Urvi M. Parikh

Tenofovir-Based Preexposure Prophylaxis for HIV Infection among African Women

BACKGROUND Reproductive-age women need effective interventions to prevent the acquisition of human immunodeficiency virus type 1 (HIV-1) infection. METHODS We conducted a randomized, placebo-controlled trial to assess daily treatment with oral tenofovir disoproxil fumarate (TDF), oral tenofovir-emtricitabine (TDF-FTC), or 1% tenofovir (TFV) vaginal gel as preexposure prophylaxis against HIV-1 infection in women in South Africa, Uganda, and Zimbabwe. HIV-1 testing was performed monthly, and plasma TFV levels were assessed quarterly. RESULTS Of 12,320 women who were screened, 5029 were enrolled in the study. The rate of retention in the study was 91% during 5509 person-years of follow-up. A total of 312 HIV-1 infections occurred; the incidence of HIV-1 infection was 5.7 per 100 person-years. In the modified intention-to-treat analysis, the effectiveness was -49.0% with TDF (hazard ratio for infection, 1.49; 95% confidence interval [CI], 0.97 to 2.29), -4.4% with TDF-FTC (hazard ratio, 1.04; 95% CI, 0.73 to 1.49), and 14.5% with TFV gel (hazard ratio, 0.85; 95% CI, 0.61 to 1.21). In a random sample, TFV was detected in 30%, 29%, and 25% of available plasma samples from participants randomly assigned to receive TDF, TDF-FTC, and TFV gel, respectively. Independent predictors of TFV detection included being married, being older than 25 years of age, and being multiparous. Detection of TFV in plasma was negatively associated with characteristics predictive of HIV-1 acquisition. Elevations of serum creatinine levels were seen more frequently among participants randomly assigned to receive oral TDF-FTC than among those assigned to receive oral placebo (1.3% vs. 0.2%, P=0.004). We observed no significant differences in the frequencies of other adverse events. CONCLUSIONS None of the drug regimens we evaluated reduced the rates of HIV-1 acquisition in an intention-to-treat analysis. Adherence to study drugs was low. (Funded by the National Institutes of Health; VOICE ClinicalTrials.gov number, NCT00705679.).

The K65R Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Exhibits Bidirectional Phenotypic Antagonism with Thymidine Analog Mutations

ABSTRACT The K65R mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is selected in vitro by many d-nucleoside analog RT inhibitors (NRTI) but has been rarely detected in treated patients. In recent clinical trials, the K65R mutation has emerged frequently in patients experiencing virologic failure on antiretroviral combinations that do not include 3′-azidothymidine (AZT). The reason for this change is uncertain. To gain insight, we examined trends in the frequency of K65R in a large genotype database, the association of K65R with thymidine analog mutations (TAMs) and other NRTI mutations, and the viral susceptibility profile of HIV-1 with K65R alone and in combination with TAMs. Among >60,000 clinical samples submitted for genotype analysis that contained one or more NRTI resistance mutations, the frequency of K65R increased from 0.4% in 1998 to 3.6% in 2003. Among samples with K65R, a strong negative association was evident with the TAMs M41L, D67N, L210W, T215Y/F, and K219Q/E (P < 0.005) but not with other NRTI mutations, including the Q151M complex. This suggested that K65R and TAMs are antagonistic. To test this possibility, we generated recombinant HIV-1 encoding K65R in two different TAM backgrounds: M41L/L210W/T215Y and D67N/K70R/T215F/K219Q. K65R reduced AZT resistance from >50-fold to <2.5-fold in both backgrounds. In addition, TAMs antagonized the phenotypic effect of K65R, reducing resistance to tenofovir, abacavir, 2′,3′-dideoxycytidine, dideoxyinosine, and stavudine. In conclusion, K65R and TAMs exhibit bidirectional phenotypic antagonism. This antagonism likely explains the negative association of these mutations in genotype databases, the rare emergence of K65R with antiretroviral therapies that contain AZT, and its more frequent emergence with combinations that exclude AZT.

