Abstract

CCR5 is the primary coreceptor for HIV entry. Early after infection, the HIV viral population diversifies rapidly into a quasispecies. It is not known whether the initial efficiency of the viral quasispecies to utilize CCR5 is associated with HIV disease progression or if it changes in an infected individual over time. The CCR5 and CXCR4 utilization efficiencies (R5-UE and X4-UE) of the HIV quasispecies were examined using a pseudovirus, single-round infection assay for samples obtained from known seroconverters from Rakai district, Uganda (n=88). Initial and longitudinal R5-UE values were examined to assess the association of R5-UE with HIV disease progression using multivariate Cox proportional hazard models. Longitudinal samples were analyzed for 35 seroconverters who had samples available from multiple time points. There was no association between initial or longitudinal changes in R5-UE and the hazard of HIV disease progression (p=0.225 and p=0.942, respectively). In addition, R5-UE increased significantly over time after HIV seroconversion (p<0.001), regardless of HIV subtype or the emergence of CXCR4-tropic virus. These data demonstrate that the R5-UE of the viral quasispecies early in HIV infection is not associated with disease progression, and that R5-UE levels increase in HIV-infected individuals over time.

Next-generation sequencing (NGS) has recently been used for analysis of HIV diversity, but this method is labor-intensive, costly, and requires complex protocols for data analysis. We compared diversity measures obtained using NGS data to those obtained using a diversity assay based on high-resolution melting (HRM) of DNA duplexes. The HRM diversity assay provides a single numeric score that reflects the level of diversity in the region analyzed. HIV gag and env from individuals in Rakai, Uganda, were analyzed in a previous study using NGS (n = 220 samples from 110 individuals). Three sequence-based diversity measures were calculated from the NGS sequence data (percent diversity, percent complexity, and Shannon entropy). The amplicon pools used for NGS were analyzed with the HRM diversity assay. HRM scores were significantly associated with sequence-based measures of HIV diversity for both gag and env (P < 0.001 for all measures). The level of diversity measured by the HRM diversity assay and NGS increased over time in both regions analyzed (P < 0.001 for all measures except for percent complexity in gag), and similar amounts of diversification were observed with both methods (P < 0.001 for all measures except for percent complexity in gag). Diversity measures obtained using the HRM diversity assay were significantly associated with those from NGS, and similar increases in diversity over time were detected by both methods. The HRM diversity assay is faster and less expensive than NGS, facilitating rapid analysis of large studies of HIV diversity and evolution.

Male circumcision reduces acquisition of herpes simplex virus type 2 (HSV-2) in men. We assessed whether male circumcision reduces HSV-2 infection among female partners. HSV-2–negative, human immunodeficiency virus–negative female partners of 368 males who were and 372 males who were not randomized to receive male circumcision were enrolled. The incidence of HSV-2 infection among females over a period of 2 years was 6.09 cases per 100 person-years in the intervention arm and 6.32 cases per 100 person-years in the control arm (incidence rate ratio [IRR], 0.96 [95% confidence interval {CI}, .62–1.49]; P = .87). Among female partners of HSV-2–positive males, the incidence of HSV-2 infection was 9.55 cases per 100 person-years in the intervention arm and 11.17 cases per 100 person-years in the control arm (IRR, 0.85 [95% CI, .44–1.67]; P = .62). Contrary to findings in males, male circumcision did not affect HSV-2 acquisition among female partners.

Background. The HIV Prevention Trials Network (HPTN) 052 trial demonstrated that early initiation of antiretroviral therapy (ART) reduces human immunodeficiency virus (HIV) transmission from HIV-infected adults (index participants) to their HIV-uninfected sexual partners. We analyzed HIV from 38 index-partner pairs and 80 unrelated index participants (controls) to assess the linkage of seroconversion events.

Methods. Linkage was assessed using phylogenetic analysis of HIV pol sequences and Bayesian analysis of genetic distances between pol sequences from index-partner pairs and controls. Selected samples were also analyzed using next-generation sequencing (env region).

Results. In 29 of the 38 (76.3%) cases analyzed, the index was the likely source of the partner’s HIV infection (linked). In 7 cases (18.4%), the partner was most likely infected from a source other than the index participant (unlinked). In 2 cases (5.3%), linkage status could not be definitively established.

