High prevalence of vaccine-preventable anal human papillomavirus infections is associated with HIV infection among gay, bisexual, and men who have sex with men in Nairobi, Kenya.

BACKGROUND: Human papillomavirus (HPV) infection is associated with anal cancers and is more prevalent in gay, bisexual, and men who have sex with men (gbMSM), partly due to their vulnerability to HIV infection. Baseline HPV genotype distributions and risk factors can inform the design of next-generation HPV vaccines to prevent anal cancer. METHODS: A cross-sectional study was conducted among gbMSM receiving care at a HIV/STI clinic in Nairobi, Kenya. Anal swabs were genotyped using a Luminex microsphere array. Multiple logistic regression methods were used to identify risk factors for four HPV outcomes (any HPV, any HR-HPV, and 4- and 9-valent vaccine-preventable HPVs). RESULTS: Among 115 gbMSM, 51 (44.3%) were HIV-infected. Overall HPV prevalence was 51.3%; 84.3% among gbMSM living with HIV and 24.6% among gbMSM without HIV (p < 0.001). One-third (32.2%) had HR-HPV and the most prevalent vaccine-preventable HR-HPV genotypes were 16, 35, 45, and 58. HPV-18 was uncommon (n = 2). The 9-valent Gardasil vaccine would have prevented 61.0% of HPV types observed in this population. In multivariate analyses, HIV status was the only significant risk factor for any HPV (adjusted odds ratio [aOR]:23.0, 95% confidence interval [95% CI]: 7.3-86.0, p < 0.001) and for HR-HPV (aOR: 8.9, 95% CI: 2.8-36.0, p < 0.001). Similar findings were obtained for vaccine-preventable HPVs. Being married to a woman significantly increased the odds of having HR-HPV infections (aOR: 8.1, 95% CI: 1.6-52.0, p = 0.016). CONCLUSIONS: GbMSM living with HIV in Kenya are at higher risk of anal HPV infections including genotypes that are preventable with available vaccines. Our findings support the need for a targeted HPV vaccination campaign in this population.

Evaluation of a collaborative model for successful implementation of a National CD4 enumeration EQA program in Cameroon.

Participation in an EQA program is critical to the quality assurance process. Reliable and precise CD4 T-cells enumeration are essential to improve the clinical management of patients by evaluating the disease progression and by monitoring the effectiveness of ART in HIV-patients. The CIRCB, CD4 reference laboratory, in collaboration with the Canadian QASI-program, recruited sites, distributed and analyzed CD4-panels in 61 sites across Cameroon. A trend and performance analysis in the pre-analytical, analytical and post-analytical phases was performed. Continuous training and corrective actions carried out from 2014 to 2018 increased the number of participating sites from 15 to 61 sites, the number of unacceptable results decreased from 50 to 10%. Specific challenges included errors in pre analytic (17.5%), analytic (77.0%) and post-analytic (5.5%) phases. This EQA requires the application of good laboratory practices, fluidic communication between all the stakeholders, continuous training, application of specific on-site corrective measures, and timely equipment maintenance in order to avoid repetitive errors and to increase laboratory performance. It could be extended to other HIV-1 testing like viral load and EID point-of-care. Partnership with QASI serve as a model for implementation of a successful EQA model for resource limited countries wanting to implement EQA for HIV testing and monitoring in alignment with 90-90-90 targets.

A naturally occurring Vif mutant (I107T) attenuates anti-APOBEC3G activity and HIV-1 replication.

The human immunodeficiency virus type 1 (HIV-1) Vif protein counteracts the antiviral activity of the apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3 (APOBEC3) family of proteins by targeting the proteins for degradation through the ubiquitin-proteasome pathway. Previous mutagenic studies have shown that multiple domains of Vif are required for interacting with APOBEC3G proteins and the proteasome pathway. However, very few mutagenesis and functional analyses of patient-derived Vif proteins have been conducted. In this study, we amplified and cloned the HIV-1 vif genes from the peripheral blood mononuclear cells (PBMCs) of five HIV-1-infected individuals in Nairobi and further tested the impact of the genes on anti-A3G activity and HIV-1 replication. The gene sequence analysis revealed high genetic variation of vif genes from different HIV-1-infected individuals. Interestingly, the Vif proteins derived from two of the three long-term survivors (LTSs) displayed a significantly impaired ability to mediate the degradation of A3G. In particular, a single amino acid change (I107T) in one of the non-functional LTS Vif variants, which has not been previously identified in the Los Alamos databases of vif sequences, was found to be responsible for the lack of anti-A3G activity. Further study demonstrated that HIV-1 carrying an I107T Vif mutation displayed significantly reduced fitness in A3G(+) T cells and PBMCs. Moreover, co-infecting A3G(+) T cells with both the wild-type and I107T Vif viruses resulted in decreased viral replication. Overall, the results of this study indicate that the HIV-1 Vif residue I107 is important for its anti-APOBEC3G activity and viral replication, which may have implications for viral fitness in vivo.

