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1.  Molecular mechanism of HIV-1 resistance to 3’-azido-2’,3’-dideoxyguanosine 
Antiviral research  2013;101:62-67.
We reported that 3’-azido-2’,3’-dideoxyguanosine (3’-azido-ddG) selected for the L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain of HIV-1 reverse transcriptase (RT). In this study, we have defined the molecular mechanisms of 3’-azido-ddG resistance by performing in-depth biochemical analyses of HIV-1 RT containing mutations L74V, F77L, V106I, L214F, R277K and K476N (SGS3). The SGS3 HIV-1 RT was from a single-genome-derived full-length RT sequence obtained from 3’-azido-ddG resistant HIV-1 selected in vitro. We also analyzed two additional constructs that either lacked the L74V mutation (SGS3-L74V) or the K476N mutation (SGS3-K476N). Pre-steady-state kinetic experiments revealed that the L74V mutation allows RT to effectively discriminate between the natural nucleotide (dGTP) and 3’-azido-ddG-triphosphate (3’-azido-ddGTP). 3’-azido-ddGTP discrimination was primarily driven by a decrease in 3’-azido-ddGTP binding affinity (Kd) and not by a decreased rate of incorporation (kpol). The L74V mutation was found to severely impair RT’s ability to excise the chain-terminating 3’-azido-ddG-monophosphate (3’-azido-ddGMP) moiety. However, the K476N mutation partially restored the enzyme’s ability to excise 3’-azido-ddGMP on an RNA/DNA, but not on a DNA/DNA, template/primer by selectively decreasing the frequency of secondary RNase H cleavage events. Collectively, these data provide strong additional evidence that the nucleoside base structure is major determinant of HIV-1 resistance to the 3’-azido-2’,3’-dideoxynucleosides.
doi:10.1016/j.antiviral.2013.10.017
PMCID: PMC3889670  PMID: 24211331
HIV-1; reverse transcriptase; nucleoside; 3’-azido-2’,3’-dideoxyguanosine; resistance; excision; discrimination
2.  Development of multiplex microsatellite PCR panels for the seagrass Thalassia hemprichii (Hydrocharitaceae)1 
Applications in Plant Sciences  2014;2(11):apps.1400078.
• Premise of the study: New microsatellites were developed for the seagrass Thalassia hemprichii (Hydrocharitaceae), a long-lived seagrass species that is found throughout the shallow waters of tropical and subtropical Indo-West Pacific. Three multiplex PCR panels were designed utilizing new and previously developed markers, resulting in a toolkit for generating a 16-locus genotype.
• Methods and Results: Through the use of microsatellite enrichment and next-generation sequencing, 16 new, validated, polymorphic microsatellite markers were isolated. Diversity was between two and four alleles per locus totaling 36 alleles. These markers, plus previously developed microsatellite markers for T. hemprichii and T. testudinum, were tested for suitability in multiplex PCR panels.
• Conclusions: The generation of an easily replicated suite of multiplex panels of codominant molecular markers will allow for high-resolution and detailed genetic structure analysis and clonality assessment with minimal genotyping costs. We suggest the establishment of a T. hemprichii primer convention for the unification of future data sets.
doi:10.3732/apps.1400078
PMCID: PMC4222546  PMID: 25383269
clonality; Hydrocharitaceae; microsatellites; population genetics; seagrass; Thalassia hemprichii
3.  Three Distinct Phases of HIV-1 RNA Decay in Treatment-Naive Patients Receiving Raltegravir-Based Antiretroviral Therapy: ACTG A5248 
The Journal of Infectious Diseases  2013;208(6):884-891.
Objective. The goal of this study was to define viral kinetics after initiation of raltegravir (RAL)–based antiretroviral therapy (ART).
Methods. ART-naive patients received RAL, tenofovir disoproxil fumarate, and emtricitabine for 72 weeks. Human immunodeficiency virus type 1 (HIV-1) RNA were measured by ultrasensitive and single-copy assays, and first (d1)–, second (d2)–, and, third (d3)–phase decay rates were estimated by mixed-effects models. Decay data were compared to historical estimates for efavirenz (EFV)– and ritonavir/lopinavir (LPV/r)–based regimens.
Results. Bi- and tri-exponential models for ultrasensitive assay (n = 38) and single-copy assay (n = 8) data, respectively, provided the best fits over 8 and 72 weeks. The median d1 with ultrasensitive data was 0.563/day (interquartile range [IQR], 0.501–0.610/day), significantly slower than d1 for EFV-based regimens [P < .001]). The median duration of d1 was 15.1 days, transitioning to d2 at an HIV-1 RNA of 91 copies/mL, indicating a longer duration of d1 and a d2 transition at lower viremia levels than with EFV. Median patient-specific decay estimates with the single-copy assay were 0.607/day (IQR, 0.582–0.653) for d1, 0.070/day (IQR, 0.042–0.079) for d2, and 0.0016/day (IQR, 0.0005–0.0022) for d3; the median d1 duration was 16.1 days, transitioning to d2 at 69 copies/mL. d3 transition occurred at 110 days, at 2.6 copies/mL, similar to values for LPV/r-based regimens.
Conclusions. Models using single-copy assay data revealed 3 phases of decay with RAL-containing ART, with a longer duration of first-phase decay consistent with RAL-mediated blockade of productive infection from preintegration complexes.
