HIV-1 superinfection occurs less frequently than initial infection in a cohort of high-risk Kenyan women.

HIV superinfection (reinfection) has been reported in several settings, but no study has been designed and powered to rigorously compare its incidence to that of initial infection. Determining whether HIV infection reduces the risk of superinfection is critical to understanding whether an immune response to natural HIV infection is protective. This study compares the incidence of initial infection and superinfection in a prospective seroincident cohort of high-risk women in Mombasa, Kenya. A next-generation sequencing-based pipeline was developed to screen 129 women for superinfection. Longitudinal plasma samples at <6 months, >2 years and one intervening time after initial HIV infection were analyzed. Amplicons in three genome regions were sequenced and a median of 901 sequences obtained per gene per timepoint. Phylogenetic evidence of polyphyly, confirmed by pairwise distance analysis, defined superinfection. Superinfection timing was determined by sequencing virus from intervening timepoints. These data were combined with published data from 17 additional women in the same cohort, totaling 146 women screened. Twenty-one cases of superinfection were identified for an estimated incidence rate of 2.61 per 100 person-years (pys). The incidence rate of initial infection among 1910 women in the same cohort was 5.75 per 100 pys. Andersen-Gill proportional hazards models were used to compare incidences, adjusting for covariates known to influence HIV susceptibility in this cohort. Superinfection incidence was significantly lower than initial infection incidence, with a hazard ratio of 0.47 (CI 0.29-0.75, p = 0.0019). This lower incidence of superinfection was only observed >6 months after initial infection. This is the first adequately powered study to report that HIV infection reduces the risk of reinfection, raising the possibility that immune responses to natural infection are partially protective. The observation that superinfection risk changes with time implies a window of protection that coincides with the maturation of HIV-specific immunity.

A species-specific amino acid difference in the macaque CD4 receptor restricts replication by global circulating HIV-1 variants representing viruses from recent infection.

HIV-1 replicates poorly in macaque cells, and this had hindered the advancement of relevant nonhuman primate model systems for HIV-1 infection and pathogenesis. Several host restriction factors have been identified that contribute to this species-specific restriction to HIV-1 replication, but these do not fully explain the poor replication of most strains of HIV-1 in macaque cells. Only select HIV-1 envelope variants, typically those derived from viruses that have been adapted in cell culture, result in infectious chimeric SIVs encoding HIV-1 envelope (SHIVs). Here we demonstrate that most circulating HIV-1 variants obtained directly from infected individuals soon after virus acquisition do not efficiently mediate entry using the macaque CD4 receptor. The infectivity of these viruses is ca. 20- to 50-fold lower with the rhesus and pig-tailed macaque versus the human CD4 receptor. In contrast, culture-derived HIV-1 envelope variants that facilitate efficient replication in macaques showed similar infectivity with macaque and human CD4 receptors (within ∼2-fold). The ability of an envelope to mediate entry using macaque CD4 correlated with its ability to mediate entry of cells expressing low levels of the human CD4 receptor and with soluble CD4 sensitivity. Species-specific differences in the functional capacity of the CD4 receptor to mediate entry mapped to a single amino acid difference at position 39 that is under strong positive selection, suggesting that the evolution of CD4 may have been influenced by its function as a viral receptor. These results also suggest that N39 in human CD4 may be a critical residue for interaction of transmitted HIV-1 variants. These studies provide important insights into virus-host cell interactions that have hindered the development of relevant nonhuman primate models for HIV-1 infection and provide possible markers, such as sCD4 sensitivity, to identify potential HIV-1 variants that could be exploited for development of better SHIV/macaque model systems.

Valacyclovir suppressive therapy reduces plasma and breast milk HIV-1 RNA levels during pregnancy and postpartum: a randomized trial.