In Vitro Activity of Structurally Diverse Nucleoside Analogs against Human Immunodeficiency Virus Type 1 with the K65R Mutation in Reverse Transcriptase

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) with a lysine-to-arginine substitution at codon 65 (HIV-165R) of reverse transcriptase (RT) can rapidly emerge in patients being treated with specific combinations of nucleoside analog RT inhibitors (NRTIs). A better understanding of the activity of approved and investigational NRTIs against HIV-165R is needed to select optimal therapy for patients infected with this mutant and to devise strategies to prevent its emergence. Therefore, we tested a broad panel of NRTIs that differed by enantiomer, pseudosugar, and base component against HIV-165R to determine how NRTI structure affects activity. Drug susceptibilities of recombinant wild-type (HIV-165K) or mutant HIV-165R were determined using a single-replication-cycle susceptibility assay with P4/R5 cells and/or a multiple-replication-cycle susceptibility assay with MT-2 cells. All d, l, and acyclic NRTIs were significantly less active against HIV-165R than against HIV-165K except for analogs containing a 3′-azido moiety. Pseudosugar structure and base component but not enantiomer influenced NRTI activity against HIV-165R. These findings support the inclusion of 3′-azido-3′-deoxythymidine in drug combinations to treat patients having HIV-165R and to prevent its emergence.

Molecular Mechanism by Which the K70E Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Confers Resistance to Nucleoside Reverse Transcriptase Inhibitors

ABSTRACT The K70E mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) has become more prevalent in clinical samples, particularly in isolates derived from patients for whom triple-nucleoside regimens that include tenofovir (TNV), abacavir, and lamivudine (3TC) failed. To elucidate the molecular mechanism by which this mutation confers resistance to these nucleoside RT inhibitors (NRTI), we conducted detailed biochemical analyses comparing wild-type (WT), K70E, and K65R HIV-1 RT. Pre-steady-state kinetic experiments demonstrate that the K70E mutation in HIV-1 RT allows the enzyme to discriminate between the natural deoxynucleoside triphosphate substrate and the NRTI triphosphate (NRTI-TP). Compared to the WT enzyme, K70E RT showed 2.1-, 2.3-, and 3.5-fold-higher levels of resistance toward TNV-diphosphate, carbovir-TP, and 3TC-TP, respectively. By comparison, K65R RT demonstrated 12.4-, 12.0-, and 13.1-fold-higher levels of resistance, respectively, toward the same analogs. NRTI-TP discrimination by the K70E (and K65R) mutation was primarily due to decreased rates of NRTI-TP incorporation and not to changes in analog binding affinity. The K65R and K70E mutations also profoundly impaired the ability of RT to excise 3′-azido-2′,3′-dideoxythymidine monophosphate (AZT-MP) and other NRTI-MP from the 3′ end of a chain-terminated primer. When introduced into an enzyme with the thymidine analog mutations (TAMs) M41L, L210W, and T215Y, the K70E mutation inhibited ATP-mediated excision of AZT-MP. Taken together, these findings indicate that the K70E mutation, like the K65R mutation, reduces susceptibility to NRTI by selectively decreasing NRTI-TP incorporation and is antagonistic to TAM-mediated nucleotide excision.

Antagonism between the HIV-1 Reverse-Transcriptase Mutation K65R and Thymidine-Analogue Mutations at the Genomic Level

Prior virologic and biochemical studies have shown phenotypic antagonism between K65R and multiple thymidine-analogue mutations (TAMs) in site-directed mutants tested in vitro. We hypothesized, on the basis of this observed antagonism, that K65R and T215Y/F with multiple TAMs would not be selected on the same human immunodeficiency virus type 1 genome in vivo. We searched a large database of patient genotypes (n=59,262) for the frequency of K65R in combination with >or=3 TAMs as determined by standard population sequencing. K65R and multiple TAMs were rarely detected (<0.1%) in the same plasma sample. Samples with both K65R and >or=3 TAMs (n=21) were further analyzed by use of single-genome sequencing. K65R was never found on the same genome with T215F/Y and >or=2 other TAMs, except in the presence of the Q151M multiple nucleoside reverse-transcriptase inhibitor (NRTI)--resistance complex. These results indicate that antagonism between the K65R and T215Y/F pathways of NRTI resistance occurs at the genomic level. Therapy with NRTI combinations that select both pathways simultaneously may delay the emergence of NRTI resistance and prolong treatment response.

Performance of swabs, lavage, and diluents to quantify biomarkers of female genital tract soluble mucosal mediators

