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1.  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.
doi:10.1097/QAI.0b013e3182874d41
PMCID: PMC3625514  PMID: 23344546
antibody-dependent cell-mediated virus inhibition; ADCVI; HIV-1; superinfection; primary infection; clade A
2.  Long-term Virologic Response and Genotypic Resistance Mutations in HIV-1 Infected Kenyan Children on Combination Antiretroviral Therapy 
Background
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.
Methods
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.
Results
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.
Discussion
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.
doi:10.1097/QAI.0b013e31827b4ac8
PMCID: PMC3593972  PMID: 23196827
3.  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.
doi:10.1097/QAI.0b013e31826c2edd
PMCID: PMC3494808  PMID: 22878424
HIV-1; inflammation; cytokine; genital; shedding; transmission
4.  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.
Objective
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.
Methods
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.
Results
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).
Conclusion
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.
doi:10.1097/QAD.0b013e328359b99b
PMCID: PMC3799883  PMID: 22948273
HIV; immunoglobulin A; discordant couple; exposed uninfected; Africa; neutralization; viral load
5.  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.
doi:10.1128/JVI.03203-12
PMCID: PMC3571393  PMID: 23269813
6.  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.
doi:10.1371/journal.ppat.1003593
PMCID: PMC3757054  PMID: 24009513
7.  Antiretroviral Treatment Interruptions Predict Female Genital Shedding of Genotypically Resistant HIV-1 RNA 
Objectives
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.
Design
We conducted a prospective cohort study with periodic evaluation of plasma and genital swab samples for HIV-1 RNA levels and antiretroviral resistance mutations.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1097/QAI.0b013e31825bd703
PMCID: PMC3404237  PMID: 22592588
HIV drug resistance; virus shedding; female; antiretroviral therapy; adherence
8.  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.
doi:10.1016/j.vaccine.2012.06.042
PMCID: PMC3447634  PMID: 22749601
9.  Breast milk cellular HIV-specific interferon γ responses are associated with protection from peripartum HIV transmission 
AIDS (London, England)  2012;26(16):2007-2016.
Objective
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.
Design
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.
Methods
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.
Results
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).
Conclusion
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.
doi:10.1097/QAD.0b013e328359b7e0
PMCID: PMC3718292  PMID: 22948269
breastfeeding; breast milk cytotoxic T lymphocytes; cytokines; early postnatal transmission; infant; MIP-1β; pediatric; sub-Saharan Africa
10.  Low-frequency nevirapine resistance at multiple sites may predict treatment failure in infants on nevirapine-based treatment 
Background
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.
Methods
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.
Results
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).
Conclusions
Low frequencies of multiple NVP resistance mutations, in addition to K103N and Y181C, present in infants before NVP-based treatment may predict treatment outcome.
doi:10.1097/QAI.0b013e3182515730
PMCID: PMC3383885  PMID: 22395670
HIV; infants; nevirapine; resistance; HAART; treatment failure
11.  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.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2334-13-263
PMCID: PMC3679794  PMID: 23734875
HIV-1; HAART; ICAM-1; VCAM-1; E-selectin; Angiopoietin-1; Angiopoietin-2; Endothelium
12.  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.
doi:10.1128/JVI.02176-12
PMCID: PMC3497638  PMID: 22973036
13.  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.
doi:10.1016/j.virol.2012.02.001
PMCID: PMC3321740  PMID: 22369748
Human Immunodeficiency Virus; Neutralizing Antibodies; Producer Cell; Pseudovirus
14.  Rapid Detection of HIV-1 Proviral DNA for Early Infant Diagnosis Using Recombinase Polymerase Amplification 
mBio  2013;4(2):e00135-13.
ABSTRACT
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.
IMPORTANCE
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.
doi:10.1128/mBio.00135-13
PMCID: PMC3622927  PMID: 23549916
15.  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.
doi:10.1128/JVI.01414-12
PMCID: PMC3457273  PMID: 22837204
16.  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.
doi:10.1097/QAD.0b013e3283509a0b
PMCID: PMC3511787  PMID: 22210637
HIV-1; cellular immunity; T cells; superinfection; re-infection
17.  Loss to Follow-Up as a Competing Risk in an Observational Study of HIV-1 Incidence 
PLoS ONE  2013;8(3):e59480.
