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1.  Safety and Immunogenicity of 2009 pH1N1 Vaccination in HIV-Infected Pregnant Women 
Two 30-μg doses of unadjuvanted pH1N1 vaccine were moderately immunogenic in human immunodeficiency virus–infected pregnant women, and no serious vaccine-related adverse events were observed. Seroprotection persisted in most women postpartum. Efficient transplacental antibody transfer occurred, but seroprotection in infants waned rapidly.
Background. Pregnant women infected with human immunodeficiency virus (HIV) may have particular vulnerability to 2009 pandemic H1N1 influenza (pH1N1) infection. The safety and immunogenicity of pH1N1 vaccination in HIV-infected pregnant women are unknown.
Methods. HIV-infected women 18–39 years of age and 14–34 weeks’ gestation on antiretroviral therapy received two 30-μg doses of unadjuvanted, inactivated pH1N1 vaccine 21 days apart. Hemagglutination inhibition titers were measured at entry, 21 days after dose 1, and 10 and 21 days after dose 2, and, in mothers and infants, at delivery and 3 and 6 months postdelivery.
Results. No severe vaccine-related adverse events were observed among 127 subjects. At entry, 21% had seroprotective (≥1:40) titers. Seroprotection and seroresponse (≥4-fold rise) occurred in 73% and 66% after dose 1 and 80% and 72% after dose 2, respectively. Of women lacking seroprotection at entry, 66% attained seroprotection after dose 1 and 75% after dose 2. Seroprotective titers were present in 67% of mothers and 65% of infants at delivery (median 66 days after dose 2), 60% of mothers and 26% of infants at 3 months postdelivery, and 59% of mothers and 12% of infants at 6 months postdelivery.
Conclusions. Two 30-μg doses were moderately immunogenic in HIV-infected pregnant women. No concerning vaccine-related safety signals were observed. Seroprotection persisted in most women postpartum. Efficient transplacental antibody transfer occurred, but seroprotection in infants waned rapidly. Vaccination to protect HIV-infected pregnant women and their newborns from new influenza strains is feasible, but more immunogenic platforms should be evaluated.
Clinical Trials Registration. NCT00992017.
doi:10.1093/cid/cit057
PMCID: PMC3634309  PMID: 23378284
pH1N1; vaccine; HIV-infected; pregnancy; immunogenicity
2.  Correction: Association of pol Diversity with Antiretroviral Treatment Outcomes among HIV-Infected African Children 
PLoS ONE  2013;8(12):10.1371/annotation/dd945f7c-c50b-461d-ab38-15e8b0966458.
doi:10.1371/annotation/dd945f7c-c50b-461d-ab38-15e8b0966458
PMCID: PMC3873195
3.  Correction: Association of pol Diversity with Antiretroviral Treatment Outcomes among HIV-Infected African Children 
PLoS ONE  2013;8(12):10.1371/annotation/6937ccda-2f54-430d-94d0-b71d1ecc5273.
doi:10.1371/annotation/6937ccda-2f54-430d-94d0-b71d1ecc5273
PMCID: PMC3861509
4.  Association of pol Diversity with Antiretroviral Treatment Outcomes among HIV-Infected African Children 
PLoS ONE  2013;8(11):e81213.
Background
In HIV-infected children, viral diversity tends to increase with age in the absence of antiretroviral treatment (ART). We measured HIV diversity in African children (ages 6–36 months) enrolled in a randomized clinical trial comparing two ART regimens (Cohort I of the P1060 trial). Children in this cohort were exposed to single dose nevirapine (sdNVP) at birth.
Methods
HIV diversity was measured retrospectively using a high resolution melting (HRM) diversity assay. Samples were obtained from 139 children at the enrollment visit prior to ART initiation. Six regions of the HIV genome were analyzed: two in gag, one in pol, and three in env. A single numeric HRM score that reflects HIV diversity was generated for each region; composite HRM scores were also calculated (mean and median for all six regions).
Results
In multivariable median regression models using backwards selection that started with demographic and clinical variables, older age was associated with higher HRM scores (higher HIV diversity) in pol (P = 0.005) and with higher mean (P = 0.014) and median (P<0.001) HRM scores. In multivariable models adjusted for age, pre-treatment HIV viral load, pre-treatment CD4%, and randomized treatment regimen, higher HRM scores in pol were associated with shorter time to virologic suppression (P = 0.016) and longer time to study endpoints (virologic failure [VF], VF/death, and VF/off study treatment; P<0.001 for all measures).