Clinical Trials Registration. NCT00660972.
doi:10.1093/infdis/jit272
PMCID: PMC3749011  PMID: 23801609
raltegravir; viral decay; single copy assay; Roche ultrasensitive assay; ACTG A5248; ACTG A5160s; ACTG A5166s
4.  Plasma Viremia and Cellular HIV-1 DNA Persist Despite Autologous Hematopoietic Stem Cell Transplantation for HIV-Related Lymphoma 
A cure of HIV-1 has been achieved in one individual through allogeneic stem cell transplantation with a CCR5delta32 homozygous donor. Whether myeloablation and autologous stem cell transplantation for lymphoma in patients on suppressive ART can eliminate HIV-1 reservoirs is unknown. Low-level plasma viremia and total HIV-1 DNA and 2-LTR circles in blood mononuclear cells were quantified after autologous transplantation in 10 patients on suppressive ART using quantitative PCR assays capable of single copy nucleic acid detection. Plasma viremia was detectable in 9 patients and HIV-1 DNA was detectable in all 10 patients, indicating that HIV-1 had not been eliminated.
doi:10.1097/QAI.0b013e31828e6163
PMCID: PMC3699958  PMID: 23493152
HIV-1 pathogenesis; HIV-1 persistence; AIDS-related lymphoma
5.  Antiretroviral Therapy and Pre-exposure Prophylaxis: Combined Impact on HIV Transmission and Drug Resistance in South Africa 
The Journal of Infectious Diseases  2013;208(2):224-234.
Background. The potential impact of antiretroviral therapy (ART) and pre-exposure prophylaxis (PrEP) with overlapping and nonoverlapping antiretrovirals (ARVs) on human immunodeficiency virus (HIV) transmission and drug resistance is unknown.
Methods. A detailed mathematical model was used to simulate the epidemiological impact of ART alone, PrEP alone, and combined ART + PrEP in South Africa.
Results. ART alone initiated at a CD4 lymphocyte cell count <200 cells/µL (80% coverage and 96% effectiveness) prevents 20% of HIV infections over 10 years but increases drug resistance prevalence to 6.6%. PrEP alone (30% coverage and 75% effectiveness) also prevents 21% of infections but with lower resistance prevalence of 0.5%. The ratio of cumulative infections prevented to prevalent drug-resistant cases after 10 years is 7-fold higher for PrEP than for ART. Combined ART + PrEP with overlapping ARVs prevents 35% of infections but increases resistance prevalence to 8.2%, whereas ART + PrEP with nonoverlapping ARVs prevents slightly more infections (37%) and reduces resistance prevalence to 7.2%.
Conclusions. Combined ART + PrEP is likely to prevent more HIV infections than either strategy alone, but with higher prevalence of drug resistance. ART is predicted to contribute more to resistance than is PrEP. Optimizing both ART and PrEP effectiveness and delivery are the keys to preventing HIV transmission and drug resistance.
doi:10.1093/infdis/jit150
PMCID: PMC3895950  PMID: 23570850
antiretroviral therapy; ART; chemoprophylaxis; HIV drug resistance; HIV epidemic; HIV transmission; mathematical model; pre-exposure prophylaxis; PrEP; South Africa
6.  CD4+CD73+ T cells are associated with lower T-cell activation and C reactive protein levels and are depleted in HIV-1 infection regardless of viral suppression 
AIDS (London, England)  2013;27(10):1545-1555.
Background
The role of the adenosine (ADO) suppression pathway, specifically CD39-expressing and CD73-expressing CD4+ T cells in HIV-1 infection is unclear.
Methods
We evaluated the frequency and numbers of CD4+CD39+ and CD4+CD73+ T cells, activated T cells, and plasma C reactive protein (CRP) levels in 36 HIV-1-positive individuals and 10 normal controls (NC). Low-level plasma viremia was evaluated using single copy assay. Mass spectrometry was used to measure hydrolysis of ATP by ectoenzyme-expressing CD4+ T cells, whereas cyclic adenosine monophosphate (cAMP) levels were measured using enzyme immunoassay. Suppression of T-cell function by exogenous ADO and CD4+CD73+ T cells was tested by flow cytometry.
Results
CD39 and CD73 are expressed in different CD4+ T-cell subsets. CD4+CD73+ T cells do not express CD25 and FOXP3, and their frequency and numbers were lower in HIV-1-positive individuals regardless of virologic suppression (P = 0.005 and P < 0.001, respectively). CD4+CD73+ numbers inversely correlated with CD4+CD38+DR+ (P = 0.002), CD8+CD38+DR+ T-cell frequency (P = 0.05), and plasma CRP levels (P = 0.01). Both subsets are required for hydrolysis of exogenous ATP to ADO and can increase CD4+ T-cell cAMP levels when incubated with exogenous ATP. Low-level viremia did not correlate with activated T-cell frequency. In vitro, ADO suppressed T-cell activation and cytokine expression. CD4+CD73+ T cells suppressed T-cell proliferation only in the presence of exogenous 5′-AMP.
Conclusion
The ADO-producing CD4+CD73+ subset of T cells is depleted in HIV-1-positive individuals regardless of viral suppression and may play a key role in controlling HIV-1-associated immune activation.
doi:10.1097/QAD.0b013e328360c7f3
PMCID: PMC3939796  PMID: 24005375
adenosine; CD73; HIV; immune activation; suppression
7.  Enhanced patient support services improve patient persistence with multiple sclerosis treatment 
Background
Subcutaneous interferon beta-1a (sc IFN β-1a) therapy (44 µg or 22 µg, three times weekly) improves relapse rates and disability progression in patients with relapsing multiple sclerosis (MS). While early treatment with disease-modifying drugs may maximize therapeutic benefit, patients with low adherence or long treatment gaps are at increased risk of relapse. MySupport is an industry-sponsored program that provides support to patients with MS who have been prescribed sc IFN β-1a in the UK or Republic of Ireland (ROI), via telephone and text messaging, website access, and (in some cases) face-to-face support from a dedicated MySupport Nurse. The aim of this audit was to assess if the MySupport program in the ROI could improve persistence to sc IFN β-1a therapy.
