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2.  Very early combination antiretroviral therapy in infants: prospects for cure 
Purpose of review
A single case of sustained HIV control in the absence of antiretroviral therapy or HIV-specific immune responses ensued following 18 months of combination antiretroviral therapy initiated at 30 h of age in a perinatally HIV-infected child (the Mississippi child). This case provides proof-of-concept that delay in HIV viremic rebound may ensue following very early treatment (VET) in perinatal infection, likely through marked reduction of latent replication-competent HIV reservoirs.
Recent findings
The latent HIV reservoir remains the critical barrier to remission. Several studies indicate that the earlier effective combination antiretroviral therapy is initiated, the smaller the size of the HIV reservoir. The unique ability of perinatally infected neonates to initiate VET at the time of birth maximizes the potential benefits of limiting latent reservoir size and permitting reservoir decay, likely lengthening the duration of remission and limiting the capacity for re-establishment of viremia.
Summary
This article covers the rationale and feasibility of VET to achieve sustained virologic remission in perinatal infection. Recent studies highlighting the effects of VET on biomarkers of HIV persistence in perinatal HIV infection are reviewed as well as implications and challenges for cure research in pediatric populations.
doi:10.1097/COH.0000000000000127
PMCID: PMC4351817  PMID: 25402708
HIV cure; HIV remission; latent reservoir; perinatal HIV
3.  HIV Type 1 (HIV-1) Proviral Reservoirs Decay Continuously Under Sustained Virologic Control in HIV-1–Infected Children Who Received Early Treatment 
The Journal of Infectious Diseases  2014;210(10):1529-1538.
Background. Early initiation of combination antiretroviral therapy (cART) to human immunodeficiency virus type 1 (HIV-1)–infected infants controls HIV-1 replication and reduces mortality.
Methods. Plasma viremia (lower limit of detection, <2 copies/mL), T-cell activation, HIV-1–specific immune responses, and the persistence of cells carrying replication-competent virus were quantified during long-term effective combination antiretroviral therapy (cART) in 4 perinatally HIV-1–infected youth who received treatment early (the ET group) and 4 who received treatment late (the LT group). Decay in peripheral blood mononuclear cell (PBMC) proviral DNA levels was also measured over time in the ET youth.
Results. Plasma viremia was not detected in any ET youth but was detected in all LT youth (median, 8 copies/mL; P = .03). PBMC proviral load was significantly lower in ET youth (median, 7 copies per million PBMCs) than in LT youth (median, 181 copies; P = .03). Replication-competent virus was recovered from all LT youth but only 1 ET youth. Decay in proviral DNA was noted in all 4 ET youth in association with limited T-cell activation and with absent to minimal HIV-1–specific immune responses.
Conclusions. Initiation of early effective cART during infancy significantly limits circulating levels of proviral and replication-competent HIV-1 and promotes continuous decay of viral reservoirs. Continued cART with reduction in HIV-1 reservoirs over time may facilitate HIV-1 eradication strategies.
doi:10.1093/infdis/jiu297
PMCID: PMC4215073  PMID: 24850788
continuous HIV-1 decay; proviral reservoirs; early antiretroviral therapy; prolonged viral suppression; reduced HIV reservoirs
4.  Raltegravir Pharmacokinetics in Neonates Following Maternal Dosing 
IMPAACT P1097 was a multicenter trial to determine washout pharmacokinetics and safety of in utero/intrapartum exposure to raltegravir in infants born to HIV-infected pregnant women receiving raltegravir-based antiretroviral therapy. Twenty-two mother-infant pairs were enrolled; evaluable pharmacokinetic data was available from 19 mother-infant pairs. Raltegravir readily crossed the placenta, with median cord blood/maternal delivery plasma raltegravir concentration ratio 1.48 (range, 0.32–4.33). Raltegravir elimination was highly variable and extremely prolonged in some infants; [median t½ 26.6 hours (range 9.3–184 hours)]. Prolonged raltegravir elimination likely reflects low neonatal UGT1A1 enzyme activity and enterohepatic recirculation. Excessive raltegravir concentrations must be avoided in the neonate, since raltegravir at high plasma concentrations may increase the risk of bilirubin neurotoxicity. Sub-therapeutic concentrations, which could lead to inadequate viral suppression and development of raltegravir resistance, must be avoided as well. Two ongoing IMPAACT studies are investigating further the pharmacology of raltegravir in neonates.
doi:10.1097/QAI.0000000000000316
PMCID: PMC4197108  PMID: 25162819
5.  Concordance between allele-specific PCR and ultra-deep pyrosequencing for the detection of HIV-1 non-nucleoside reverse transcriptase inhibitor resistance mutations 
Journal of virological methods  2014;207:182-187.