BACKGROUND: The effect of herpes simplex virus type 2 (HSV-2) suppression on human immunodeficiency virus type 1 (HIV-1) RNA in the context of prevention of mother-to-child transmission (PMTCT) interventions is unknown. METHODS: Between April 2008 and August 2010, we conducted a randomized, double-blind trial of twice daily 500 mg valacyclovir or placebo beginning at 34 weeks gestation in 148 HIV-1/HSV-2 coinfected pregnant Kenyan women ineligible for highly active antiretroviral therapy (CD4 > 250 cells/mm(3)). Women received zidovudine and single dose nevirapine for PMTCT and were followed until 12 months postpartum. RESULTS: Mean baseline plasma HIV-1 RNA was 3.88 log(10) copies/mL. Mean plasma HIV-1 was lower during pregnancy (-.56 log(10) copies/mL; 95% confidence interval [CI], -.77 to -.34) and after 6 weeks postpartum (-.51 log(10) copies/mL; 95% CI, -.73 to -.30) in the valacyclovir arm than the placebo arm. Valacyclovir reduced breast milk HIV-1 RNA detection at 6 and 14 weeks postpartum compared with placebo (30% lower, P = .04; 46% lower, P = .01, respectively), but not after 14 weeks. Cervical HIV-1 RNA detection was similar between arms (P = .91). CONCLUSIONS: Valacyclovir significantly decreased early breast milk and plasma HIV-1 RNA among women receiving PMTCT. CLINICAL TRIALS REGISTRATION: NCT00530777.

Evaluation of a single round polymerase chain reaction assay using dried blood spots for diagnosis of HIV-1 infection in infants in an African setting.

BACKGROUND: The aim of this study was to develop an economical 'in-house' single round polymerase chain reaction (PCR) assay using filter paper-dried blood spots (FP-DBS) for early infant HIV-1 diagnosis and to evaluate its performance in an African setting. METHODS: An 'in-house' single round PCR assay that targets conserved regions in the HIV-1 polymerase (pol) gene was validated for use with FP-DBS; first we validated this assay using FP-DBS spiked with cell standards of known HIV-1 copy numbers. Next, we validated the assay by testing the archived FP-DBS (N=115) from infants of known HIV-1 infection status. Subsequently this 'in-house' HIV-1 pol PCR FP-DBS assay was then established in Nairobi, Kenya for further evaluation on freshly collected FP-DBS (N=186) from infants, and compared with findings from a reference laboratory using the Roche Amplicor® HIV-1 DNA Test, version 1.5 assay. RESULTS: The HIV-1 pol PCR FP-DBS assay could detect one HIV-1 proviral copy in 38.7% of tests, 2 copies in 46.9% of tests, 5 copies in 72.5% of tests and 10 copies in 98.1% of tests performed with spiked samples. Using the archived FP-DBS samples from infants of known infection status, this assay was 92.8% sensitive and 98.3% specific for HIV-1 infant diagnosis. Using 186 FP-DBS collected from infants recently defined as HIV-1 positive using the commercially available Roche Amplicor v1.5 assay, 178 FP-DBS tested positive by this 'in-house' single-round HIV-1 pol PCR FP-DBS PCR assay. Upon subsequent retesting, the 8 infant FP-DBS samples that were discordant were confirmed as HIV-1 negative by both assays using a second blood sample. CONCLUSIONS: HIV-1 was detected with high sensitivity and specificity using both archived and more recently collected samples. This suggests that this 'in-house' HIV-1 pol FP-DBS PCR assay can provide an alternative cost-effective, reliable and rapid method for early detection of HIV-1 infection in infants.

Correlates of HIV detection among breastfeeding postpartum Kenyan women eligible under Option B.

