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author:("ziegler, Teri")
1.  HIV Pre-Exposure Prophylaxis Integrated with Municipal and Community Based Sexual Health Services 
JAMA internal medicine  2016;176(1):75-84.
Importance
Several randomized trials have demonstrated the efficacy of pre-exposure prophylaxis (PrEP) in preventing human immunodeficiency virus (HIV) acquisition. Little is known about adherence, sexual practices, and overall effectiveness when PrEP is implemented in sexual transmitted infection (STI) and community-based clinics.
Objective
To assess PrEP adherence, sexual behaviors, and incidence of STIs and HIV in a cohort of men who have sex with men (MSM) and transgender women initiating PrEP in the United States.
Design, Setting, and Participants
Demonstration project conducted from October 2012 to February 2015, among MSM and transgender women in 2 STI clinics in San Francisco, California and Miami, Florida, and a community health center in Washington, DC.
Intervention
Daily, oral tenofovir disoproxil fumarate/emtricitabine was provided free of charge for 48 weeks. All participants received HIV testing, brief client-centered counseling, and clinical monitoring.
Main Outcomes and Measures
Tenofovir diphosphate (TFV-DP) concentrations in dried blood spots (DBS); self-reported numbers of anal sex partners and episodes of condomless receptive anal sex; STI/HIV incidence.
Results
Overall, 557 participants initiated PrEP, with 78% retained through 48 weeks. Based on 294 participants with DBS testing, 80-86% had protective DBS levels (consistent with ≥4 doses/week) at follow-up visits. African-Americans (57%, p=0.003) and those from the Miami site (65%, p<0.001) were less likely to have protective levels, while those with stable housing (87%, p=0.02) and reporting ≥2 condomless anal sex partners in the past 3 months (89%, p=0.01) were more likely to have protective levels. Mean anal sex partners declined during follow-up, while the proportion engaging in condomless receptive anal sex remained stable. Overall STI incidence was high (90/100 person-years), but did not increase over time. Two individuals became HIV-infected during follow-up (HIV incidence 0.43%, 95% CI 0.05-1.54); both had TFV-DP levels consistent with <2 doses/week at seroconversion.
Conclusions and Relevance
HIV incidence was extremely low, despite a high incidence of STIs in a large US PrEP demonstration project. Adherence was higher among those with higher reported risk behavior. Interventions that address racial and geographic disparities and housing instability may increase PrEP impact.
doi:10.1001/jamainternmed.2015.4683
PMCID: PMC5042323  PMID: 26571482
Pre-exposure prophylaxis (PrEP); HIV prevention; Men who have sex with men (MSM); sexually transmitted diseases (STD); implementation; adherence; retention; disparities
2.  Recent Infection, Sexually Transmitted Infections and Transmission Clusters Frequently Observed Among Persons Newly-Diagnosed with HIV in San Francisco 
There were 1,311 newly-diagnosed HIV cases in San Francisco between 2005 and 2011 that were linked to care at publicly-funded facilities and had viral sequences available for analysis. Of the 214 cases characterized as recently-infected with HIV at time of diagnosis, 25% had a recent sexually transmitted infection (STI) diagnosis (vs. 10% among longer-standing HIV infections, p<0.001) and 57% were part of a phylogenetic transmission cluster (vs. 42% among longer-standing HIV infection, p<0.001). The association observed between recent HIV infection and having a STI diagnosis during the interval overlapping likely HIV acquisition points to potential opportunities to interrupt HIV transmission.
doi:10.1097/QAI.0000000000000681
PMCID: PMC4501865  PMID: 25967271
HIV; recent infection; acute infection; sexually transmitted infections; phylogenetics; transmission clusters
4.  Adherence to Pre-Exposure Prophylaxis for HIV Prevention in a Clinical Setting 
PLoS ONE  2016;11(6):e0157742.
Background
The HIV epidemic in the United States (US) disproportionately affects gay, bisexual, and other men who have sex with men (MSM). Pre-exposure prophylaxis (PrEP) using co-formulated tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) has demonstrated high efficacy in reducing HIV incidence among MSM. However, low adherence was reported in major efficacy trials and may present a substantial barrier to successful PrEP implementation. Rates of adherence to PrEP in “real-world” clinical settings in the US remain largely unknown.
Methods
We reviewed demographic and clinical data for the first 50 patients to enroll in a clinical PrEP program in Providence, Rhode Island. We analyzed self-reported drug adherence as well as drug concentrations in dried blood spots (DBS) from patients who attended either a three- or six-month follow-up appointment. We further assessed drug concentrations and the resistance profile of a single patient who seroconverted while taking PrEP.