Conclusions
In this cohort of sdNVP-exposed, ART-naïve African children, higher levels of HIV diversity in the HIV pol region prior to ART initiation were associated with better treatment outcomes.
doi:10.1371/journal.pone.0081213
PMCID: PMC3842253  PMID: 24312277
5.  Drug-Induced Reactivation of Apoptosis Abrogates HIV-1 Infection 
PLoS ONE  2013;8(9):e74414.
HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose) polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP) in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of selectively cytocidal antivirals that eliminate viral infection by destroying infected cells. A drug-based drug discovery program, based on these compounds, is warranted to determine the potential of such agents in clinical trials of HIV-infected patients.
doi:10.1371/journal.pone.0074414
PMCID: PMC3781084  PMID: 24086341
6.  Dynamics of the resting CD4+ T-cell latent HIV reservoir in infants initiating HAART less than 6 months of age 
AIDS (London, England)  2012;26(12):1483-1490.
Objectives
Identification of HIV infection in exposed infants facilitates early therapy, which may limit viral reservoirs that maintain HIV infection under HAART.
Methods
The dynamics of the resting CD4+ T-cell latent HIV reservoir was determined over the first 2 years of life in 17 HIV-infected infants initiating lopinavir/ritonavir-based HAART at a median age of 8.1 weeks and achieving adequate suppression of plasma viral load by 24 weeks.
Results
The resting CD4+ T-cell latent HIV reservoir was detected in 12 of 14 (86%) infants tested at 24 weeks of HAART [median frequency 1.88 infectious units per million (IUPM); range <0.22 to 81.7), and remained measurable (median IUPM =0.32; range < 0.22 to 3.25) in six of 10 (60%) children retested at 96 weeks. The reservoir declined, from 24 to 96 weeks of HAART, at an estimated mean rate of 0.028 log10 IUPM/month, corresponding to a half-life of 11 months (95% confidence interval 6–30 months]. A strong relationship was found between the frequency of latently infected CD4+T cells at 96 weeks of HAART and time to first undetectable plasma viral load (Spearman r =0.91, P <0.001).
Conclusion
Although the resting CD4+ T-cell latent reservoir remains detectable over the first 2 years of HAART in a substantial proportion of infants, its size is associated with time to first undetectable viral load. To minimize HIV reservoirs in infants, rapid curtailment of viremia may limit HIV reservoirs and should be a therapeutic goal of early HAART in infants.
doi:10.1097/QAD.0b013e3283553638
PMCID: PMC3495308  PMID: 22555165
early HAART; infants; latent reservoir dynamics
7.  Gut microbial colonisation in premature neonates predicts neonatal sepsis 
Background
Neonatal sepsis due to intestinal bacterial translocation is a major cause of morbidity and mortality. Understanding microbial colonisation of the gut in prematurity may predict risk of sepsis to guide future strategies to manipulate the microbiome.
Methods
Prospective longitudinal study of premature infants. Stool samples were obtained weekly. DNA was extracted and the V6 hypervariable region of 16S rRNA was amplified followed by high throughput pyrosequencing, comparing subjects with and without sepsis.
Results
Six neonates were 24–27 weeks gestation at birth and had 18 samples analysed. Two subjects had no sepsis during the study period, two developed late-onset culture-positive sepsis and two had culture-negative systemic inflammation. 324 350 sequences were obtained. The meconium was not sterile and had predominance of Lactobacillus, Staphylococcus and Enterobacteriales. Overall, infants who developed sepsis began life with low microbial diversity, and acquired a predominance of Staphylococcus, while healthy infants had more diversity and predominance of Clostridium, Klebsiella and Veillonella.
Conclusions
In very low birth weight infants, the authors found that meconium is not sterile and is less diverse from birth in infants who will develop late-onset sepsis. Empiric, prolonged antibiotics profoundly decrease microbial diversity and promote a microbiota that is associated not only with neonatal sepsis, but the predominant pathogen previously identified in the microbiome. Our data suggest that there may be a ‘healthy microbiome’ present in extremely premature neonates that may ameliorate risk of sepsis. More research is needed to determine whether altered antibiotics, probiotics or other novel therapies can re-establish a healthy microbiome in neonates.
doi:10.1136/fetalneonatal-2011-301373
PMCID: PMC3724360  PMID: 22562869
8.  Evaluation of Tuberculosis Diagnostics in Children: 2. Methodological Issues for Conducting and Reporting Research Evaluations of Tuberculosis Diagnostics for Intrathoracic Tuberculosis in Children. Consensus From an Expert Panela 
The Journal of Infectious Diseases  2012;205(Suppl 2):S209-S215.
