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1.  Early development of broad neutralizing antibodies in HIV-1 infected infants 
Nature medicine  2014;20(6):655-658.
Eliciting protective neutralizing antibodies (NAbs) against HIV-1 is daunting because of the extensive genetic and antigenic diversity of HIV-1. Moreover, broad and potent responses are uncommon even during persistent infection, with only a subset of adults developing broadly neutralizing antibodies (bNAbs) that recognize variants from different HIV-1 clades1–8. It is not known whether bNAbs can also arise in HIV-1-infected infants, who typically progress to disease faster than adults9, presumably in part due to an immature immune system10. Here, we show that bNAbs develop at least as commonly in infants as in adults. Cross-clade NAb responses were detected in 20/28 infected infants, in some cases, within 1 year of infection. Among infants with the top quartile of responses, neutralization of Tier 2–3 variants from multiple clades was detected at 20 months post-infection. These findings suggest that, even in early life, there is sufficient B-cell functionality to mount bNAbs against HIV-1. Additionally, the relatively early appearance of bNAbs in infants may provide a unique setting for understanding the pathways of B-cell maturation leading to bNAbs.
PMCID: PMC4060046  PMID: 24859529
2.  Association between Cellular Immune Activation, Target Cell Frequency, and Risk of Human Immunodeficiency Virus Type 1 Superinfection 
Journal of Virology  2014;88(10):5894-5899.
We performed a case-control study of women at risk of HIV-1 superinfection to understand the relationship between immune activation and HIV-1 acquisition. An increase in the frequency of HIV-1 target cells, but not in other markers of T cell activation, was associated with a 1.7-fold increase in the odds of superinfection. This suggests that HIV-1 acquisition risk is influenced more by the frequency of target cells than by the generalized level of immune activation.
PMCID: PMC4019101  PMID: 24623424
3.  Evidence for Efficient Vertical Transfer of Maternal HIV-1 Envelope-Specific Neutralizing Antibodies but No Association of Such Antibodies with Reduced Infant Infection 
Little is known about the efficiency of vertical transfer of HIV-1-specific antibodies. We compared antibody levels in plasma from 60 mother-infant pairs near the time of birth, including 14 breastfeeding transmission pairs. The Envelope binding titers were strongly correlated (r=0.91, p<0.0001) and similar (1.4-fold greater in maternal plasma) between a mother and her corresponding infant as were the neutralizing antibody (Nab) levels (r = 0.80, p<0.0001; 1.3-fold higher), suggesting efficient transfer. There was no significant difference in Nab responses between transmitting and non-transmitting mothers, although there was a trend for transmitting mothers to have higher HIV-1-specific Nabs.
PMCID: PMC3805370  PMID: 23774880
Mother-to-child transfer; HIV-1 envelope; Neutralizing antibodies
4.  Non-Instrumented Incubation of a Recombinase Polymerase Amplification Assay for the Rapid and Sensitive Detection of Proviral HIV-1 DNA 
PLoS ONE  2014;9(9):e108189.
Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25–43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS.
PMCID: PMC4180440  PMID: 25264766
5.  Mother–Infant HIV Transmission: Do Maternal HIV-Specific Antibodies Protect the Infant? 
PLoS Pathogens  2014;10(8):e1004283.
PMCID: PMC4140838  PMID: 25144453
6.  Performance of the Integrated Management of Childhood Illness (IMCI) Algorithm for Diagnosis of HIV-1 Infection among Kenyan Infants 
AIDS (London, England)  2012;26(15):1935-1941.
Early infant HIV-1 diagnosis and treatment substantially improve survival. Where virologic HIV-1 testing is unavailable, Integrated Management of Childhood Illness (IMCI) clinical algorithms may be used for infant HIV-1 screening. We evaluated the performance of the 2008 WHO IMCI HIV algorithm in a cohort of HIV-exposed Kenyan infants.
From 1999–2003, 444 infants had monthly clinical assessments and quarterly virologic HIV-1 testing. Using archived clinical data, IMCI sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were calculated using virologic testing as a gold standard. Linear regression and survival analyses were used to determine the effect of age on IMCI performance and timing of diagnosis.