Recent advances in genotyping technologies have allowed for detection of HIV-1 drug resistance mutations present at low levels. The presence and percentage of Y181C and K103N drug-resistant variants in the blood of 105 subtype C HIV-infected infants who failed single-dose nevirapine prophylaxis for HIV transmission were compared using two highly sensitive genotyping methods, allele-specific PCR (AS-PCR) and ultra-deep pyrosequencing. Significant correlations in detection between both methods were found for both Y181C (correlation coefficients of 0.94 [95% CI 0.91-0.96]) and K103N (0.89 [95% CI 0.84 – 0.92]) mutations. The majority of discordant specimens (3/5 Y181C and 8/11 K103N) had wild-type variants when population sequencing was used, but mutant variants were detectable at very low levels (≤5%) with either assay. This difference is most likely due to stochastic variations in the appearance of mutant variants. Overall, both AS-PCR and ultra-deep pyrosequencing methods have proven to be sensitive and accurate, and may confidently be used where feasible.
doi:10.1016/j.jviromet.2014.07.010
PMCID: PMC4150030  PMID: 25034127
HIV-1 drug resistance; Allele-specific PCR; Ultra-deep sequencing; low-frequency NNRTI variants
6.  Research into a functional cure for HIV in neonates: the need for ethical foresight 
The Lancet. Infectious diseases  2014;14(9):893-898.
In 2013, researchers announced that a newborn child from Mississippi, USA might have been functionally cured of HIV by being given combination antiretroviral therapy within hours of birth. Public and media attention has since been captured by the possibility of finding a cure for HIV transmitted from mother to child. Research into the strategy used for the Mississippi patient is crucially important to establish whether it can be replicated and shown to work in diverse populations. At the same time, any ethical issues likely to arise in such studies should be addressed and not ignored in the pursuit of a functional cure. In this Personal View we identify ethical issues that could arise in research towards achievment of a functional cure for HIV in neonates, including difficult trade-offs associated with choosing the study population and questions about the broader social implications of the research, and propose ways to resolve them.
doi:10.1016/S1473-3099(14)70766-2
PMCID: PMC4219548  PMID: 24906850
8.  Switching children previously exposed to nevirapine to nevirapine-based treatment after initial suppression with a protease-inhibitor-based regimen: long-term follow-up of a randomised, open-label trial 
The Lancet. Infectious diseases  2012;12(7):521-530.
Background
Protease inhibitor (PI)-based therapy is recommended as first-line treatment for HIV-infected infants exposed to nevirapine prophylaxis. However, long-term use poses adherence challenges, is associated with metabolic toxicities, limits second-line options, and is costly. We report long-term outcomes of switching nevirapine-exposed children to nevirapine-based therapy after effective suppression of virus replication on a PI-based regimen.
Methods
At one site in Johannesburg, South Africa, 195 nevirapine-exposed children under 24 months of age who achieved virologic suppression <400 copies/ml while treated with a lopinavir/ritonavir (LPV/r)-based regimen were randomized to switch to nevirapine-based therapy (n=96) or remain on the LPV/r-based regimen (n=99). Children were followed a median of 156 weeks and 3 deaths occurred in each group. HIV RNA was quantified every 3 months.
Findings
By 156 weeks post-randomization, more children in the switch group (23.9%) than in the controls (11.1%) had viral failure (p=0.01) defined as confirmed viremia >1000 copies/ml. However, all (22/22) failures in the switch group were detected by 52 weeks compared with 50% in the controls. Children in the switch group (40.5%) were more likely to suppress to <50 copies/ml (primary endpoint of study) than controls (31.3%, p=0.01) and had better CD4 and growth responses initially after switching. Viral failure was related to non-adherence and pre-treatment drug resistance. In those without pre-treatment drug resistance, no significant difference in viral failure between the switch (14.0%) and control (9.5%) groups (p=0.34) was observed.
