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HIV-1 Drug Resistance in the iPrEx Pre-Exposure Prophylaxis Trial
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Journal of Infectious Diseases Advance Access published April 16, 2014
Teri Liegler1,2, Mohamed Abdel-Mohsen1,2, L. Gordon Bentley3, Robert Atchison3, Timothy Schmidt2, Jacqueline Javier2, Megha Mehrotra3, Christopher Eden3, David V. Glidden4, Vanessa McMahan3, Peter L. Anderson5, Peilin Li6, Joseph K. Wong6, Susan Buchbinder7, Juan V. Guanira8, and Robert M. Grant1,3, for the iPrEx Study Team 1Department of Medicine, University of California, San Francisco CA 94143, USA 2AIDS Research Institute, University of California, San Francisco CA 94143, USA 3J. David Gladstone Institutes, San Francisco CA 94158, USA 4Department of Epidemiology, University of California, San Francisco CA 94143, USA 5Department of Pharmaceutical Sciences, University of Colorado Denver, Aurora CO 80045, USA 6Veterans Affairs Medical Center, San Francisco CA 94143 USA 7Bridge HIV, San Francisco Department of Public Health, San Francisco CA 94102 USA 8Asociaci—n Civil Impacta Salud y Educaci—n, Lima Peru
The iPrEx study demonstrated that oral FTC/TDF as pre-exposure 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, M184 V/I were measured using 454 deep sequencing and a novel 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 131 on-study infections (48 randomized to FTC/TDF), none showed FTC/TDF mutations by clinical assays despite detectable drug within the infection window 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 one of which had low but detectable drug prior to seroconversion. Among those with acute infection at randomization to FTC/TDF, M184 V 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.
We have performed a comprehensive analysis of FTC/TDF-selected DR in 131 participants who seroconverted post-randomization in the iPrEx study, and in 2 participants with exposure to FTC/TDF during unrecognized acute infection. Overall, detection of PrEP-selected DR mutations was infrequent. Bulk genotypic and phenotypic resistance assays revealed FTC-associated mutations (M184V or I) exclusively in participants exposed to FTC/TDF during acute infection, of which 1 had confirmed WT virus at entry [3]. TDF-selected mutations were not observed in PrEP recipients. Of the 48 infected participants randomized to FTC/TDF, minor variant FTC-selected DR (M184I) was detected in 2 participants, one by qMVA (0.53%) and another by deep sequencing (0.75%), of which only the former had detectable FTC within the estimated infection window. Taken together, clinically significant DR selected by PrEP was limited to those who initiated drug after established infection, and was associated with FTC exposure.
The emergence of PrEP-associated viral mutations where PrEP was initiated during acute infection was also reported for men and women in TDF2 and HIV serodiscordant couples in Partners PrEP, respectively [2, 4]. Consistent with the findings in iPrEx, and the Bangkok Tenofovir study, PrEP-selected mutations were absent among seroconverters infected post-randomization. This may reflect drug concentrations or exposure duration that were insufficient to prevent infection and select for drug resistance. In all PrEP study reports, efficacy significantly increases when measured drug exposure is taken into account [6]. By contrast, in the Fem-PrEP trial, PrEP-associated DR in SC randomized to the FTC/TDF arm was noted exclusively in those infected post-randomization[9]. Seroconversion in 3 of 4 women occurred within 8 weeks of study entry, raising the possibility of emergent infection during PrEP initiation. Taken together, these results underscore the importance of recognizing the rare individual with acute seronegative infection, through sensitive HIV RNA testing, or 4th generation Ag/Ab combination testing. Other potential strategies where such diagnostic testing is impractical include delaying PrEP initiation in individuals with symptoms consistent with acute viral infection syndromes.
In the absence of continued drug selection, the FTC-resistant viruses proportionately waned within the infected host over time, reaching background levels by 24 weeks. This is consistent with our findings measuring the time-course of transmitted M184V reversion and WT outgrowth in ARV na•ve subjects [36, 37], and reflects the impaired replication capacity in viruses carrying FTC-selected mutations [38-40]. The contribution of archived, low frequency M184V/I in the proviral DNA reservoir to treatment response is not known. Long-term persistence of K103N is detected as minor variants in untreated infants exposed to sdNVP at birth [24]. In a meta analysis [41], there is increased risk of treatment failure with pre-existing NNRTI minor variant resistance, but reduced risk is noted with minor variant NRTI resistance comparable to frequencies seen in our study (~1%).