Confirming the diagnosis of childhood tuberculosis is a major challenge. However, research on childhood tuberculosis as it relates to better diagnostics is often neglected because of technical difficulties, such as the slow growth in culture, the difficulty of obtaining specimens, and the diverse and relatively nonspecific clinical presentation of tuberculosis in this age group. Researchers often use individually designed criteria for enrollment, diagnostic classifications, and reference standards, thereby hindering the interpretation and comparability of their findings. The development of standardized research approaches and definitions is therefore needed to strengthen the evaluation of new diagnostics for detection and confirmation of tuberculosis in children.
In this article we present consensus statements on methodological issues for conducting research of Tuberculosis diagnostics among children, with a focus on intrathoracic tuberculosis. The statements are complementary to a clinical research case definition presented in an accompanying publication and suggest a phased approach to diagnostics evaluation; entry criteria for enrollment; methods for classification of disease certainty, including the rational use of culture within the case definition; age categories and comorbidities for reporting results; and the need to use standard operating procedures. Special consideration is given to the performance of microbiological culture in children and we also recommend for alternative methodological approaches to report findings in a standardized manner to overcome these limitations are made. This consensus statement is an important step toward ensuring greater rigor and comparability of pediatric tuberculosis diagnostic research, with the aim of realizing the full potential of better tests for children.
doi:10.1093/infdis/jir879
PMCID: PMC3334504  PMID: 22476719
9.  Assessment of Lopinavir Pharmacokinetics With Respect to Developmental Changes in Infants and the Impact on Weight Band–Based Dosing 
Improved antiretroviral therapies are needed for the treatment of HIV infected infants due to rapid disease progression and drug resistance from perinatal exposure to antiretrovirals. We examined longitudinal pharmacokinetic (PK) data from a clinical trial of lopinavir/ritonavir in HIV-infected infants initiating therapy less than 6 months of age. A population PK analysis was performed using NONMEM to characterize changes in lopinavir (LP V) PK relating to maturational changes in infants, and to assess dosing requirements in this population. We also investigated the relationship between LPV PK and viral dynamic response. Age and ritonavir concentrations were the only significant covariates found. Population PK of LPV was characterized by high apparent clearance in young infants which decreased with age. Although younger infants had lower LPV concentrations, viral dynamics did not correlate with initial LPV exposure. Monte Carlo simulations demonstrated that WHO weight band-based dosing recommendations predicted therapeutic LPV concentrations and provided comparable drug exposure levels comparable to those resulting from US Food and Drug Administration (FDA)-suggested dosing regimens.
doi:10.1038/clpt.2011.218
PMCID: PMC3356582  PMID: 22190064
population pharmacokinetics; HIV; WHO; AIDS; antiretroviral
10.  Nevirapine versus Ritonavir-Boosted Lopinavir for HIV-Infected Children 
The New England journal of medicine  2012;366(25):2380-2389.
BACKGROUND
Nevirapine-based antiretroviral therapy is the predominant (and often the only) regimen available for children in resource-limited settings. Nevirapine resistance after exposure to the drug for prevention of maternal-to-child human immunodeficiency virus (HIV) transmission is common, a problem that has led to the recommendation of ritonavir-boosted lopinavir in such settings. Regardless of whether there has been prior exposure to nevirapine, the performance of nevirapine versus ritonavir-boosted lopinavir in young children has not been rigorously established.
METHODS
In a randomized trial conducted in six African countries and India, we compared the initiation of HIV treatment with zidovudine, lamivudine, and either nevirapine or ritonavir-boosted lopinavir in HIV-infected children 2 to 36 months of age who had no prior exposure to nevirapine. The primary end point was virologic failure or discontinuation of treatment by study week 24.
RESULTS
A total of 288 children were enrolled; the median percentage of CD4+ T cells was 15%, and the median plasma HIV type 1 (HIV-1) RNA level was 5.7 log10 copies per milliliter. The percentage of children who reached the primary end point was significantly higher in the nevirapine group than in the ritonavir-boosted lopinavir group (40.8% vs. 19.3%; P<0.001). Among the nevirapine-treated children with virologic failure for whom data on resistance were available, more than half (19 of 32) had resistance at the time of virologic failure. In addition, the time to a protocol-defined toxicity end point was shorter in the nevirapine group (P = 0.04), as was the time to death (P = 0.06).