Overall IMCI sensitivity, specificity, PPV, and NPV value were 58%, 87%, 52%, and 90%, respectively. Sensitivity (1.4%) and PPV (14%) were lowest at 1 month of age, when 81% of HIV-infections already had occurred. Sensitivity increased with age (p<0.0001), but remained low throughout infancy (range=1.4–35%). Specificity (range=97–100%) was high at each time point and was not associated with age. Fifty-eight percent of HIV-1 infected infants (50/86) were eventually diagnosed by IMCI, and use of IMCI was estimated to delay diagnosis in HIV-infected infants by a median of 5.9 months (p<0.0001).
IMCI had low sensitivity during the first month of life, when the majority of HIV-1 infections had already occurred, and initiation of treatment is most critical. Although sensitivity increased with age, the substantial delay in HIV-1 diagnosis using IMCI limits its utility in early infant HIV-1 diagnosis.
PMCID: PMC4113472  PMID: 22824627
IMCI; HIV; infant; Africa; clinical algorithm; pediatric
7.  Endothelial Activation Biomarkers Increase after HIV-1 Acquisition: Plasma VCAM-1 Predicts Disease Progression 
AIDS (London, England)  2013;27(11):10.1097/QAD.0b013e328360e9fb.
We aimed to determine whether endothelial activation biomarkers increase after HIV-1 acquisition, and whether biomarker levels measured in chronic infection would predict disease progression and death in HIV-1 seroconverters.
HIV-1-seronegative Kenyan women were monitored monthly for seroconversion, and followed prospectively after HIV-1 acquisition.
Plasma levels of angiopoietins-1 and -2 (ANG-1, ANG-2) and soluble vascular cell adhesion marker-1 (VCAM-1), intercellular adhesion marker-1 (ICAM-1), and E-selectin were tested in stored samples from before infection, acute infection, and at two points during chronic infection. We used non-parametric tests to compare biomarkers before and after HIV-1 acquisition, and Cox proportional-hazards regression to analyze associations with disease progression (CD4 <200 cells/μL, Stage IV disease, or ART initiation) or death.
Soluble ICAM-1 and VCAM-1 were elevated relative to baseline in all post-infection periods assessed (p<0.0001). Soluble E-selectin and the ANG-2:ANG-1 ratio increased in acute infection (p=0.0001), and ANG-1 decreased in chronic infection (p=0.0004). Among 228 subjects followed over 1,028 person-years, 115 experienced disease progression or death. Plasma VCAM-1 levels measured during chronic infection were independently associated with time to HIV progression or death (aHR 5.36, 95% confidence interval 1.99–14.44 per log10 increase), after adjustment for set point plasma viral load, age at infection, and soluble ICAM-1 levels.
HIV-1 acquisition was associated with endothelial activation, with sustained elevations of soluble ICAM-1 and VCAM-1 post-infection. Soluble VCAM-1 may be an informative biomarker for predicting the risk of HIV-1 disease progression, morbidity, and mortality.
PMCID: PMC3883757  PMID: 23807276
HIV-1; VCAM-1; ICAM-1; angiopoietin-1; angiopoietin-2; E-selectin; endothelial activation
8.  Antibody-dependent cell-mediated virus inhibition (ADCVI) antibody activity does not correlate with risk of HIV-1 superinfection 
Previous studies of HIV-infected women with high risk behavior have indicated that neither neutralizing antibody nor cellular immunity elicited by an initial HIV-1 infection is associated with protection against superinfection with a different HIV-1 strain. Here, we measured antibody-dependent cell-mediated virus inhibition (ADCVI) antibody activity in the plasma of 12 superinfected cases and 36 singly infected matched controls against 2 heterologous viruses. We found no association between plasma ADCVI activity and superinfection status. ADCVI antibody activity against heterologous virus elicited by the original infection may not contribute to preventing a superinfecting HIV-1.
PMCID: PMC3625514  PMID: 23344546
antibody-dependent cell-mediated virus inhibition; ADCVI; HIV-1; superinfection; primary infection; clade A
9.  Long-term Virologic Response and Genotypic Resistance Mutations in HIV-1 Infected Kenyan Children on Combination Antiretroviral Therapy 
HIV-infected children may require the use of combination antiretroviral treatment (cART) into adulthood. However, regimens are limited to first- and second-line in many African settings. Therefore, understanding the long-term rate of virologic failure and drug resistance during prolonged antiretroviral treatment is important for establishing treatment strategies in African pediatric cohorts.