Conclusions. Nearly one-fifth of the seroconversion events in HPTN 052 were unlinked. The association of early ART and reduced HIV transmission was stronger when the analysis included only linked events. This underscores the importance of assessing the genetic linkage of HIV seroconversion events in HIV prevention studies involving serodiscordant couples.

Abstract

To investigate HIV-1 molecular epidemiology in Singapore, we sequenced portions of three regions of the HIV-1 genome (protease HXB2: 2163 to 2620, gp120 HXB2: 6904 to 7628, and gp41 HXB2: 7817 to 8264) from 212 plasma samples collected between February 2008 and August 2009. From these samples, 109 (51.4%) generated interpretable data in all regions. Sixty-one (56.0%) were identified as CRF01_AE, 26 (23.9%) as subtype B and 14 (12.8%) as possible novel recombinant forms. The main novel recombinant pattern, detected in 13 sequences, had subtype B in protease and gp41 and CRF01_AE in gp120. There was intermixing of subtypes within transmission risk groups. However, 85% of subjects infected with the novel recombinant forms self-identified as men who have sex with men or bisexuals compared with only 41% of individuals infected with CRF01_AE and 62% infected with subtype B (p = 0.001).

Herpes simplex virus type 2 (HSV-2) infection is one of the most commonly sexually transmitted infections worldwide. While glycoprotein G-2 ELISA based assays are commonly used for the serologic detection of HSV-2 infections, they have low specificity in developing countries. Euroline Western blot (WB) is a commercially available assay that is easy to perform; however, little is known about its performance characteristics. This study evaluated Euroline WB for the detection of HSV-2 antibodies compared to University of Washington Western blot in three geographically different regions, Baltimore, Maryland, Rakai, Uganda, and Kunming, China. Among the 135 American men attending an STD clinic in Baltimore, Maryland, 72% (n=97) were HSV-2 positive by Euroline WB. The Euroline WB had a sensitivity of 97.8% and a specificity of 81.8%. Among the 273 commercial sex workers in Kunming, 62.3% were HSV-2 positive by Euroline WB. The Euroline WB had a sensitivity of 96.9% and a specificity of 89.1%. Among the 437 Ugandans in Rakai, 67.3% were HSV-2 positive by Euroline WB. The Euroline WB had a sensitivity of 98.7% and a specificity of 65.4%. The Euroline WB has a consistently high sensitivity, but specificity varied significantly among the different locations.

Background. Male circumcision reduces human immunodeficiency virus (HIV) and herpes simplex virus type 2 (HSV-2) acquisition, and HSV-2 infection is associated with an increased risk of HIV acquisition. To assess the cellular basis for these associations, we estimated immunologic cellular densities in foreskin tissue.

Methods. Immunostained CD1a+ dendritic cell and CD4+ and CD8+ T cell densities were quantified in foreskin samples obtained from medical circumcision in Rakai, Uganda (35 HIV-infected, HSV-2-infected men; 5 HIV-infected, HSV-2-uninfected men; 22 HIV-uninfected, HSV-2-infected men; and 29 HIV-uninfected, HSV-2-uninfected men.

Results. CD1A+ dendritic cell densities did not vary by HIV or HSV-2 status. Compared with densities in HIV-uninfected, HSV-2-uninfected men (mean, 26.8 cells/mm2), CD4+ T cell densities were similar in the HIV-infected, HSV-2-infected group (mean, 28.7 cells/mm2), were significantly decreased in the HIV-infected, HSV-2-uninfected group (mean, 11.2; P < .05), and were increased in the HIV-uninfected, HSV-2-infected group (mean, 68.7; P < .05). Dermal CD8+ T cell densities were higher in the HIV and HSV-2-coinfected group (mean, 102.9) than in the HIV-uninfected, HSV-2-uninfected group (mean, 10.0; P < .001), the HIV-infected, HSV-2-uninfected group (mean, 27.3; P < .001), and the HIV-uninfected, HSV-2-infected group (mean, 25.3; P < .005).

Discussion. The increased CD4+ cellular density in the HIV-uninfected, HSV-2-infected men may help to explain why HSV-2–infected men are at increased risk of HIV acquisition. The absence of this increase in men coinfected with both HIV and HSV-2 is likely in part the result of the progressive loss of CD4+ cells in HIV infection. Conversely, HIV and HSV-2 coinfection appears to synergistically increase CD8+ T cell densities.