CONCLUSIONS
Outcomes were superior with ritonavir-boosted lopinavir among young children with no prior exposure to nevirapine. Factors that may have contributed to the sub-optimal results with nevirapine include elevated viral load at baseline, selection for nevirapine resistance, background regimen of nucleoside reverse-transcriptase inhibitors, and the standard ramp-up dosing strategy. The results of this trial present policymakers with difficult choices. (Funded by the National Institute of Allergy and Infectious Diseases and others; P1060 ClinicalTrials.gov number, NCT00307151.)
doi:10.1056/NEJMoa1113249
PMCID: PMC3443859  PMID: 22716976
11.  Mortality Trends in the US Perinatal AIDS Collaborative Transmission Study (1986–2004) 
A significant highly active antiretroviral therapy-associated decrease in annual mortality and a prolongation in survival were seen in this US perinatal cohort of human immunodeficiency virus-infected children. Temporal reductions in opportunistic infection (OI)-associated mortality were replaced by non-OI-associated deaths.
(See the Editorial Commentary by Nachman, on pages 1035–6.)
Background. Highly active antiretroviral therapy (HAART) has improved human immunodeficiency virus (HIV)–associated morbidity and mortality. The bimodal mortality distribution in HIV-infected children makes it important to evaluate temporal effects of HAART among a birth cohort with long-term, prospective follow-up.
Methods. Perinatal AIDS Collaborative Transmission Study (PACTS)/PACTS–HIV Follow-up of Perinatally Exposed Children (HOPE) study was a Centers for Disease Control and Prevention–sponsored multicenter, prospective birth cohort study of HIV-exposed uninfected and infected infants from 1985 until 2004. Mortality was evaluated for the no/monotherapy, mono-/dual-therapy, and HAART eras, that is, 1 January 1986 through 31 December 1990, from 1 January 1991 through 31 December 1996, and 1 January 1997 through 31 December 2004.
Results. Among 364 HIV-infected children, 56% were female and 69% black non-Hispanic. Of 98 deaths, 79 (81%) and 61 (62%) occurred in children ≤3 and ≤2 years old, respectively. The median age at death increased significantly across the eras (P < .0001). The average annual mortality rates were 18 (95% confidence interval [CI], 11.6–26.8), 6.9 (95% CI, 5.4–8.8), and 0.8 (95% CI, 0.4–1.5) events per 100 person-years for the no/monotherapy, mono-/dual-therapy and HAART eras, respectively. The corresponding 6-year survival rates for children born in these eras were 57%, 76%, and 91%, respectively (P < .0001). Among children who received HAART in the first 6 months of age, the probability of 6-year survival was 94%. Ten-year survival rates for HAART and non-HAART recipients were 94% and 45% (P < .05). HAART-associated reductions in mortality remained significant after adjustment for confounders (hazard ratio, 0.3; 95% CI, .08–.76). Opportunistic infections (OIs) caused 31.8%, 16.9%, and 9.1% of deaths across the respective eras (P = .051).
Conclusions. A significant decrease in annual mortality and a prolongation in survival were seen in this US perinatal cohort of HIV-infected children. Temporal decreases in OI-associated mortality resulted in relative proportional increases of non–OI-associated deaths.
doi:10.1093/cid/cir641
PMCID: PMC3202314  PMID: 22002982
12.  Use of Dried-Blood-Spot Samples and In-House Assays To Identify Antiretroviral Drug Resistance in HIV-Infected Children in Resource-Constrained Settings ▿  
Journal of Clinical Microbiology  2011;49(12):4077-4082.
Monitoring HIV drug resistance is an important component of the World Health Organization's global HIV program. HIV drug resistance testing is optimal with commercially available clinically validated test kits using plasma; however, that type of testing may not be feasible or affordable in resource-constrained settings. HIV genotyping from dried blood spots (DBS) with noncommercial (in-house) assays may facilitate the capture of HIV drug resistance outcomes in resource-constrained settings but has had varying rates of success. With in-house assays for HIV reverse transcriptase, we evaluated the yield of genotyping DBS samples collected from HIV-infected children who were enrolled in two clinical trials conducted in sub-Saharan Africa (median HIV viral load, 5.88 log10 HIV RNA copies/ml; range, 4.04 to 6.99). Overall, HIV genotypes were obtained for 94 (89.5%) of 105 samples tested (95% and 84% from clinical trials #1 and #2, respectively); however, successful analysis of 15 (16.1%) of the 94 samples required repeat testing using a different set of primers on previously synthesized cDNA. The yield of genotyping was lower on the DBS that were stored suboptimally from clinical trial #2 (56% versus 88% for optimally stored). Concordance with plasma genotypes derived using a clinically validated, commercial kit-based assay (ViroSeq HIV-1 genotyping system) was also assessed in a subset of children with paired testing. For 34 samples with paired DBS and plasma genotypes, there was 100% concordance for major drug resistance mutations. DBS genotyping using in-house assays provides an alternative for antiretroviral drug resistance testing in children in resource-constrained regions but may require region-specific optimization before widespread use.
