Terry B. Ball

HIV-1-specific mucosal IgA in a cohort of HIV-1-resistant Kenyan sex workers.

OBJECTIVES Most HIV-1 transmission is sexual; therefore, immune responses in the genital mucosa may be important in mediating protection against HIV infection. This study examined HIV-1-specific mucosal IgA in a cohort of HIV-1-resistant Kenyan female sex workers. METHODS HIV-1-specific immune responses were compared in HIV-1-resistant and HIV-1-infected sex workers, and in lower risk uninfected women. Cervical and vaginal samples from each group were tested for HIV-1-specific IgA and IgG by enzyme immunoassay. Systemic T-helper lymphocyte cell responses to HIV-1 envelope peptide epitopes were assayed using an interleukin 2 bioassay. HIV-1 risk-taking behaviours were assessed using standardized questionnaires. RESULTS HIV-1-specific IgA was present in the genital tract of 16 out of 21 (76%) HIV-1-resistant sex workers, five out of 19 (26%) infected women, and three out of 28 (11%) lower risk women (P < 0.0001). Among lower risk women, the presence of HIV-1-specific IgA was associated with HIV-1 risk-taking behaviour. Systemic T-helper lymphocyte responses to HIV-1 envelope peptides were present in 11 out of 20 (55%) HIV-1-resistant women, four out of 18 (22%) infected women, and one out of 25 (4%) lower risk women (P < 0.001). T-helper lymphocyte responses did not correlate with the presence or titre of virus-specific mucosal IgA in any study group. CONCLUSIONS HIV-1-specific IgA is present in the genital tract of most HIV-1-resistant Kenyan sex workers, and of a minority of lower risk uninfected women, where it is associated with risk-taking behaviour. These data suggest a role for mucosal HIV-1-specific IgA responses in HIV-1 resistance, independent of host cellular responses.

Elevated elafin/trappin-2 in the female genital tract is associated with protection against HIV acquisition.

Objectives:Globally, heterosexual intercourse is the primary route of HIV-1 (HIV) transmission. It follows that mechanisms that protect against HIV infection are likely operative at the genital mucosa. In HIV-resistant Kenyan sex workers who are highly exposed to HIV infection yet remain uninfected, protection correlates with HIV-specific immune responses and genetic factors. However, these factors do not entirely explain this model of natural immunity to HIV. We hypothesized that protection may be mediated by innate immune proteins in the genital tract of HIV-resistant sex workers. Design and methods:The genital proteome of mucosal secretions from HIV-resistant women was examined using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry. Cervical lavage samples were collected from 315 HIV-resistant, HIV-uninfected and HIV-infected commercial sex workers. Results:Univariate analysis identified a 6 kDa biomarker of HIV resistance in genital secretions from these women. This protein was identified by tandem mass spectrometry as elafin and was found to be overexpressed in HIV-resistant women compared with HIV-uninfected (P = 0.001) and infected (P = 0.002) women. The elevated levels of elafin/trappin-2 in HIV-resistant women were confirmed using ELISA. The prospective association of elevated cervicovaginal elafin/trappin-2 levels with protection from HIV acquisition was then confirmed in an independent cohort of high-risk female sex workers. Conclusion:Using a unique proteomics approach in a large scale, cross-sectional cohort study, we identified elafin/trappin-2 as a novel innate immune factor, which is highly associated with resistance. This association was confirmed within an independent, prospective cohort study. Genital tract elafin/trappin-2 levels constitute a natural correlate of HIV protection in humans.

Elevated T Cell Counts and RANTES Expression in the Genital Mucosa of HIV-1–Resistant Kenyan Commercial Sex Workers

The initial site of exposure to human immunodeficiency virus (HIV)-1 during heterosexual transmission occurs in the genital tract. Although the majority of immunological studies have focused on the immune response to HIV-1 at the systemic level, our understanding of tissue-specific immunity is deficient. The goal of the present study was to characterize T cell populations found in the cervix of women shown to be resistant to infection by HIV-1. Levels of both systemic and cervical mucosal lymphocytes were compared between HIV-1-resistant, HIV-1-uninfected, and HIV-1-infected commercial sex workers (CSWs) as well as HIV-1-uninfected non-CSW control subjects at low risk for exposure. The HIV-1-resistant CSWs had increased cervical CD4+ and CD8+ T cell counts, compared with the HIV-1-uninfected CSWs; importantly, these increases were not reflected in the systemic lymphocyte compartment. There was a 2-fold increase in CD4+ T cell counts in the HIV-1-resistant CSWs, compared with both the HIV-1-infected and the HIV-1-uninfected CSWs. Expression of the HIV-1 coreceptors CCR5 and CXCR4 was also determined, and cytokine and beta chemokine levels in the genital mucosa were assessed. The HIV-1-resistant CSWs had a 10-fold increase in RANTES expression, compared with the HIV-1-uninfected CSWs. This is the first study to show elevated levels of beta chemokines and CD4+ T cells in the genital tracts of women who are exposed to HIV-1 and yet are uninfected.

