The Epidemiology of HIV and HSV-2 Infections among Women Participating in Microbicide and Vaccine Feasibility Studies in Northern Tanzania

Objectives

To prepare for future HIV prevention trials, we conducted prospective cohort studies among women working in food and recreational facilities in northern Tanzania. We examined the prevalence and incidence of HIV and HSV-2, and associated risk factors.

Methods

Women aged 18–44 years working in food and recreational facilities were screened to determine their eligibility for the studies. Between 2008–2010, HIV-negative women were enrolled and followed for 12 months. At enrolment and 3-monthly, we collected socio-demographic and behavioural data, and performed clinical examinations for collection of biological specimens that were tested for reproductive tract infections. Risk factors for HIV and HSV-2 incidence were investigated using Poisson regression models.

Results

We screened 2,229 and enrolled 1,378 women. The median age was 27 years (interquartile range, IQR 22, 33), and median duration working at current facility was 2 years. The prevalences of HIV at screening and HSV-2 at enrolment were 16% and 67%, respectively. Attendance at the 12-month visit was 86%. HIV and HSV-2 incidence rates were 3.7 (95% confidence interval, CI: 2.8,5.1) and 28.6 (95% CI: 23.5,35.0)/100 person-years, respectively. Women who were separated, divorced, or widowed were at increased risk of HIV (adjusted incidence rate ratio, aRR = 6.63; 95% CI: 1.97,22.2) and HSV-2 (aRR = 2.00; 95% CI: 1.15,3.47) compared with married women. Women reporting ≥3 partners in the past 3 months were at higher HIV risk compared with women with 0–1 partner (aRR = 4.75; 95% CI: 2.10,10.8), while those who had reached secondary education or above were at lower risk of HSV-2 compared with women with incomplete primary education (aRR = 0.42; 95% CI: 0.22,0.82).

Conclusions

HIV and HSV-2 rates remain substantially higher in this cohort than in the general population, indicating urgent need for effective interventions. These studies demonstrate the feasibility of conducting trials to test new interventions in this highly-mobile population.     

Protection Provided by a Herpes Simplex Virus 2 (HSV-2) Glycoprotein C and D Subunit Antigen Vaccine against Genital HSV-2 Infection in HSV-1-Seropositive Guinea Pigs

A prophylactic vaccine for genital herpes disease remains an elusive goal. We report the results of two studies performed collaboratively in different laboratories that assessed immunogenicity and vaccine efficacy in herpes simplex virus 1 (HSV-1)-seropositive guinea pigs immunized and subsequently challenged intravaginally with HSV-2. In study 1, HSV-2 glycoproteins C (gC2) and D (gD2) were produced in baculovirus and administered intramuscularly as monovalent or bivalent vaccines with CpG and alum. In study 2, gD2 was produced in CHO cells and given intramuscularly with monophosphoryl lipid A (MPL) and alum, or gC2 and gD2 were produced in glycoengineered Pichia pastoris and administered intramuscularly as a bivalent vaccine with Iscomatrix and alum to HSV-1-naive or -seropositive guinea pigs. In both studies, immunization boosted neutralizing antibody responses to HSV-1 and HSV-2. In study 1, immunization with gC2, gD2, or both immunogens significantly reduced the frequency of genital lesions, with the bivalent vaccine showing the greatest protection. In study 2, both vaccines were highly protective against genital disease in naive and HSV-1-seropositive animals. Comparisons between gD2 and gC2/gD2 in study 2 must be interpreted cautiously, because different adjuvants, gD2 doses, and antigen production methods were used; however, significant differences invariably favored the bivalent vaccine. Immunization of naive animals with gC2/gD2 significantly reduced the number of days of vaginal shedding of HSV-2 DNA compared with that for mock-immunized animals. Surprisingly, in both studies, immunization of HSV-1-seropositive animals had little effect on recurrent vaginal shedding of HSV-2 DNA, despite significantly reducing genital disease.     