doi:10.1128/JCM.01004-11
PMCID: PMC3232965  PMID: 21956987
13.  Assessing Medication Adherence of Perinatally HIV-Infected Children Using Caregiver Interviews 
Medication adherence is critical for children’s HIV treatment success, but obtaining accurate assessments is challenging when complex measurement technologies are not feasible. Our goal was to evaluate a multidimensional adherence interview designed to improve upon existing adherence measures. Data from caregivers (N = 126) of perinatally infected children were analyzed to determine the ability of the revised interview guide to detect potential treatment non-adherence. Questions related to viral load (VL) on a bivariate level included proportion of doses taken in the previous 3 days and 6 months, caregivers’ knowledge of prescribed dosing frequencies, and caregivers’ reports of problems associated with medication administration. VL was not associated with 3-day recall of missed doses. In multivariate analyses, only caregiver knowledge of prescribed dosing frequencies was uniquely associated with VL. Our modified interview appears to successfully identify family struggles with adherence and to have the capacity to help clinicians address medication adherence challenges.
doi:10.1016/j.jana.2010.02.006
PMCID: PMC2964376  PMID: 20452242
adherence assessment; HIV disease; knowledge of prescribed dosing frequency; pediatrics
15.  Antiretroviral Treatment for Children with Peripartum Nevirapine Exposure 
The New England journal of medicine  2010;363(16):1510-1520.
BACKGROUND
Single-dose nevirapine is the cornerstone of the regimen for prevention of mother-to-child transmission of human immunodeficiency virus (HIV) in resource-limited settings, but nevirapine frequently selects for resistant virus in mothers and children who become infected despite prophylaxis. The optimal antiretroviral treatment strategy for children who have had prior exposure to single-dose nevirapine is unknown.
METHODS
We conducted a randomized trial of initial therapy with zidovudine and lamivudine plus either nevirapine or ritonavir-boosted lopinavir in HIV-infected children 6 to 36 months of age, in six African countries, who qualified for treatment according to World Health Organization (WHO) criteria. Results are reported for the cohort that included children exposed to single-dose nevirapine prophylaxis. The primary end point was virologic failure or discontinuation of treatment by study week 24. Enrollment in this cohort was terminated early on the recommendation of the data and safety monitoring board.
RESULTS
A total of 164 children were enrolled. The median percentage of CD4+ lymphocytes was 19%; a total of 56% of the children had WHO stage 3 or 4 disease. More children in the nevirapine group than in the ritonavir-boosted lopinavir group reached a primary end point (39.6% vs. 21.7%; weighted difference, 18.6 percentage-points; 95% confidence interval, 3.7 to 33.6; nominal P = 0.02). Baseline resistance to nevirapine was detected in 18 of 148 children (12%) and was predictive of treatment failure. No significant between-group differences were seen in the rate of adverse events.
CONCLUSIONS
Among children with prior exposure to single-dose nevirapine for perinatal prevention of HIV transmission, antiretroviral treatment consisting of zidovudine and lamivudine plus ritonavir-boosted lopinavir resulted in better outcomes than did treatment with zidovudine and lamivudine plus nevirapine. Since nevirapine is used for both treatment and perinatal prevention of HIV infection in resource-limited settings, alternative strategies for the prevention of HIV transmission from mother to child, as well as for the treatment of HIV infection, are urgently required. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT00307151.)
doi:10.1056/NEJMoa1000931
PMCID: PMC3021781  PMID: 20942667
16.  Long-term outcomes for HIV-infected infants less than 6 months of age at initiation of lopinavir/ritonavir combination antiretroviral therapy 
AIDS (London, England)  2011;25(5):643-649.
Objective
To investigate the longitudinal pharmacokinetics, safety and efficacy of lopinavir/ritonavir (LPV/r) in HIV-infected infants initiating combination antiretroviral therapy (cART) between 2 weeks and 6 months of age.
Method
A prospective, open-label, multicenter Phase I/II study of LPV/r-based cART at a dose of 300/75 mg/m2/dose LPV/r twice daily. Intensive pharmacokinetic sampling at 12 months of age and quarterly predose LPV concentrations were collected and safety, virologic and immunologic responses were monitored every 4–12 weeks up to 252 weeks.