Results
Of the first 50 patients to be prescribed PrEP, 62% attended a follow-up appointment at three months and 38% at six months. Of those who attended an appointment at either time point (70%, n = 35), 92% and 95% reported taking ±4 doses/week at three and six months, respectively. Drug concentrations were performed on a random sample of 20 of the 35 patients who attended a follow-up appointment. TDF levels consistent with ±4 doses/week were found in 90% of these patients. There was a significant correlation between self-reported adherence and drug concentrations (r = 0.49, p = 0.02). One patient who had been prescribed PrEP seroconverted at his three-month follow-up visit. The patient’s drug concentrations were consistent with daily dosing. Population sequencing and ultrasensitive allele-specific PCR detected the M184V mutation, but no other TDF- or FTC-associated mutations, including those present as minor variants.
Conclusion
In this clinical PrEP program, adherence was high, and self-reported drug adherence accurately reflected drug concentrations as measured by DBS.
doi:10.1371/journal.pone.0157742
PMCID: PMC4917105  PMID: 27333000
5.  Select Host Restriction Factors Are Associated with HIV Persistence During Antiretroviral Therapy 
AIDS (London, England)  2015;29(4):411-420.
Objective
The eradication of HIV necessitates elimination of the HIV latent reservoir. Identifying host determinants governing latency and reservoir size in the setting of antiretroviral therapy (ART) is an important step in developing strategies to cure HIV infection. We sought to determine the impact of cell-intrinsic immunity on the HIV latent reservoir.
Design
We investigated the relevance of a comprehensive panel of established anti-HIV-1 host restriction factors to multiple established virologic and immunologic measures of viral persistence in HIV-1-infected, ART-suppressed individuals.
Methods
We measured the mRNA expression of 42 anti-HIV-1 host restriction factors, levels of cell-associated HIV-1 RNA, levels of total pol and 2-LTR circle HIV-1 DNA, and immunophenotypes of CD4+ T cells in 72 HIV-1-infected subjects on suppressive ART (23 subjects initiated ART <1 year post-infection, and 49 subjects initiated ART >1 year post-infection). Correlations were analyzed using non-parametric tests.
Results
The enhanced expression of a few select host restriction factors, p21, schlafen 11, and PAF1, was strongly associated with reduced CD4+ T cell-associated HIV RNA during ART (p<0.001). In addition, our data suggested that ART perturbs the regulatory relationship between CD4+ T cell activation and restriction factor expression. Lastly, cell-intrinsic immune responses were significantly enhanced in subjects who initiated ART during early versus chronic infection, and may contribute to the reduced reservoir size observed in these individuals.
Conclusions
Intrinsic immune responses modulate HIV persistence during suppressive ART, and may be manipulated to enhance the efficacy of ART and promote viral eradication through reversal of latency in vivo.
doi:10.1097/QAD.0000000000000572
PMCID: PMC4385712  PMID: 25602681
HIV latency; antiretroviral therapy; intrinsic immunity; host restriction factors; p21; schlafen 11; PAF1 complex
7.  Deep sequencing of HIV-1 variants from paired plasma and cerebrospinal fluid during primary HIV infection 
Journal of virus eradication  2015;1(4):264-268.
Background
Limited data exist comparing viral quasispecies between cerebrospinal fluid (CSF) and plasma compartments during primary HIV infection. Deep sequencing is a new method to examine the HIV plasma and CSF quasispecies.
Methods
In this pilot study, deep sequencing of protease (PR) and reverse transcriptase (RT) was performed in plasma and CSF from participants during primary HIV infection. Estimated mutational load was calculated by mutant variant frequency multiplied by HIV-RNA level.
Results
Paired plasma and CSF samples were studied from five antiretroviral therapy-naïve male participants with median 109 days post estimated transmission, age 32 years, CD4 cell count 580 cells/μL, HIV-RNA 5.18 log10 copies/mL in plasma and 3.67 log10 copies/mL in CSF. Plasma samples averaged 7,124 reads of PR and 2,448 reads of RT, whereas CSF samples averaged 7,082 and 2,792 reads, respectively. A distinct drug-resistance pattern with linked mutations present at significant levels (5–10%) was detected in one participant in CSF. Other low abundance variants (>0.2%) were detected in plasma and CSF of four out of five participants.
Conclusions
Deep sequencing of CSF HIV is technically possible with sufficient HIV-RNA levels. Differences between the quasispecies in the two compartments detected in one participant, which were present with a high mutational load in CSF at an estimated 3.6 months after HIV infection, suggest that early CNS compartmentalisation may be revealed by sensitive deep-sequencing methods. The presence of distinct low abundance (<1%) resistance variants in plasma and CSF of three other subjects may be significant, but further investigation is needed.
PMCID: PMC4743659  PMID: 26855971
HIV-1; resistance mutations; cerebrospinal fluid; compartmentalisation; HIV-associated neurocognitive disorder; primary HIV infection
8.  TIGIT Marks Exhausted T Cells, Correlates with Disease Progression, and Serves as a Target for Immune Restoration in HIV and SIV Infection 
PLoS Pathogens  2016;12(1):e1005349.