A systems biology examination of the human female genital tract shows compartmentalization of immune factor expression.

Many mucosal factors in the female genital tract (FGT) have been associated with HIV susceptibility, but little is known about their anatomical distribution in the FGT compartments. This study comprehensively characterized global immune factor expression in different tissue sites of the lower and upper FGT by using a systems biology approach. Tissue sections from the ectocervix, endocervix, and endometrium from seven women who underwent hysterectomy were analyzed by a combination of quantitative mass spectrometry and immunohistochemical staining. Of the >1,000 proteins identified, 281 were found to be differentially abundant in different tissue sites. Hierarchical clustering identified four major functional pathways distinguishing compartments, including innate immune pathways (acute-phase response, LXR/RXR) and development (RhoA signaling, gluconeogenesis), which were enriched in the ectocervix/endocervix and endometrium, respectively. Immune factors important for HIV susceptibility, including antiproteases, immunoglobulins, complement components, and antimicrobial factors, were most abundant in the ectocervix/endocervix, while the endometrium had a greater abundance of certain factors that promote HIV replication. Immune factor abundance is heterogeneous throughout the FGT and shows unique immune microenvironments for HIV based on the exposure site. This may have important implications for early events in HIV transmission and site-specific susceptibility to HIV in the FGT.

Salivary basic proline-rich proteins are elevated in HIV-exposed seronegative men who have sex with men.

OBJECTIVE: Innate mucosal factors are associated with protection in HIV-exposed seronegative (HESN) individuals, but studies of MSM have been very limited. We performed proteomic analysis of saliva from a cohort of HESN MSM who have regular unprotected oral receptive intercourse with their HIV-infected partner. METHODS: Saliva samples from HESN (n = 25) and non-exposed male controls (n = 22) were analyzed by 2D-LC mass spectrometry. An overexpressed innate protein factor was further characterized by immunoblot, and compared with CC-chemokine expression, HIV-neutralizing activity, clinical factors, and sexual behavior. RESULTS: Of 337 total proteins, seven were identified as differentially abundant in the HESN group. The five overabundant proteins (Basic salivary proline-rich proteins (bPRP) 2 and 3, Histatin-3, Lysozyme C, and SLPI) have known antimicrobial activity. bPRP2 showed the highest overabundance (>six-fold) in HESN individuals compared with controls (P = 0.009), including multiple isoforms. Salivary bPRP2 correlated with CC-chemokine levels in HESN individuals including RANTES (P = 0.02), MIP-1-alpha (P = 0.01), MIP-1-beta (P = 0.0002), MCP-1 (P = 0.005) and Eotaxin (P = 0.003) but not with frequency of HIV neutralizing activity, oral sexual practices, or viral load of the sexual partner. CONCLUSION: This study identifies salivary bPRP2 as a novel soluble factor elevated in the oral compartment of HIV-exposed MSM.

Microarray analysis of HIV resistant female sex workers reveal a gene expression signature pattern reminiscent of a lowered immune activation state.