Results
Thirty-one HIV-infected infants enrolled into two age cohorts, 14 days to <6 weeks and 6 weeks to <6 months; 29 completed ≥48 weeks of follow-up (median=123 weeks, range 4–252). At 12 months of age, median LPV area under the curve was comparable for both age cohorts and similar to older children and adults. At week 48, 22 of 31 patients (71%) had HIV-1 RNA <400 copies/ml and 11 of 15 (73%) had <50 copies/ml; 29 of 31 achieved HIV-1 RNA <400 copies/ml on study treatment and 19 (66%) remained durably suppressed until the end of study; viral suppression correlated with a higher percentage of predose time points exceeding the LPV target of 1 μg/ml (92 vs. 71%, P=0.002).
Conclusion
LPV/r at 300/75 mg/m2/dose as part of a cART regimen resulted in viral suppression through 96 weeks of treatment in >65% of young infants. Due to initially low LPV exposure in infants <6 weeks of age, frequent dose adjustment for weight gain is advisable and consideration should be given to studying a higher dose for very young infants.
doi:10.1097/QAD.0b013e32834403f6
PMCID: PMC3068474  PMID: 21297419
AIDS; HIV-1; initiation of antiretroviral therapy in young infants; lopinavir/ritonavir; pharmacokinetics of antiretrovirals
17.  Inhibition of HIV-1 gene expression by Ciclopirox and Deferiprone, drugs that prevent hypusination of eukaryotic initiation factor 5A 
Retrovirology  2009;6:90.
Background
Eukaryotic translation initiation factor eIF5A has been implicated in HIV-1 replication. This protein contains the apparently unique amino acid hypusine that is formed by the post-translational modification of a lysine residue catalyzed by deoxyhypusine synthase and deoxyhypusine hydroxylase (DOHH). DOHH activity is inhibited by two clinically used drugs, the topical fungicide ciclopirox and the systemic medicinal iron chelator deferiprone. Deferiprone has been reported to inhibit HIV-1 replication in tissue culture.
Results
Ciclopirox and deferiprone blocked HIV-1 replication in PBMCs. To examine the underlying mechanisms, we investigated the action of the drugs on eIF5A modification and HIV-1 gene expression in model systems. At early times after drug exposure, both drugs inhibited substrate binding to DOHH and prevented the formation of mature eIF5A. Viral gene expression from HIV-1 molecular clones was suppressed at the RNA level independently of all viral genes. The inhibition was specific for the viral promoter and occurred at the level of HIV-1 transcription initiation. Partial knockdown of eIF5A-1 by siRNA led to inhibition of HIV-1 gene expression that was non-additive with drug action. These data support the importance of eIF5A and hypusine formation in HIV-1 gene expression.
Conclusion
At clinically relevant concentrations, two widely used drugs blocked HIV-1 replication ex vivo. They specifically inhibited expression from the HIV-1 promoter at the level of transcription initiation. Both drugs interfered with the hydroxylation step in the hypusine modification of eIF5A. These results have profound implications for the potential therapeutic use of these drugs as antiretrovirals and for the development of optimized analogs.
doi:10.1186/1742-4690-6-90
PMCID: PMC2770518  PMID: 19825182
18.  Correlates of Nontransmission in US Women at High Risk of Human Immunodeficiency Virus Type 1 Infection through Sexual Exposure 
The Journal of infectious diseases  2002;185(4):428-438.
Seventeen women who were persistently uninfected by human immunodeficiency virus type 1 (HIV-1), despite repeated sexual exposure, and 12 of their HIV-positive male partners were studied for antiviral correlates of nontransmission. Thirteen women had ≥1 immune response in the form of CD8 cell noncytotoxic HIV-1 suppressive activity, proliferative CD4 cell response to HIV antigens, CD8 cell production of macrophage inflammatory protein–1β, or ELISPOT assay for HIV-1–specific interferon-γ secretion. The male HIV-positive partners without AIDS had extremely high CD8 cell counts. All 8 male partners evaluated showed CD8 cell–related cytotoxic HIV suppressive activity. Reduced CD4 cell susceptibility to infection, neutralizing antibody, single-cell cytokine production, and local antibody in the women played no apparent protective role. These observations suggest that the primary protective factor is CD8 cell activity in both the HIV-positive donor and the HIV-negative partner. These findings have substantial implications for vaccine development.
doi:10.1086/338830
PMCID: PMC2743095  PMID: 11865394
19.  Diminished Human Immunodeficiency Virus Type 1 DNA Yield from Dried Blood Spots after Storage in a Humid Incubator at 37°C Compared to −20°C▿  
Journal of Clinical Microbiology  2008;46(9):2945-2949.