Methods
Anonymized data were supplied retrospectively from the MySupport program, for ROI patients who were registered in January 2010 to receive sc IFN β-1a three times weekly. Patients were recorded as “new” at their first drug delivery; “active”, if they continued to receive scheduled deliveries; “interrupted”, if their medication delivery was halted; or “stopped”, if no deliveries were made for 12 months. The number of “active” patients was recorded monthly for 24 months. Results were compared with data from UK patients with MS, who were receiving National Health Service (NHS) support only, or this support plus MySupport.
Results
A greater proportion of ROI patients receiving MySupport (compared against UK patients receiving NHS support only) were on treatment at 12 months (87.8% versus 79.3%) and at 24 months (76.2% versus 61.8%). The odds of being on treatment were significantly greater, at all time points, for ROI patients receiving MySupport, versus UK patients receiving NHS support only (P<0.0001).
Conclusion
A personalized support program, utilizing one-to-one nursing support and additional support materials, can increase the probability of patients with MS remaining on disease-modifying drug treatment.
doi:10.2147/PPA.S59496
PMCID: PMC4062553  PMID: 24966668
multiple sclerosis; interferon beta-1a; persistence; adherence
8.  Baseline Natural Killer and T Cell Populations Correlation with Virologic Outcome after Regimen Simplification to Atazanavir/Ritonavir Alone (ACTG 5201) 
PLoS ONE  2014;9(5):e95524.
Objectives
Simplified maintenance therapy with ritonavir-boosted atazanavir (ATV/r) provides an alternative treatment option for HIV-1 infection that spares nucleoside analogs (NRTI) for future use and decreased toxicity. We hypothesized that the level of immune activation (IA) and recovery of lymphocyte populations could influence virologic outcomes after regimen simplification.
Methods
Thirty-four participants with virologic suppression ≥48 weeks on antiretroviral therapy (2 NRTI plus protease inhibitor) were switched to ATV/r alone in the context of the ACTG 5201 clinical trial. Flow cytometric analyses were performed on PBMC isolated from 25 patients with available samples, of which 24 had lymphocyte recovery sufficient for this study. Assessments included enumeration of T-cells (CD4/CD8), natural killer (NK) (CD3+CD56+CD16+) cells and cell-associated markers (HLA-DR, CD's 38/69/94/95/158/279).
Results
Eight of the 24 patients had at least one plasma HIV-1 RNA level (VL) >50 copies/mL during the study. NK cell levels below the group median of 7.1% at study entry were associated with development of VL >50 copies/mL following simplification by regression and survival analyses (p = 0.043 and 0.023), with an odds ratio of 10.3 (95% CI: 1.92–55.3). Simplification was associated with transient increases in naïve and CD25+ CD4+ T-cells, and had no impact on IA levels.
Conclusions
Lower NK cell levels prior to regimen simplification were predictive of virologic rebound after discontinuation of nucleoside analogs. Regimen simplification did not have a sustained impact on markers of IA or T lymphocyte populations in 48 weeks of clinical monitoring.
Trial Registration
ClinicalTrials.gov NCT00084019
doi:10.1371/journal.pone.0095524
PMCID: PMC4011688  PMID: 24802242
9.  Histone Deacetylase Inhibitor Romidepsin Induces HIV Expression in CD4 T Cells from Patients on Suppressive Antiretroviral Therapy at Concentrations Achieved by Clinical Dosing 
PLoS Pathogens  2014;10(4):e1004071.
Persistent latent reservoir of replication-competent proviruses in memory CD4 T cells is a major obstacle to curing HIV infection. Pharmacological activation of HIV expression in latently infected cells is being explored as one of the strategies to deplete the latent HIV reservoir. In this study, we characterized the ability of romidepsin (RMD), a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, to activate the expression of latent HIV. In an in vitro T-cell model of HIV latency, RMD was the most potent inducer of HIV (EC50 = 4.5 nM) compared with vorinostat (VOR; EC50 = 3,950 nM) and other histone deacetylase (HDAC) inhibitors in clinical development including panobinostat (PNB; EC50 = 10 nM). The HIV induction potencies of RMD, VOR, and PNB paralleled their inhibitory activities against multiple human HDAC isoenzymes. In both resting and memory CD4 T cells isolated from HIV-infected patients on suppressive combination antiretroviral therapy (cART), a 4-hour exposure to 40 nM RMD induced a mean 6-fold increase in intracellular HIV RNA levels, whereas a 24-hour treatment with 1 µM VOR resulted in 2- to 3-fold increases. RMD-induced intracellular HIV RNA expression persisted for 48 hours and correlated with sustained inhibition of cell-associated HDAC activity. By comparison, the induction of HIV RNA by VOR and PNB was transient and diminished after 24 hours. RMD also increased levels of extracellular HIV RNA and virions from both memory and resting CD4 T-cell cultures. The activation of HIV expression was observed at RMD concentrations below the drug plasma levels achieved by doses used in patients treated for T-cell lymphomas. In conclusion, RMD induces HIV expression ex vivo at concentrations that can be achieved clinically, indicating that the drug may reactivate latent HIV in patients on suppressive cART.