Children ages 18 months to 12 years initiated first-line cART and were followed every 1–3 months, for up to 5.5 years. Treatment was switched to second-line based on clinical and immunologic criteria according to national guidelines. Virologic failure was determined retrospectively as defined by ≥2 viral loads >5000 copies/mL. Drug resistance was assessed during viral failure by population-based sequencing.
Among 100 children on first-line cART followed for a median 49 months, 34% experienced virologic failure. Twenty-three (68%) of the 34 children with viral failure had detectable resistance mutations, of whom 14 (61%) had multi-class resistance. Fourteen (14%) children were switched to second-line regimens and followed for a median of 28 months. Retrospective analysis revealed that virologic failure had occurred a median of 12 months prior to the switch to second-line. During prolonged first-line treatment in the presence of viral failure, additional resistance mutations accumulated, however, only 1 (7%) of 14 children had persistent viremia during second-line treatment.
Virologic suppression was maintained on first-line cART in two-thirds of HIV-infected children for up to 5 years. Switch to second-line based on clinical/immunologic criteria occurred ~1 year after viral failure, but the delay did not consistently compromise second-line treatment.
PMCID: PMC3593972  PMID: 23196827
10.  Genital Inflammation Predicts HIV-1 Shedding Independent of Plasma Viral Load and Systemic Inflammation 
In women, genital HIV-1 RNA levels predict the risk of HIV-1 transmission independent of plasma viral load. To better understand the factors that contribute to genital HIV-1 shedding, we evaluated the relationships between genital and plasma cytokine concentrations and HIV-1 RNA levels. Vaginal, but not plasma, levels of interferon gamma-induced protein 10 (IP-10) were significantly associated with vaginal viral load, independent of plasma viral load. Thus, efforts to decrease HIV-1 transmission must take into account the role of local inflammation, which is not necessarily reflected in plasma measurements.
PMCID: PMC3494808  PMID: 22878424
HIV-1; inflammation; cytokine; genital; shedding; transmission
11.  Cervicovaginal HIV-1 Neutralizing IgA Detected among HIV-1-Exposed Seronegative Female Partners in HIV-1-Discordant Kenyan Couples 
AIDS (London, England)  2012;26(17):2155-2163.
Cervicovaginal HIV-1-neutralizing IgA was associated with reduced HIV-1 acquisition in a cohort of commercial sex workers. We aimed to define the prevalence and correlates of HIV-1-neutralizing IgA from HIV-1-exposed seronegative (HESN) women in HIV-1-serodiscordant relationships.
HIV-1-serodiscordant couples in Nairobi were enrolled and followed quarterly up to two years, and women in concordant HIV-1-negative relationships were enrolled as controls. Cervicovaginal, seminal, and blood samples were collected at enrollment and follow-up. Cervicovaginal IgA was assessed for HIV-1-neutralizing activity by a peripheral blood mononuclear cell-based assay using an HIV-1 clade A primary isolate.
HESN women in discordant relationships had significantly more HIV-1-neutralizing IgA detected in genital secretions compared to control women (36 of 155 [23%] vs. 4 of 70 [6%], respectively; odds ratio [OR] 5.0; 95% confidence interval [CI] 1.70–14.64; P=0.003). These responses persisted over time in all available follow-up cervicovaginal samples from women with detectable HIV-1-neutralizing IgA at baseline. Partner median HIV-1 plasma viral load was lower among women who had HIV-1-neutralizing IgA compared to women without detectable activity (4.3 vs. 4.8 log10 copies/ml, respectively; OR 0.70; 95% CI 0.51–0.94; P=0.02). A similar trend was found with partner seminal viral load (OR 0.57; 95% CI 0.32–1.02; P=0.06).
HESN women were 5-times more likely to have neutralizing IgA in cervicovaginal secretions than low-risk control women, and these responses were inversely associated with partner viral load. These observations support the existence of antiviral activity in the mucosal IgA fraction following sexual HIV-1 exposure.