Collecting whole blood on filter paper simplifies the processing, transport, and storage of specimens used for the diagnosis of human immunodeficiency virus type 1 (HIV-1) and other tests. Specimens may be collected in tropical or rural areas with minimal facilities for handling specimens. To compare simulated tropical conditions with freezer storage, we examined the stability of HIV-1 DNA in dried blood spots (DBS) stored in humid heat and at −20°C. DBS were created by spotting 50-μl aliquots of whole blood on 903 filter paper. DNA was extracted from DBS at baseline and after 2, 6, or 12 months of storage at −20°C or at 37°C with ∼85% humidity. The DNA was tested undiluted or diluted using the Amplicor HIV-1 DNA PCR (Roche), version 1.5. Each reaction was scored positive, negative, or indeterminate based on optical density. Results were compared between storage conditions and over time. A total of 1,832 reactions from 916 DBS were analyzed, including 100 DBS at baseline, 418 stored at −20°C, and 398 stored at 37°C. A chi-square test showed fewer positive reactions for DBS stored at 37°C (55%) than for those stored at −20°C (78%) (P < 0.0001). Samples stored at −20°C showed little change in the probability of detection of HIV-1 DNA over time; the odds ratio (OR) was 0.93 after storage for 1 year. Samples stored at 37°C demonstrated a significant change in detection at 1 year (OR, 0.29). We conclude that exposure of DBS to 37°C and high humidity impaired the recovery of HIV-1 DNA from DBS, whereas DNA recovery was preserved when DBS were stored frozen.
doi:10.1128/JCM.00359-08
PMCID: PMC2546762  PMID: 18614660
21.  Model for Assessment of Proficiency of Human Immunodeficiency Virus Type 1 Sequencing-Based Genotypic Antiretroviral Assays 
Journal of Clinical Microbiology  2005;43(8):3963-3970.
Use of sequencing-based genotyping as a diagnostic assay for human immunodeficiency virus (HIV) antiretroviral resistance is increasing. Periodic evaluation of the proficiency of laboratories performing this assay should be established. It is important to identify components of the assay that influence the generation of reliable sequencing data and that should and can be monitored. A model was developed to determine what parameters were reasonable and feasible for assessing the performance of genotyping assays. Ten laboratories using the genotyping platform, HIV-1 Genotyping System (HGS) v. 1 and software versions 1.1 or 2.0, participated in two rounds of testing. For each round, each group was sent a panel consisting of three clinical samples to sequence in real time. Six months later, seven laboratories using the TRUGENE HIV-1 Genotyping Kit participated in a separate round, working with both panels at the same time. Analysis of the data showed that one main indicator of genotyping proficiency was achievement of ≥98% sequence homology of a sample tested to a group consensus sequence for that sample. A second was concordant identification of codons at sites identified with resistance mutations in the sample, although scoring of these criteria is still undetermined from this study. These criteria are applicable to all sequence-based genotyping platforms and have been used as a baseline for assessing the performance of genotyping for the determination of antiretroviral resistance in our ongoing proficiency program.
doi:10.1128/JCM.43.8.3963-3970.2005
PMCID: PMC1233968  PMID: 16081937
22.  Evaluation of the Editing Process in Human Immunodeficiency Virus Type 1 Genotyping 
Journal of Clinical Microbiology  2003;41(7):3265-3272.
Sequencing-based human immunodeficiency virus type 1 (HIV-1) genotyping assays require subjective interpretation (editing) of sequence data from multiple primers to form consensus sequences and identify antiretroviral drug resistance mutations. We assessed interlaboratory variations in editing and their impact on the recognition of resistance mutations. Six samples were analyzed in a central laboratory by using a research-use-only HIV-1 genotyping system previously produced by Applied Biosystems. The electronic files of individual primer sequences from the samples were sent to 10 laboratories to compare sequence editing strategies. Each sequence data set included sequences from seven primers spanning protease codons 1 to 99 and reverse transcriptase codons 1 to 320. Each laboratory generated a consensus sequence for each sample and completed a questionnaire about editing strategy. The amount of editing performed, the concordance of consensus sequences among the laboratories, and the identification of resistance mutations were evaluated. Sequence agreement was high among the laboratories despite wide variations in editing strategies. All laboratories identified 66 (88%) of 75 resistance mutations in the samples. Nonconcordant identifications were made for 9 (12%) of the 75 mutations, all of which required editing for identification. These results indicate a need for standardized editing guidelines in genotyping assays. Proficiency in editing should be assessed in training and included in quality control programs for HIV-1 genotyping.
doi:10.1128/JCM.41.7.3265-3272.2003
PMCID: PMC165383  PMID: 12843074
23.  Selective Loss of Innate CD4+ Vα24 Natural Killer T Cells in Human Immunodeficiency Virus Infection 
Journal of Virology  2002;76(15):7528-7534.