Author Summary
Combination antiretroviral therapy has greatly improved the clinical outcome of HIV infection treatment. However, latent viral reservoirs established primarily in memory CD4 T cells persist even after long periods of suppressive antiretroviral therapy, which hinders the ability to achieve a prolonged drug-free remission or a cure of the HIV infection. Activation of HIV expression from latent reservoirs is a part of proposed strategies that may potentially lead to virus elimination and ultimately cure of the infection. In this study, we show that romidepsin, a histone deacetylase inhibitor approved for the treatment of T-cell lymphomas, is a potent activator of HIV expression in an in vitro model of viral latency as well as ex vivo in resting and memory CD4 T cells isolated from HIV-infected patients with suppressed viremia. Importantly, the ex vivo activation of latent HIV occurred at romidepsin concentrations lower than those achieved in drug-treated lymphoma patients. In addition, romidepsin exhibited a more potent effect than other drugs in the same class that have already been shown to activate HIV expression in vivo. Together, these results support the clinical assessment of romidepsin in HIV-infected patients on suppressive antiretroviral therapy.
doi:10.1371/journal.ppat.1004071
PMCID: PMC3983056  PMID: 24722454
10.  Greater Suppression of Nevirapine Resistance With 21- vs 7-Day Antiretroviral Regimens After Intrapartum Single-Dose Nevirapine for Prevention of Mother-to-Child Transmission of HIV 
Seven- or 21-day regimens of tenofovir/emtricitabine, zidovudine/lamivudine, or lopinavir/ritonavir after single-dose nevirapine (NVP) were effective in suppressing NVP resistance detected by population genotype. Allele-specific polymerase chain reaction revealed that the 21-day regimens were significantly better at preventing the emergence of minor NVP resistance.
Background. Nevirapine (NVP) resistance emerges in up to 70% of women exposed to single-dose (sd) NVP for prevention of mother-to-child transmission of human immunodeficiency virus (HIV).
Methods. HIV-infected pregnant women were randomized to receive sdNVP and either zidovudine/lamivudine (3TC), tenofovir/emtricitabine (FTC), or lopinavir/ritonavir for either 7 or 21 days. The primary endpoint was the emergence of new NVP resistance mutations as detected by standard population genotype at 2 and 6 weeks after treatment. Low-frequency NVP- or 3TC/FTC-resistant mutants at codons 103, 181, and 184 were sought using allele-specific polymerase chain reaction (ASP).
Results. Among 484 women randomized, 422 (87%) received study treatment. Four hundred twelve (98%) women had primary endpoint results available; of these, 5 (1.2%) had new NVP resistance detected by population genotype: 4 of 215 in the 7-day arms (1.9%; K103N in 4 women with Y181C, Y188C, or G190A in 3 of 4) and 1 of 197 (0.5%; V108I) in the 21-day arms (P = .37). Among women with ASP results, new NVP resistance mutations emerged significantly more often in the 7-day arms (13/74 [18%]) than in the 21-day arms (3/66 [5%], P = .019). 3TC/FTC-resistant mutants (M184V/I) emerged infrequently (7/134 [5%]), and their occurrence did not differ by arm.
Conclusions. Three short-term antiretroviral strategies, begun simultaneously with the administration of sdNVP, resulted in a low rate (1.2%) of new NVP-resistance mutations when assessed at 2 and 6 weeks following completion of study treatment by standard genotype. ASP revealed that 21-day regimens were significantly better than 7-day regimens at preventing the emergence of minor NVP resistance variants.
Clinical Trials Registration. NCT00099632.
doi:10.1093/cid/cis1219
PMCID: PMC3588119  PMID: 23300238
nevirapine; mother-to-child transmission; pregnancy; resistance; HIV
11.  Lack of Detectable HIV-1 Molecular Evolution during Suppressive Antiretroviral Therapy 
PLoS Pathogens  2014;10(3):e1004010.
A better understanding of changes in HIV-1 population genetics with combination antiretroviral therapy (cART) is critical for designing eradication strategies. We therefore analyzed HIV-1 genetic variation and divergence in patients' plasma before cART, during suppression on cART, and after viral rebound. Single-genome sequences of plasma HIV-1 RNA were obtained from HIV-1 infected patients prior to cART (N = 14), during suppression on cART (N = 14) and/or after viral rebound following interruption of cART (N = 5). Intra-patient population diversity was measured by average pairwise difference (APD). Population structure was assessed by phylogenetic analyses and a test for panmixia. Measurements of intra-population diversity revealed no significant loss of overall genetic variation in patients treated for up to 15 years with cART. A test for panmixia, however, showed significant changes in population structure in 2/10 patients after short-term cART (<1 year) and in 7/10 patients after long-term cART (1–15 years). The changes consisted of diverse sets of viral variants prior to cART shifting to populations containing one or more genetically uniform subpopulations during cART. Despite these significant changes in population structure, rebound virus after long-term cART had little divergence from pretherapy virus, implicating long-lived cells infected before cART as the source for rebound virus. The appearance of genetically uniform virus populations and the lack of divergence after prolonged cART and cART interruption provide strong evidence that HIV-1 persists in long-lived cells infected before cART was initiated, that some of these infected cells may be capable of proliferation, and that on-going cycles of viral replication are not evident.