PMCID: PMC3799883  PMID: 22948273
HIV; immunoglobulin A; discordant couple; exposed uninfected; Africa; neutralization; viral load
12.  Disruption of Thiamine Uptake and Growth of Cells by Feline Leukemia Virus Subgroup A 
Journal of Virology  2013;87(5):2412-2419.
Feline leukemia virus (FeLV) is still a major cause of morbidity and mortality in domestic cats and some wild cats despite the availability of relatively effective vaccines against the virus. FeLV subgroup A (FeLV-A) is transmitted in natural infections, and FeLV subgroups B, C, and T can evolve directly from FeLV-A by mutation and/or recombination with endogenous retroviruses in domestic cats, resulting in a variety of pathogenic outcomes. The cell surface entry receptor for FeLV-A is a putative thiamine transporter (THTR1). Here, we have addressed whether FeLV-A infection might disrupt thiamine uptake into cells and, because thiamine is an essential nutrient, whether this disruption might have pathological consequences. First, we cloned the cat ortholog of the other of the two known thiamine transporters in mammals, THTR2, and we show that feline THTR1 (feTHTR1) and feTHTR2 both mediate thiamine uptake, but feTHTR2 does not function as a receptor for FeLV-A. We found that feTHTR1 is widely expressed in cat tissues and in cell lines, while expression of feTHTR2 is restricted. Thiamine uptake mediated by feTHTR1 was indeed blocked by FeLV-A infection, and in feline fibroblasts that naturally express feTHTR1 and not feTHTR2, this blockade resulted in a growth arrest at physiological concentrations of extracellular thiamine. The growth arrest was reversed at high extracellular concentrations of thiamine. Our results show that FeLV-A infection can indeed disrupt thiamine uptake with pathological consequences. A prediction of these experiments is that raising the plasma levels of thiamine in FeLV-infected cats may ameliorate the pathogenic effects of infection.
PMCID: PMC3571393  PMID: 23269813
13.  HIV-1 Superinfection Occurs Less Frequently Than Initial Infection in a Cohort of High-Risk Kenyan Women 
PLoS Pathogens  2013;9(8):e1003593.
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 100pys. 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.
Author Summary
HIV-infected individuals with continued exposure are at risk of acquiring a second infection, a process known as superinfection. Superinfection has been reported in various at-risk populations, but how frequently it occurs remains unclear. Determining the frequency of superinfection compared with initial infection can help clarify whether the immune response developed against HIV can protect from reinfection – critical information for understanding whether such responses should guide HIV vaccine design. In this study, we developed a sensitive high-throughput method to identify superinfection and used this to conduct a screen for superinfection in 146 women in a high-risk cohort. This enabled us to determine if first HIV infection affects the risk of second infection by comparing the incidence of superinfection in this group to the incidence of initial infection in 1910 women in the larger cohort. We found that the incidence of superinfection was approximately half that of initial infection after controlling for behavioral and clinical differences that might affect infection risk. These results suggest that the immune response elicited in natural HIV infection may provide partial protection against subsequent infection and indicate the setting of superinfection may shed light on the features of a protective immune response and inform vaccine design.
PMCID: PMC3757054  PMID: 24009513
14.  Antiretroviral Treatment Interruptions Predict Female Genital Shedding of Genotypically Resistant HIV-1 RNA 
Resistant viruses may emerge in the female genital tract during antiretroviral therapy (ART). Our objective was to identify predictors of drug-resistant HIV-1 RNA in genital secretions after initiation of non-nucleoside reverse transcriptase inhibitor (NNRTI)-based therapy.
We conducted a prospective cohort study with periodic evaluation of plasma and genital swab samples for HIV-1 RNA levels and antiretroviral resistance mutations.
First-line ART was initiated in 102 women. Plasma and genital HIV-1 RNA levels were measured at months 0, 3, 6, and 12. Genotypic resistance testing was performed for samples from all participants with RNA >1,000 copies/mL at month 6 or 12. Cox regression analysis was used to identify factors associated with incident genital tract resistance.
