Joshua Kimani

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.

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.

Identification of preferential CD4 + T-cell targets for HIV infection in the cervix

A better understanding of the cellular targets of HIV infection in the female genital tract may inform HIV prevention efforts. Proposed correlates of cellular susceptibility include the HIV co-receptor CCR5, peripheral homing integrins, and immune activation. We used a CCR5-tropic pseudovirus to quantify HIV entry into unstimulated endocervical CD4(+) T cells collected by cytobrush. Virus entry was threefold higher into cervix-derived CD4(+) T cells than blood, but was strongly correlated between these two compartments. Cervix-derived CD4(+) T cells expressing CD69, α(4)β(7), or α(4)β(1) were preferential HIV targets; this enhanced susceptibility was strongly correlated with increased CCR5 expression in α(4)β(7)(+) and CD69(+) CD4(+) T cells, and to a lesser extent in α(4)β(1)(+) CD4(+) T cells. Direct binding of gp140 to integrins was not observed, integrin inhibitors had no effect on virus entry, and pseudotypes with an env that preferentially binds α(4)β(7) still demonstrated enhanced entry into α(4)β(1)(+) cells. In summary, a rapid and sensitive HIV entry assay demonstrated enhanced susceptibility of activated endocervical CD4(+) T cells, and those expressing α(4)β(7) or α(4)β(1). This may relate to increased CCR5 expression by these cell subsets, but did not appear to be due to direct interaction of α(4)β(7) or α(4)β(1) with HIV envelope.

Acting locally: innate mucosal immunity in resistance to HIV-1 infection in Kenyan commercial sex workers

Cohort studies of female commercial sex workers (CSWs) in Kenya were among the first to identify highly HIV-1-exposed seronegative (HESN) individuals. As natural resistance is usually mediated by innate immune mechanisms, we focused on determining whether expression and function of innate signaling pathways were altered locally in the genital mucosa of HESN CSWs. Our results demonstrated that selected pattern-recognition receptors (PRRs) were significantly reduced in expression in cervical mononuclear cells (CMCs) from HESN compared with the new HIV-negative (HIV-N) and HIV-positive (HIV-P) groups. Although baseline levels of secreted cytokines were reduced in CMCs of HESN, they were highly stimulated following exposure to ssRNA40 in vitro. Importantly, cervical epithelial cells from HESN also expressed reduced levels of PRRs, but Toll-like receptor 3 (TLR3) and TLR7 as well as nuclear factor-κB and activator protein 1 were highly expressed and activated. Lastly, inflammatory cytokines interleukin (IL)-1β, IL-8, and RANTES (regulated and normal T cell expressed and secreted) were detected at lower levels in cervicovaginal lavage of HESN compared with the HIV-N and HIV-P groups. Overall, our study reveals a local microenvironment of HIV resistance in the genital mucosa consisting of a finely controlled balance of basal immune quiescence with a focused and potent innate anti-viral response critical to resistance to sexual transmission of HIV-1.

Stable CD4 Expression and Local Immune Activation in the Ectocervical Mucosa of HIV-Infected Women

Studies using genital tissue samples from HIV-infected women might provide important information about HIV susceptibility and transmission. In this study, ectocervical biopsies were obtained from 20 HIV-seropositive (HIV+) Kenyan female sex workers (FSW) and 20 HIV-seronegative lower risk (HIV− LR) women. To control for the impact of sex work, 20 HIV− FSW were also recruited. Immune molecules were assessed in situ by immunohistochemistry and for mRNA expression by quantitative PCR. The HIV+ women were reportedly infected for a median of 3 y (1–21 y), with a median viral load of 11,735 copies/ml (20–648,000 copies/ml). These women had significantly lower CD4 blood cell counts than the HIV− LR women but comparable levels of CD4 expression in ectocervix. Whereas cellular markers were similar between the HIV+ group and the HIV− LR women, the HIV-binding molecules CCR5, dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin, and mannose receptor as well as the inflammatory markers CD69, IFN-γ, IL-6, and IL-22 were significantly upregulated in the HIV+ group. As compared with the HIV− FSW women, the HIV+ women had significantly upregulated levels of CD4, CD3, CCR5, Langerin, dendritic cell–specific intercellular adhesion molecule-3–grabbing nonintegrin, and mannose receptor as well as inflammatory cytokines. The CD4 cell depletion previously seen in the gut mucosa of HIV-infected individuals was thus not observed in the ectocervical mucosa. Stable CD4 cell expression and local immune activation in the lower female genital tract may promote viral replication and genital shedding and increase the risk of sexual HIV transmission.

Subtype-Specific Differences in Gag-Protease-Driven Replication Capacity are Consistent with Inter-Subtype Differences in HIV-1 Disease Progression.