Vα24 natural killer T (NKT) cells are innate immune cells involved in regulation of immune tolerance, autoimmunity, and tumor immunity. However, the effect of human immunodeficiency virus type 1 (HIV-1) infection on these cells is unknown. Here, we report that the Vα24 NKT cells can be subdivided into CD4+ or CD4− subsets that differ in their expression of the homing receptors CD62L and CD11a. Furthermore, both CD4+ and CD4− NKT cells frequently express both CXCR4 and CCR5 HIV coreceptors. We find that the numbers of NKT cells are reduced in HIV-infected subjects with uncontrolled viremia and marked CD4+ T-cell depletion. The number of CD4+ NKT cells is inversely correlated with HIV load, indicating depletion of this subset. In contrast, CD4− NKT-cell numbers are unaffected in subjects with high viral loads. HIV infection experiments in vitro show preferential depletion of CD4+ NKT cells relative to regular CD4+ T cells, in particular with virus that uses the CCR5 coreceptor. Thus, HIV infection causes a selective loss of CD4+ lymph node homing (CD62L+) NKT cells, with consequent skewing of the NKT-cell compartment to a predominantly CD4− CD62L− phenotype. These data indicate that the key immunoregulatory NKT-cell compartment is compromised in HIV-1-infected patients.
doi:10.1128/JVI.76.15.7528-7534.2002
PMCID: PMC136353  PMID: 12097565
24.  Anti-TAR Polyamide Nucleotide Analog Conjugated with a Membrane-Permeating Peptide Inhibits Human Immunodeficiency Virus Type 1 Production 
Journal of Virology  2002;76(8):3881-3891.
The emergence of drug-resistant variants has posed a significant setback against effective antiviral treatment for human immunodeficiency virus (HIV) infections. The choice of a nonmutable region of the viral genome such as the conserved transactivation response element (TAR element) in the 5′ long terminal repeat (LTR) may potentially be an effective target for drug development. We have earlier demonstrated that a polyamide nucleotide analog (PNA) targeted to the TAR hairpin element, when transfected into cells, can effectively inhibit Tat-mediated transactivation of HIV type 1 (HIV-1) LTR (T. Mayhood et al., Biochemistry 39:11532-11539, 2000). Here we show that this anti-TAR PNA (PNATAR), upon conjugation with a membrane-permeating peptide vector (transportan) retained its affinity for TAR in vitro similar to the unconjugated analog. The conjugate was efficiently internalized into the cells when added to the culture medium. Examination of the functional efficacy of the PNATAR-transportan conjugate in cell culture using luciferase reporter gene constructs resulted in a significant inhibition of Tat-mediated transactivation of HIV-1 LTR. Furthermore, PNATAR-transportan conjugate substantially inhibited HIV-1 production in chronically HIV-1-infected H9 cells. The mechanism of this inhibition appeared to be regulated at the level of transcription. These results demonstrate the efficacy of PNATAR-transportan as a potential anti-HIV agent.
doi:10.1128/JVI.76.8.3881-3891.2002
PMCID: PMC136084  PMID: 11907228
25.  Destabilization of tRNA3Lys from the primer-binding site of HIV-1 genome by anti-A loop polyamide nucleotide analog 
Nucleic Acids Research  2001;29(24):5099-5106.
Initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs by extension of the cellular tRNA3Lys which anneals to the primer-binding site (PBS) on the 5′ non-translated region of the viral RNA genome. The A-rich sequence (A-loop) upstream of the PBS interacts with the anticodon loop of tRNA3Lys and has been proposed to be essential for conferring specificity to tRNA3Lys for priming the initiation of HIV-1 reverse transcription. We observed that polyamide nucleic acid targeted to the A-loop sequence (PNAal) exhibits high binding specificity for its target sequence. The PNAal pre-bound to the A-loop sequence prevents tRNA3Lys priming on the viral RNA consequently blocking in vitro initiation of reverse transcription. Further, PNAal can efficiently disrupt the preformed [tRNA3Lys–viral RNA] complex thereby rendering it non-functional for reverse transcription. The endogenous reverse transcription in disrupted HIV-1 virions containing packaged tRNA3Lys and its replicating enzyme RT was significantly inhibited by PNAal, thus providing direct evidence of the involvement of the A-loop region of viral RNA genome in tRNA3Lys priming process. These findings suggest the potential of the A-loop region as a critical target for blocking HIV-1 replication.
PMCID: PMC97570  PMID: 11812842

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