Detectable genital tract resistance developed in 5 women, all with detectable plasma resistance (estimated incidence, 5.5/100 person-years of observation). Treatment interruption >48 hours, adherence by pill count, adherence by visual analog scale, and baseline plasma viral load were associated with incident genital tract resistance. In multivariate analysis, only treatment interruption was associated with risk of detectable genital tract resistance (adjusted hazard ratio 14.2, 95% CI 1.3–158.4).
Treatment interruption >48 hours during NNRTI-based therapy led to a significantly increased risk of detecting genotypically resistant HIV-1 RNA in female genital tract secretions. Patient- and program-level interventions to prevent treatment interruptions could reduce the risk of shedding resistant HIV-1 during ART.
PMCID: PMC3404237  PMID: 22592588
HIV drug resistance; virus shedding; female; antiretroviral therapy; adherence
15.  Motif-Optimized Subtype A HIV Envelope-based DNA Vaccines Rapidly Elicit Neutralizing Antibodies When Delivered Sequentially 
Vaccine  2012;30(37):5519-5526.
HIV-1 infection results in the development of a diverging quasispecies unique to each infected individual. Envelope (Env)-specific neutralizing antibodies (NAbs) typically develop over months to years after infection and initially are limited to the infecting virus. In some subjects, antibody responses develop that neutralize heterologous isolates (HNAbs), a phenomenon termed broadening of the NAb response. Studies of co-crystalized antibodies and proteins have facilitated the identification of some targets of broadly neutralizing monoclonal antibodies (NmAbs) capable of neutralizing many or most heterologous viruses; however, the ontogeny of these antibodies in vivo remains elusive. We hypothesize that Env protein escape variants stimulate broad NAb development in vivo and could generate such NAbs when used as immunogens. Here we test this hypothesis in rabbits using HIV Env vaccines featuring: (1) use of individual quasispecies env variants derived from an HIV-1 subtype A-infected subject exhibiting high levels of NAbs within the first year of infection that increased and broadened with time; (2) motif optimization of envs to enhance in vivo expression of DNA formulated as vaccines; and (3) a combined DNA plus protein boosting regimen. Vaccines consisted of multiple env variants delivered sequentially and a simpler regimen that utilized only the least and most divergent clones. The simpler regimen was as effective as the more complex approach in generating modest HNAbs and was more efficient when modified, motif-optimized DNA was used in combination with trimeric gp140 protein. This is a rationally designed strategy that facilitates future vaccine design by addressing the difficult problem of generating HNAbs to HIV by empirically testing the immunogenicity of naturally occurring quasispecies env variants.
PMCID: PMC3447634  PMID: 22749601
16.  Breast milk cellular HIV-specific interferon γ responses are associated with protection from peripartum HIV transmission 
AIDS (London, England)  2012;26(16):2007-2016.
Breast milk is a major route of infant HIV infection, yet the majority of breast-fed, HIV-exposed infants escape infection by unknown mechanisms. This study aimed to investigate the role of HIV-specific breast milk cells in preventing infant HIV infection.
A prospective study was designed to measure associations between maternal breast milk HIV-specific interferon-γ (IFN-γ) responses and infant HIV-1 detection at 1 month of age.
In a Kenyan cohort of HIV-infected mothers, blood and breastmilk HIV-gag IFN-γ ELISpot responses were measured. Logistic regression was used to measure associations between breast milk IFN-γ responses and infant HIV infection at 1 month of age.
IFN-γ responses were detected in breast milk from 117 of 170 (69%) women. IFN-γ responses were associated with breast milk viral load, levels of macrophage inflammatory protein (MIP) 1α, MIP-1β, regulated upon activation, normal T-cell expressed, and secreted and stromal-cell derived factor 1 and subclinical mastitis. Univariate factors associated with infant HIV infection at 1 month postpartum included both detection and breadth of breast milk IFN-γ response (P =0.08, P =0.04, respectively), breast milk MIP-1β detection (P =0.05), and plasma (P =0.004) and breast milk (P =0.004) viral load. In multivariate analyses adjusting for breast milk viral load and MIP-1β, breast milk IFN-γ responses were associated with an approximately 70% reduction in infant HIV infection [adjusted odds ratio (aOR) 0.29, 95% confidence interval (CI) 0.092–0.91], and each additional peptide pool targeted was associated with an approximately 35% reduction in infant HIV (aOR 0.65, 95% CI 0.44–0.97).