HIV superinfection, which occurs when a previously infected individual acquires a new distinct HIV strain, has been described in a number of populations. Previous methods to detect superinfection have involved a combination of labor-intensive assays with various rates of success. We designed and tested a next-generation sequencing (NGS) protocol to identify HIV superinfection by targeting two regions of the HIV viral genome, p24 and gp41. The method was validated by mixing control samples infected with HIV subtype A or D at different ratios to determine the inter- and intrasubtype sensitivity by NGS. This amplicon-based NGS protocol was able to consistently identify distinct intersubtype strains at ratios of 1% and intrasubtype variants at ratios of 5%. By using stored samples from the Rakai Community Cohort Study (RCCS) in Uganda, 11 individuals who were HIV seroconcordant but virally unlinked from their spouses were then tested by this method to detect superinfection between 2002 and 2005. Two female cases of HIV intersubtype superinfection (18.2%) were identified. These results are consistent with other African studies and support the hypothesis that HIV superinfection occurs at a relatively high rate. Our results indicate that NGS can be used for detection of HIV superinfection within large cohorts, which could assist in determining the incidence and the epidemiologic, virologic, and immunological correlates of this phenomenon.

Abstract

HIV-1 subtype D (HIV-1D) progresses to disease faster and has lower transmissibility than subtype A (HIV-1A). We examined whether these differences could lead to a population level change in the distribution of these subtypes over time. HIV-1 viral RNA was extracted from stored serum samples from HIV-positive subjects participating in a population-based cohort study in Rakai, Uganda in 1994 and 2002. Portions of the viral proteins gag and gp41 were sequenced and subtyped. HIV-1 subtype assignments were generated for 773 subjects in 1994 and 812 subjects in 2002. The change in subtype distribution of the population as a whole as well as quartile age groups were examined for significant changes using a linear model. There was a significant decrease in the proportion of subjects infected with HIV-1D from 70.2% to 62.4% and a significant increase in subjects infected with HIV-1A from 16.7% to 23.3% over the 8-year period (p = 0.005). The most marked changes in proportion of HIV-1D and A were seen in the younger individuals (<25 and 25–30 years; p < 0.05). The percentages of subjects infected with HIV-1C and recombinant subtypes did not change significantly. Over this 8-year period, the overall viral population in this region evolved toward the less virulent HIV-1A strain, most likely as a consequence of the faster disease progression and lower transmissibility of HIV-1D.

Introduction

Microbial translocation has been implicated as a contributing factor to the heightened immune activation observed during HIV-1 disease progression. When examined in a longitudinal study of HIV-1 seroconverters in Rakai, Uganda microbial translocation was not associated with HIV-1 disease progression. However, the role of general immune activation in HIV disease progression in this population was not fully examined.

Methods

Longitudinal serum samples of HIV-1 seroconverters in three HIV-1 disease progression groups [long-term nonprogressors (LTNP), standard progressors (SP), and rapid progressors (RP)] from Rakai, Uganda, were tested for levels of C-reactive protein (CRP), a marker for immune activation.

Results

CRP levels significantly increased in the SP group (p<0.0001), but not in the RP group or the LTNP group. CRP levels during the first year post-HIV-seroconversion in the RP group were significantly higher than those observed in the LTNP group (p<0.05). For the entire population CRP levels negatively correlated with Lipopolysaccharide levels (p<0.05) and were not associated with endotoxin antibody levels.

Conclusions

This study suggests that in this population increased immune activation is significantly associated with HIV-1 disease progression, but not microbial translocation.

BACKGROUND

Since the identification of xenotropic murine leukemia virus–related virus (XMRV) in prostate cancer patients in 2006 and in chronic fatigue syndrome patients in 2009, conflicting findings have been reported regarding its etiologic role in human diseases and prevalence in general populations. In this study, we screened both plasma and peripheral blood mononuclear cells (PBMNCs) collected in Africa from blood donors and human immunodeficiency virus Type 1 (HIV-1)-infected individuals to gain evidence of XMRV infection in this geographic region.