A distinct cytokine and chemokine profile at the genital mucosa is associated with HIV-1 protection among HIV-exposed seronegative commercial sex workers.

The predominance of HIV-1 sexual transmission requires a greater understanding of the interaction between HIV-1 and the mucosal immune system. The study of HIV-1-exposed seronegative (HESN) individuals serves as a model to identify the correlates of protection and to aid in microbicide development. A total of 22 cytokines/chemokines were analyzed at the systemic and mucosal compartments in 57 HESN, 51 HIV-1-negative, and 67 HIV-1-infected commercial sex workers from Nairobi, Kenya. HESN individuals had significantly lower expression of monokine induced by interferon-γ (MIG), interferon-γ-induced protein 10 (IP-10), and interleukin-1α (IL-1α) in their genital mucosa compared with controls. HESN cytokine expression also distinctly correlates with mucosal antiproteases, suggesting that HESN individuals have a unique pattern of mucosal chemokine/cytokine expression, which may result in reduced trafficking at the mucosa. These data support the immune quiescence model of protection, whereby lower T-cell activation/recruitment at the mucosal compartment reduces HIV-1 target cell numbers and is an important component of natural protection from HIV-1.

Polymorphisms in IRF-1 associated with resistance to HIV-1 infection in highly exposed uninfected Kenyan sex workers

Objective:To determine the correlation between polymorphisms in the IL-4 gene cluster and resistance to HIV-1 infection. Design:A cross-sectional genetic analysis of polymorphisms within the IL-4 gene cluster was conducted in a well-described female sex worker cohort from Nairobi, Kenya, known to exhibit differential susceptibility to HIV-1 infection. Methods:Microsatellite genotyping was used to screen six microsatellite markers in the IL-4 gene cluster for associations with HIV-1 resistance. Further analysis of the interferon regulatory factor 1 (IRF-1) gene was conducted by genomic sequencing. Associations between IRF-1 gene polymorphisms and the HIV-1 resistance phenotype were determined using the chi-square test and Kaplan–Meier survival analysis. The functional consequence of IRF-1 polymorphism was conducted by quantitative Western blot. Results:Three polymorphisms in IRF-1, located at 619, the microsatellite region and 6516 of the gene, showed associations with resistance to HIV-1 infection. The 619A, 179 at IRF-1 microsatellite and 6516G alleles were associated with the HIV-1-resistant phenotype and a reduced likelihood of seroconversion. Peripheral blood mononuclear cells from patients with protective IRF-1 genotypes exhibited significantly lower basal IRF-1 expression and reduced responsiveness to exogenous IFN-γ stimulation. Conclusion:Polymorphisms in the IRF-1 gene are associated with resistance to infection by HIV-1 and a lowered level of IRF-1 protein expression. This study adds IRF-1, a transcriptional immunoregulatory gene, to the list of genetic correlates of altered susceptibility to HIV-1. This is the first report suggesting that a viral transcriptional regulator might contribute to resistance to HIV-1. Further functional analysis on the role of IRF-1 polymorphisms and HIV-1 resistance is underway.

Acute sexually transmitted infections increase human immunodeficiency virus type 1 plasma viremia, increase plasma type 2 cytokines, and decrease CD4 cell counts.

In Kenya, the median incubation time to AIDS in seroconverting sex workers is 4 years; this incubation time is specific to female sex workers. We studied the influence of acute sexually transmitted infections (STIs) on several immunologic parameters in 32 human immunodeficiency virus type 1 (HIV-1)-positive and 10 HIV-1-negative women sex workers who were followed for 1-5 months. Plasma cytokines, soluble cytokine receptors, CD4 and CD8 T cell counts, and HIV-1 plasma viremia were quantitated before, during, and after episodes of STI. Increases in interleukin (IL)-4, IL-6, IL-10, soluble tumor necrosis factor (TNF)-alpha, and viremia and a decline in CD4(+) T cell counts occurred during gonococcal cervicitis and returned to baseline after treatment. Increases in viremia correlated with increased IL-4 and decreased IL-6 concentrations. Similar changes were seen among women with acute pelvic inflammatory disease. Acute bacterial STI resulted in increased HIV-1 viremia. This may be mediated through increased inflammatory cytokines or through modulation of immune responses that control HIV-1 viremia.

Resistance to HIV-1 infection among highly exposed sex workers in Nairobi: what mediates protection and why does it develop?

Variability in susceptibility to infection and disease caused by infectious agents is a characteristic of all populations. Among susceptible individuals exposed to an infection, not all become infected and among infected individuals, not all develop disease. It seems logical that variability in susceptibility to infection and disease would apply to infection and disease with human immunodeficiency viruses. However, until recently, it has been generally held that there is no natural immunity to HIV-1 and that once infected, all individuals would ultimately succumb to AIDS.

Identification of differentially expressed proteins in the cervical mucosa of HIV-1-resistant sex workers.