Semi-Parametric Spatial Joint Modeling of HIV and HSV-2 among Women in Kenya

Several diseases have common risk factors. The joint modeling of disease outcomes within a spatial statistical context may provide more insight on the interaction of diseases both at individual and at regional level. Spatial joint modeling allows for studying of the relationship between diseases and also between regions under study. One major approach for joint spatial modeling is the multivariate conditional autoregressive approach. In this approach, it is assumed that all the covariates in the study have linear effects on the multiple response variables. In this study, we relax this linearity assumption and allow some covariates to have nonlinear effects using the penalized regression splines. This model was used to jointly model the spatial variation of human immunodeficiency virus (HIV) and herpes simplex virus-type 2 (HSV-2) among women in Kenya. The model was applied to HIV and HSV-2 prevalence data among women aged 15–49 years in Kenya, derived from the 2007 Kenya AIDS indicator survey. A full Bayesian approach was used and the models were implemented in WinBUGS software. Both diseases showed significant spatial variation with highest disease burdens occurring around the Lake Victoria region. There was a nonlinear association between age of an individual and HIV and HSV-2 infection. The peak age for HIV was around 30 years while that of HSV-2 was about 40 years. A positive significant spatial correlation between HIV and HSV-2 was observed with a correlation of 0.6831(95% CI: 0.3859, 0.871).     

Modeling the synergy between HSV-2 and HIV and potential impact of HSV-2 therapy

Mounting evidence indicates that genital HSV-2 infection may increase susceptibility to HIV infection and that co-infection may increase infectiousness. Accordingly, antiviral treatment of people with HSV-2 may mitigate the incidence of HIV in populations where both pathogens occur. To better understand the epidemiological synergy between HIV and HSV-2, we formulate a deterministic compartmental model that describes the transmission dynamics of these pathogens. Unlike earlier models, ours incorporates gender and heterogeneous mixing between activity groups. We derive explicit expressions for the reproduction numbers of HSV-2 and HIV, as well as the invasion reproduction numbers via next generation matrices. A qualitative analysis of the system includes the local and global behavior of the model. Simulations reinforce these analytical results and demonstrate epidemiological synergy between HSV-2 and HIV. In particular, numerical results show that HSV-2 favors the invasion of HIV, may dramatically increase the peak as well as reducing the time-to-peak of HIV prevalence, and almost certainly has exacerbated HIV epidemics. The potential population-level impact of HSV-2 on HIV is demonstrated by calculating the fraction of HIV infections attributable to HSV-2 and the difference between HIV prevalence in the presence and absence of HSV-2. The potential impact of treating people with HSV-2 on HIV control is demonstrated by comparing HIV prevalence with and without HSV-2 therapy. Most importantly, we illustrate that the aforementioned aspects of the population dynamics can be significantly influenced by the sexual structure of the population.     

Herpes simplex virus 2 (HSV-2) evolves faster in cell culture than HSV-1 by generating greater genetic diversity

Herpes simplex virus type 1 and 2 (HSV-1 and HSV-2, respectively) are prevalent human pathogens of clinical relevance that establish long-life latency in the nervous system. They have been considered, along with the Herpesviridae family, to exhibit a low level of genetic diversity during viral replication. However, the high ability shown by these viruses to rapidly evolve under different selective pressures does not correlates with that presumed genetic stability. High-throughput sequencing has revealed that heterogeneous or plaque-purified populations of both serotypes contain a broad range of genetic diversity, in terms of number and frequency of minor genetic variants, both in vivo and in vitro. This is reminiscent of the quasispecies phenomenon traditionally associated with RNA viruses. Here, by plaque-purification of two selected viral clones of each viral subtype, we reduced the high level of genetic variability found in the original viral stocks, to more genetically homogeneous populations. After having deeply characterized the genetic diversity present in the purified viral clones as a high confidence baseline, we examined the generation of de novo genetic diversity under culture conditions. We found that both serotypes gradually increased the number of de novo minor variants, as well as their frequency, in two different cell types after just five and ten passages. Remarkably, HSV-2 populations displayed a much higher raise of nonconservative de novo minor variants than the HSV-1 counterparts. Most of these minor variants exhibited a very low frequency in the population, increasing their frequency over sequential passages. These new appeared minor variants largely impacted the coding diversity of HSV-2, and we found some genes more prone to harbor higher variability. These data show that herpesviruses generate de novo genetic diversity differentially under equal in vitro culture conditions. This might have contributed to the evolutionary divergence of HSV-1 and HSV-2 adapting to different anatomical niche, boosted by selective pressures found at each epithelial and neuronal tissue.     