HIV infection induces phenotypic and functional changes to CD8+ T cells defined by the coordinated upregulation of a series of negative checkpoint receptors that eventually result in T cell exhaustion and failure to control viral replication. We report that effector CD8+ T cells during HIV infection in blood and SIV infection in lymphoid tissue exhibit higher levels of the negative checkpoint receptor TIGIT. Increased frequencies of TIGIT+ and TIGIT+ PD-1+ CD8+ T cells correlated with parameters of HIV and SIV disease progression. TIGIT remained elevated despite viral suppression in those with either pharmacological antiretroviral control or immunologically in elite controllers. HIV and SIV-specific CD8+ T cells were dysfunctional and expressed high levels of TIGIT and PD-1. Ex-vivo single or combinational antibody blockade of TIGIT and/or PD-L1 restored viral-specific CD8+ T cell effector responses. The frequency of TIGIT+ CD4+ T cells correlated with the CD4+ T cell total HIV DNA. These findings identify TIGIT as a novel marker of dysfunctional HIV-specific T cells and suggest TIGIT along with other checkpoint receptors may be novel curative HIV targets to reverse T cell exhaustion.
Author Summary
HIV-1 infection contributes substantially to global morbidity and mortality, with no immediate promise of an effective vaccine. One major obstacle to vaccine development and therapy is to understand why HIV-1 replication persists in a person despite the presence of viral specific immune responses. The emerging consensus has been that these immune cells are functionally ‘exhausted’ or anergic, and thus, although they can recognize HIV-1 specific target cells, they are unable to effectively keep up with rapid and dynamic viral replication in an individual. We have identified a novel combination pathway that can be targeted, TIGIT and PD-L1which may be responsible, at least in part, for making these immune cells dysfunctional and exhausted and thus unable to control the virus. We show that by blocking the TIGIT and PD-L1 pathway, we can reverse the defects of these viral specific immune cells. Our findings will give new directions to vaccines and therapies that will potentially reverse these dysfunctional cells and allow them to control HIV-1 replication, but also serve in “Shock and Kill” HIV curative strategies.
doi:10.1371/journal.ppat.1005349
PMCID: PMC4704737  PMID: 26741490
9.  Decreased HIV Type 1 Transcription in CCR5-Δ32 Heterozygotes During Suppressive Antiretroviral Therapy 
The Journal of Infectious Diseases  2014;210(11):1838-1843.
Individuals who are heterozygous for the CCR5-Δ32 mutation provide a natural model to examine the effects of reduced CCR5 expression on human immunodeficiency virus (HIV) persistence. We evaluated the HIV reservoir in 18 CCR5-Δ32 heterozygotes and 54 CCR5 wild-type individuals during suppressive antiretroviral therapy. Cell-associated HIV RNA levels (P = .035), RNA to DNA transcriptional ratios (P = .013), and frequency of detectable HIV 2–long terminal repeat circular DNA (P = .013) were significantly lower in CD4+ T cells from CCR5-Δ32 heterozygotes. Cell-associated HIV RNA was significantly correlated with CCR5 surface expression on CD4+ T cells (r2 = 0.136; P = .002). Our findings suggest that curative strategies should further explore manipulation of CCR5.
doi:10.1093/infdis/jiu338
PMCID: PMC4271057  PMID: 24935955
HIV; CCR5; Δ32; coreceptor; reservoir; latency; eradication; NF-κB; HIV transcription; HIV replication
10.  Deep sequencing of HIV-1 variants from paired plasma and cerebrospinal fluid during primary HIV infection 
Journal of Virus Eradication  null;1(4):264-268.
Abstract
Background: 
Limited data exist comparing viral quasispecies between cerebrospinal fluid (CSF) and plasma compartments during primary HIV infection. Deep sequencing is a new method to examine the HIV plasma and CSF quasispecies.
Methods: 
In this pilot study, deep sequencing of protease (PR) and reverse transcriptase (RT) was performed in plasma and CSF from participants during primary HIV infection. Estimated mutational load was calculated by mutant variant frequency multiplied by HIV-RNA level.
Results: 
Paired plasma and CSF samples were studied from five antiretroviral therapy-naïve male participants with median 109 days post estimated transmission, age 32 years, CD4 cell count 580 cells/μL, HIV-RNA 5.18 log10 copies/mL in plasma and 3.67 log10 copies/mL in CSF. Plasma samples averaged 7,124 reads of PR and 2,448 reads of RT, whereas CSF samples averaged 7,082 and 2,792 reads, respectively. A distinct drug-resistance pattern with linked mutations present at significant levels (5–10%) was detected in one participant in CSF. Other low abundance variants (>0.2%) were detected in plasma and CSF of four out of five participants.
Conclusions: 
Deep sequencing of CSF HIV is technically possible with sufficient HIV-RNA levels. Differences between the quasispecies in the two compartments detected in one participant, which were present with a high mutational load in CSF at an estimated 3.6 months after HIV infection, suggest that early CNS compartmentalisation may be revealed by sensitive deep-sequencing methods. The presence of distinct low abundance (<1%) resistance variants in plasma and CSF of three other subjects may be significant, but further investigation is needed.