Interpretation
Viral load testing through 52 weeks can identify all children likely to fail this PI-switch strategy. Switching children once suppressed to a nevirapine-based regimen may be a valuable treatment option provided that adequate viral load monitoring can be done.
doi:10.1016/S1473-3099(12)70051-8
PMCID: PMC4539971  PMID: 22424722
9.  Age at Virologic Control Influences Peripheral Blood HIV Reservoir Size and Serostatus in Perinatally-Infected Adolescents 
JAMA pediatrics  2014;168(12):1138-1146.
Importance
Combination antiretroviral therapy (cART) initiated within several weeks of HIV infection in adults limits proviral reservoirs that preclude HIV cure. Biomarkers of restricted proviral reservoirs may aid in the monitoring of HIV remission or cure.
Objectives
To quantify peripheral blood proviral reservoir size in perinatally HIV-infected adolescents and to identify correlates of limited proviral reservoirs.
Design, Setting, and Participants
A cross-sectional study including 144 perinatally HIV-infected (PHIV+) youth (median age: 14.3 years), enrolled in the US-based Pediatric HIV/AIDS Cohort Study, on durable (median: 10.2 years) cART, stratified by age at virologic control.
Main Outcome and Measures
The primary endpoint was peripheral blood mononuclear cell (PBMC) proviral load following virologic control at different ages. Correlations between proviral load and markers of active HIV production (HIV-specific antibodies, 2-long terminal repeat (2-LTR) circles), and markers of immune activation and inflammation were also assessed.
Results
Proviral reservoir size was markedly reduced in the PHIV+ youth who achieved virologic control by age 1 year (4.2 [interquartile range, 2.6-8 6] copies per 1 million PBMCs) compared to those who achieved virologic control between 1-5 years of age (19.4 [interquartile range, 5.5-99.8] copies per 1 million PBMCs) or after age 5 years (−(70.7 [interquartile range, 23.2-209.4] copies per 1 million PBMCs; P < .00l). A proviral burden <10 copies/million PBMCs was measured in 11 (79%), 20 (40%), and 13 (18%) participants with virologic control at ages <1 year, 1-5 years, and >5 years, respectively (p<0.001). Lower proviral load was associated with undetectable 2-LTR circles (p<0.001) and HIV negative or indeterminate serostatus (p<0.001), but not with concentrations of soluble immune activation markers CD14 and CD163.
Conclusions and Relevance
Early effective cART along with prolonged virologic suppression after perinatal HIV infection leads to negligible peripheral blood proviral reservoirs in adolescence and is associated with negative or indeterminate HIV serostatus. These findings highlight the long-term effect of early effective control of HIV replication on biomarkers of HIV persistence in perinatal infection and the utility of HIV serostatus as a biomarker for small proviral reservoir size, though not necessarily of cure.
doi:10.1001/jamapediatrics.2014.1560
PMCID: PMC4324476  PMID: 25286283
10.  Therapeutic Vaccination Expands and Improves the Function of the HIV-Specific Memory T-Cell Repertoire 
The Journal of Infectious Diseases  2013;207(12):1829-1840.
Background. The licensing of herpes zoster vaccine has demonstrated that therapeutic vaccination can help control chronic viral infection. Unfortunately, human trials of immunodeficiency virus (HIV) vaccine have shown only marginal efficacy.
Methods. In this double-blind study, 17 HIV-infected individuals with viral loads of <50 copies/mL and CD4+ T-cell counts of >350 cells/µL were randomly assigned to the vaccine or placebo arm. Vaccine recipients received 3 intramuscular injections of HIV DNA (4 mg) coding for clade B Gag, Pol, and Nef and clade A, B, and C Env, followed by a replication-deficient adenovirus type 5 boost (1010 particle units) encoding all DNA vaccine antigens except Nef. Humoral, total T-cell, and CD8+ cytotoxic T-lymphocyte (CTL) responses were studied before and after vaccination. Single-copy viral loads and frequencies of latently infected CD4+ T cells were determined.
Results. Vaccination was safe and well tolerated. Significantly stronger HIV-specific T-cell responses against Gag, Pol, and Env, with increased polyfunctionality and a broadened epitope-specific CTL repertoire, were observed after vaccination. No changes in single-copy viral load or the frequency of latent infection were observed.
Conclusions. Vaccination of individuals with existing HIV-specific immunity improved the magnitude, breadth, and polyfunctionality of HIV-specific memory T-cell responses but did not impact markers of viral control.