Herpes Simplex Virus 2 (HSV-2) Infected Cell Proteins Are among the Most Dominant Antigens of a Live-Attenuated HSV-2 Vaccine

Virion glycoproteins such as glycoprotein D (gD) are believed to be the dominant antigens of herpes simplex virus 2 (HSV-2). We have observed that mice immunized with a live HSV-2 ICP0^- mutant virus, HSV-2 0ΔNLS, are 10 to 100 times better protected against genital herpes than mice immunized with a HSV-2 gD subunit vaccine (PLoS ONE 6:e17748). In light of these results, we sought to determine which viral proteins were the dominant antibody-generators (antigens) of the live HSV-2 0ΔNLS vaccine. Western blot analyses indicated the live HSV-2 0ΔNLS vaccine elicited an IgG antibody response against 9 or more viral proteins. Many antibodies were directed against infected-cell proteins of >100 kDa in size, and only 10 ± 5% of antibodies were directed against gD. Immunoprecipitation (IP) of total HSV-2 antigen with 0ΔNLS antiserum pulled down 19 viral proteins. Mass spectrometry suggested 44% of immunoprecipitated viral peptides were derived from two HSV-2 infected cells proteins, RR-1 and ICP8, whereas only 14% of immunoprecipitated peptides were derived from HSV-2’s thirteen glycoproteins. Collectively, the results suggest the immune response to the live HSV-2 0ΔNLS vaccine includes antibodies specific for infected cell proteins, capsid proteins, tegument proteins, and glycoproteins. This increased breadth of antibody-generating proteins may contribute to the live HSV-2 vaccine’s capacity to elicit superior protection against genital herpes relative to a gD subunit vaccine.     

Epidemiological Impact of a Genital Herpes Type 2 Vaccine for Young Females

Genital Herpes, which is caused by Herpes Simplex Virus-1 or -2 (HSV-1, -2, predominantly HSV-2) is a sexually transmitted infection (STI) that causes a chronic latent infection with outbreak episodes linked to transmission. Antiviral therapies are effective in reducing viral shedding during these episodes, but are ineffective as a whole since many outbreaks are asymptomatic or have mild symptoms. Thus, the development of a vaccine for genital herpes is needed to control this disease. The question of how to implement such a vaccine program is an important one, and may be similar to the vaccination program for Human Papilloma Virus (HPV) for young females. We have developed a mathematical model to describe the epidemiology of vaccination targeting young females against HSV-2. The model population is delineated with respect to age group, sexual activity and infection status including oral infection of HSV-1, which may affect vaccine efficacy. A threshold parameter , which determines the level of vaccine uptake needed to eradicate HSV-2, is found. Computer simulation shows that an adolescent-only vaccination program may be effective in eliminating HSV-2 disease, however, the success of extinction greatly depends on the level of vaccine uptake, the vaccine efficacy, the age of sexual maturity and safe sex practices. However, the time course of eradication would take many years. We also investigate the prevalence of infection in the total population and in women between 16–30 years of age before and after vaccination has been introduced, and show that the adolescent-only vaccination program can be effective in reducing disease prevalence in these populations depending on the level of vaccine uptake and vaccine efficacy. This will also result in a decrease of maternal-fetal transmission of HSV-2 infection. Another important, if commonsense, conclusion is that vaccination of some females reduces infection in men, which then reduces infection in women.     