Author Summary
Anti-HIV compounds are highly effective for preventing the onset of AIDS but they do not cure infected individuals. Very low levels of virus remain detectable in the blood of most patients despite antiviral treatment and levels surge if treatment is stopped. It is crucial to understand why current treatments are not equipped to cure HIV infection so that new therapies addressing these shortcomings can be developed. By characterizing genetic sequences of HIV in patients before and during antiviral treatment, we found that the low levels of virus detected in the blood of treated patients did not result from newly infected cells but originated from cells, or the daughters of cells, that were already infected when treatment was initiated. This finding demonstrates that HIV present in blood after prolonged antiviral treatment is derived from cells infected prior to treatment which likely expanded over time through cell division. Such long lived, infected cells are likely the critical target for developing strategies to cure HIV infection.
doi:10.1371/journal.ppat.1004010
PMCID: PMC3961343  PMID: 24651464
12.  Impact of Chemotherapy for HIV-1 Related Lymphoma on Residual Viremia and Cellular HIV-1 DNA in Patients on Suppressive Antiretroviral Therapy 
PLoS ONE  2014;9(3):e92118.
The first cure of HIV-1 infection was achieved through complex, multimodal therapy including myeloablative chemotherapy, total body irradiation, anti-thymocyte globulin, and allogeneic stem cell transplantation with a CCR5 delta32 homozygous donor. The contributions of each component of this therapy to HIV-1 eradication are unclear. To assess the impact of cytotoxic chemotherapy alone on HIV-1 persistence, we longitudinally evaluated low-level plasma viremia and HIV-1 DNA in PBMC from patients in the ACTG A5001/ALLRT cohort on suppressive antiretroviral therapy (ART) who underwent chemotherapy for HIV-1 related lymphoma without interrupting ART. Plasma HIV-1 RNA, total HIV-1 DNA and 2-LTR circles (2-LTRs) in PBMC were measured using sensitive qPCR assays. In the 9 patients who received moderately intensive chemotherapy for HIV-1 related lymphoma with uninterrupted ART, low-level plasma HIV-1 RNA did not change significantly with chemotherapy: median HIV-1 RNA was 1 copy/mL (interquartile range: 1.0 to 20) pre-chemotherapy versus 4 copies/mL (interquartile range: 1.0 to 7.0) post-chemotherapy. HIV-1 DNA levels also did not change significantly, with median pre-chemotherapy HIV-1 DNA of 355 copies/106 CD4+ cells versus 228 copies/106 CD4+ cells post-chemotherapy. 2-LTRs were detectable in 2 of 9 patients pre-chemotherapy and in 3 of 9 patients post-chemotherapy. In summary, moderately intensive chemotherapy for HIV-1 related lymphoma in the context of continuous ART did not have a prolonged impact on HIV-1 persistence.
Clinical Trials Registration Unique Identifier:
NCT00001137
doi:10.1371/journal.pone.0092118
PMCID: PMC3956871  PMID: 24638072
13.  HIV Populations Are Large and Accumulate High Genetic Diversity in a Nonlinear Fashion 
Journal of Virology  2013;87(18):10313-10323.
HIV infection is characterized by rapid and error-prone viral replication resulting in genetically diverse virus populations. The rate of accumulation of diversity and the mechanisms involved are under intense study to provide useful information to understand immune evasion and the development of drug resistance. To characterize the development of viral diversity after infection, we carried out an in-depth analysis of single genome sequences of HIV pro-pol to assess diversity and divergence and to estimate replicating population sizes in a group of treatment-naive HIV-infected individuals sampled at single (n = 22) or multiple, longitudinal (n = 11) time points. Analysis of single genome sequences revealed nonlinear accumulation of sequence diversity during the course of infection. Diversity accumulated in recently infected individuals at rates 30-fold higher than in patients with chronic infection. Accumulation of synonymous changes accounted for most of the diversity during chronic infection. Accumulation of diversity resulted in population shifts, but the rates of change were low relative to estimated replication cycle times, consistent with relatively large population sizes. Analysis of changes in allele frequencies revealed effective population sizes that are substantially higher than previous estimates of approximately 1,000 infectious particles/infected individual. Taken together, these observations indicate that HIV populations are large, diverse, and slow to change in chronic infection and that the emergence of new mutations, including drug resistance mutations, is governed by both selection forces and drift.
doi:10.1128/JVI.01225-12
PMCID: PMC3754011  PMID: 23678164
14.  Discovery of a Small Molecule Agonist of Phosphatidylinositol 3-Kinase p110α That Reactivates Latent HIV-1 
PLoS ONE  2014;9(1):e84964.
Combination antiretroviral therapy (cART) can effectively suppress HIV-1 replication, but the latent viral reservoir in resting memory CD4+ T cells is impervious to cART and represents a major barrier to curing HIV-1 infection. Reactivation of latent HIV-1 represents a possible strategy for elimination of this reservoir. In this study we describe the discovery of 1,2,9,10-tetramethoxy-7H-dibenzo[de,g]quinolin-7-one (57704) which reactivates latent HIV-1 in several cell-line models of latency (J89GFP, U1 and ACH-2). 57704 also increased HIV-1 expression in 3 of 4 CD8+-depleted blood mononuclear cell preparations isolated from HIV-1-infected individuals on suppressive cART. In contrast, vorinostat increased HIV-1 expression in only 1 of the 4 donors tested. Importantly, 57704 does not induce global T cell activation. Mechanistic studies revealed that 57704 reactivates latent HIV-1 via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. 57704 was found to be an agonist of PI3K with specificity to the p110α isoform, but not the p110β, δ or γ isoforms. Taken together, our work suggests that 57704 could serve as a scaffold for the development of more potent activators of latent HIV-1. Furthermore, it highlights the involvement of the PI3K/Akt pathway in the maintenance of HIV-1 latency.
doi:10.1371/journal.pone.0084964
PMCID: PMC3906007  PMID: 24489654
15.  An exploratory survey measuring stigma and discrimination experienced by people living with HIV/AIDS in South Africa: the People Living with HIV Stigma Index 
BMC Public Health  2014;14:80.