To identify novel biomarkers for HIV-1 resistance, including pathways that may be critical in anti-HIV-1 vaccine design, we carried out a gene expression analysis on blood samples obtained from HIV-1 highly exposed seronegatives (HESN) from a commercial sex worker cohort in Nairobi and compared their profiles to HIV-1 negative controls. Whole blood samples were collected from 43 HIV-1 resistant sex workers and a similar number of controls. Total RNA was extracted and hybridized to the Affymetrix HUG 133 Plus 2.0 micro arrays (Affymetrix, Santa Clara CA). Output data was analysed through ArrayAssist software (Agilent, San Jose CA). More than 2,274 probe sets were differentially expressed in the HESN as compared to the control group (fold change ≥1.3; p value ≤0.0001, FDR <0.05). Unsupervised hierarchical clustering of the differentially expressed genes readily distinguished HESNs from controls. Pathway analysis through the KEGG signaling database revealed a majority of the impacted pathways (13 of 15, 87%) had genes that were significantly down regulated. The most down expressed pathways were glycolysis/gluconeogenesis, pentose phosphate, phosphatidyl inositol, natural killer cell cytotoxicity and T-cell receptor signaling. Ribosomal protein synthesis and tight junction genes were up regulated. We infer that the hallmark of HIV-1 resistance is down regulation of genes in key signaling pathways that HIV-1 depends on for infection.

A comparison of parallel pyrosequencing and sanger clone-based sequencing and its impact on the characterization of the genetic diversity of HIV-1.

BACKGROUND: Pyrosequencing technology has the potential to rapidly sequence HIV-1 viral quasispecies without requiring the traditional approach of cloning. In this study, we investigated the utility of ultra-deep pyrosequencing to characterize genetic diversity of the HIV-1 gag quasispecies and assessed the possible contribution of pyrosequencing technology in studying HIV-1 biology and evolution. METHODOLOGY/PRINCIPAL FINDINGS: HIV-1 gag gene was amplified from 96 patients using nested PCR. The PCR products were cloned and sequenced using capillary based Sanger fluorescent dideoxy termination sequencing. The same PCR products were also directly sequenced using the 454 pyrosequencing technology. The two sequencing methods were evaluated for their ability to characterize quasispecies variation, and to reveal sites under host immune pressure for their putative functional significance. A total of 14,034 variations were identified by 454 pyrosequencing versus 3,632 variations by Sanger clone-based (SCB) sequencing. 11,050 of these variations were detected only by pyrosequencing. These undetected variations were located in the HIV-1 Gag region which is known to contain putative cytotoxic T lymphocyte (CTL) and neutralizing antibody epitopes, and sites related to virus assembly and packaging. Analysis of the positively selected sites derived by the two sequencing methods identified several differences. All of them were located within the CTL epitope regions. CONCLUSIONS/SIGNIFICANCE: Ultra-deep pyrosequencing has proven to be a powerful tool for characterization of HIV-1 genetic diversity with enhanced sensitivity, efficiency, and accuracy. It also improved reliability of downstream evolutionary and functional analysis of HIV-1 quasispecies.

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.

Reduced rates of HIV acquisition during unprotected sex by Kenyan female sex workers predating population declines in HIV prevalence.

OBJECTIVES: Female sex workers (FSWs) form a core group at high risk of both sexual HIV acquisition and secondary transmission. The magnitude of these risks may vary by sexual risk taking, partner HIV prevalence, host immune factors and genital co-infections. We examined temporal trends in HIV prevalence and per-act incidence, adjusted for behavioral and other variables, in FSWs from Nairobi, Kenya. METHODS: An open cohort of FSWs followed since 1985. Behavioral and clinical data were collected six monthly from 1985 to 2005, and sexually transmitted infection (STI) diagnostics and HIV serology performed. A Cox proportional hazards model with time-dependent covariables was used to estimate infection risk as a function of calendar time. RESULTS: HIV prevalence in new FSW enrollees peaked at 81% in 1986, and was consistently below 50% after 1997. Initially uninfected FSWs remained at high risk of acquiring HIV throughout the study period, but the rate of HIV acquisition during unprotected sex with a casual client declined by over four-fold. This reduction correlated closely with decreases in gonorrhea prevalence, and predated reductions in the Kenyan HIV population prevalence by over a decade. CONCLUSIONS: The per-act rate of HIV acquisition in high-risk Nairobi FSWs fell dramatically between 1985 and 2005. This decline may represent the impact of improved STI prevention/therapy, immunogenetic shifts in at-risk women, or changes in the proportion of HIV exposures occurring with clients who had acute HIV infection. Declining HIV incidence in high-risk cohorts may predict and/or be causally related to future reductions in population prevalence.

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.