HIV-infected sex workers with beneficial HLA-variants are potential hubs for selection of HIV-1 recombinants that may affect disease progression

Cytotoxic T lymphocyte (CTL) responses against the HIV Gag protein are associated with lowering viremia; however, immune control is undermined by viral escape mutations. The rapid viral mutation rate is a key factor, but recombination may also contribute. We hypothesized that CTL responses drive the outgrowth of unique intra-patient HIV-recombinants (URFs) and examined gag sequences from a Kenyan sex worker cohort. We determined whether patients with HLA variants associated with effective CTL responses (beneficial HLA variants) were more likely to carry URFs and, if so, examined whether they progressed more rapidly than patients with beneficial HLA-variants who did not carry URFs. Women with beneficial HLA-variants (12/52) were more likely to carry URFs than those without beneficial HLA variants (3/61) (p < 0.0055; odds ratio = 5.7). Beneficial HLA variants were primarily found in slow/standard progressors in the URF group, whereas they predominated in long-term non-progressors/survivors in the remaining cohort (p = 0.0377). The URFs may sometimes spread and become circulating recombinant forms (CRFs) of HIV and local CRF fragments were over-represented in the URF sequences (p < 0.0001). Collectively, our results suggest that CTL-responses associated with beneficial HLA variants likely drive the outgrowth of URFs that might reduce the positive effect of these CTL responses on disease progression

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 \u3c 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 \u3c 0.001) and for HR-HPV (aOR: 8.9, 95% CI: 2.8–36.0, p \u3c 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 Quantitative Real-Time PCR Assay to Measure HIV-Specific Mucosal CD8+ T Cell Responses in the Cervix

Several candidate HIV vaccines aim to induce virus-specific cellular immunity particularly in the genital tract, typically the initial site of HIV acquisition. However, standardized and sensitive methods for evaluating HIV-specific immune responses at the genital level are lacking. Therefore we evaluated real-time quantitative PCR (qPCR) as a potential platform to measure these responses. β-Actin and GAPDH were identified as the most stable housekeeping reference genes in peripheral blood mononuclear cells (PBMCs) and cervical mononuclear cells (CMCs) respectively and were used for normalizing transcript mRNA expression. HIV-specific cellular T cell immune responses to a pool of optimized CD8+ HIV epitopes (HIV epitope pool) and Staphylococcal enterotoxin B (SEB) superantigen control were assayed in HIV infected PBMC by qPCR, with parallel assessment of cytokine protein production. Peak HIV-specific mRNA expression of IFNγ, IL-2 and TNFα occurred after 3, 5 and 12 hours respectively. PBMCs were titrated to cervical appropriate cell numbers to determine minimum required assay input cell numbers; qPCR retained sensitivity with input of at least 2.5×104 PBMCs. This optimized qPCR assay was then used to assess HIV-specific cellular T cell responses in cytobrush-derived cervical T cells from HIV positive individuals. SEB induced IFNγ mRNA transcription was detected in CMCs and correlated positively with IFNγ protein production. However, qPCR was unable to detect HIV-induced cytokine mRNA production in the cervix of HIV-infected women despite robust detection of gene induction in PBMCs. In conclusion, although qPCR can be used to measure ex vivo cellular immune responses to HIV in blood, HIV-specific responses in the cervix may fall below the threshold of qPCR detection. Nonetheless, this platform may have a potential role in measuring mitogen-induced immune responses in the genital tract

A Comparative Analysis of Gene Expression Patterns and Cell Phenotypes between Cervical and Peripheral Blood Mononuclear Cells

Studies of the immunological environment in the female genital tract (FGT) are critical for the development of vaccines or microbicides to halt the spread of sexually transmitted infections. Challenges arise due to the difficulties of sampling from this site, and the majority of studies have been conducted utilising peripheral blood mononuclear cells. Identifying functional differences between immune cells of the FGT and peripheral blood would aid in our understanding of mucosal immunology. We compared the gene expression profile of mononuclear cells at these two sites. Messenger RNA expression analysis was performed using gene expression arrays on matched cervical mononuclear cells and peripheral blood mononuclear cells. Further cellular phenotyping was done by 10 colour flow cytometry. Of the 22,185 genes expressed by these samples, 5345 genes were significantly differentially expressed between the cell populations. Most differences can be explained by significantly lower levels of T and B cells and higher levels of macrophages and dendritic cells in the FGT compared with peripheral blood. Several immunologically relevant pathways such as apoptosis and innate immune signalling, and a variety of cytokines and cytokine receptors were differentially expressed. This study highlights the importance of the unique immunological environment of the FGT and identifies important differences between systemic and mucosal immune compartments

Chemokine Receptor 5 Δ32 Allele in Patients with Severe Pandemic (H1N1) 2009

Because chemokine receptor 5 (CCR5) may have a role in pulmonary immune response, we explored whether patients with severe pandemic (H1N1) 2009 were more likely to carry the CCR5Δ32 allele than were members of the general population. We found a large proportion of heterozygosity for the CCR5Δ32 allele among white patients with severe disease

Very high energy observations of the BL Lac objects 3C 66A and OJ 287

Using the Solar Tower Atmospheric Cherenkov Effect Experiment (STACEE), we have observed the BL Lac objects 3C 66A and OJ 287. These are members of the class of low-frequency-peaked BL Lac objects (LBLs) and are two of the three LBLs predicted by Costamante and Ghisellini to be potential sources of very high energy (>100 GeV) gamma-ray emission. The third candidate, BL Lacertae, has recently been detected by the MAGIC collaboration. Our observations have not produced detections; we calculate a 99% CL upper limit of flux from 3C 66A of 0.15 Crab flux units and from OJ 287 our limit is 0.52 Crab. These limits assume a Crab-like energy spectrum with an effective energy threshold of 185 GeV.Comment: 24 pages, 15 figures, Accepted for publication in Astroparticle Physic

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

<p>Abstract</p> <p>Background</p> <p>The <it>GNB3 C825T </it>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 <it>GNB3 </it>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. <it>GNB3 </it>825 genotyping was performed for enrolees in both a commercial sex worker cohort and a perinatal HIV transmission (PHT) cohort in Nairobi, Kenya. <it>Ex vivo </it>immune activation was quantified by flow cytometry, and plasma chemokine levels were assessed by cytokine bead array.</p> <p>Results</p> <p><it>GNB3 </it>genotype was not associated with sexual or vertical HIV-1 acquisition within these cohorts. Within the Pumwani cohort, <it>GNB3 </it>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-1β and TRAIL levels quantified by cytokine bead array were also similar between groups.</p> <p>Conclusions</p> <p>In contrast to previous reports, we were unable to provide evidence to suggest that the <it>GNB3 C825T </it>polymorphism affects HIV-1 acquisition or disease progression within African populations. <it>Ex vivo </it>immune activation and plasma chemokine levels were similarly unaffected by <it>GNB3 </it>genotype in both HIV-1-negative and HIV-1-positive individuals. The paucity of studies investigating the impact of <it>GNB3 </it>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.</p

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