These data show breast milk HIV-gag-specific IFN-γ cellular immune responses are prevalent and may contribute to protection from early HIV transmission. More broadly, these data suggest breast milk cellular responses are potentially influential in decreasing mother-to-child transmission of viruses.
PMCID: PMC3718292  PMID: 22948269
breastfeeding; breast milk cytotoxic T lymphocytes; cytokines; early postnatal transmission; infant; MIP-1β; pediatric; sub-Saharan Africa
17.  Low-frequency nevirapine resistance at multiple sites may predict treatment failure in infants on nevirapine-based treatment 
Resistance commonly arises in infants exposed to single-dose nevirapine (sdNVP) for prevention of mother to child transmission (PMTCT). While K103N and Y181C are common following sdNVP, multiple other mutations also confer NVP-resistance. It remains unclear whether specific NVP-resistance mutations or combinations of mutations predict virologic failure in infants when present at low frequencies prior to NVP-based treatment.
Twenty sdNVP-exposed infants who were subsequently treated with NVP-based highly active antiretroviral therapy (HAART) were examined. Pre-treatment plasma samples were tested for the presence of NVP-resistance mutations by allele-specific PCR (ASPCR) for K103N and Y181C and ultra-deep pyrosequencing (UDPS) for all primary NVP mutations. Viral levels were determined every 3 months for up to 24months on NVP-HAART. Cox proportional hazard models were used to determine correlates of viral failure.
The NVP resistance mutations K103N or Y181C were detected in pre-treatment plasma samples in 6 infants by ASPCR. NVP resistance at these or other sites was detectable by UDPS in 10 out of 20 infants tested. Virologic failure occurred in 50% of infants with any NVP resistance mutations detected, while only 20% of infants without resistance experienced viral failure, but the difference was not significant (p=0.19). An increase in the number of NVP resistance mutations detectable by UDPS in an infant was significantly associated with an increased risk of virologic failure (HR=1.79 (95%CI: 1.07, 2.99), p=0.027).
Low frequencies of multiple NVP resistance mutations, in addition to K103N and Y181C, present in infants before NVP-based treatment may predict treatment outcome.
PMCID: PMC3383885  PMID: 22395670
HIV; infants; nevirapine; resistance; HAART; treatment failure
18.  A prospective study of endothelial activation biomarkers, including plasma angiopoietin-1 and angiopoietin-2, in Kenyan women initiating antiretroviral therapy 
BMC Infectious Diseases  2013;13:263.
HIV-1-related inflammation is associated with increased levels of biomarkers of vascular adhesion and endothelial activation, and may increase production of the inflammatory protein angiopoietin-2 (ANG-2), an adverse prognostic biomarker in severe systemic infection. We hypothesized that antiretroviral therapy (ART) initiation would decrease endothelial activation, reducing plasma levels of ANG-2.
Antiretroviral-naïve Kenyan women with advanced HIV infection were followed prospectively. Endothelial activation biomarkers including soluble intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1), and E-selectin, and plasma ANG-2 and angiopoietin-1 (ANG-1) were tested in stored plasma samples from 0, 6, and 12 months after ART initiation. We used Wilcoxon matched-pairs signed rank tests to compare endothelial activation biomarkers across time-points, generalized estimating equations to analyze associations with change in log10-transformed biomarkers after ART initiation, and Cox proportional-hazards regression to analyze associations with mortality.
The 102 HIV-1-seropositive women studied had advanced infection (median CD4 count, 124 cells/μL). Soluble ICAM-1 and plasma ANG-2 levels decreased at both time-points after ART initiation, with concomitant increases in the beneficial protein ANG-1. Higher ANG-2 levels after ART initiation were associated with higher plasma HIV-1 RNA, oral contraceptive pill use, pregnancy, severe malnutrition, and tuberculosis. Baseline ANG-2 levels were higher among five women who died after ART initiation than among women who did not (median 2.85 ng/mL [inter-quartile range (IQR) 2.47–5.74 ng/mL] versus median 1.32 ng/mL [IQR 0.35–2.18 ng/mL], p = 0.01). Both soluble ICAM-1 and plasma ANG-2 levels predicted mortality after ART initiation.
