLA escape mutant. This method classifies many positively selected mutations across the entire gag region according to their potential for immune escape and their effect on disease progression.

Negative mucosal synergy between Herpes simplex type 2 and HIV in the female genital tract.

OBJECTIVE: There is substantial epidemiological evidence that infection by Herpes simplex virus type 2 (HSV2) enhances both HIV susceptibility and subsequent sexual transmission. Both infections are extremely common in female sex workers (FSWs) in sub-Saharan Africa, and up to 80% of new HIV infections in urban men in the region are acquired via transactional sex. The present study aimed to elucidate the mucosal immune interactions between HIV and HSV2 in the genital tract. METHODS: Endocervical immune cell populations, cytokine/chemokine protein levels in cervico-vaginal secretions and cervical immune gene expression profiles were measured in a well-defined cohort of HIV-infected and uninfected Kenyan FSWs. Associations between the genital immune milieu and infection by and/or shedding of common genital co-pathogens were examined. RESULTS: HIV-infected FSWs were much more likely to be infected by HSV2, and to shed HSV2 DNA in the genital tract. There was also a profound negative 'mucosal synergy' between these viruses. In HIV uninfected FSWs, HSV2 infection was associated with a ten-fold increase in cervical immature dendritic cells (iDC) expressing DC-SIGN, and a three-fold increase in cervical CD4+ T cells expressing CCR5. HIV infection was associated with iDC depletion in the cervix, and with increased HSV2 genital reactivation, which in turn was associated with HIV shedding levels. CONCLUSIONS: The findings suggest a mucosal vicious circle in which HSV2 infection increases HIV target cells in the genital mucosa, subsequent HIV infection impairs HSV2 mucosal immune control, and local HSV2 reactivation enhances both HSV2 and HIV transmission.

"How I Wish This Thing Was Initiated 100 Years Ago!" Willingness to Take Daily Oral Pre-Exposure Prophylaxis among Men Who Have Sex with Men in Kenya.

BACKGROUND: The MSM population in Kenya contributes to 15% of HIV incidence. This calls for innovative HIV prevention interventions. Pre-exposure prophylaxis (PrEP) has been efficacious in preventing HIV among MSM in trials. There is limited data on the willingness to take daily oral PrEP in sub-Sahara Africa. PrEP has not been approved for routine use in most countries globally. This study aimed to document the willingness to take PrEP and barriers to uptake and adherence to PrEP in Kenya. The findings will inform the design of a PrEP delivery program as part of the routine HIV combination prevention. METHODS: Eighty MSM were recruited in 2 Counties in December 2013. Quantitative data on sexual behaviour and willingness to take PrEP were collected using semi-structured interviews and analysed using SPSS. Qualitative data on knowledge of PrEP, motivators and barriers to uptake and adherence to PrEP were collected using in-depth interviews and FGDs and analysed using Nvivo. Analysis of data in willingness to take PrEP was conducted on the HIV negative participants (n = 55). RESULTS: 83% of MSM were willing to take daily oral HIV PrEP. Willingness to take PrEP was higher among the bi-sexual and younger men. Motivators for taking PrEP were the need to stay HIV negative and to protect their partners. History of poor medication adherence, fear of side effects and HIV stigma were identified as potential barriers to adherence. Participants were willing to buy PrEP at a subsidized price. CONCLUSIONS: There is willingness to take PrEP among MSM in Kenya and there is need to invest in targeted education and messaging on PrEP to enhance adherence, proper use and reduce stigma in the general population and among policy makers.

Influence of HLA class I haplotypes on HIV-1 seroconversion and disease progression in Pumwani sex worker cohort.