BACKGROUND: The Option B+ strategy streamlines delivery of HIV antiretroviral therapy (ART) to pregnant women, but concerns remain about ART treatment adherence and long term outcomes. METHODS: We conducted a retrospective analysis of a cohort of HIV-positive, postpartum breastfeeding women receiving ART via Option B+ in Nairobi, Kenya. The primary outcome was virologic failure in plasma (HIV RNA >1000 copies/mL), and detection in breast milk (>150 copies/mL) and endocervical secretions (>100 copies/mL) at 2 postpartum timepoints. Correlates of virologic failure were assessed using univariate tests and multivariate logistic regression. RESULTS: Of 42 women at 6-14 weeks postpartum, 21.4% of women had HIV RNA detected in plasma; 14.3% in breast milk, and 23.7% in endocervical secretions. At 18-24 weeks postpartum, the percentages were 21.1%, 7.1%, and 14.3%, respectively. Younger maternal age, intent to breastfeed for longer, and later ART start in pregnancy were significantly associated with plasma virologic failure (p < 0.05 for each). Odds of plasma virologic failure at 6-14 weeks postpartum were 1.25 times higher (95% CI 1.04, 1.51) for each increase in week of gestation at ART initiation. Only 3 women had resistance mutations to their regimen. CONCLUSIONS: Despite months of ART, nearly one-quarter of the women in our cohort did not achieve plasma virologic suppression in the postpartum period. After adjusting for time on ART, earlier ART initiation in pregnancy was significantly associated with plasma suppression. Our findings suggest that postpartum HIV RNA monitoring in Option B+ programs will be needed to achieve elimination of MTCT.

Effects of valacyclovir on markers of disease progression in postpartum women co-infected with HIV-1 and herpes simplex virus-2.

OBJECTIVE: Herpes simplex virus type 2 (HSV-2) suppression has been shown to reduce HIV-1 disease progression in non-pregnant women and men, but effects on pregnant and postpartum women have not been described. METHODS: We analyzed data from a cohort of Kenyan women participating in a randomized clinical trial of HSV-2 suppression. Pregnant HIV-1-seropositive, HSV-2-seropositive women who were not eligible for antiretroviral therapy (WHO stage 1-2, CD4>250 cells/µl) were randomized to either 500 mg valacyclovir or placebo twice daily from 34 weeks gestation through 12 months postpartum. Women received zidovudine and single-dose nevirapine for prevention of mother-to-child HIV-1 transmission. HIV-1 progression markers, including CD4 count and plasma HIV-1 RNA levels, were measured serially. Multivariate linear regression was used to compare progression markers between study arms. RESULTS: Of 148 women randomized, 136 (92%) completed 12 months of postpartum follow-up. While adjusted mean CD4 count at 12 months (565 cells/µl placebo arm, 638 cells/µl valacyclovir arm) increased from antenatal levels in both arms, the mean CD4 count increase was 73 cells/µl higher in the valacyclovir arm than placebo arm (p = 0.03). Mean increase in CD4 count was 154 cells/µl in the valacyclovir arm, almost double the increase of 78 cells/µl in the placebo arm. At 12 months, adjusted HIV-1 RNA levels in the placebo arm increased by 0.66 log(10) copies/ml from baseline, and increased by only 0.21 log(10) copies/ml in the valacyclovir arm (0.40 log(10) copies/ml difference, p = 0.001). CONCLUSION: Women randomized to valacyclovir suppressive therapy during pregnancy and postpartum had greater increases in CD4 counts and smaller increases in plasma HIV-1 RNA levels than women in the placebo arm. Valacyclovir suppression during pregnancy and breastfeeding may improve outcomes and delay antiretroviral therapy for HIV-1/HSV-2 co-infected women.

Pediatric HIV-1 in Kenya: pattern and correlates of viral load and association with mortality.

BACKGROUND: There is limited information regarding the pattern and correlates of viral replication in vertically HIV-1-infected children and its role on their outcomes in resource-limited settings. METHODS: HIV-1-infected infants were followed from birth to 24 months. Serial HIV-1 RNA levels were compared in infants infected in utero (<48 hours), peripartum (48 hours-1 month), and late postnatal (after 1 month). Cofactors for viral peak [highest viral load (VL) within 6 months of infection] and set point and mortality were determined. RESULTS: Among 85 HIV-1-infected infants, 24 were infected in utero, 41 peripartum, 13 late postnatal; 7 had no 48-hour assay. HIV-1 VL set point was significantly lower in infants infected >1 month vs. < or = 1 month (5.59 vs. 6.24 log10 copies per milliliter, P = 0.01). Maternal VL correlated with peak infant VL (P < 0.001). Univariately, infant peak and set point VL and 6-month CD4% <15% predicted mortality; and 6-month CD4% <15% remained independently predictive in multivariate analyses (hazard ratio = 4.85, 95% confidence interval: 1.90 to 12.36). CONCLUSIONS: Infants infected after the age of 1 month contained virus better than infants infected before 1 month of age. Maternal VL predicted infant VL, which, in turn was associated with early mortality.