Objective
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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1371/journal.pone.0059480
PMCID: PMC3595247  PMID: 23555041
18.  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.
doi:10.1128/JVI.00953-12
PMCID: PMC3446598  PMID: 22740394
19.  Valacyclovir Suppressive Therapy Reduces Plasma and Breast Milk HIV-1 RNA Levels During Pregnancy and Postpartum: A Randomized Trial 
The Journal of Infectious Diseases  2011;205(3):366-375.
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/mm3). 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 log10 copies/mL. Mean plasma HIV-1 was lower during pregnancy (−.56 log10 copies/mL; 95% confidence interval [CI], −.77 to −.34) and after 6 weeks postpartum (−.51 log10 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.
doi:10.1093/infdis/jir766
PMCID: PMC3256951  PMID: 22147786
20.  The role of amino acid changes in the human immunodeficiency virus type 1 transmembrane domain in antibody binding and neutralization 
Virology  2011;421(2):235-244.
The detailed interactions between antibodies and the HIV-1 envelope protein that lead to neutralization are not well defined. Here, we show that several conservative substitutions in the envelope gp41 led to a ~ 100 fold increase in neutralization sensitivity to monoclonal antibodies (MAbs) that target gp41: 4E10 and 2F5. Substitution at position 675 alone did not impact neutralization susceptibility to MAbs that recognize more distal sites in gp120 (b12, VRC01, PG9). However, changes at position 675 in conjunction with Thr to Ala at position 569 increased the neutralization sensitivity to all gp41 and gp120 MAbs and plasma, in some cases by more than 1000-fold. Interestingly, the T569A change had a dramatic effect on b12 binding, but no effect on neutralization sensitivity. This finding suggests that antibody neutralization may occur through a multi-step pathway that includes distinct changes in envelope conformation that may affect binding but not neutralization susceptibility.
doi:10.1016/j.virol.2011.09.032
PMCID: PMC3249416  PMID: 22029936
21.  Breast Milk HIV-1 RNA Levels and Female Sex Are Associated With HIV-1–Specific CD8+ T-Cell Responses in HIV-1–Exposed, Uninfected Infants in Kenya 
The Journal of Infectious Diseases  2011;204(11):1806-1810.
Background. Although evidence supports a relationship between human immunodeficiency virus (HIV)–1 exposure and HIV-1−specific CD8+ T cell responses, studies have not demonstrated a direct association between the quantity of HIV-1 to which a person is exposed and the presence or absence of a response.
Methods. From 1999 to 2005, maternal HIV-1 RNA levels were measured in blood, cervical secretions, and breast milk at delivery and 1 month after delivery. HIV-1−specific interferon (IFN)–γ Elispot assays were conducted to determine infant CD8+ T-cell responses at 3 months of age.
Results. Among 161 infants tested with Elispot assays, 23 (14%) had positive results. Mothers whose infants had a positive assay had higher breast milk HIV-1 RNA levels at month 1 compared with mothers whose infants had negative Elispot assays (3.1 vs 2.5 log10 copies/mL; P = .017). Female infants were also more likely to have positive Elispot assays than male infants (P = .046), and in multivariate analyses, both female sex and high breast milk HIV-1 levels remained important predictors of a positive response (P = .022 and P = .015, respectively).
Conclusions. Exposure to breast milk HIV-1 and sex were associated with development of HIV-1−specific CD8+ T-cell responses in infants. These data support a role for mucosal exposure via the oral route in induction of systemic HIV-1−specific cellular immunity.
doi:10.1093/infdis/jir643
PMCID: PMC3203234  PMID: 21984736
22.  Increased Levels of HIV-1–Infected Cells in Endocervical Secretions After the Luteinizing Hormone Surge 
Summary
Levels of HIV-1 RNA in endocervical specimens fluctuate with the menstrual cycle, suggesting that cell-free HIV-1 levels may vary during the cycle, which could influence infectivity. Here, we examined daily changes in endocervical HIV-1–infected cells during 1 cycle. There were significant positive associations between the number of days from the luteinizing hormone surge and the number of HIV-1 DNA copies/swab (P = 0.001) and the number of total cells/swab (P < 0.001) in endocervical specimens. These data suggest that sampling of cell-associated endocervical HIV-1 increases after the periovulatory period, which could result in increased exposure to HIV-1–infected cells during sexual contact.
doi:10.1097/QAI.0b013e318165b952
PMCID: PMC3412868  PMID: 18209681
cervical; genital shedding; HIV-1; HIV-1 DNA; hormones; infectivity
23.  HIV-1 Disease Progression in Breast-Feeding and Formula-Feeding Mothers: A Prospective 2-Year Comparison of T Cell Subsets, HIV-1 RNA Levels, and Mortality 
The Journal of Infectious Diseases  2006;195(2):220-229.