We examined the effect of HLA class I haplotypes on HIV-1 seroconversion and disease progression in the Pumwani sex worker cohort. This study included 595 HIV-1 positive patients and 176 HIV negative individuals. HLA-A, -B, and -C were typed to 4-digit resolution using sequence-based typing method. HLA class I haplotype frequencies were estimated using PyPop 32-0.6.0. The influence of haplotypes on time to seroconversion and CD4+ T cell decline to <200 cells/mm3 were analyzed by Kaplan-Meier analysis using SPSS 13.0. Before corrections for multiple comparisons, three 2-loci haplotypes were significantly associated with faster seroconversion, including A*23∶01-C*02∶02 (p = 0.014, log rank(LR) = 6.06, false-discovery rate (FDR) = 0.056), B*42∶01-C*17∶01 (p = 0.01, LR = 6.60, FDR = 0.08) and B*07∶02-C*07∶02 (p = 0.013, LR = 6.14, FDR = 0.069). Two A*74∶01 containing haplotypes, A*74∶01-B*15∶03 (p = 0.047, LR = 3.942, FDR = 0.068) and A*74∶01-B*15∶03-C*02∶02 (p = 0.045, LR = 4.01, FDR = 0.072) and B*14∶02-C*08∶02 (p = 0.021, LR = 5.36, FDR = 0.056) were associated with slower disease progression. Five haplotypes, including A*30∶02-B*45∶01 (p = 0.0008, LR = 11.183, FDR = 0.013), A*30∶02-C*16∶01 (p = 0.015, LR = 5.97, FDR = 0.048), B*53∶01-C*04∶01 (p = 0.010, LR = 6.61, FDR = 0.08), B*15∶10-C*03∶04 (p = 0.031, LR = 4.65, FDR = 0.062), and B*58∶01-C*03∶02 (p = 0.037, LR = 4.35, FDR = 0.066) were associated with faster progression to AIDS. After FDR corrections, only the associations of A*30∶02-B*45∶01 and A*30∶02-C*16∶01 with faster disease progression remained significant. Cox regression and deconstructed Kaplan-Meier survival analysis showed that the associations of haplotypes of A*23∶01-C*02∶02, B*07∶02-C*07∶02, A*74∶01-B*15∶03, A*74∶01-B*15∶03-C*02∶02, B*14∶02-C*08∶02 and B*58∶01-C*03∶02 with differential seroconversion or disease progression are due to the dominant effect of a single allele within the haplotypes. The true haplotype effect was observed with A*30∶02-B*45∶01, A*30∶02-C*16∶02, B*53∶01-C*04∶01 B*15∶10-C*03∶04, and B*42∶01-C*17∶01. In these cases, the presence of both alleles accelerated the disease progression or seroconversion than any of the single allele within the haplotypes. Our study showed that the true effects of HLA class I haplotypes on HIV seroconversion and disease progression exist and the associations of HLA class I haplotype can also be due to the dominant effect of a single allele within the haplotype.

Mucosal serpin A1 and A3 levels in HIV highly exposed sero-negative women are affected by the menstrual cycle and hormonal contraceptives but are independent of epidemiological confounders.

OBJECTIVE: Serpins (serine protease inhibitors) are associated with protection against HIV infection. Here, we characterized mucosal serpin expression in the genital tract of HIV highly exposed sero-negative (HESN) women meeting our epidemiological definition of HIV resistance in relation to epidemiological variables. METHODS: Cervicovaginal lavage (CVL) fluid and plasma were collected from 84 HIV-resistant, 54 HIV-uninfected, and 66 HIV-infected female commercial sex workers. Serpin A1 and A3 concentrations were measured by ELISA and compared with clinical information. RESULTS: Mucosal serpin A1 was elevated during proliferative phase over secretory phase (P = 0.017*), while A3 remained similar (P = 0.25). Plasma and mucosal serpin A1/A3 levels were not associated with each other and appeared compartment specific (r = 0.21, r = 0.056). Serpin A1/A3 expression did not associate with age (r = 0.009, r = -0.06), duration of sex work (r = 0.13, r = -0.10), clients per day (r = -0.11, r = -0.02), concurrent STIs (P = 0.36, P = 0.15), but was lower in women using hormonal contraceptives (P = 0.034, P = 0.008). Mucosal serpin A1/A3 levels in HIV-infected individuals were not significantly different with disease status as determined by plasma CD4(+) T-cell counts (P = 0.94, P = 0.30). CONCLUSION: This study shows the relationship of serpins to the menstrual cycle and hormonal contraceptives, as well as their independence to epidemiological sexual confounders. This information provides a broader understanding of innate components of the mucosal immune system in women.

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

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.

Immunogenicity of sequences around HIV-1 protease cleavage sites: potential targets and population coverage analysis for a HIV vaccine targeting protease cleavage sites.