Background
The continued presence of stigma and its persistence even in areas where HIV prevalence is high makes it an extraordinarily important, yet difficult, issue to eradicate. The study aimed to assess current and emerging HIV/AIDS stigma and discrimination trends in South Africa as experienced by people living with HIV/AIDS (PLHIV).
Methods
The PLHIV Stigma Index, a questionnaire that measures and detects changing trends in relation to stigma and discrimination experienced by PLHIV, was used as the survey tool. The study was conducted in 10 clinics in four provinces supported by the Foundation for Professional Development (FPD), with an interview total of 486 PLHIV. A cross-sectional design was implemented in the study, and both descriptive and inferential analysis was conducted on the data.
Results
Findings suggest that PLHIV in this population experience significant levels of stigma and discrimination that negatively impact on their health, working and family life, as well as their access to health services. Internalised stigma was prominent, with many participants blaming themselves for their status.
Conclusion
The findings can be used to develop and inform programmes and interventions to reduce stigma experienced by PLHIV. The current measures for dealing with stigma should be expanded to incorporate the issues related to health, education and discrimination experienced in the workplace, that were highlighted by the study.
doi:10.1186/1471-2458-14-80
PMCID: PMC3909177  PMID: 24461042
HIV/AIDS; Stigma; Discrimination; South African context
16.  Residual Plasma Viremia and Infectious HIV-1 Recovery from Resting Memory CD4 Cells in Patients on Antiretroviral Therapy: Results from ACTG A5173 
Antiviral therapy  2013;18(4):10.3851/IMP2543.
Background
In HIV-1-infected patients receiving antiretroviral therapy (ART), the relationship between residual viremia and ex vivo recovery of infectious virus from latently-infected CD4 cells is uncertain.
Methods
We measured residual viremia (HIV-1 RNA copies/mL) by single-copy assay (SCA) and the latent reservoir by infectious virus recovery from resting memory CD4 cells (infectious units per million cells [IUPM]) in patients who initiated ART. We assessed immune activation by measuring CD38 expression on T cells.
Results
Ten patients who initiated ART and maintained a plasma HIV-1 RNA level <200 copies/mL had residual viremia and IUPM measured every 24 weeks. Five of 10 patients had longitudinal IUPM measured at weeks 24–96; the remainder had IUPM measured 1–3 times over 24–72 weeks. Analyses of 29 paired measurements revealed a positive association between level of residual viremia and IUPM (0.56 higher log10 HIV-1 RNA copies/mL per 1 log10 higher IUPM, p=0.005). Residual viremia level was positively associated with CD38 density and percentage on CD8+ T-cells in concurrent samples and with pre-ART HIV-1 RNA levels.
Conclusions
In patients with HIV-1 RNA levels <200 copies/mL 24–96 weeks after initiating ART, the level of viremia is positively associated with infectious virus recovery from resting memory CD4 cells. Whether this association persists after longer-term suppressive ART needs to be determined. If additional studies show that residual viremia measured by SCA reflects the size of the latent reservoir in patients who have had virologic suppression for longer periods of time, this could facilitate testing of potentially curative strategies to reduce this important reservoir.
doi:10.3851/IMP2543
PMCID: PMC3887470  PMID: 23411421
HIV-1; reservoir; residual viremia; single-copy assay
17.  Class-Sparing Regimens for Initial Treatment of HIV-1 Infection 
The New England journal of medicine  2008;358(20):10.1056/NEJMoa074609.
BACKGROUND
The use of either efavirenz or lopinavir–ritonavir plus two nucleoside reverse-transcriptase inhibitors (NRTIs) is recommended for initial therapy for patients with human immunodeficiency virus type 1 (HIV-1) infection, but which of the two regimens has greater efficacy is not known. The alternative regimen of lopinavir–ritonavir plus efavirenz may prevent toxic effects associated with NRTIs.
METHODS
In an open-label study, we compared three regimens for initial therapy: efavirenz plus two NRTIs (efavirenz group), lopinavir–ritonavir plus two NRTIs (lopinavir–ritonavir group), and lopinavir–ritonavir plus efavirenz (NRTI-sparing group). We randomly assigned 757 patients with a median CD4 count of 191 cells per cubic millimeter and a median HIV-1 RNA level of 4.8 log10 copies per milliliter to the three groups.
RESULTS
At a median follow-up of 112 weeks, the time to virologic failure was longer in the efavirenz group than in the lopinavir–ritonavir group (P = 0.006) but was not significantly different in the NRTI-sparing group from the time in either of the other two groups. At week 96, the proportion of patients with fewer than 50 copies of plasma HIV-1 RNA per milliliter was 89% in the efavirenz group, 77% in the lopinavir–ritonavir group, and 83% in the NRTI-sparing group (P = 0.003 for the comparison between the efavirenz group and the lopinavir–ritonavir group). The groups did not differ significantly in the time to discontinuation because of toxic effects. At virologic failure, antiretroviral resistance mutations were more frequent in the NRTI-sparing group than in the other two groups.