High Rate of Hypothyroidism in Multidrug-Resistant Tuberculosis Patients Co-Infected with HIV in Mumbai,India

Background

Adverse events (AEs) among HIV-infected patients with multidrug-resistant tuberculosis (MDR-TB) receiving anti-TB and antiretroviral treatments (ART) are under-researched and underreported. Hypothyroidism is a common AE associated with ethionamide, p-aminosalicylic acid (PAS), and stavudine. The aim of this study was to determine the frequency of and risk factors associated with hypothyroidism in HIV/MDR-TB co-infected patients.

Methods

This was a prospective, observational cohort study, using routine laboratory data in a Médecins Sans Frontières (MSF) clinic in collaboration with Sewri TB Hospital, Mumbai, India. Hypothyroidism was defined as a thyroid stimulating hormone (TSH) result >10 mIU/L at least once during treatment. Patients having a baseline result and one additional result after 3 months were eligible for enrolment.

Results

Between October 2006 and March 2013, 116 patients were enrolled, 69 of whom were included. The median (IQR) age was 38 years (34-43) and 61% were male. By March 2013, 37/69 (54%) had hypothyroidism after at least 90 days of treatment. Age, gender, CD4 counts and stavudine-based ART were not associated with the occurrence of hypothyroidism in multivariate models. The co-administration of PAS and ethionamide was found to double the risk of hypothyroidism (RR: 1.93, 95% CI: 1.06-3.54).

Discussion

High rate of hypothyroidism was recorded in a Mumbai cohort of MDR-TB/HIV co-infected patients on treatment. This is a treatable and reversible AE, however, it may go undiagnosed in the absence of regular monitoring. Care providers should not wait for clinical symptoms, as this risks compromising treatment adherence. Simple, affordable and reliable point-of-care tools for measuring TSH are needed, especially in high MDR-TB burden countries. Our findings suggest the need for TSH screening at baseline, three months, six months, and every six months thereafter for HIV-infected patients on MDR-TB treatment regimens containing PAS and/or ethionamide, until newer, safer and more efficacious MDR-TB regimens become available.     

Prevalence of Herpes Simplex Virus 2 (HSV-2) infection and associated risk factors in a cohort of HIV negative women in Durban,South Africa

Background

Herpes Simplex Virus 2 (HSV-2) is one of the most common sexually transmitted infections (STIs) worldwide and is a risk factor for the acquisition and transmission of other STIs, including HIV. We determined the prevalence and predictors of HSV-2 infection among women screened for a HIV prevention trial in Durban, South Africa. Univariate and multivariate logistic and Cox regression models were used to determine the correlates and predictors of HSV-2 infection at enrolment and seroconversion during the study respectively.

Results

Prevalence of HSV-2 at screening was 65% and crude incidence was 22.3 per 100 person-years (PY) (95% CI 20.4–24.3). The HIV seroconversion was significantly higher among those testing positive for HSV-2 at baseline compared to women who were negative [8.7 per 100 person years (PY) versus 5.2 per 100 PY; (p < 0.001)]. In univariate analysis, age was determined to be the most significant predictor for HSV-2 diagnosis, while co-infection with syphilis was also a significant predictor, while age and co-infection with syphilis remained the two most significant predictors of having HSV-2 in multivariate analysis at baseline. Consistent with these results, along with HIV seroconversion, age was also identified as a significant predictor for incidence of HSV-2.

Conclusion

Given the unacceptably high prevalence and incidence rates of HSV-2 infection reported here, HSV-2 and general STI education needs to be reinforced in these communities, with a focus on condom education for prevention. HSV-2 has emerged as the most prevalent STI which is most often asymptomatic and unrecognized, and which increases women’s risk of acquiring other STIs, including HIV.