The FTC-resistant viruses associated with PrEP exposure had phenotypic susceptibility profiles predicting durable suppression with current second-line regimens based on World Health Organization recommendations [42]. The response to regimens containing FTC or 3TC for viruses harboring isolated M184VI mutations is difficult to predict, although use of a boosted PI is an option to minimize the risk of virological failure. The mutations M184VI confer hypersusceptibility to AZT and TDF [39, 43], raising the possibility of successful AZT- or TDF-based dual NRTI regimens combined with a fully active NNRTI or integrase inhibitor, thus sparing 2nd-line boosted protease-inhibitor based regimens.
In iPrEx, antiretrovirals were withdrawn at first evidence of infection, with clinical followup consistent with the in-country standard of care. However, recent insights into the potential clinical benefit of very early treatment initiation warrants reconsideration of this strategy as therapy intensification during hyper-acute infection might attenuate the course of HIV spread and decrease viral reservoir size [44, 45]. Indeed, the decreased frequency of seroconversions in subjects undergoing acute infection at randomization detected in the active arms compared with placebo in iPrEx (2 versus 8) and CAPRISA 004 (1 versus 7) may reflect attenuated infection when antiretrovirals are started during the Fiebig 1 infection stage or earlier [46].
A better understanding of the FTC/TDF dose and dosing patterns that are associated with prophylactic effects and selection for drug resistance is important for evaluating risk benefit ratios of PrEP. In a case-control substudy of iPrEx participants, the frequency of drug detection in infected participants within a 90 day infection window was significantly lower (11%) than that seen in uninfected controls (51%), but equivalent at time points farther from infection, indicating increased risk of infection during periods of low drug exposure [8]. We find that such periods of low drug exposure were not sufficient for selection of drug resistance. One subject who had detectable HIV-1 RNA when starting PrEP rapidly developed FTC resistance; his drug concentrations were TFV-DP 18.7 fmol/106 PBMC and FTC-TP 0.95 pmol/106 PBMC at the serconversion visit 4 weeks later, by which time M184V was selected. This indicates that these concentrations of drug were sufficient to select for resistance, while being in the prophylactic range for those who are uninfected when starting PrEP [8, 47]. This case, and the lack of FTC or TDF resistance among those with incident infections, suggests that concentrations of drug required to overcome viral fitness barriers to DR selection are also sufficient to prevent HIV infection.
There are limitations in directly extrapolating our findings to other settings of PrEP use. The iPrEx study design included monthly serologic monitoring of PrEP recipients and termination of PrEP at seroconversion. Current guidelines for PrEP in clinical settings indicate HIV-1 monitoring at at least 3 month intervals [48], potentially increasing drug exposure duration and risk of DR if infection occurs . The absence K65R or K70E in iPrEx may be due to insufficient duration of TDF exposure. Among PrEP users with pre-existing infection in other trials, K65R appeared after 4 weeks of TDF PrEP in one person [2] and after 7 months of FTC/TDF PrEP in another [4]. In GS-934, a randomized study of FTC/TDF/EFV vs 3TC/TDF/EFV in infected ART-na•ve subjects, 2/19 developed M184V or I while none developed K65R or K70E after treatment for a median of 16 weeks post-virologic failure [49]. In clinical practice, PrEP may be used intermittently, started and stopped as people change their sexual practices; the frequency and nature of DR in these or other settings may differ from that reported here.
In summary, we found DR in iPrEx SC was limited to those initiating PrEP with unrecognized infection. In 8 participants with measurable systemic drug levels near the infection window, PrEP-selected DR with a potential clinical impact was absent, due to insufficient levels or duration. Continued surveillance of PrEP exposure and DR in seroconverters from ongoing PrEP demonstration projects will extend these findings from randomized controlled trials.

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