HV-1-specific cytotoxic T lymphocytes and breast milk HIV-1 transmission.

BACKGROUND: Breast-feeding by infants exposed to human immunodeficiency virus type 1 (HIV-1) provides an opportunity to assess the role played by repeated HIV-1 exposure in eliciting HIV-1-specific immunity and in defining whether immune responses correlate with protection from infection. METHODS: Breast-feeding infants born to HIV-1-seropositive women were assessed for HLA-selected HIV-1 peptide-specific cytotoxic T lymphocyte interferon (IFN)-gamma responses by means of enzyme-linked immunospot (ELISpot) assays at 1, 3, 6, 9, and 12 months of age. Responses were deemed to be positive when they reached > or = 50 HIV-1-specific sfu/1 x 10(6) peripheral blood mononuclear cells (PBMCs) and were at least twice those of negative controls. RESULTS: A total of 807 ELISpot assays were performed for 217 infants who remained uninfected with HIV-1 at approximately 12 months of age; 101 infants (47%) had at least 1 positive ELISpot result (median, 78-170 sfu/1 x 10(6) PBMCs). The prevalence and magnitude of responses increased with age (P = .01 and P = .007, respectively); the median log(10) value for HIV-1-specific IFN-gamma responses increased by 1.0 sfu/1 x 10(6) PBMCs/month (P < .001) between 1 and 12 months of age. Of 141 HIV-1-uninfected infants with 1-month ELISpot results, 10 (7%) acquired HIV-1 infection (0/16 with positive vs. 10/125 [8%] with negative ELISpot results; P = .6). Higher values for log(10) HIV-1-specific spot-forming units at 1 month of age were associated with a decreased risk of HIV-1 infection, adjusted for maternal HIV-1 RNA level (adjusted hazard ratio, 0.09 [95% confidence interval, 0.01-0.72]). CONCLUSION: . Breast-feeding HIV-1-exposed uninfected infants frequently had HIV-1-specific IFN-gamma responses. Greater early HIV-1-specific IFN-gamma responses were associated with decreased HIV-1 acquisition.

Acute cytomegalovirus infection in Kenyan HIV-infected infants.

OBJECTIVE: Cytomegalovirus (CMV) coinfection may influence HIV-1 disease progression during infancy. Our aim was to describe the incidence of CMV infection and the kinetics of viral replication in Kenyan HIV-infected and HIV-exposed uninfected infants. METHODS: HIV-1 and CMV plasma viral loads were serially measured in 20 HIV-exposed uninfected and 44 HIV-infected infants born to HIV-infected mothers. HIV-infected children were studied for the first 2 years of life, and HIV-exposed uninfected infants were studied for 1 year. RESULTS: CMV DNA was detected frequently during the first months of life; by 3 months of age, CMV DNA was detected in 90% of HIV-exposed uninfected infants and 93% of infants who had acquired HIV-1 in utero. CMV viral loads were highest in the 1-3 months following the first detection of virus and declined rapidly thereafter. CMV peak viral loads were significantly higher in the HIV-infected infants compared with the HIV-exposed uninfected infants (mean 3.2 versus 2.7 log10 CMV DNA copies/ml, respectively, P = 0.03). The detection of CMV DNA persisted to 7-9 months post-CMV infection in both the HIV-exposed uninfected (8/17, 47%) and HIV-infected (13/18, 72%, P = 0.2) children. Among HIV-infected children, CMV DNA was detected in three of the seven (43%) surviving infants tested between 19 and 21 months post-CMV infection. Finally, a strong correlation was found between peak CMV and HIV-1 viral loads (rho = 0.40, P = 0.008). CONCLUSION: Acute CMV coinfection is common in HIV-infected Kenyan infants. HIV-1 infection was associated with impaired containment of CMV replication.