ABSTRACT There are marked differences in the spread and prevalence of HIV-1 subtypes worldwide, and differences in clinical progression have been reported. However, the biological reasons underlying these differences are unknown. Gag-protease is essential for HIV-1 replication, and Gag-protease-driven replication capacity has previously been correlated with disease progression. We show that Gag-protease replication capacity correlates significantly with that of whole isolates (r = 0.51; P = 0.04), indicating that Gag-protease is a significant contributor to viral replication capacity. Furthermore, we investigated subtype-specific differences in Gag-protease-driven replication capacity using large well-characterized cohorts in Africa and the Americas. Patient-derived Gag-protease sequences were inserted into an HIV-1 NL4-3 backbone, and the replication capacities of the resulting recombinant viruses were measured in an HIV-1-inducible reporter T cell line by flow cytometry. Recombinant viruses expressing subtype C Gag-proteases exhibited substantially lower replication capacities than those expressing subtype B Gag-proteases (P < 0.0001); this observation remained consistent when representative Gag-protease sequences were engineered into an HIV-1 subtype C backbone. We identified Gag residues 483 and 484, located within the Alix-binding motif involved in virus budding, as major contributors to subtype-specific replicative differences. In East African cohorts, we observed a hierarchy of Gag-protease-driven replication capacities, i.e., subtypes A/C < D < intersubtype recombinants (P < 0.0029), which is consistent with reported intersubtype differences in disease progression. We thus hypothesize that the lower Gag-protease-driven replication capacity of subtypes A and C slows disease progression in individuals infected with these subtypes, which in turn leads to greater opportunity for transmission and thus increased prevalence of these subtypes. IMPORTANCE HIV-1 subtypes are unevenly distributed globally, and there are reported differences in their rates of disease progression and epidemic spread. The biological determinants underlying these differences have not been fully elucidated. Here, we show that HIV-1 Gag-protease-driven replication capacity correlates with the replication capacity of whole virus isolates. We further show that subtype B displays a significantly higher Gag-protease-mediated replication capacity than does subtype C, and we identify a major genetic determinant of these differences. Moreover, in two independent East African cohorts we demonstrate a reproducible hierarchy of Gag-protease-driven replicative capacity, whereby recombinants exhibit the greatest replication, followed by subtype D, followed by subtypes A and C. Our data identify Gag-protease as a major determinant of subtype differences in disease progression among HIV-1 subtypes; furthermore, we propose that the poorer viral replicative capacity of subtypes A and C may paradoxically contribute to their more efficient spread in sub-Saharan Africa.

Clade-Specific Evolution Mediated by HLA-B*57/5801 in Human Immunodeficiency Virus Type 1 Clade A1 p24

ABSTRACT 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.

The Role of Chlamydia trachomatis in High-Risk Human Papillomavirus Persistence Among Female Sex Workers in Nairobi, Kenya.

Background Little is known about risk factors for persistent high-risk human papillomavirus (hrHPV) infection in low-income settings, and prior research has not quantified the relative duration of hrHPV infections stratified by risk factors. We compared the duration of hrHPV infection among female sex workers (FSWs) by exposure to sexually transmitted infections (STIs), using a highly sensitive biomarker assay. Methods From 2009 to 2011, 350 FSWs enrolled in this longitudinal study. Every 3 months, sociodemographic and sexual behavior data were collected via questionnaire, and APTIMA assays were used to detect the rRNA of Chlamydia trachomatis (CT), Neisseria gonorrhea, Trichomonas vaginalis, Mycoplasma genitalium, and messenger RNA of the E6/E7 oncoproteins expressed by hrHPV. Among 173 FSW who were infected with hrHPV during the observation period, accelerated failure time models estimated time ratios (TRs) for duration of hrHPV infection, comparing FSW infected with STIs at baseline to STI-uninfected FSWs. Results Median follow-up time was 26.2 months (interquartile range, 18.8–27.5 months). The median duration of hrHPV infection among all FSWs was 9.3 months (95% confidence interval [CI], 9.3–11.5). The duration of hrHPV infection among FSW infected with CT at baseline was greater than that among FSWs who were uninfected (adjusted TR, 1.7; 95% CI, 1.2–2.6). Among FSWs who were coinfected with hrHPV and CT at baseline, the adjusted TR was 3.4 (95% CI, 2.5–5.4) compared with FSWs infected with hrHPV only. No other STI was associated with hrHPV duration. Conclusions Recent or concurrent CT infection was associated with prolonged hrHPV infection among a cohort of Nairobi FSWs. Management of CT could reduce risk for hrHPV persistence.

Non-Cationic Proteins Are Associated with HIV Neutralizing Activity in Genital Secretions of Female Sex Workers

Objective Cationic proteins found in cervicovaginal secretions (CVS) are known to contribute to the early antiviral immune response against HIV-infection in vitro. We here aimed to define additional antiviral factors that are over-expressed in CVS from female sex workers at high risk of infection. Methods CVS were collected from Kenyan HIV-seronegative (n = 34) and HIV-seropositive (n = 12) female sex workers, and were compared with those from HIV-seronegative low-risk women (n = 12). The highly exposed seronegative (HESN) sex workers were further divided into those with less (n = 22) or more (n = 12) than three years of documented sex work. Cationic protein-depleted CVS were assessed for HIV-neutralizing activity by a PBMC-based HIV-neutralizing assay, and then characterized by proteomics. Results HIV neutralizing activity was detected in all unprocessed CVS, however only CVS from the female sex worker groups maintained its HIV neutralizing activity after cationic protein-depletion. Differentially abundant proteins were identified in the cationic protein-depleted secretions including 26, 42, and 11 in the HESN>3yr, HESN<3yr, and HIV-positive groups, respectively. Gene ontology placed these proteins into functional categories including proteolysis, oxidation-reduction, and epidermal development. The proteins identified in this study include proteins previously associated with the HESN phenotype in other cohorts as well as novel proteins not yet associated with anti-HIV activities. Conclusion While cationic proteins appear to contribute to the majority of the intrinsic HIV neutralizing activity in the CVS of low-risk women, a broader range of non-cationic proteins were associated with HIV neutralizing activity in HESN and HIV-positive female sex workers. These results indicate that novel protein factors found in CVS of women with high-risk sexual practices may have inherent antiviral activity, or are involved in other aspects of anti-HIV host defense, and warrant further exploration into their mode of action.

High‐risk HPV‐RNA screening of physician‐ and self‐collected specimens for detection of cervical lesions among female sex workers in Nairobi, Kenya

To compare accuracy of detecting high‐grade cervical lesions (squamous intraepithelial lesions or greater, HSIL+) by high‐risk HPV messenger RNA (hrHPV‐RNA) testing between physician‐ and self‐collected specimens, and by conventional cytology.