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CLINICAL SCIENCE: CONCISE COMMUNICATION

Drug resistance among patients who acquired HIV infection in a preexposure prophylaxis trial

Delaugerre, Constancea,b; Rodriguez, Christophec,d; Capitant, Catherinee; Nere, Marie-Laurea,b; Mercier-Darty, Mélaniec,d; Carette, Dianee; Pialoux, Gillesf; Cotte, Laurentg; Charreau, Isabellee; Molina, Jean-Michelb,h and the IPERGAY study group

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doi: 10.1097/QAD.0000000000001960
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Abstract

Introduction

Antiretroviral drugs are now used for preexposure prophylaxis (PrEP) among uninfected persons highly exposed to HIV [1]. Drugs chosen for PrEP should have a high genetic barrier and a low prevalence of primary resistance. Most randomized trials have shown that PrEP with oral tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) is well tolerated and effective in this setting [2–8].

TDF has a high-genetic barrier to resistance and rarely selects mutations in the HIV reverse transcriptase (RT) gene at amino acids K65R and K70E [9,10]. In contrast, the RT-M184V or I mutations are frequently and rapidly selected under FTC pressure [11]. Data from PrEP efficacy trials indicate that antiretroviral resistance rarely emerges, mainly involves M184V/I, and occurs predominantly in individuals who started PrEP during unrecognized, seronegative acute HIV infection [3–7,12–17].

The ANRS IPERGAY trial assessed the efficacy of the TDF/FTC combination taken before and after sexual activity by seronegative MSM. It showed a relative 86% reduction in the incidence of HIV-1 infection [95% confidence interval (CI) 40–98] in the TDF/FTC arm compared with the placebo arm [8]. This rose to 97% (95% CI 81–100) during the open-label phase with TDF/FTC [18].

Standard sequencing only detects resistant variants present at high frequencies (>20%) within the viral population, whereas ultradeep sequencing (UDS) has proven capable of detecting minority resistant variants with a threshold of 1% [19]. Here, we assessed the presence of resistance-associated mutations (RAMs) among participants who acquired HIV infection during the IPERGAY trial, using both standard and ultradeep sequencing.

Methods

Study cohort

Samples and data were obtained from the IPERGAY ANRS study, a double-blind, randomized trial of PrEP with oral TDF/FTC (300 mg TDF/200 mg FTC tablet) versus placebo among HIV-uninfected MSM before and after sex. As on-demand PrEP proved more effective than placebo on the incidence of HIV infection, the blind phase was prematurely discontinued in November 2014 and followed by an open-label (OLE) phase in which on-demand TDF/FTC was offered to all participants [8,18].

During the blind phase, participants were randomized after a screening visit. Day 0 (D0) was the date of the visit where participants received pills of TDF/FTC or placebo. Study visits were scheduled at month 1 (M1), M2 and every 2 months thereafter. The OLE phase was proposed to participants already enrolled in the blind phase and also to screened participants who had not yet been randomized. The OLE day 0 corresponded either to a blind-phase visit (last visit of blind phase, i.e. M4) or to a new visit.

Blood samples were banked at each visit and also at a supplementary visit closest to the HIV diagnosis (2–10 days) for drug-resistance testing, if HIV-1 infection was diagnosed.

Drug adherence measurements

Participants were asked to return their study-drug bottles at each visit, and remaining pills were counted to estimate the number taken during each time period [7].

Plasma was tested at each visit for tenofovir (TFV) and FTC, using a validated liquid chromatography–tandem mass spectrometry method with a detection limit of 1 ng/ml for TFV and 2 ng/ml for FTC [20].

HIV diagnosis

HIV testing was performed at each visit by using the antigen/antibody immunoassays (EIA-4G) Architect HIV Ag/Ab Combo assay (Abbott, Rungis, France). In case of suspected primary HIV infection, plasma HIV-1 RNA load was measured concomitantly. In a specific analysis [21], HIV-1 RNA load was retrospectively measured in plasma stored at the visit preceding the EIA-4G result.

Analysis of drug resistance

The RT gene was amplified (∼600 base pairs) using ANRS consensus primers (www.hivfrenchresistance.org/). Sequencing used both population-based and UDS technology on the 454 platform (GS FLX; Roche, Mylan, France) [19]. Sanger and UDS sequences were aligned with the HIV-1 subtype B HXB2 reference strain to detect IAS resistance mutations (https://www.iasusa.org). UDS sequences were demultiplexed, filtered and aligned using two pipelines, Pyropack and AVA software (Roche) [22].

Results

Among the 478 high-risk MSM who were screened for the IPERGAY trial, 31 were diagnosed with HIV-1 infection (Fig. 1). Ten participants had HIV-1 infection at the screening visit (#1–10), one between randomization and D0 in the TDF/FTC arm (#11) and two at D0 in the placebo arm (#12 and #13). Two participants (#14, #15) who had received TDF/FTC and 14 participants (#16–29) who had received placebo acquired HIV-1 infection during the double-blind phase. Two participants who had received TDF/FTC in the OLE phase acquired HIV-1 infection, one at OLE D0 (#30) and one at OLE M1 (#31). Of note, two participants (#13 and #16) initially randomized to the placebo arm were then enrolled in the OLE phase but were found to be infected at OLE D0.

Fig. 1
Fig. 1:
Flow chart of the 31 participants with HIV-1 infection in the ANRS IPERGAY trial.Distribution of the HIV-1-infected participants during the double-blind randomized phase (BLIND, light grey) and the open-label phase (OLE, dark grey) among ANRS IPERGAY trial participants. In bold: participants who had been exposed to TDF/FTC. Underlined: participants first randomized to the placebo arm and then exposed to TDF/FTC in the OLE phase at the time of resistance sampling.