PMCID: PMC4743659  PMID: 26855971
HIV-1; resistance mutations; cerebrospinal fluid; compartmentalisation; HIV-associated neurocognitive disorder; primary HIV infection
11.  Design and Implementation of an External Quality Assessment Program for HIV Viral Load Measurements Using Dried Blood Spots 
Journal of Clinical Microbiology  2014;53(3):964-966.
An external quality assurance program was developed for HIV-1 RNA viral load measurements taken from dried blood spots using a reference panel and field-collected specimens. The program demonstrated that accurate and reproducible quantitation can be obtained from field-collected specimens. Residual proviral DNA may confound interpretation in virologically suppressed subjects.
doi:10.1128/JCM.02698-14
PMCID: PMC4390657  PMID: 25520449
12.  Newly Exerted T Cell Pressures on Mutated Epitopes following Transmission Help Maintain Consensus HIV-1 Sequences 
PLoS ONE  2015;10(4):e0120787.
CD8+ T cells are important for HIV-1 virus control, but are also a major contributing factor that drives HIV-1 virus sequence evolution. Although HIV-1 cytotoxic T cell (CTL) escape mutations are a common aspect during HIV-1 infection, less is known about the importance of T cell pressure in reversing HIV-1 virus back to a consensus sequences. In this study we aimed to assess the frequency with which reversion of transmitted mutations in T cell epitopes were associated with T cell responses to the mutation. This study included 14 HIV-1 transmission pairs consisting of a ‘source’ (virus-donor) and a ‘recipient’ (newly infected individual). Non-consensus B sequence amino acids (mutations) in T cell epitopes in HIV-1 gag regions p17, p24, p2 and p7 were identified in each pair and transmission of mutations to the recipient was verified with population viral sequencing. Longitudinal analyses of the recipient’s viral sequence were used to identify whether reversion of mutations back to the consensus B sequence occurred. Autologous 12-mer peptides overlapping by 11 were synthesized, representing the sequence region surrounding each reversion and longitudinal analysis of T cell responses to source-derived mutated and reverted epitopes were assessed. We demonstrated that mutations in the source were frequently transmitted to the new host and on an average 17 percent of mutated epitopes reverted to consensus sequence in the recipient. T cell responses to these mutated epitopes were detected in 7 of the 14 recipients in whom reversion occurred. Overall, these findings indicate that transmitted non-consensus B epitopes are frequently immunogenic in HLA-mismatched recipients and new T cell pressures to T cell escape mutations following transmission play a significant role in maintaining consensus HIV-1 sequences.
doi:10.1371/journal.pone.0120787
PMCID: PMC4412704  PMID: 25919393
13.  Changes in Population HIV RNA Levels in Mbarara, Uganda During Scale-Up of HIV Antiretroviral Therapy Access 
Objective
In a rural Ugandan community scaling up antiretroviral therapy (ART), we sought to determine if population based HIV RNA levels (population viral load) decreased from 2011-2012.
Design
Serial cross-sectional analyses (May 2011, May 2012) of a defined study community of 6,300 persons in a district with HIV prevalence of 8%.
Methods
We measured HIV-1 RNA (viral load, VL) levels on all individuals testing positive for HIV during a five-day high-throughput multi-disease community health campaign in May, 2012 that recruited two-thirds of the population. We aggregated individual-level VL results into population viral load metrics including the proportion of individuals with an undetectable VL, and compared these VL metrics to those we previously reported for this geographic region in 2011.
Results
In 2012, 223/2,179 adults were HIV-seropositive (10%). Overall, among 208/223 HIV-seropositive adults in whom VL was tested, 53% had an undetectable VL (95% CI 46-60%), up from 37% (95% CI 30-45%; p=0.02) in 2011. Seven (3%) individuals had a VL>100,000 copies/mL in 2012, down from 21 (13%) in 2011 (p=0.0007). Mean log(VL) [geometric mean] was 3.18 log (95% CI 3.06-3.29 log) in 2012, down from 3.62 log (95% CI 3.46-3.78 log) in 2011 (p<0.0001). Similar reductions in population VL were seen amongst males and females.
Conclusions
Reductions in population VL metrics and a substantial increase in persons with an undetectable VL were observed in a rural Ugandan community from 2011-2012. These findings from a resource-limited setting experiencing rapid ART scale-up may reflect a population-level effectiveness of expanding ART access.
doi:10.1097/QAI.0000000000000021
PMCID: PMC4172444  PMID: 24146022
Population HIV RNA levels; viral load; HIV antiretroviral therapy (ART); ART effectiveness; epidemiology
14.  Weighing the Risk of Drug Resistance With the Benefits of HIV Preexposure Prophylaxis 
The Journal of Infectious Diseases  2015;211(8):1202-1204.
doi:10.1093/infdis/jiu678
PMCID: PMC4371769  PMID: 25587019
HIV; preexposure prophylaxis; antiretroviral; drug resistance
15.  The Immunologic Effects of Mesalamine in Treated HIV-Infected Individuals with Incomplete CD4+ T Cell Recovery: A Randomized Crossover Trial 
PLoS ONE  2014;9(12):e116306.