Biomarkers of endothelial activation decreased after ART initiation in women with advanced HIV-1 infection. Changes in plasma ANG-2 were associated with HIV-1 RNA levels over 12 months of follow-up. Soluble ICAM-1 and plasma ANG-2 levels represent potential biomarkers for adverse outcomes in advanced HIV-1 infection.
PMCID: PMC3679794  PMID: 23734875
HIV-1; HAART; ICAM-1; VCAM-1; E-selectin; Angiopoietin-1; Angiopoietin-2; Endothelium
19.  A Species-Specific Amino Acid Difference in the Macaque CD4 Receptor Restricts Replication by Global Circulating HIV-1 Variants Representing Viruses from Recent Infection 
Journal of Virology  2012;86(23):12472-12483.
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.
PMCID: PMC3497638  PMID: 22973036
20.  The Neutralization Sensitivity of Viruses Representing Human Immunodeficiency Virus Type 1 Variants of Diverse Subtypes from Early in Infection is Dependent on Producer Cell, as well as Characteristics of the Specific Antibody and Envelope Variant 
Virology  2012;427(1):25-33.
Neutralization properties of human immunodeficiency virus (HIV-1) are often defined using pseudoviruses grown in transformed cells, which are not biologically relevant HIV-1 producer cells. Little information exists on how these viruses compare to viruses produced in primary lymphocytes, particularly for globally relevant HIV-1 strains. Therefore, replication-competent chimeras encoding envelope variants from the dominant HIV-1 subtypes (A, C, and D) obtained early after infection were generated and the neutralization properties explored. Pseudoviruses generated in 293T cells were the most sensitive to antibody neutralization. Replicating viruses generated in primary lymphocytes were most resistant to neutralization by plasma antibodies and most monoclonal antibodies (b12, 4E10, 2F5, VRC01). These differences were not associated with differences in envelope content. Surprisingly, the virus source did not impact neutralization sensitivity of most viruses to PG9. These findings suggest that producer cell type has a major effect on neutralization sensitivity, but in an antibody dependent manner.
PMCID: PMC3321740  PMID: 22369748
Human Immunodeficiency Virus; Neutralizing Antibodies; Producer Cell; Pseudovirus
21.  Rapid Detection of HIV-1 Proviral DNA for Early Infant Diagnosis Using Recombinase Polymerase Amplification 
mBio  2013;4(2):e00135-13.
Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes.
Diagnosis of HIV-1 infection in infants cannot rely on the antibody-based tests used in adults because of the transfer of maternal HIV-1 antibodies from mother to child. Therefore, infant diagnostics rely on detection of the virus itself. However, current infant HIV-1 diagnostic methods require a laboratory setting with complex equipment. Here we describe the initial development of an HIV-1 diagnostic for infants that may be performed at the point of care in rural health clinics. We utilize a method that can amplify and detect HIV-1 DNA at an incubation temperature within the range of 25 to 42°C, eliminating the need for thermocycling equipment. HIV-1 diagnostics are challenging to develop due to the high diversity seen in HIV-1 strains worldwide. Here we show that this method detects the major HIV-1 strains circulating globally.
PMCID: PMC3622927  PMID: 23549916
22.  A Combination of Broadly Neutralizing HIV-1 Monoclonal Antibodies Targeting Distinct Epitopes Effectively Neutralizes Variants Found in Early Infection 
Journal of Virology  2012;86(19):10857-10861.
Neutralizing antibody protection against HIV-1 may require broad and potent antibodies targeting multiple epitopes. We tested 7 monoclonal antibodies (MAbs) against 45 viruses of diverse subtypes from early infection. The CD4 binding site MAb NIH45-46W was most broad and potent (91% coverage; geometric mean 50% inhibitory concentration [IC50], 0.09 μg/ml). Combining NIH45-46W and a V3-specific MAb, PGT128, neutralized 96% of viruses, while PGT121, another V3-specific MAb, neutralized the remainder. Thus, 2 or 3 antibody specificities may prevent infection by most HIV-1 variants.