Developing an effective preventative vaccine against HIV-1 has proved to be a great challenge. The classical and proven vaccine approach has failed so far or produced a modest effect, new approaches are needed. In this study we evaluated the immunogenicity of the sequences around the protease cleavage sites (PCS) and the population coverage of a vaccine targeting HIV-1 PCS. The sequence conservation was evaluated by comparing entropy score of sequences around PCS with Gag and Pol. The immunogenicity of sequences around the 12 PCS (+10/-10 amino acids) was analyzed by identifying epitopes of HLA class I alleles in PCS region using four approaches: (1) identification of previously reported HLA class I allele epitopes around PCS region; (2) screening and validating epitopes of 8 HLA class I alleles common to most world populations using iTopia Epitope Discovery system and IFN-γ ELISpot assays; (3) screening of 151 patients of Pumwani cohort for PBMC IFN-γ ELISPOT responses to the subtype A and D consensus around PCS region; and (4) prediction of HLA alleles with epitopes around the PCS using NetMHCpan. Population coverage was calculated using the web-based analysis tool of the Immune Epitope Database based on HLA class I genotype frequencies from dbMHC database. The results showed that many HLA class I alleles have multiple epitopes in the 12 PCS regions, indicating sequence immunogenicity around PCS. Multiple epitopes of many HLA class I alleles common to >95% world populations have been identified around the 12 PCS region. Targeting these sites is a feasible vaccine approach.

Enumeration of sex workers in the central business district of Nairobi, Kenya.

Accurate program planning for populations most at risk for HIV/STI acquisition requires knowledge of the size and location where these populations can best be reached. To obtain this information for sex workers operating at 137 hotspots in the central business district (CBD) in Nairobi, Kenya, we utilized a combined mapping and capture-recapture enumeration exercise. The majority of identified hotspots in this study were bars. Based on this exercise, we estimate that 6,904 male and female sex workers (95% confidence intervals, 6690 and 7118) were working nightly in the Nairobi CBD in April 2009. Wide ranges of captures per spot were obtained, suggesting that relatively few hot spots (18%) contain a relatively high proportion of the area's sex workers (65%). We provide geographic data including relatively short distances from hotspots to our dedicated sex worker outreach program in the CBD (mean<1 km), and clustering of hotspots within a relatively small area. Given the size covered and areas where sex work is likely taking place in Nairobi, the estimate is several times lower than what would be obtained if the entire metropolitan area was enumerated. These results have important practical and policy implications for enhancing HIV/STI prevention efforts.

HIV postexposure prophylaxis in an urban population of female sex workers in Nairobi, Kenya.

OBJECTIVES: To assess biological and behavioral factors in HIV-uninfected female sex workers (FSWs) accessing postexposure prophylaxis (PEP) and to characterize the circumstances preceding PEP, time to access, and adherence. METHODS: Participants were HIV-uninfected FSWs enrolled in an HIV care and prevention program in Nairobi, Kenya. Those accessing PEP between 2009 and 2010 were enrolled and compared with controls. Multiple logistic regression models were used to compare PEP-related biological and behavioral parameters. RESULTS: PEP users (n = 326) had been involved in sex work for a shorter duration than nonusers [n = 2570; 3.3 vs. 5.1 yrs, AOR: 0.92, 95% confidence interval (CI): 0.89 to 0.95] and were less likely to report a regular partner (54.9% vs. 72.5%, AOR: 0.52, 0.39 to 0.68). PEP use was associated with gonorrhea infection (6.9% vs. 2.6%, AOR: 2.37, 95% CI: 1.34 to 4.21) and alcohol use (84.3% vs. 76.1%, AOR: 1.58, 95% CI: 1.09 to 2.31), but with increased condom use (85.1% vs. 68.2%, AOR 1.80, 95% CI: 1.38-2.35) and a history of prior HIV testing (89.2 vs. 76.2%, AOR: 2.22, 95% CI: 1.45 to 3.40). Reasons for PEP access centered on issues of client mistrust. The median time from exposure to PEP was 18 hours, with an estimated adherence of 49%. Precise PEP efficacy could not be calculated, but HIV incidence was 0.6% in users (2/326) compared with 2.1% (30/1417) in nonusers (Cox regression P = 0.35). CONCLUSIONS: "PEP services were accessed by 10% of FSWs during the study period and were not implicated in any incident HIV cases." Users had indicators of increased sexual risk and higher health care literacy. Increasing PEP access and compliance in FSWs may be an important HIV prevention strategy.