The anti-inflammatory agent, mesalamine (5-aminosalicylic acid) has been shown to decrease mucosal inflammation in ulcerative colitis. The effect of mesalamine in HIV-infected individuals, who exhibit abnormal mucosal immune activation and microbial translocation (MT), has not been established in a placebo-controlled trial. We randomized 33 HIV-infected subjects with CD4 counts <350 cells/mm3 and plasma HIV RNA levels <40 copies/ml on antiretroviral therapy (ART) to add mesalamine vs. placebo to their existing regimen for 12 weeks followed by a 12 week crossover to the other arm. Compared to placebo-treated subjects, mesalamine-treated subjects did not experience any significant change in the percent CD38+HLA-DR+ peripheral blood CD4+ and CD8+ T cells at week 12 (P  = 0.38 and P  = 0.63, respectively), or in the CD4+ T cell count at week 12 (P  = 0.83). The percent CD38+HLA-DR+ CD4+ and CD8+ T cells also did not change significantly in rectal tissue (P  = 0.86, P  = 0.84, respectively). During the period of mesalamine administration, plasma sCD14, IL-6, D-dimer, and kynurenine to tryptophan ratio were not changed significantly at week 12 and were similarly unchanged at week 24. This study suggests that, at least under the conditions studied, the persistent immune activation associated with HIV infection is not impacted by the anti-inflammatory effects of mesalamine.
Trial Registration
ClinicalTrials.gov NCT01090102
doi:10.1371/journal.pone.0116306
PMCID: PMC4283685  PMID: 25545673
16.  Role of MicroRNA Modulation in the Interferon-α/Ribavirin Suppression of HIV-1 In Vivo 
PLoS ONE  2014;9(10):e109220.
Background
Interferon-α (IFN-α) treatment suppresses HIV-1 viremia and reduces the size of the HIV-1 latent reservoir. Therefore, investigation of the molecular and immunologic effects of IFN-α may provide insights that contribute to the development of novel prophylactic, therapeutic and curative strategies for HIV-1 infection. In this study, we hypothesized that microRNAs (miRNAs) contribute to the IFN-α-mediated suppression of HIV-1. To inform the development of novel miRNA-based antiretroviral strategies, we investigated the effects of exogenous IFN-α treatment on global miRNA expression profile, HIV-1 viremia, and potential regulatory networks between miRNAs and cell-intrinsic anti-HIV-1 host factors in vivo.
Methods
Global miRNA expression was examined in longitudinal PBMC samples obtained from seven HIV/HCV-coinfected, antiretroviral therapy-naïve individuals before, during, and after pegylated interferon-α/ribavirin therapy (IFN-α/RBV). We implemented novel hybrid computational-empirical approaches to characterize regulatory networks between miRNAs and anti-HIV-1 host restriction factors.
Results
miR-422a was the only miRNA significantly modulated by IFN-α/RBV in vivo (p<0.0001, paired t test; FDR<0.037). Our interactome mapping revealed extensive regulatory involvement of miR-422a in p53-dependent apoptotic and pyroptotic pathways. Based on sequence homology and inverse expression relationships, 29 unique miRNAs may regulate anti-HIV-1 restriction factor expression in vivo.
Conclusions
The specific reduction of miR-422a is associated with exogenous IFN-α treatment, and likely contributes to the IFN-α suppression of HIV-1 through the enhancement of anti-HIV-1 restriction factor expression and regulation of genes involved in programmed cell death. Moreover, our regulatory network analysis presents additional candidate miRNAs that may be targeted to enhance anti-HIV-1 restriction factor expression in vivo.
doi:10.1371/journal.pone.0109220
PMCID: PMC4183579  PMID: 25275557
17.  Association of Cervical Biopsy with HIV Type 1 Genital Shedding Among Women on Highly Active Antiretroviral Therapy 
AIDS Research and Human Retroviruses  2013;29(7):1000-1005.
Abstract
HIV-1 genital shedding is associated with increased HIV-1 transmission risk. Inflammation and ulceration are associated with increased shedding, while highly active antiretroviral therapy (HAART) has been shown to have a protective effect. We sought to examine the impact of cervical biopsies, a routine component of cervical cancer screening, on HIV-1 genital RNA levels in HIV-infected women on HAART. We enrolled HIV-1-infected women undergoing cervical biopsy for diagnosis of cervical intraepithelial neoplasia (CIN) 2/3 in this prospective cohort study. All were stable on HAART for at least 3 months. Clinical and demographic information as well as plasma HIV-1 viral load were collected at the baseline visit. Specimens for cervical HIV-1 RNA were collected immediately prior to biopsy, and 2 and 7 days afterward. Quantitative PCR determined HIV-1 concentration in cervical specimens at each time point to a lower limit of detection of 40 copies/specimen. Among the 30 participants, five (16.6%) women had detectable cervical HIV-1 RNA at baseline, of whom four (80%) had detectable HIV-1 RNA after cervical biopsy, with no significant increase in viral load in the follow-up specimens. Only one woman (3.3%) with undetectable baseline cervical HIV-1 RNA had detection postbiopsy. Detectable plasma HIV-1 RNA was the only factor associated with baseline cervical HIV-1 RNA. In women on HAART, an increase in cervical HIV-1 RNA detection or concentration was not associated with cervical biopsy. These findings help provide safety data regarding cervical cancer screening and diagnosis in HIV-infected women and inform postprocedure counseling.
doi:10.1089/aid.2012.0341
PMCID: PMC3685685  PMID: 23594240
18.  Is there an association between HIV-1 genital shedding and cervical intraepithelial neoplasia 2/3 among women on antiretroviral therapy? 