Novel tools are necessary to understand mechanisms of altered susceptibility to HIV-1 infection in women of the Pumwani Sex Worker cohort, Kenya. In this cohort, more than 140 of the 2000 participants have been characterized to be relatively resistant to HIV-1 infection. Given that sexual transmission of HIV-1 occurs through mucosal surfaces such as that in the cervicovaginal environment, our hypothesis is that innate immune factors in the genital tract may play a role in HIV-1 infection resistance. Understanding this mechanism may help develop microbicides and/or vaccines against HIV-1. A quantitative proteomics technique (2D-DIGE: two-dimensional difference in-gel electrophoresis) was used to examine cervical mucosa of HIV-1 resistant women ( n = 10) for biomarkers of HIV-1 resistance. Over 15 proteins were found to be differentially expressed between HIV-1-resistant women and control groups ( n = 29), some which show a greater than 8-fold change. HIV-1-resistant women overexpressed several antiproteases, including those from the serpin B family, and also cystatin A, a known anti-HIV-1 factor. Immunoblotting for a selection of the identified proteins confirmed the DIGE volume differences. Validation of these results on a larger sample of individuals will provide further evidence these biomarkers are associated with HIV-1 resistance and could help aid in the development of effective microbicides against HIV-1.

Comprehensive proteomic study identifies serpin and cystatin antiproteases as novel correlates of HIV-1 resistance in the cervicovaginal mucosa of female sex workers.

Not all individuals exposed to HIV-1 become infected, and evidence from HIV-1 highly exposed seronegative women (HIV-1-resistant) suggests that mucosal factors in the female genital tract, the first site of contact for the virus, are playing a role. To better understand factors mediating protection from HIV-1, we performed a large clinical study using the tools of systems biology to fully characterize the cervicovaginal mucosa proteome in HIV-1-resistant women. Cervicovaginal lavage fluid was collected from 293 HIV-1-resistant, uninfected, and infected sex workers and analyzed by 2D-LC LTQ-FT-MS. Of the more than 360 unique proteins identified, 41 were differentially abundant (>3-fold cutoff) in HIV-1-resistant women. The majority of over-abundant proteins were antiproteases (>40%), some with described anti-inflammatory and anti-HIV-1 activity. Quantification of specific anti-HIV-1 antiproteases Serpin A1, Serpin A3, and Cystatin B and an epithelial antiprotease A2ML1 found them to be significantly over-abundant in HIV-1-resistant women (p = 0.004; p = 0.046; p = 0.0003; and p = 0.04, respectively). Expression levels were not correlated to sexual practices or other epidemiological factors. Mucosal antiprotease levels correlated with pro-inflammatory cytokine concentration (p = <0.0001), but independently of pro-inflammatory cytokine levels in HIV-1-resistant women including TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, and IL-8. This comprehensive systems biology approach identifies mucosal serpins and cystatins as novel correlates of HIV-1-resistance. This represents the first study characterizing these factors in the female genital tract.

A trim5α exon 2 polymorphism is associated with protection from Hiv-1 infection in the Pumwani sex worker cohort

Objective:The innate immune component TRIM5α has the ability to restrict retrovirus infection in a species-specific manner. TRIM5α of some primate species restricts infection by HIV-1, whereas human TRIM5α lacks this specificity. Previous studies have suggested that certain polymorphisms in human TRIM5α may enhance or impair the proteins affinity for HIV-1. This study investigates the role of TRIM5α polymorphisms in resistance/susceptibility to HIV-1 within the Pumwani sex worker cohort in Nairobi, Kenya. A group of women within this cohort remain HIV-1-seronegative and PCR-negative despite repeated exposure to HIV-1 through active sex work. Design:A 1 kb fragment of the TRIM5α gene, including exon 2, from 1032 women enrolled in the Pumwani sex worker cohort was amplified and sequenced. Single-nucleotide polymorphisms (SNPs) and haplotypes were compared between HIV-1-positive and resistant women. Methods:The TRIM5α exon 2 genomic fragment was amplified, sequenced and genotyped. Pypop32-0.6.0 was used to determine SNP and haplotype frequencies and statistical analysis was carried out using SPSS-13.0 for Windows. Results:A TRIM5α SNP (rs10838525) resulting in the amino acid change from arginine to glutamine at codon 136, was enriched in HIV-1-resistant individuals [P = 1.104E-05; odds ratio (OR) 2.991; 95% confidence interval (CI) 1.806–4.953] and women with 136Q were less likely to seroconvert (P = 0.002; log-rank 12.799). Wild-type TRIM5α exon 2 was associated with susceptibility to HIV-1 (P = 0.006; OR 0.279; 95% CI 0.105–0.740) and rapid seroconversion (P = 0.001; log-rank 14.475). Conclusions:Our findings suggest that a shift from arginine to glutamine at codon 136 in the coiled-coil region of TRIM5α confers protection against HIV-1 in the Pumwani sex worker cohort.