Clinical Trials Registration. NCT00270465
doi:10.1093/infdis/jit098
PMCID: PMC3654747  PMID: 23482645
HIV; vaccination; therapy; cytotoxic T lymphocytes; humoral immunity; viral latency
11.  Absence of Detectable HIV-1 Viremia after Treatment Cessation in an Infant 
The New England journal of medicine  2013;369(19):1828-1835.
SUMMARY
An infant born to a woman with human immunodeficiency virus type 1 (HIV-1) infection began receiving antiretroviral therapy (ART) 30 hours after birth owing to high-risk exposure. ART was continued when detection of HIV-1 DNA and RNA on repeat testing met the standard diagnostic criteria for infection. After therapy was discontinued (when the child was 18 months of age), levels of plasma HIV-1 RNA, proviral DNA in peripheral-blood mononuclear cells, and HIV-1 antibodies, as assessed by means of clinical assays, remained undetectable in the child through 30 months of age. This case suggests that very early ART in infants may alter the establishment and long-term persistence of HIV-1 infection.
doi:10.1056/NEJMoa1302976
PMCID: PMC3954754  PMID: 24152233
12.  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
13.  World Health Organization Generic Protocol to Assess Drug-Resistant HIV Among Children <18 Months of Age and Newly Diagnosed With HIV in Resource-Limited Countries 
Increased use of nonnucleoside reverse transcriptase inhibitors (NNRTIs) in pregnant and breastfeeding women will result in fewer children infected with human immunodeficiency virus (HIV). However, among children infected despite prevention of mother-to-child transmission (PMTCT), a substantial proportion will acquire NNRTI-resistant HIV, potentially compromising response to NNRTI-based antiretroviral therapy (ART). In countries scaling up PMTCT and pediatric ART programs, it is crucial to assess the proportion of young children with drug-resistant HIV to improve health outcomes and support national and global decision making on optimal selection of pediatric first-line ART. This article summarizes a new World Health Organization surveillance protocol to assess resistance using remnant dried blood spot specimens from a representative sample of children aged <18 months being tested for early infant diagnosis.
doi:10.1093/cid/cis003
PMCID: PMC3338310  PMID: 22544184
14.  Slower Clearance of Nevirapine Resistant Virus in Infants Failing Extended Nevirapine Prophylaxis for Prevention of Mother-to-Child HIV Transmission 
Abstract
Nevirapine resistance mutations arise commonly following single or extended-dose nevirapine (ED-NVP) prophylaxis to prevent mother-to-child transmission (PMTCT) of human immunodeficiency virus (HIV), but decay within 6–12 months of single-dose exposure. Use of ED-NVP prophylaxis in infants is expected to rise, but data on decay of nevirapine resistance mutations in infants in whom ED-NVP failed remain limited. We assessed, in Ethiopian infants participating in the Six-Week Extended Nevirapine (SWEN) Trial, the prevalence and persistence of nevirapine resistance mutations at 6 and 12 months following single-dose or up to 6 weeks of ED-NVP, and correlated their presence with the timing of infection and the type of resistance mutations. Standard population genotyping followed by high-throughput cloning were done on dried blood spot samples collected during the trial. More infants who received ED-NVP had nevirapine resistance detected by standard population genotyping (high frequencies) at age 6 months compared with those who received single-dose nevirapine (SD-NVP) (58% of 24 vs. 26% of 19, respectively; p = 0.06). Moreover, 56% of ED-NVP-exposed infants with nevirapine resistance at age 6 months still had nevirapine resistance mutations present at high frequencies at age 1 year. Infants infected before 6 weeks of age who received either SD- or ED-NVP were more likely to have Y181C or K103N; these mutations were also more likely to persist at high frequencies through 1 year of age. HIV-infected infants in whom ED-NVP prophylaxis fails are likely to experience delayed clearance of nevirapine-resistant virus in the first year of life, which in turn places them at risk for early selection of multidrug-resistant HIV after initial therapy with nonnucleoside reverse transcriptase inhibitor-based regimens.
doi:10.1089/aid.2010.0346
PMCID: PMC3149453  PMID: 21241214
15.  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
16.  Effect of therapeutic HIV recombinant poxvirus vaccines on the size of the resting CD4+ T-cell latent HIV reservoir 
AIDS (London, England)  2011;25(18):2227-2234.
Objectives
Therapeutic HIV vaccinations may alter the size of the resting memory CD4+ T-cell latent HIV reservoir as HIV establishes latency when memory responses are formed, including those toward HIV. Alternatively, latently infected CD4+ T cells maybe killed, while exiting the reservoir upon activation.