Objective
Given the high prevalence of cervical intraepithelial neoplasia (CIN) grade 2/3 among HIV-infected women, we sought to examine the relationship between CIN 2/3 and HIV-1 genital shedding among women on highly active antiretroviral therapy (HAART).
Materials and Methods
Paired plasma and cervical wick specimens for HIV-1 RNA measurements were obtained from 44 HIV-infected women (cases) with biopsy-confirmed CIN2/3 and 44 age-matched HIV-infected women with normal cervical findings on colposcopy (controls). All subjects tested negative for sexually transmitted infections and had been stable on HAART for at least three months. HIV-1 viral load was measured in both blood and cervical specimens using commercial real-time PCR assays.
Results
CIN2/3 was not significantly associated with the detection or magnitude of plasma or cervical HIV-1 RNA shedding. HIV was detected in the plasma in 10 (23%) cases and 10 (25%) controls (OR=1.0 95% CI 0.33–3.1). Cervical HIV-1 was detected in 6 (13.6%) cases and 9 (20.4%) controls (OR= 0.61 95% CI=0.20–1.90). Mean HIV-1 concentration in cervical secretions among women with CIN2/3 who shed was 2.93 log10 copies versus 2.72 among controls (p=0.65).
Conclusions
Among women on HAART, we found no relationship between CIN 2/3 and HIV-1 genital shedding.
doi:10.1097/LGT.0b013e3182712286
PMCID: PMC3690152  PMID: 23486070
HIV-1 Genital Shedding; Highly Active Anti-Retroviral Therapy; Cervical Intraepithelial Neoplasia 2/3
19.  Effects of Alpha Interferon Treatment on Intrinsic Anti-HIV-1 Immunity In Vivo 
Journal of Virology  2014;88(1):763-767.
Alpha interferon (IFN-α) suppresses human immunodeficiency virus type 1 (HIV-1) replication in vitro by inducing cell-intrinsic retroviral restriction mechanisms. We investigated the effects of IFN-α/ribavirin (IFN-α/riba) treatment on 34 anti-HIV-1 restriction factors in vivo. Expression of several anti-HIV-1 restriction factors was significantly induced by IFN-α/riba in HIV/hepatitis C virus (HCV)-coinfected individuals. Fold induction of cumulative restriction factor expression in CD4+ T cells was significantly correlated with viral load reduction during IFN-α/riba treatment (r2 = 0.649; P < 0.016). Exogenous IFN-α induces supraphysiologic restriction factor expression associated with a pronounced decrease in HIV-1 viremia.
doi:10.1128/JVI.02687-13
PMCID: PMC3911728  PMID: 24155399
20.  HIV-1 Drug Resistance in the iPrEx Preexposure Prophylaxis Trial 
The Journal of Infectious Diseases  2014;210(8):1217-1227.
Background. The iPrEx study demonstrated that combination oral emtricitabine and tenofovir disoproxil fumarate (FTC/TDF) as preexposure prophylaxis (PrEP) protects against HIV acquisition in men who have sex with men and transgender women. Selection for drug resistance could offset PrEP benefits.
Methods. Phenotypic and genotypic clinical resistance assays characterized major drug resistant mutations. Minor variants with FTC/TDF mutations K65R, K70E, M184V/I were measured using 454 deep sequencing and a novel allele-specific polymerase chain reaction (AS-PCR) diagnostic tolerant to sequence heterogeneity.
Results. Control of primer-binding site heterogeneity resulted in improved accuracy of minor variant measurements by AS-PCR. Of the 48 on-study infections randomized to FTC/TDF, none showed FTC/TDF mutations by clinical assays despite detectable drug levels in 8 participants. Two randomized to FTC/TDF had minor variant M184I detected at 0.53% by AS-PCR or 0.75% by deep sequencing, only 1 of which had low but detectable drug levels. Among those with acute infection at randomization to FTC/TDF, M184V or I mutations that were predominant at seroconversion waned to background levels within 24 weeks after discontinuing drug.
Conclusions. Drug resistance was rare in iPrEx on-study FTC/TDF-randomized seroconverters, and only as low-frequency minor variants. FTC resistance among those initiating PrEP with acute infection waned rapidly after drug discontinuation.