     

Recognition of Herpes Simplex Virus Type 2 Tegument Proteins by CD4 T Cells Infiltrating Human Genital Herpes Lesions

The local cellular immune response to herpes simplex virus (HSV) is important in the control of recurrent HSV infection. The antiviral functions of infiltrating CD4-bearing T cells may include cytotoxicity, inhibition of viral growth, lymphokine secretion, and support of humoral and CD8 responses. The antigens recognized by many HSV-specific CD4 T cells localizing to genital HSV-2 lesions are unknown. T cells recognizing antigens encoded within map units 0.67 to 0.73 of HSV DNA are frequently recovered from herpetic lesions. Expression cloning with this region of DNA now shows that tegument protein VP22 and the viral dUTPase, encoded by genes U_L49 and U_L50, respectively, are T-cell antigens. Separate epitopes in VP22 were defined for T-cell clones from each of three patients. Reactivity with the tegument protein encoded by U_L21 was identified for an additional patient. Three new epitopes were identified in VP16, a tegument protein associated with VP22. Some tegument-specific CD4 T-cell clones exhibited cytotoxic activity against HSV-infected cells. These results suggest that herpes simplex tegument proteins are processed for antigen presentation in vivo and are possible candidate compounds for herpes simplex vaccines.     

Prevalence and Incidence Estimation of HSV-2 by Two IgG ELISA Methods among South African Women at High Risk of HIV

Introduction

Previous comparison studies of the Kalon and HerpeSelect 2 ELISA IgG assays on sub-Saharan samples have found differences in the sensitivity and specificity of these assays. Using longitudinal samples from an HIV prevention study, we compared both assays and determined the HSV-2 prevalence and incidence in a South African young female population at elevated risk of acquiring HIV.

Methods

Samples at baseline were tested in both assays using the manufacturers’ guidelines (cut-off > 1.10). When non-reactive in one assay, the final visit samples were tested to determine the incidence rate. Using correlation and regression analyses, the intra- and inter-assay variabilities were assessed.

Results

The prevalence rate was 41.1% and 44.9% for Kalon and HerpeSelect using the manufacturer guidelines, respectively. Agreement between the two tests were high (kappa = 0.92). The original optical density values of both assays were highly correlated (R = 0.94), but the calibrator and correspondingly cut-off index values differed between the assays. Lowering the index value cut-off for the Kalon assay by 40% (to 0.66) resulted in a HSV-2 prevalence of 43.2%, and increased agreement between the assays (to kappa = 0.96). The incidence rate was 16.3/100 Person Years using the lower cut-off for the Kalon assay.

Discussion

In this longitudinal study, we showed that the performance of the two assays was very similar. After lowering the cut-off for the Kalon assay to 0.66 early infections were detected without impairing its specificity. The prevalence and incidence rates are in line with previously described rates for sub-Saharan African cohorts.     

Incidence of Liver Damage of Uncertain Origin in HIV Patients Not Co-Infected with HCV/HBV

Background and Aims

Several studies have reported that a significant number of HIV patients not co-infected with HCV/HBV develop liver damage of uncertain origin (LDUO). The objective of our study was to evaluate the incidence of and risk factors for the development of LDUO in HIV infected patients not co-infected with HCV/HBV.

Methods

Prospective longitudinal study that included HIV-infected patients free of previous liver damage and viral hepatitis B or C co-infections. Patients were followed up at 6-monthly intervals. Liver stiffness was measured at each visit. Abnormal liver stiffness (ALS) was defined as a liver stiffness value greater than 7.2 kPa at two consecutive measurements. For patients who developed ALS, a protocol was followed to diagnose the cause of liver damage. Those patients who could not be diagnosed with any specific cause of liver disease were diagnosed as LDUO and liver biopsy was proposed.

Results

210 patients matched the inclusion criteria and were included. 198 patients completed the study. After a median (Q1–Q3) follow-up of 18 (IQR 12–26) months, 21 patients (10.6%) developed ALS. Of these, fifteen patients were diagnosed as LDUO. The incidence of LDUO was 7.64 cases/100 patient-years. Histological studies were performed on ten (66.6%) patients and all showed liver steatosis. A higher HOMA-IR value and body mass index were independently associated with the development of LDUO.