STUDY DESIGN AND METHODS

A total of 199 plasma samples, 19 PBMNC samples, and 50 culture supernatants from PBMNCs of blood donors from Cameroon found to be infected with HIV-1 and HIV-1 patients from Uganda were screened for XMRV infection using a sensitive nested polymerase chain reaction (PCR) or reverse transcription (RT)-PCR assay.

RESULTS

Using highly sensitive nested PCR or RT-PCR and real-time PCR assays capable of detecting at least 10 copies of XMRV plasmid DNA per reaction, none of the 268 samples tested were found to be XMRV DNA or RNA positive.

CONCLUSIONS

Our results failed to demonstrate the presence of XMRV infection in African blood donors or individuals infected with HIV-1. More studies are needed to understand the prevalence, epidemiology, and geographic distribution of XMRV infection worldwide.

Significant hurdles remain to large-scale implementation of medical interventions in the developing world due to the lack of a modern diagnostic infrastructure. This is especially pertinent to the international roll-out of antiretroviral drugs to treat HIV, which ideally includes a CD4 t-cell to determine eligibility. We designed a novel technique to estimate mature t-cell numbers by calculating the amount of rearranged t-cell receptor β genes from dried blood spots of HIV-infected individuals in the United States and Uganda. It was observed that the rearranged t-cell receptor β count correlated well with total lymphocyte counts from both study populations (Baltimore R=0.602, Uganda R=0.497; p<0.001) and the ability for this measurement to determine antiretroviral initiation was similar to total lymphocyte counts, which can be used to determine eligibility in HIV+ children. This technique as well as other dried blood spot based technologies could increase the diagnostic and monitoring capabilities in resource-limited settings.

Abstract

To analyze HIV-1 subtype distribution, sequence analysis was performed on serum specimens obtained in 1994 from the Rakai Health Sciences community cohort in Uganda. Portions of gag-p24 and env-gp41 were sequenced and HIV subtype was determined for 773 subjects residing in 10 community clusters in rural Uganda. Subtypes A (17%) and D (70%) were the most common strains in the population. Subtype distribution varied by geographic region with significantly more subtype A in northern community clusters compared with southern clusters (21% vs. 8%, p < 0.001) and more subtype D in southern clusters compared with northern clusters (78% vs. 65%, p < 0.008). These data illustrate the geographic complexity of subtype variation, which has important implications for HIV-1 vaccine design.

Objective

Studies on long-term nonprogressors (LTNP) have been conducted in the USA and Europe. This study examined the frequency of LTNPs and HIV controllers among 637 HIV-1 seroconverters in rural Uganda.

Design and Methods

LTNPs were defined as being infected for more than 7 years with a CD4+ T-cell count above 600 cells per microliter, and HIV controllers as having undetectable viral loads on 3 separate occasions without antiretroviral treatment. HIV-1 viral load and subtype distribution between LTNP and non-LTNP populations were determined.

Results

Of the HIV seroconverters, 9.1% (58/637) were LTNPs and 1.4% (9/637) were HIV controllers. LTNPs had a significantly lower viral load at set point than non-LTNP participants (P < 0.001). The Kaplan–Meier joint probability of surviving to 7 years with a CD4 count >600 was 19.2%. Individuals who survived 7 years had a significantly higher frequency of HIV-1 subtype A (P < 0.05), but seroconverters infected with HIV-1A did not have a significantly higher probability of becoming an LTNP.

Conclusions

The frequency of LTNPs appears to be relatively high in Uganda and it may be important to take this into account when designing studies of viral pathogenesis and performing HIV vaccine trials in sub-Saharan Africa.

To analyze HIV-1 subtype distribution, sequence analysis was performed on serum specimens obtained in 1994 from the Rakai Health Sciences community cohort in Uganda. Portions of gag-p24 and env-gp41 were sequenced and HIV subtype was determined for 773 subjects residing in 10 community clusters in rural Uganda. Subtypes A (17%) and D (70%) were the most common strains in the population. Subtype distribution varied by geographic region with significantly more subtype A in northern community clusters compared with southern clusters (21% vs. 8%, p < 0.001) and more subtype D in southern clusters compared with northern clusters (78% vs. 65%, p < 0.008). These data illustrate the geographic complexity of subtype variation, which has important implications for HIV-1 vaccine design.