Clinical Trials Registration. NCT00458393.
doi:10.1093/infdis/jiu233
PMCID: PMC4176446  PMID: 24740633
454 deep sequencing; AS-PCR; drug resistance; HIV-1; minor variant; preexposure prophylaxis; PrEP; FTC/TDF
21.  Targeting of Conserved Gag-Epitopes in Early HIV Infection Is Associated with Lower Plasma Viral Load and Slower CD4+ T Cell Depletion 
Abstract
We aimed to investigate whether the character of the immunodominant HIV-Gag peptide (variable or conserved) targeted by CD8+ T cells in early HIV infection would influence the quality and quantity of T cell responses, and whether this would affect the rate of disease progression. Treatment-naive HIV-infected study subjects within the OPTIONS cohort at the University of California, San Francisco, were monitored from an estimated 44 days postinfection for up to 6 years. CD8+ T cells responses targeting HLA-matched HIV-Gag-epitopes were identified and characterized by multicolor flow cytometry. The autologous HIV gag sequences were obtained. We demonstrate that patients targeting a conserved HIV-Gag-epitope in early infection maintained their epitope-specific CD8+ T cell response throughout the study period. Patients targeting a variable epitope showed decreased immune responses over time, although there was no limitation of the functional profile, and they were likely to target additional variable epitopes. Maintained immune responses to conserved epitopes were associated with no or limited sequence evolution within the targeted epitope. Patients with immune responses targeting conserved epitopes had a significantly lower median viral load over time compared to patients with responses targeting a variable epitope (0.63 log10 difference). Furthermore, the rate of CD4+ T cell decline was slower for subjects targeting a conserved epitope (0.85% per month) compared to subjects targeting a variable epitope (1.85% per month). Previous studies have shown that targeting of antigens based on specific HLA types is associated with a better disease course. In this study we show that categorizing epitopes based on their variability is associated with clinical outcome.
doi:10.1089/aid.2012.0171
PMCID: PMC3581067  PMID: 23140171
22.  Assessment of Population-Based HIV RNA Levels in a Rural East African Setting Using a Fingerprick-Based Blood Collection Method 
Population-based human immunodeficiency virus (HIV) RNA metrics can help estimate antiretroviral therapy effectiveness within a community. We developed a fingerprick-based viral load technique and measured population HIV RNA levels in a rural Ugandan community, providing the first report from a resource limited setting.
Background. Population-based human immunodeficiency virus type 1 (HIV-1) RNA levels (viral load [VL]) are proposed metrics for antiretroviral therapy (ART) program effectiveness. We estimated population-based HIV RNA levels using a fingerprick-based approach in a rural Ugandan community implementing rapid ART scale-up.
Methods. A fingerprick-based HIV RNA measurement technique was validated against standard phlebotomy. This technique was deployed during a 5-day community-wide health campaign in a 6300-person community. Assessments included rapid HIV antibody testing, VL, and CD4+ T-cell count via fingerprick. We estimated population HIV RNA levels and the prevalence of undetectable RNA, assessed predictors of VL via linear regression, and mapped RNA levels within community geographic units.
Results. During the community-wide health campaign, 179 of 2282 adults (7.8%) and 10 of 1826 children (0.5%) tested seropositive for HIV. Fingerprick VL was determined in 174 of 189 HIV-positive persons (92%). The mean log(VL) was 3.67 log (95% confidence interval [CI], 3.50–3.83 log copies/mL), median VL was 2720 copies/mL (interquartile range, <486–38 120 copies/mL), and arithmetic mean VL was 64 064 copies/mL. Overall, 64 of 174 of individuals had undetectable RNA (37% [95% CI, 30%–44%]), 24% had VL 486–10 000; 25% had VL 10 001–100 000; and 15% had VL>100 000 copies/mL. Among participants taking ART, 83% had undetectable VL.
Conclusions. We developed and implemented a fingerprick VL testing method and provide the first report of population HIV RNA levels in Africa. In a rural Ugandan community experiencing ART scale-up, we found evidence of population-level ART effectiveness, but found a substantial population to be viremic, in need of ART, and at risk for transmission.
doi:10.1093/cid/cis881
PMCID: PMC3552523  PMID: 23243180
population HIV-RNA levels; viral load; fingerprick; ART effectiveness; epidemiology
23.  Modification of the Abbott RealTime assay for detection of HIV-1 plasma RNA viral loads less than one copy per milliliter 
Journal of virological methods  2011;175(2):10.1016/j.jviromet.2011.04.015.