Quantification of genital human immunodeficiency virus type 1 (HIV-1) DNA in specimens from women with low plasma HIV-1 RNA levels typical of HIV-1 nontransmitters.

Studies of human immunodeficiency virus type 1 (HIV-1) transmission suggest that genital HIV-1 RNA and DNA may both be determinants of HIV-1 infectivity. Despite its potential role in HIV-1 transmission, there are limited quantitative data on genital HIV-1 DNA. Here we validated an in-house real-time PCR method for quantification of HIV-1 DNA in genital specimens. In reactions with 100 genomes to 1 genome isolated from a cell line containing one HIV-1 provirus/cell, this real-time PCR assay is linear and agrees closely with a commercially available real-time PCR assay specific for a cellular housekeeping gene. In mock genital samples spiked with low numbers of HIV-1-infected cells such that the expected HIV-1 DNA copy number/reaction was 100, 10, or 5, the average copy number/reaction was 80.2 (standard deviation [SD], 28.3), 9.1 (SD, 5.4), or 3.1 (SD, 2.1), respectively. We used this method to examine genital HIV-1 DNA levels in specimens from women whose low plasma HIV-1 RNA levels are typical of HIV-1 nontransmitters. The median HIV-1 DNA copy number in endocervical secretions from these women (1.8 HIV-1 DNA copies/10,000 cells) was lower than that for women with higher plasma HIV-1 RNA levels (16.6 HIV-1 DNA copies/10,000 cells) (P=0.04), as was the median HIV-1 DNA copy number in vaginal secretions (undetectable versus 1.0 HIV-1 DNA copies/10,000 cells). These data suggest that women with low plasma HIV-1 RNA and thus a predicted low risk of HIV-1 transmission have low levels of genital HIV-1 cell-associated virus. The assay described here can be utilized in future efforts to examine the role of cell-associated HIV-1 in transmission.

A comparison of genital HIV-1 shedding and sexual risk behavior among Kenyan women based on eligibility for initiation of HAART according to WHO guidelines.

BACKGROUND: Guidelines for initiating antiretrovirals are based on markers of advanced disease and are not directly linked to markers of HIV-1 transmission such as viral shedding. METHODS: We evaluated genital HIV-1 shedding and risk behavior among 650 antiretroviral-naïve women stratified by WHO criteria for initiating antiretrovirals based on CD4 count and symptoms. RESULTS: Genital HIV-1 concentrations increased in stepwise fashion with declining CD4 counts and the presence of symptoms. Compared with the reference group (asymptomatic with CD4 >350 cells/microL), those with advanced immunosuppression (CD4 <200 cells/microL) had significantly higher cervical HIV-1 RNA concentrations (2.4 log10 copies/swab vs. 3.8 log10 copies/swab, P < 0.001). However, women with CD4 counts <200 cells/microL were also less likely than the reference group to report intercourse during the past week (58% vs. 26%, P < 0.001). CONCLUSIONS: Antiretroviral guidelines focusing on individuals with the most advanced immunosuppression will target those with the highest genital HIV-1 concentrations. However, individuals with less advanced immunosuppression also have high levels of genital HIV-1 and may be more sexually active. The effect of increased antiretroviral availability on the spread of HIV-1 might be enhanced by extending treatment, in addition to other risk reduction services, to those with less advanced disease.

Comparison of human immunodeficiency virus type 1 viral loads in Kenyan women, men, and infants during primary and early infection.

Steady-state levels of human immunodeficiency virus type 1 (HIV-1) RNA in plasma reached at approximately 4 months postinfection are highly predictive of disease progression. Several studies have investigated viral levels in adults or infants during primary and early infection. However, no studies have directly compared these groups. We compared differences in peak and set point plasma HIV-1 RNA viral loads among antiretrovirus-naive Kenyan infants and adults for whom the timing of infection was well defined. Peak and set point viral loads were significantly higher in infants than in adults. We did not observe any gender-specific differences in viral set point in either adults or infants. However, infants who acquired HIV-1 in the first 2 months of life, either in utero, intrapartum, or through early breast milk transmission, had significantly higher set point HIV-1 RNA levels than infants who were infected after 2 months of age through late breast milk transmission or adults who were infected through heterosexual transmission.