CONCLUSIONS
Virologic failure was less likely in the efavirenz group than in the lopinavir–ritonavir group. The virologic efficacy of the NRTI-sparing regimen was similar to that of the efavirenz regimen but was more likely to be associated with drug resistance. (ClinicalTrials.gov number, NCT00050895.)
doi:10.1056/NEJMoa074609
PMCID: PMC3885902  PMID: 18480202
19.  Predictors of Residual Viremia in Patients on Long-term Suppressive Antiretroviral Therapy 
Antiviral therapy  2012;18(1):39-43.
Background
HIV-1-infected individuals with plasma RNA <50 copies/mL on antiretroviral therapy (ART) may have residual, low-level viremia detectable by PCR assays which can detect a single copy of viral RNA (single-copy assay, SCA). The clinical predictors of residual viremia in patients on long-term suppressive ART are incompletely understood.
Methods
We evaluated factors associated with residual viremia in patients on suppressive ART who underwent screening for a raltegravir intensification trial (ACTG A5244). The screened population was HIV-1-infected adults receiving ART for ≥12 months with pre-ART HIV-1 RNA >100,000 copies/mL and on-therapy RNA levels below detection limits of commercial assays for ≥6 months.
Results
Of 103 patients eligible for analysis, the median age was 46 years and the median duration of viral suppression was 4.8 years. Sixty-two percent had detectable viremia (>0.2 copies/mL) by SCA (median 0.2 copies/mL; quartile [Q] 1, Q3 [<0.2, 1.8]). Younger patients had lower HIV-1 RNA levels than older individuals (r=0.27, p=0.005). Patients with virologic suppression on ART for 2 years or less had higher residual viremia than those with suppression for more than 2 years (median 2.3 vs. 0.2 copies/mL, p=0.016).
Conclusions
Among HIV-1-infected patients with pre-ART HIV-1 RNA >100,000 copies/mL, residual viremia was detectable in the majority (62%) despite many years of suppressive ART. Higher level viremia was associated with older age and less than 2 years of virologic suppression on ART. These findings should help in selection of candidates for clinical trials of interventions designed to eliminate residual viremia.
doi:10.3851/IMP2323
PMCID: PMC3578982  PMID: 22914318
HIV-1; Single-copy assay; residual viremia
20.  Dynamics of Immune Reconstitution and Activation Markers in HIV+ Treatment-Naïve Patients Treated with Raltegravir, Tenofovir Disoproxil Fumarate and Emtricitabine 
PLoS ONE  2013;8(12):e83514.
Background
The dynamics of CD4+ T cell reconstitution and changes in immune activation and inflammation in HIV-1 disease following initiation of antiretroviral therapy (ART) are incompletely defined and their underlying mechanisms poorly understood.
Methods
Thirty-nine treatment-naïve patients were treated with raltegravir, tenofovir DF and emtricitabine. Immunologic and inflammatory indices were examined in persons with sustained virologic control during 48 weeks of therapy.
Results
Initiation of ART increased CD4+ T cell numbers and decreased activation and cell cycle entry among CD4+ and CD8+ T cell subsets, and attenuated markers of coagulation (D-dimer levels) and inflammation (IL-6 and TNFr1). These indices decayed at different rates and almost all remained elevated above levels measured in HIV-seronegatives through 48 weeks of viral control. Greater first and second phase CD4+ T cell restoration was related to lower T cell activation and cell cycling at baseline, to their decay with treatment, and to baseline levels of selected inflammatory indices, but less so to their changes on therapy.
Conclusions
ART initiation results in dynamic changes in viral replication, T cell restoration, and indices of immune activation, inflammation, and coagulation. These findings suggest that determinants of T cell activation/cycling and inflammation/coagulation may have distinguishable impact on immune homeostasis.
Trial Registration
Clinicaltrials.gov NCT00660972
doi:10.1371/journal.pone.0083514
PMCID: PMC3867440  PMID: 24367599
21.  Persistence of Viral Reservoirs in Multiple Tissues after Antiretroviral Therapy Suppression in a Macaque RT-SHIV Model 
PLoS ONE  2013;8(12):e84275.
Although antiretroviral therapy (ART) can suppress HIV-1 replication sufficiently to eliminate measurable plasma viremia, infected cells remain and ensure viral recrudescence after discontinuation of ART. We used a macaque model of HIV-1/AIDS to evaluate the location of infected cells during ART. Twelve macaques were infected with RT-SHIVmne, a SIV containing HIV-1 reverse transcriptase, conferring sensitivity to non-nucleoside reverse transcriptase inhibitors (NNRTIs). Ten to fourteen weeks post-infection, 6 animals were treated with 3 or 4 antiretroviral drugs for 17-20 weeks; 6 control animals remained untreated. Viral DNA (vDNA) and RNA (vRNA) were measured in peripheral blood mononuclear cells (PBMC) and at necropsy in multiple tissues by quantitative PCR and RT-PCR. The majority of virally infected cells were located in lymphoid tissues with variable levels in the gastrointestinal tract of both treated and untreated animals. Tissue viral DNA levels correlated with week 1 plasma viremia, suggesting that tissues that harbor proviral DNA are established within the first week of infection. PBMC vDNA levels did not correlate with plasma viremia or tissue levels of vDNA. vRNA levels were high in lymphoid and gastrointestinal tissues of the untreated animals; animals on ART had little vRNA expressed in tissues and virus could not be cultured from lymph node resting CD4+ cells after 17-20 weeks on ART, indicating little or no ongoing viral replication. Strategies for eradication of HIV-1 will need to target residual virus in ART suppressed individuals, which may not be accurately reflected by frequencies of infected cells in blood.
doi:10.1371/journal.pone.0084275
PMCID: PMC3867492  PMID: 24367650
22.  Frequent Emergence of N348I in HIV-1 Subtype C Reverse Transcriptase with Failure of Initial Therapy Reduces Susceptibility to Reverse-Transcriptase Inhibitors 
N348I emerges frequently with failure of first-line antiretroviral therapy (ART) in subtype C human immunodeficiency virus type 1 infection and affects susceptibility to nevirapine, efavirenz, etravirine, and zidovudine. This finding has implications for cross-resistance to subsequent ART regimens in resource-limited settings.