Abstract/Summary
Although commercial tests are approved for detection of HIV-1 plasma viral loads ≥20 copies per milliliter (ml), only one specialized research assay has been reported to detect plasma viral loads as low as 1 copy/ml. This manuscript describes a method of concentrating HIV-1 virions from up to 30ml of plasma, which can be combined with a commercial viral load test to create a widely-available, reproducible assay for quantifying plasma HIV RNA levels less than 1 copy/ml. Using this pre-analytically modified assay, samples with a known level of 0.5 copy/ml were detected in 8 of 12 replicates (mean 0.47 copy/ml; 95% confidence interval (CI) 0.14-0.81 copy/ml) and samples with a known level of 1.0 copy/ml were detected in 13 of 13 replicates (mean 1.96 copy/ml; 95% CI 1.42-2.50 copy/ml). By concentrating virus from 30ml of plasma, HIV RNA could be measured in 16 of 19 samples (84%) from 12 of 12 subjects (mean 2.77 copy/ml; 95% CI 0.86-4.68 copy/ml). The measured viral load correlated inversely (r= −0.78; p=0.028) with the total duration of viral suppression (viral load<40 copies/ml).
doi:10.1016/j.jviromet.2011.04.015
PMCID: PMC3827908  PMID: 21536073
HIV; plasma RNA; viral load; single copy; Abbott
24.  Expression profile of host restriction factors in HIV-1 elite controllers 
Retrovirology  2013;10:106.
Background
Several host-encoded antiviral factors suppress HIV-1 replication in a cell-autonomous fashion in vitro. The relevance of these defenses to the control of HIV-1 in vivo remains to be elucidated. We hypothesized that cellular restriction of HIV-1 replication plays a significant role in the observed suppression of HIV-1 in "elite controllers", individuals who maintain undetectable levels of viremia in the absence of antiretroviral therapy (ART). We comprehensively compared the expression levels of 34 host restriction factors and cellular activation levels in CD4+ T cells and sorted T cell subsets between elite controllers, HIV-1-infected (untreated) non-controllers, ART-suppressed, and uninfected individuals.
Results
Expression of schlafen 11, a codon usage-based inhibitor of HIV-1 protein synthesis, was significantly elevated in CD4+ T cells from elite controllers as compared to both non-controllers (p = 0.048) and ART-suppressed individuals (p = 0.024), with this effect most apparent in central memory CD4+ T cells. Schlafen 11 expression levels were comparable between controllers and uninfected individuals. Cumulative restriction factor expression was positively correlated with CD4+ T cell activation (r2 = 0.597, p < 0.0001), viral load (r2 = 0.34, p = 0.015), and expression of ISG15 (r2 = 0.73, p < 0.0001), a marker of interferon exposure. APOBEC3C, APOBEC3D, CTR9, TRIM26, and TRIM32 were elevated in elite controllers with respect to ART-suppressed individuals, while levels were comparable to uninfected individuals and non-controllers.
Conclusions
Host restriction factor expression typically scales with cellular activation levels. However, the elevated mRNA and protein expression of schlafen 11, despite low activation and viral load, violates the global pattern and may be a signature characteristic of HIV-1 elite control.
doi:10.1186/1742-4690-10-106
PMCID: PMC3827935  PMID: 24131498
Elite controllers; Intrinsic immunity; Retroviral restriction factors; APOBEC3; BST2/tetherin; TRIM; schlafen 11; p21; T cell activation
25.  Prospective Antiretroviral Treatment of Asymptomatic, HIV-1 Infected Controllers 
PLoS Pathogens  2013;9(10):e1003691.
The study of HIV-infected “controllers” who are able to maintain low levels of plasma HIV RNA in the absence of antiretroviral therapy (ART) may provide insights for HIV cure and vaccine strategies. Despite maintaining very low levels of plasma viremia, controllers have elevated immune activation and accelerated atherosclerosis. However, the degree to which low-level replication contributes to these phenomena is not known. Sixteen asymptomatic controllers were prospectively treated with ART for 24 weeks. Controllers had a statistically significant decrease in ultrasensitive plasma and rectal HIV RNA levels with ART. Markers of T cell activation/dysfunction in blood and gut mucosa also decreased substantially with ART. Similar reductions were observed in the subset of “elite” controllers with pre-ART plasma HIV RNA levels below conventional assays (<40 copies/mL). These data confirm that HIV replication persists in controllers and contributes to a chronic inflammatory state. ART should be considered for these individuals (ClinicalTrials.gov NCT01025427).
Author Summary
HIV-infected “controllers” are rare individuals who are HIV-seropositive but are able to maintain low levels of plasma HIV RNA in the absence of antiretroviral therapy (ART). There has been intense interest in characterizing these unique individuals because they have been considered as a potential model for a “functional cure” of HIV. Previously, our group has shown that controllers have elevated levels of T cell activation and accelerated atherosclerosis, suggesting that very low levels of viral replication may lead to disproportionately high levels of immune activation. However, the degree to which viral replication contributes to these outcomes is not known. We therefore conducted the first, prospective study of ART initiation in a cohort of asymptomatic HIV-infected controllers, in order to determine the virologic and immunologic effects of treating controllers with ART. Controllers had a significant decreases in ultrasensitive plasma HIV RNA, rectal HIV RNA, and markers of T cell activation/dysfunction in blood and gut mucosa with ART. Similar reductions were observed in the subset of “elite” controllers with extremely low pre-ART plasma HIV RNA levels (<40 copies/mL). These data suggest that HIV replication persists in controllers and contributes to a chronic inflammatory state.
doi:10.1371/journal.ppat.1003691
PMCID: PMC3795031  PMID: 24130489

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