PMCID: PMC3457273  PMID: 22837204
23.  Cellular Immune Responses and Susceptibility to HIV-1 Superinfection: A Case-Control Study 
AIDS (London, England)  2012;26(5):643-646.
A case control study was performed to determine the effects of HIV-1-specific cellular immune responses on the odds of acquiring a second HIV-1 infection (superinfection). Changes in the frequency of cytokine-producing or cytolytic CD8+ or CD4+ T cells were not associated with significant alterations in the odds of superinfection, suggesting that HIV-1 specific cellular immune responses at the level induced by chronic infection do not appear to significantly contribute to protection from HIV-1 superinfection.
PMCID: PMC3511787  PMID: 22210637
HIV-1; cellular immunity; T cells; superinfection; re-infection
24.  Loss to Follow-Up as a Competing Risk in an Observational Study of HIV-1 Incidence 
PLoS ONE  2013;8(3):e59480.
Conventional survival estimates may be biased if loss to follow-up (LTF) is associated with the outcome of interest. Our goal was to assess whether the association between sexual risk behavior and HIV-1 acquisition changed after accounting for LTF with competing risks regression.
HIV-1-seronegative women who enrolled in a Kenyan sex worker cohort from 1993–2007 were followed prospectively and tested for HIV at monthly clinic visits. Our primary predictor was self-reported sexual risk behavior in the past week, analyzed as a time-dependent covariate. Outcomes included HIV-1 acquisition and LTF. We analyzed the data using Cox proportional hazards regression and competing risks regression, in which LTF was treated as a competing event.
A total of 1,513 women contributed 4,150 person-years (py), during which 198 (13.1%) acquired HIV-1 infection (incidence, 4.5 per 100 py) and 969 (64.0%) were LTF (incidence, 23.4 per 100 py). After adjusting for potential confounders, women reporting unprotected sex with multiple partners were less likely to be lost to follow-up (adjusted sub-hazard ratio (aSHR) 0.50, 95% confidence interval (CI) 0.32–0.76, relative to no sexual activity). The risk of HIV-1 acquisition after reporting unprotected sex with multiple partners was similar with Cox regression (adjusted hazard ratio (aHR) 2.41, 95% CI 1.36–4.27) and competing risks regression (aSHR 2.47, 95% CI 1.33–4.58).
Unprotected sex with multiple partners was associated with higher HIV-1 acquisition risk, but lower attrition. This differential attrition did not substantially bias Cox regression estimates when compared to competing risks regression results.
PMCID: PMC3595247  PMID: 23555041
25.  Neutralizing Antibody Escape during HIV-1 Mother-to-Child Transmission Involves Conformational Masking of Distal Epitopes in Envelope 
Journal of Virology  2012;86(18):9566-9582.
HIV-1 variants transmitted to infants are often resistant to maternal neutralizing antibodies (NAbs), suggesting that they have escaped maternal NAb pressure. To define the molecular basis of NAb escape that contributes to selection of transmitted variants, we analyzed 5 viruses from 2 mother-to-child transmission pairs, in which the infant virus, but not the maternal virus, was resistant to neutralization by maternal plasma near transmission. We generated chimeric viruses between maternal and infant envelope clones obtained near transmission and examined neutralization by maternal plasma. The molecular determinants of NAb escape were distinct, even when comparing two maternal variants to the transmitted infant virus within one pair, in which insertions in V4 of gp120 and substitutions in HR2 of gp41 conferred neutralization resistance. In another pair, deletions and substitutions in V1 to V3 conferred resistance, but neither V1/V2 nor V3 alone was sufficient. Although the sequence determinants of escape were distinct, all of them involved modifications of potential N-linked glycosylation sites. None of the regions that mediated escape were major linear targets of maternal NAbs because corresponding peptides failed to compete for neutralization. Instead, these regions disrupted multiple distal epitopes targeted by HIV-1-specific monoclonal antibodies, suggesting that escape from maternal NAbs occurred through conformational masking of distal epitopes. This strategy likely allows HIV-1 to utilize relatively limited changes in the envelope to preserve the ability to infect a new host while simultaneously evading multiple NAb specificities present in maternal plasma.
PMCID: PMC3446598  PMID: 22740394

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