Background
There is conflicting evidence regarding the effects of breast-feeding on maternal mortality from human immunodeficiency virus type 1 (HIV-1) infection, and little is known about the effects of breast-feeding on markers of HIV-1 disease progression.
Methods
HIV-1–seropositive women were enrolled during pregnancy and received short-course zidovudine. HIV-1 RNA levels and CD4 cell counts were determined at baseline and at months 1, 3, 6, 12, 18, and 24 postpartum and were compared between breast-feeding and formula-feeding mothers.
Results
Of 296 women, 98 formula fed and 198 breast-fed. At baseline, formula-feeding women had a higher education level and prevalence of HIV-1–related illness than did breast-feeding women; however, the groups did not differ with respect to CD4 cell counts and HIV-1 RNA levels. Between months 1 and 24 postpartum, CD4 cell counts decreased 3.9 cells/µL/month (P< .001), HIV-1 RNA levels increased 0.005 log10 copies/mL/month (P = .03), and body mass index (BMI) decreased 0.03 kg/m2/month (P< .001). The rate of CD4 cell count decline was higher in breast-feeding mothers (7.2 cells/µL/month) than in mothers who never breast-fed (4.0 cells/µL/month) (P = .01). BMI decreased more rapidly in breast-feeding women (P = .04), whereas HIV-1 RNA levels and mortality did not differ significantly between breast-feeding and formula-feeding women.
Conclusions
Breast-feeding was associated with significant decreases in CD4 cell counts and BMI. HIV-1 RNA levels and mortality were not increased, suggesting a limited adverse impact of breast-feeding in mothers receiving extended care for HIV-1 infection.
doi:10.1086/510245
PMCID: PMC3394541  PMID: 17191167
24.  Subtype C Is Associated with Increased Vaginal Shedding of HIV-1 
The Journal of Infectious Diseases  2005;192(3):492-496.
The prevalence of human immunodeficiency virus (HIV)–1–infected cells and HIV-1 RNA levels in genital secretions and breast milk and the risk of mother-to-child transmission of HIV-1 were compared among subtypes A, C, and D in a Kenyan cohort. Pregnant women infected with subtype C were significantly more likely to shed HIV-1-infected vaginal cells than were those infected with subtype A or D (odds ratio [OR], 3.6 [95% confidence interval {CI}, 1.4–8.8]; P = .006). This relationship held after adjusting for age, CD4 cell count, and plasma HIV-1 RNA load (OR, 3.1 [95% CI, 1.1–8.6]; P = .03). These observations suggest that HIV-1 subtype influences mucosal shedding of HIV-1.
doi:10.1086/431514
PMCID: PMC3387274  PMID: 15995964
25.  Longitudinal Analysis of Human Immunodeficiency Virus Type 1 RNA in Breast Milk and of Its Relationship to Infant Infection and Maternal Disease 
The Journal of Infectious Diseases  2003;187(5):741-747.
Transmission of human immunodeficiency virus type 1 (HIV-1) via breast-feeding can occur throughout lactation. Defining both fluctuation in breast-milk virus level over time and how breast-milk virus correlates with mother-to-child transmission is important for establishing effective interventions. We quantified breast-milk HIV-1 RNA levels in serial samples collected from 275 women for up to 2 years after delivery. Higher maternal plasma virus load, lower maternal CD4 T cell count, and detection of HIV-1 DNA in maternal genital secretions were significantly associated with elevated breast-milk HIV-1 RNA. Within women who breast-fed, median virus load in colostrum/early milk was significantly higher than that in mature breast milk collected 14 days after delivery (P ≤ .004). Breast-feeding mothers who transmitted HIV-1 to their infants had both significantly higher breast-milk viral RNA throughout lactation and more-consistent viral shedding, compared with mothers who did not transmit HIV-1. In breast-feeding women, a 2-fold-increased risk of transmission was associated with every 10-fold increase in breast-milk virus load (95% confidence interval, 1.3–3.0; P < .001). These results indicate that the risk of infant infection from breast-feeding is influenced by breast-milk virus load, which is highest early after delivery.
doi:10.1086/374273
PMCID: PMC3384731  PMID: 12599047

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