Conclusion

We found a high incidence of LDUO in HIV-infected patients associated with metabolic risk factors. The leading cause of LDUO in our study was non-alcoholic fatty liver disease.     

Dynamic Modeling of Herpes Simplex Virus Type-2 (HSV-2) Transmission: Issues in Structural Uncertainty

The sexually transmitted infection (STI) Herpes simplex virus type-2 (HSV-2) is of public health concern because it is a very common frequently unrecognized lifelong infection, which may facilitate HIV transmission. Within HIV/STI modeling, structural uncertainty has received less attention than parametric uncertainty. By merging the compartments of a “complex” model, a “simple” HSV-2 model is developed. Sexual interactions between female sex workers (FSWs) and clients are modeled using data from India. Latin Hypercube Sampling selects from parameter distributions and both models are run for each of the 10,000 parameter sets generated. Outputs are compared (except for 2,450 unrealistic simulations). The simple model is a good approximation to the complex model once the HSV-2 epidemic has reached 60% of the equilibrium prevalence (95% of the 7,550 runs produced <10% relative error). The simple model is a reduced version of the complex model that retains details implicitly. For late-stage epidemics, the simple model gives similar prevalence trends to the complex model. As HSV-2 epidemics in many populations are advanced, the simple model is accurate in most instances, although the complex model may be preferable for early epidemics. The analysis highlights the issue of structural uncertainty and the value of reducing complexity.     

Effects of Genital Ulcer Disease and Herpes Simplex Virus Type 2 on the Efficacy of Male Circumcision for HIV Prevention: Analyses from the Rakai Trials

Background

Randomized trials show that male circumcision (MC) reduces the incidence of HIV and herpes simplex virus type 2 (HSV-2) infections, and symptomatic genital ulcer disease (GUD). We assessed the role of GUD and HSV-2 in the protection against HIV afforded by MC.

Methods and Findings

HIV-uninfected men were randomized to immediate (n = 2,756) or delayed MC (n = 2,775) in two randomized trials in Rakai, Uganda. GUD symptoms, HSV-2 status, and HIV acquisition were determined at enrollment and at 6, 12, and 24 mo of follow up. Ulcer etiology was assessed by PCR. We estimated the prevalence and prevalence risk ratios (PRRs) of GUD in circumcised versus uncircumcised men and assessed the effects of HSV-2 serostatus as a risk-modifying factor for GUD. We estimated the proportion of the effect of MC on HIV acquisition that was mediated by symptomatic GUD, and by HSV-2 infection. Circumcision significantly reduced symptomatic GUD in HSV-2-seronegative men (PRR = 0.51, 95% [confidence interval] CI 0.43–0.74), HSV-2-seropositive men (PRR = 0.66, 95% CI 0.51–0.69), and in HSV-2 seroconverters (PRR = 0.48, 95% CI 0.30–0.79). The proportion of acute ulcers due to HSV-2 detected by PCR was 48.0% in circumcised men and 39.3% in uncircumcised men (χ² p = 0.62). Circumcision reduced the risk of HIV acquisition in HSV-2 seronegative men (incidence rate ratio [IRR] = 0.34, 95% CI 0.15–0.81), and potentially in HSV-2 seroconverters (IRR = 0.56, 95% CI 0.19–1.57; not significant), but not in men with prevalent HSV-2 at enrollment (IRR = 0.89, 95% CI 0.49–1.60). The proportion of reduced HIV acquisition in circumcised men mediated by reductions in symptomatic GUD was 11.2% (95% CI 5.0–38.0), and the proportion mediated by reduced HSV-2 incidence was 8.6% (95% CI −1.2 to 77.1).

Conclusions

Circumcision reduced GUD irrespective of HSV-2 status, but this reduction played only a modest role in the protective effect of circumcision on HIV acquisition.

NIH Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00425984","term_id":"NCT00425984"}}NCT00425984

Gates Foundation Trial registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00124878","term_id":"NCT00124878"}}NCT00124878 Please see later in the article for the Editors'' Summary