Methods
The effect of therapeutic immunization with modified vaccinia Ankara and Fowlpox-based HIV vaccines on the latent reservoir was examined in 19 young adults who were receiving effective antiretroviral therapy. Correlations between size of the reservoir [measured in infectious units per million (IUPM)] resting CD4+ T cells and HIV-specific immune responses, including immune activation were examined. Decay of the reservoir was assessed using random-effects model.
Results
A modest transient decrease in the size of the reservoir was observed at week 40 [mean −0.31 log10 IUPM (95% confidence interval: −0.60 to −0.03; P =0.03] following HIV vaccinations. The estimated half-life (T1/2) of the reservoir during the 40 weeks following vaccination was 9.8 months and statistically different from zero (P =0.02), but 35.3 months and not different from zero (P =0.21) over 72 weeks of study. Latent reservoir size at baseline was not correlated with HIV-specific CD4+, CD8+ responses or immune activation, but became correlated with CD4+ IFNγ (r =0.54, P =0.02) and IL-2 responses at 6 weeks after immunization (r =0.48, P =0.04).
Conclusion
Therapeutic HIV vaccinations led to a transient increase in decay of latently infected CD4+ T cells. Further studies of therapeutic HIV vaccines may provide important insights into facilitating decay of the latent reservoir.
doi:10.1097/QAD.0b013e32834cdaba
PMCID: PMC3314338  PMID: 21918423
resting memory CD4+ T-cell latent reservoir; therapeutic HIV vaccines
17.  Induction Therapy with Protease-Inhibitors Modifies the Effect of Nevirapine Resistance on Virologic Response to Nevirapine-based HAART in Children 
Low levels of nevirapine-resistant HIV after failed prophylaxis compromise first-line nevirapine-based therapy. Initial therapy with lopinavir/ritonavir in nevirapine-exposed children raised the threshold level of pretreatment resistance (≥25% of the viral population) at which reuse of nevirapine-based therapy was affected.
Background. Nevirapine resistance after failed prophylaxis to prevent mother-to-child human immunodeficiency virus (HIV) transmission can compromise subsequent nevirapine-based highly active antiretroviral therapy (HAART).
Methods. Nevirapine-exposed children who achieved virologic suppression with lopinavir/ritonavir-based induction HAART before switch to nevirapine-based HAART or who continued the lopinavir/ritonavir regimen were studied. Nevirapine-resistant HIV was quantified (≥1% frequency) in plasma before therapy and archived in peripheral blood mononuclear cells after induction HAART with ultradeep pyrosequencing. The primary endpoint was virologic failure (confirmed viremia ≥1000 copies/mL by 52 weeks) on nevirapine-based HAART, and Receiver operating characteristic analysis identified threshold levels of resistance associated with failure.
Results. Nevirapine resistance mutations were detected in plasma at a median frequency of 25.6% in 41 (33%) of 124 children starting HAART at median 9 months of age. After a median nine months of induction HAART, nevirapine-resistant HIV remained archived in cells in 59 (61%) of 96 children (median 13.6% of cells). The threshold frequency of nevirapine resistance in plasma most predictive of virologic failure on nevirapine-based HAART was 25%. Children maintaining resistance before therapy at or above this threshold frequency had a 3.5 fold higher risk of failure (95% confidence interval, 1.1–10.8) than children without detectable plasma resistance. In contrast, virologic failure was not independently associated with age, resistance in plasma below 25% frequencies, or archived in cells.
Conclusions. Virologic suppression with lopinavir/ritonavir-based HAART in nevirapine-exposed children raises the threshold level of resistance at which reuse of nevirapine-based therapy is compromised. Standard genotyping may allow identification of children likely to benefit from an induction-switch approach.
doi:10.1093/cid/ciq161
PMCID: PMC3060899  PMID: 21258105
18.  Identification of Ongoing Human Immunodeficiency Virus Type 1 (HIV-1) Replication in Residual Viremia during Recombinant HIV-1 Poxvirus Immunizations in Patients with Clinically Undetectable Viral Loads on Durable Suppressive Highly Active Antiretroviral Therapy▿  
Journal of Virology  2009;83(19):9731-9742.