Background Measurement of immune mediators and antimicrobial activity in female genital tract secretions may provide biomarkers predictive of risk for HIV-1 acquisition and surrogate markers of microbicide safety. However, optimal methods for sample collection do not exist. This study compared collection methods. Methods Secretions were collected from 48 women (24 with bacterial vaginosis [BV]) using vaginal and endocervical Dacron and flocked swabs. Cervicovaginal lavage (CVL) was collected with 10 mL of Normosol-R (n = 20), saline (n = 14), or water (n = 14). The concentration of gluconate in Normosol-R CVL was determined to estimate the dilution factor. Cytokine and antimicrobial mediators were measured by Luminex or ELISA and corrected for protein content. Endogenous anti-HIV-1 and anti-E. coli activity were measured by TZM-bl assay or E. coli growth. Results Higher concentrations of protein were recovered by CVL, despite a 10-fold dilution of secretions, as compared to swab eluents. After protein correction, endocervical swabs recovered the highest mediator levels regardless of BV status. Endocervical and vaginal flocked swabs recovered significantly higher levels of anti-HIV-1 and anti-E. coli activity than Dacron swabs (P<0.001). BV had a significant effect on CVL mediator recovery. Normosol-R tended to recover higher levels of most mediators among women with BV, whereas saline or water tended to recover higher levels among women without BV. Saline recovered the highest levels of anti-HIV-1 activity regardless of BV status. Conclusions Endocervical swabs and CVL collected with saline provide the best recovery of most mediators and would be the optimal sampling method(s) for clinical trials.

The 3'-azido group is not the primary determinant of 3'-azido-3'-deoxythymidine (AZT) responsible for the excision phenotype of AZT-resistant HIV-1

The mechanism of human immunodeficiency virus (HIV) 1 resistance to 3′-azido-3′-deoxythymidine (AZT) involves reverse transcriptase (RT)-catalyzed phosphorolytic excision of the chain-terminating AZT-5′-monophosphate (AZTMP). Primers terminated with AZTMP are generally better substrates for this reaction than those terminated with 2′,3′-dideoxynucleoside-5′-monophosphate (2′,3′-ddNMP) analogs that lack a 3′-azido moiety. This led to the hypothesis that the 3′-azido group is a major structural determinant for maintaining the primer terminus in the appropriate site for phosphorolytic excision of AZTMP by AZT-resistant (AZTR) RT. To test this hypothesis, we evaluated the incorporation, phosphorolytic excision, and antiviral activity of a panel of 3′-azido-2′,3′-ddN including 3′-azido-2′,3′-ddA (AZddA), 3′-azido-2′,3′-ddC (AZddC), 3′-azido-2′,3′-ddG (AZddG), AZT, and 3′-azido-2′,3′-ddU (AZddU). The results indicate that mutations correlated with resistance to AZT (D67N/K70R/T215F/K219Q) confer resistance to the 3′-azidopyrimidine nucleosides (AZddC, AZT, and AZddU) but not to the 3′-azidopurine nucleosides (AZddA and AZddG). The data suggest that the presence of a 3′-azido group on the 3′-terminal nucleotide of the primer does not confer increased phosphorolytic excision by AZTR RT for all 3′-azido-ddNMP analogs. Thus, the 3′-azido group cannot be the only structural determinant important for the enhanced phosphorolytic excision of AZTMP associated with HIV resistance to AZT. Other structural components, such as the base, must play a role in defining the specificity of the excision phenotype arising from AZT resistance mutations.

Risk of HIV-1 acquisition among women who use different types of injectable progestin contraception in South Africa: a prospective cohort study.

BACKGROUND Several observational studies have reported that HIV-1 acquisition seems to be higher in women who use depot medroxyprogesterone acetate (DMPA) than in those who do not use hormonal contraception. We aimed to assess whether two injectable progestin-only contraceptives, DMPA and norethisterone enanthate (NET-EN), confer different risks of HIV-1 acquisition. METHODS We included data from South African women who used injectable contraception while participating in theVOICE study, a multisite, randomised, placebo-controlled trial that investigated the safety and efficacy of three formulations of tenofovir for prevention of HIV-1 infection in women between Sept 9, 2009, and Aug 13, 2012. Women were assessed monthly for contraceptive use and incident infection. We estimated the difference in incident HIV-1infection between DMPA and NET-EN users by Cox proportional hazards regression analyses in this prospective cohort. The VOICE trial is registered with ClinicalTrials.gov, NCT00705679. FINDINGS 3141 South African women using injectable contraception were included in the present analysis: 1788 (56·9%)solely used DMPA, 1097 (34·9%) solely used NET-EN, and 256 (8·2%) used both injectable types at different times during follow-up. During 2733·7 person-years of follow-up, 207 incident HIV-1 infections occurred (incidence7·57 per 100 person-years, 95% CI 6·61–8·68). Risk of HIV-1 acquisition was higher among DMPA users (incidence 8·62 per 100 person-years, 95% CI 7·35–10·11) than among NET-EN users (5·67 per 100 person-years, 4·35–7·38;hazard ratio 1·53, 95% CI 1·12–2·08; p=0·007). This association persisted when adjusted for potential confoundingvariables (adjusted hazard ratio [aHR] 1·41, 95% CI 1·06–1·89; p=0·02). Among women seropositive for herpes simplex virus type 2 (HSV-2) at enrolment, the aHR was 2·02 (95% CI 1·26–3·24) compared with 1·09 (0·78–1·52)for HSV-2-seronegative women (pinteraction=0·07). INTERPRETATION Although moderate associations in observational analyses should be interpreted with caution, thesefi ndings suggest that NET-EN might be an alternative injectable drug with a lower HIV risk than DMPA in high HIV-1 incidence settings where NET-EN is available. FUNDING National Institutes of Health, Mary Meyer Scholars Fund, and the Ruth Freeman Memorial Fund.