Validation of performance of the gen-probe human immunodeficiency virus type 1 viral load assay with genital swabs and breast milk samples.

Human immunodeficiency type 1 (HIV-1) continues to spread at an alarming rate. The virus may be transmitted through blood, genital secretions, and breast milk, and higher levels of systemic virus in the index case, as measured by plasma RNA viral load, have been shown to correlate with increased risk of transmitting HIV-1 both vertically and sexually. Less is known about the correlation between transmission and HIV-1 levels in breast milk or genital secretions, in part because reliable quantitative assays to detect HIV-1 in these fluids are not available. Here we show that the Gen-Probe HIV-1 viral load assay can be used to accurately quantify viral load in expressed breast milk and in cervical and vaginal samples collected on swabs. Virus could be quantified from breast milk and swab samples spiked with known amounts of virus, including HIV-1 subtypes A, C, and D. As few as 10 copies of HIV-1 RNA could be detected above background threshold levels in > or =77% of assays performed with spiked breast milk supernatants and mock swabs. In genital swab samples from HIV-1-infected women, similar levels of HIV-1 RNA were consistently detected in duplicate swabs taken from the same woman on the same clinic visit, suggesting that the RNA values from a single swab sample can be used to measure genital viral load.

Micronutrient supplementation increases genital tract shedding of HIV-1 in women: results of a randomized trial.

To test the hypothesis that micronutrient supplementation decreases genital HIV-1 shedding, a double-blind, randomized, placebo-controlled trial of 6 weeks of multivitamin plus selenium supplementation vs. placebo was conducted among 400 HIV-1-seropositive, nonpregnant, antiretroviral-naive women in Mombasa, Kenya. Primary outcome measures included cervical and vaginal shedding of HIV-1-infected cells and RNA. Secondary outcomes included plasma viral load and CD4 count. Surprisingly, the odds of detection of vaginal HIV-1-infected cells were 2.5-fold higher (P = 0.001) and the quantity of HIV-1 RNA in vaginal secretions was 0.37 log10 copies/swab higher (P = 0.004) among women who received micronutrients in comparison to placebo, even after adjustment for potential confounders including baseline HIV-1 shedding and CD4 count. The increase in vaginal HIV-1 shedding was greatest among women who had normal baseline selenium levels. Micronutrient supplementation resulted in higher CD4 (+23 cells/microL, P = 0.03) and CD8 (+74 cells/microL, P = 0.005) counts compared with placebo but did not alter the plasma viral load. In this randomized trial, micronutrients resulted in higher levels of genital HIV-1 shedding compared with placebo. The potential benefit of micronutrient supplementation in HIV-1-seropositive women should be considered in relation to the potential for increased infectivity.

Vitamin A supplementation and human immunodeficiency virus type 1 shedding in women: results of a randomized clinical trial.

Observational studies have associated vitamin A deficiency with vaginal shedding of human immunodeficiency virus (HIV) type 1-infected cells and mother-to-child HIV-1 transmission. To assess the effect of vitamin A supplementation on vaginal shedding of HIV-1, a randomized, double-blind, placebo-controlled trial of 6 weeks of daily oral vitamin A (10,000 IU of retinyl palmitate) was conducted among 400 HIV-1-infected women in Mombasa, Kenya. At follow-up, there was no statistically significant difference in the prevalence of HIV-1 DNA (18% vs. 21%, P=.4) or the quantity of HIV-1 RNA (3.12 vs. 3.00 log(10) copies/swab, P=1.0) in vaginal secretions of women receiving vitamin A, compared with women receiving placebo. No significant effect of supplementation on plasma HIV-1 load or CD4 or CD8 cell counts was observed, and no effect was seen among women who were vitamin A deficient at baseline. Vitamin A supplementation is unlikely to decrease the infectivity of women infected with HIV-1.