In most human immunodeficiency virus type 1 (HIV-1)-infected individuals who achieve viral loads of <50 copies/ml during highly active antiretroviral therapy (HAART), low levels of plasma virus remain detectable for years by ultrasensitive methods. The relative contributions of ongoing virus replication and virus production from HIV-1 reservoirs to persistent low-level viremia during HAART remain controversial. HIV-1 vaccination of HAART-treated individuals provides a model for examining low-level viremia, as immunizations may facilitate virus replication and sequence evolution. In a phase 1 trial of modified vaccinia virus Ankara/fowlpox virus-based HIV-1 vaccines in 20 HIV-infected young adults receiving HAART, we assessed the prevalence of low-level viremia and sequence evolution, using ultrasensitive viral load (<6.5 copies/ml) and genotyping (five-copy sensitivity) assays. Viral evolution, consisting of new drug resistance mutations and novel amino acid changes within a relevant HLA-restricted allele (e.g., methionine, isoleucine, glutamine, or arginine for leucine at position 205 of RT), was found in 1 and 3 of 20 subjects, respectively. Sequence evolution was significantly correlated with levels of viremia of between 6.5 and <50 copies/ml (P = 0.03) and was more likely to occur within epitopes presented by relevant HLA alleles (P < 0.001). These findings suggest that ongoing virus replication contributes to low-level viremia in patients on HAART and that this ongoing replication is subject to CD8+ T-cell selective pressures.
doi:10.1128/JVI.00570-09
PMCID: PMC2748010  PMID: 19605490
19.  Safety and immunogenicity of recombinant poxvirus HIV-1 vaccines in young adults on highly active antiretroviral therapy 
Vaccine  2008;26(52):6883-6893.
A trial to evaluate the safety and immunogenicity of recombinant modified vaccinia Ankara (MVA) and fowlpox (FP) vectors expressing multiple HIV-1 proteins was conducted in twenty HIV-1 infected youth with suppressed viral replication on HAART. The MVA and FP-based multigene HIV-1 vaccines were safe and well tolerated. Increased frequencies of HIV-1 specific CD4+ proliferative responses and cytokine secreting cells were detected following immunization. Increased frequencies and breadth of HIV-1 specific CD8+ T cell responses were also detected. Plasma HIV-1–specific antibody levels and neutralizing activity were unchanged following vaccination. Poxvirus based vaccines may merit further study in therapeutic vaccine protocols.
doi:10.1016/j.vaccine.2008.09.084
PMCID: PMC2845914  PMID: 18940219
HIV-1; Vaccine; Therapeutic Immunization
20.  Analyses of HIV-1 Drug-Resistance Profiles Among Infected Adolescents Experiencing Delayed Antiretroviral Treatment Switch After Initial Nonsuppressive Highly Active Antiretroviral Therapy 
AIDS Patient Care and STDs  2008;22(7):545-552.
Abstract
Treatment failure and drug resistance create obstacles to long-term management of HIV-1 infection. Nearly 60% of infected persons fail their first highly active antiretroviral therapy (HAART) regimen, partially because of nonadherence, requiring a switch to a second regimen to prevent drug resistance. Among HIV-infected youth, a group with rising infection rates, treatment switch is often delayed; virologic and immunologic consequences of this delay are unknown. We conducted a retrospective, longitudinal study of drug resistance outcomes of initial HAART in U.S. youth enrolled between 1999–2001 in a multicenter, observational study and experiencing delayed switch in their first nonsuppressive treatment regimen for up to 3 years. HIV-1 genotyping was performed on plasma samples collected longitudinally, and changes in drug resistance mutations, CD4+ T cell numbers and viral replication capacity were assessed. Forty-four percent (n = 18) of youth in the parent study experiencing virologic nonsuppression were maintained on their initial HAART regimen for a median of 144 weeks. Drug resistance was detected in 61% (11/18) of subjects during the study. Subjects on non-nucleoside reverse transcriptase inhibitor (NNRTI) regimens developed more (8/10) drug resistance mutations than those on protease-inhibitor (PI) regimens (2/7) (p = 0.058). Subjects developing NNRTI-resistance (NNRTI-R), showed a trend toward lower CD4+ T cell gains (median: −6 cells/mm3 per year) than those without detectable NNRTI-R (median: +149 cells/mm3 per year) (p = 0.16). HIV-1–infected youth maintained on initial nonsuppressive NNRTI-based HAART regimens are more likely to develop drug-resistant viremia than with PI-based HAART. This finding may have implications for initial treatment regimens and transmission risk in HIV-infected youth, a group with rising infection rates.
doi:10.1089/apc.2007.0200
PMCID: PMC2929158  PMID: 18479228
21.  Nevirapine Resistance and Breast-Milk HIV Transmission: Effects of Single and Extended-Dose Nevirapine Prophylaxis in Subtype C HIV-Infected Infants 
PLoS ONE  2009;4(1):e4096.