Plasma and Mucosal HIV Viral Loads Are Associated with Genital Tract Inflammation In HIV-Infected Women

Background: Systemic and mucosal inflammation may play a role in HIV control. A cross-sectional comparison was conducted among women in the Womens Interagency HIV Study to explore the hypothesis that compared with HIV-uninfected participants, women with HIV, and, in particular, those with high plasma viral load (PVL) have increased levels of mucosal and systemic inflammatory mediators and impaired mucosal endogenous antimicrobial activity. Methods: Nineteen HIV-uninfected, 40 HIV-infected on antiretroviral therapy (ART) with PVL ⩽ 2600 copies/mL (low viral load) (HIV+-LVL), and 19 HIV-infected on or off ART with PVL >10,000 (high viral load) (HIV+-HVL) were evaluated. Immune mediators and viral RNA were quantified in plasma and cervicovaginal lavage (CVL). The CVL antimicrobial activity was also determined. Results: Compared to HIV-uninfected participants, HIV+-HVL women had higher levels of mucosal but not systemic proinflammatory cytokines and chemokines, higher Nugent scores, and lower Escherichia coli bactericidal activity. In contrast, there were no significant differences between HIV+-LVL and HIV-uninfected controls. After adjusting for PVL, HIV genital tract shedding was significantly associated with higher CVL concentrations of IL-6, IL-1&bgr;, MIP-1&agr;, and CCL5 (RANTES) and higher plasma concentrations of MIP-1&agr;. High PVL was associated with higher CVL levels of IL-1&bgr; and RANTES, as well as with higher Nugent scores, lower E. coli bactericidal activity, smoking, and lower CD4 counts; smoking and CD4 count retained statistical significance in a multivariate model. Conclusions: Further study is needed to determine if the relationship between mucosal inflammation and PVL is causal and to determine if reducing mucosal inflammation is beneficial.

Prevalence of HIV-1 drug resistance among women screening for HIV prevention trials in KwaZulu-Natal, South Africa (MTN-009).

Background A major concern with using antiretroviral (ARV)-based products for HIV prevention is the potential spread of drug resistance, particularly from individuals who are HIV-infected but unaware of their status. Limited data exist on the prevalence of HIV infection or drug resistance among potential users of ARV-based prevention products. Methods A cross-sectional study of reproductive-aged women who presented to screen for an HIV prevention trial was conducted at 7 clinical sites in Durban, South Africa. CD4+T cell counts, HIV-1 RNA levels and population sequencing of the protease and reverse transcriptase genes were performed for all women with 2 positive HIV rapid tests. Resistance mutations were identified using the Stanford Calibrated Population Resistance Tool. Results Of the 1073 evaluable women, 400(37%) were confirmed as HIV-infected. Of those, plasma HIV-1 RNA was detectable in 365/400(91%) and undetectable(<40 copies/ml) in 35/400(9%) women. 156 women(39%) were eligible for antiretroviral therapy (CD4+T cell counts<350 cells/mm3) and 50(13%) met criteria for AIDS(CD4<200 cells/mm3). Of 352 plasma samples(>200 copies/ml) analyzed for drug resistance, 26(7.4%) had nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside reverse transcriptase inhibitor (NNRTI) or protease inhibitor (PI) drug resistance mutations. Among those with resistance, 18/26 participants(62%) had single-class NNRTI resistance and 5/26(19%) had dual-class NRTI/NNRTI. Major mutations in reverse transcriptase included K65R(n = 1), L74I(n = 1), K103N(n = 19), V106M(n = 4), Y181C(n = 2), M184V(n = 4), and K219E/R(n = 2). Major PI-resistance mutations were rare: M46L(n = 1) and I85V(n = 1). All participants were infected with subtype C virus, except one infected with subtype A. Conclusions In women from Durban, South Africa screening for an HIV prevention trial, the HIV prevalence was high (37%) and HIV drug resistance prevalence was above 5%. This study highlights the potential challenges faced when implementing an ARV-based prevention product that overlaps with first-line antiretroviral therapy. Effective screening to exclude HIV infection among women interested in uptake of ARV-based HIV prevention will be essential in limiting the spread of ARV resistance.