Background. It is not known how often mutations in the connection and ribonuclease H domains of reverse transcriptase (RT) emerge with failure of first-line antiretroviral therapy (ART) in subtype C human immunodeficiency virus type 1 (HIV-1) infection and how these mutations affect susceptibility to other antiretrovirals.
Methods. We compared full-length RT sequences in plasma obtained before therapy and at virologic failure of initial ART among 63 participants with subtype C HIV-1 infection enrolled in the Comprehensive International Program of Research on AIDS in South Africa (CIPRA-SA) study. Recombinant viruses containing full-length plasma-derived RT sequences from participants with N348I at virologic failure were assayed for drug susceptibility.
Results. Y181C and M184V mutations in the RT polymerase domain were associated with failure of stavudine-lamivudine-nevirapine (d4T/3TC/NVP; P < .01), and K103N, V106M, and M184V with failure of d4T/3TC/efavirenz (EFV; P < .01). N348I in the RT connection domain emerged in 45% (P = .002) and 12% (P = .06) of participants receiving failing regimens containing NVP or EFV, respectively. Longitudinal analyses revealed that nonnucleoside RT inhibitor resistance mutations in the polymerase domain generally appeared first. N348I emerged at the same time, or after, M184V. N348I in the context of polymerase domain mutations reduced susceptibility to NVP (8.9–13-fold), EFV (4–56-fold), etravirine (ETV; 1.9–4.7-fold) and decreased hypersusceptibility to zidovudine (AZT; 1.4–2.2-fold).
Conclusions. N348I emerges frequently with virologic failure of first-line ART in subtype C HIV-1 infection and reduces susceptibility to NVP, EFV, ETV, and AZT. Additional studies are warranted to characterize the effects of N348I on virologic response to second- and third-line regimens in resource-limited settings where subtype C predominates.
doi:10.1093/cid/cis501
PMCID: PMC3491849  PMID: 22618567
23.  A Pilot Trial of Adding Maraviroc to Suppressive Antiretroviral Therapy for Suboptimal CD4+ T-Cell Recovery Despite Sustained Virologic Suppression: ACTG A5256 
The Journal of Infectious Diseases  2012;206(4):534-542.
Background. Despite viral suppression, antiretroviral therapy (ART) does not restore CD4+ T-cell counts in many patients infected with human immunodeficiency virus type 1 (HIV-1).
Methods. In a single-arm pilot trial involving ART recipients with suppressed plasma levels of HIV-1 RNA for at least 48 weeks and stable suboptimal CD4+ T-cell recovery, subjects added maraviroc, a CCR5 antagonist, to their existing ART for 24 weeks. After stopping maraviroc, they were followed for an additional 24 weeks. A Wilcoxon signed-rank test was used to evaluate whether maraviroc was associated with an increase of at least 20 cells/µL in the CD4+ T-cell count.
Results. A total of 34 subjects were enrolled. The median age was 50 years, and the median baseline CD4+ T-cell count was 153 cells/µL. The median increase in CD4+ T-cell count from baseline to week 22/24 was 12 cells/µL (90% confidence interval, 1–22). A CD4+ T-cell count increase of at least 20 cells/µL was not detected (P = .97). Markers of immune activation and apoptosis decreased during maraviroc intensification; this decline partially reversed after discontinuing maraviroc.
Conclusions. Adding maraviroc to suppressive ART for 24 weeks was not associated with an increase in CD4+ T-cell counts of at least 20 cells/µL. Further studies of CCR5 antagonists in the dampening of immune activation associated with HIV infection are warranted.
Clinical Trials Registration. NCT 00709111.
doi:10.1093/infdis/jis376
PMCID: PMC3491731  PMID: 22740718
25.  New Tools for Quantifying HIV-1 Reservoirs: Plasma RNA Single Copy Assays and Beyond 
Current HIV/AIDS reports  2012;9(1):91-100.
Quantification of plasma HIV-1 RNA below the limit of FDA-approved assays by a single copy quantitative PCR assays (SCA) has provided significant insights into HIV-1 persistence despite potent antiretroviral therapy as well as a means to assess the impact of therapeutic strategies, such as treatment intensification, on residual viremia. In this review, we discuss insights gained from plasma HIV-1 RNA SCA and highlight the need for additional assays to characterize better the cellular and tissue reservoirs of HIV-1. Accurate, reproducible, and sensitive assays to quantify HIV-1 reservoirs, before and after therapeutic interventions, are essential tools in the quest for a cure of HIV-1 infection.
doi:10.1007/s11904-011-0104-6
PMCID: PMC3693463  PMID: 22215419
Single copy assay; Residual viremia; HIV reservoirs; HIV quantification; Ultrasensitive PCR; HIV treatment intensification

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