Background
Daily nevirapine (NVP) prophylaxis to HIV-exposed infants significantly reduces breast-milk HIV transmission. We assessed NVP-resistance in Indian infants enrolled in the “six-week extended-dose nevirapine” (SWEN) trial who received single-dose NVP (SD-NVP) or SWEN for prevention of breast-milk HIV transmission but who also acquired subtype C HIV infection during the first year of life.
Methods/Findings
Standard population sequencing and cloning for viral subpopulations present at ≥5% frequency were used to determine HIV genotypes from 94% of the 79 infected Indian infants studied. Timing of infection was defined based on when an infant's blood sample first tested positive for HIV DNA. SWEN-exposed infants diagnosed with HIV by six weeks of age had a significantly higher prevalence of NVP-resistance than those who received SD-NVP, by both standard population sequencing (92% of 12 vs. 38% of 29; p = 0.002) and low frequency clonal analysis (92% of 12 vs. 59% of 29; p = 0.06). Likelihood of infection with NVP-resistant HIV through breast-milk among infants infected after age six weeks was substantial, but prevalence of NVP-resistance did not differ among SWEN or SD-NVP exposed infants by standard population sequencing (15% of 13 vs. 15% of 20; p = 1.00) and clonal analysis (31% of 13 vs. 40% of 20; p = 0.72). Types of NVP-resistance mutations and patterns of persistence at one year of age were similar between the two groups. NVP-resistance mutations did differ by timing of HIV infection; the Y181C variant was predominant among infants diagnosed in the first six weeks of life, compared to Y188C/H during late breast-milk transmission.
Conclusions/Significance
Use of SWEN to prevent breast-milk HIV transmission carries a high likelihood of resistance if infection occurs in the first six weeks of life. Moreover, there was a continued risk of transmission of NVP-resistant HIV through breastfeeding during the first year of life, but did not differ between SD-NVP and SWEN groups. As with SD-NVP, the value of preventing HIV infection in a large number of infants should be considered alongside the high risk of resistance associated with extended NVP prophylaxis.
Trial Registration
ClinicalTrials.gov NCT00061321
doi:10.1371/journal.pone.0004096
PMCID: PMC2606064  PMID: 19119321
22.  Residual Human Immunodeficiency Virus Type 1 Viremia in Some Patients on Antiretroviral Therapy Is Dominated by a Small Number of Invariant Clones Rarely Found in Circulating CD4+ T Cells†  
Journal of Virology  2006;80(13):6441-6457.
Antiretroviral therapy can reduce human immunodeficiency virus type 1 (HIV-1) viremia to below the detection limit of ultrasensitive clinical assays (50 copies of HIV-1 RNA/ml). However, latent HIV-1 persists in resting CD4+ T cells, and low residual levels of free virus are found in the plasma. Limited characterization of this residual viremia has been done because of the low number of virions per sample. Using intensive sampling, we analyzed residual viremia and compared these viruses to latent proviruses in resting CD4+ T cells in peripheral blood. For each patient, we found some viruses in the plasma that were identical to viruses in resting CD4+ T cells by pol gene sequencing. However, in a majority of patients, the most common viruses in the plasma were rarely found in resting CD4+ T cells even when the resting cell compartment was analyzed with assays that detect replication-competent viruses. Despite the large diversity of pol sequences in resting CD4+ T cells, the residual viremia was dominated by a homogeneous population of viruses with identical pol sequences. In the most extensively studied case, a predominant plasma sequence was also found in analysis of the env gene, and linkage by long-distance reverse transcriptase PCR established that these predominant plasma sequences represented a single predominant plasma virus clone. The predominant plasma clones were released for months to years without evident sequence change. Thus, in some patients on antiretroviral therapy, the major mechanism for residual viremia involves prolonged production of a small number of viral clones without evident evolution, possibly by cells other than circulating CD4+ T cells.
doi:10.1128/JVI.00591-06
PMCID: PMC1488985  PMID: 16775332
23.  Nosocomial Malaria and Saline Flush 
Emerging Infectious Diseases  2005;11(7):1097-1099.
An investigation of malaria in a US patient without recent travel established Plasmodium falciparum molecular genotype identity in 2 patients who shared a hospital room. P. falciparum can be transmitted in a hospital environment from patient to patient by blood inoculum if standard precautions are breached.
doi:10.3201/eid1107.050092
PMCID: PMC3371795  PMID: 16022788
molecular epidemiology; infection; case report; Malaria; nosocomial; microsatellite; plasmodium falciparum; transmission; blood-borne; mass spectrometric
24.  A Novel Assay Allows Genotyping of the Latent Reservoir for Human Immunodeficiency Virus Type 1 in the Resting CD4+ T Cells of Viremic Patients 
Journal of Virology  2005;79(8):5185-5202.
A latent reservoir for human immunodeficiency virus type 1 (HIV-1) consisting of integrated provirus in resting memory CD4+ T cells prevents viral eradication in patients on highly active antiretroviral therapy (HAART). It is difficult to analyze the nature and dynamics of this reservoir in untreated patients and in patients failing therapy, because it is obscured by an excess of unintegrated viral DNA that constitutes the majority of viral species in resting CD4+ T cells from viremic patients. Therefore, we developed a novel culture assay that stimulates virus production from latent, integrated HIV-1 in resting CD4+ T cells in the presence of antiretroviral drugs that prevent the replication of unintegrated virus. Following activation, resting CD4+ T cells with integrated HIV-1 DNA produced virus particles for several days, with peak production at day 5. Using this assay, HIV-1 pol sequences from the resting CD4+ T cells of viremic patients were found to be genetically distinct from contemporaneous plasma virus. Despite the predominance of a relatively homogeneous population of drug-resistant viruses in the plasma of patients failing HAART, resting CD4+ T cells harbored a diverse array of wild-type and archival drug-resistant viruses that were less fit than plasma virus in the context of current therapy. These results provide the first direct evidence that resting CD4+ T cells serve as a stable reservoir for HIV-1 even in the setting of high levels of viremia. The ability to analyze archival species in viremic patients may have clinical utility in detecting drug-resistant variants not present in the plasma.
doi:10.1128/JVI.79.8.5185-5202.2005
PMCID: PMC1069549  PMID: 15795302
25.  Novel Single-Cell-Level Phenotypic Assay for Residual Drug Susceptibility and Reduced Replication Capacity of Drug-Resistant Human Immunodeficiency Virus Type 1 
Journal of Virology  2004;78(4):1718-1729.
Human immunodeficiency virus type 1 (HIV-1)-infected individuals who develop drug-resistant virus during antiretroviral therapy may derive benefit from continued treatment for two reasons. First, drug-resistant viruses can retain partial susceptibility to the drug combination. Second, therapy selects for drug-resistant viruses that may have reduced replication capacities relative to archived, drug-sensitive viruses. We developed a novel single-cell-level phenotypic assay that allows these two effects to be distinguished and compared quantitatively. Patient-derived gag-pol sequences were cloned into an HIV-1 reporter virus that expresses an endoplasmic reticulum-retained Env-green fluorescent protein fusion. Flow cytometric analysis of single-round infections allowed a quantitative analysis of viral replication over a 4-log dynamic range. The assay faithfully reproduced known in vivo drug interactions occurring at the level of target cells. Simultaneous analysis of single-round infections by wild-type and resistant viruses in the presence and absence of the relevant drug combination divided the benefit of continued nonsuppressive treatment into two additive components, residual virus susceptibility to the drug combination and selection for drug-resistant variants with diminished replication capacities. In some patients with drug resistance, the dominant circulating viruses retained significant susceptibility to the combination. However, in other cases, the dominant drug-resistant viruses showed no residual susceptibility to the combination but had a reduced replication capacity relative to the wild-type virus. In this case, simplification of the regimen might still allow adequate suppression of the wild-type virus. In a third pattern, the resistant viruses had no residual susceptibility to the relevant drug regimen but nevertheless had a replication capacity equivalent to that of wild-type virus. In such cases, there is no benefit to continued treatment. Thus, the ability to simultaneously analyze residual susceptibility and reduced replication capacity of drug-resistant viruses may provide a basis for rational therapeutic decisions in the setting of treatment failure.
doi:10.1128/JVI.78.4.1718-1729.2004
PMCID: PMC369469  PMID: 14747537

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