The median [interquartile range (IQR)] plasma HIV-1 RNA level at the time of resistance sampling was 5.52 (4.47–6.79) log10 copies/ml. Population-based sequencing of the RT gene was successful in 31 participants and showed that participants were predominantly infected with HIV-1 subtype B (64.5%, 20/31) or CRF02_AG (22.6%, 7/31). Ultradeep sequencing was successful in 21/31 participants (68%), with a mean number of 3384 ± 1854 reads, a mean length of 473 ± 21, and a mean quality phred score of 30 ± 0.8.

Analysis of HIV infection in participants who were diagnosed at screening and before starting preexposureprophylaxis at the time of resistance testing

Ten participants were not randomized (#1–10) because HIV infection was diagnosed at the screening visit (Table 1a). One participant (#11) was randomized to the TDF/FTC arm but was diagnosed between screening and D0 and never received TDF/FTC pills. The last participant (#12) randomized to the placebo arm was diagnosed at D0. TFV and FTC were not detected in the plasma of these participants. Bulk sequencing showed primary RAMs in two patients: the M41L and T215L mutations associated with NRTI resistance, zidovudine, with the A98G and K103N mutations associated with NNRTI resistance, efavirenz and nevirapine, in patient #1; and mutation V179T, associated with NNRTI resistance, etravirine, in participant #11. UDS confirmed the V179T mutation, at a high level, in participant #11, and revealed the D67N mutation in 1% of sequences in participant #9. UDS failed in participant #1.

Table 1
Table 1:
Characteristics of diagnostic, adherence and drug-resistance among the 31 seroconverters.
Table 1
Table 1:
(Continued) Characteristics of diagnostic, adherence and drug-resistance among the 31 seroconverters.
Table 1
Table 1:
(Continued) Characteristics of diagnostic, adherence and drug-resistance among the 31 seroconverters.

Analysis of HIV infection in participants who had started preexposure prophylaxis at the time of resistance testing

Six participants had started to receive TDF/FTC pills at the time of resistance testing (Table 1b). Participant #13 was randomized to the placebo arm until M1 and then received TDF/FTC in the OLE phase. HIV was diagnosed by EIA 4G at OLE D0, but retrospective analysis showed detectable plasma HIV-1 RNA (450 copies/ml) in the previous sample (placebo phase D0). At the time of resistance sampling, he had detectable TFV and FTC concentrations, 162.2 and 642.1 ng/ml, respectively. Two participants (#14 and #15) were randomized to the TDF/FTC arm. EIA-4G was positive between two visits (M20–M22) in participant #14 as performed because of symptoms of primary infection, and was positive at M16 in participant #15. TFV and FTC were detected in the plasma of both participants (at M1, M4 and M6 in participant #14 and at M1, M6 and M8 in participant #15) but were undetectable in plasma at the time of resistance testing (M20 + 8 days and M16, respectively). Participant #16 was randomized to the placebo arm until M4 and then received TDF/FTC in the OLE phase. EIA-4G was positive at OLE D0, when the participant had no detectable drug. Participant #30 was diagnosed at OLE D0 after having taken five pills, as confirmed by TFV and FTC measurement (18.78 and 8.2 ng/ml, respectively). Participant #31 was first randomized to the TDF/FTC arm of the blind phase until M6 + 30 days, and then entered the OLE phase. He had detectable TFV/FTC at M2 (329.8 and 227.3 ng/ml, respectively) and at OLE D0 (6.1 and 4.5 ng/ml respectively).

No RAMs to either drug were detected in these six patients by either bulk sequencing or UDS.

Analysis of HIV infection in participants who had received placebo pills at the time of resistance testing

Among the 13 participants who had received only placebo pills, one (#17) was diagnosed retrospectively by plasma HIV-1 RNA assay (110 copies/ml), and four were diagnosed between two visits because of signs and symptoms (#19, #22–26; Table 1c). We found a detectable drug concentration in one participant (#28), with 10.4 ng/ml TFV and 3.9 ng/ml FTC at M2, but the infection occurred at M18. Bulk sequencing showed NNRTI mutations in two participants: V90I and V108I in participant #22, and E138A in participant #25. UDS confirmed that the viral quasispecies harbored these mutations at a high level (>99% of sequences) and revealed additional mutation, G190E at 6.9%, in participant #25. Moreover, UDS showed mutation L100I in 2.6% of sequences in participant #28.

Discussion

Intermittent PrEP use could lead to low and fluctuating drug levels, creating an environment in which selective pressure for resistance is not continuous and potentially eliciting low frequencies of resistant variants. Here, we analyzed drug resistance using standard and sensitive sequencing approaches among the 31 participants who acquired HIV-1 infection during the ANRS IPERGAY trial of PrEP.

We confirm that resistance to TDF and/or to FTC rarely arises during PrEP. This is mainly explained by the fact that TDF/FTC was not initiated during HIV seroconversion. Indeed, as previously reported, most cases of de novo drug resistance occurred in participants with negative antibody-rapid test results who initiated PrEP during the undiagnosed acute infection [7,12–14,17]. The use of antigen/antibody immunoassays to screen our participants enabled diagnosis at a very acute stage (47% Fiebig stage I–III [21]) and allowed us to avoid unwarranted PrEP initiation. We have previously reported that only two infections were missed by the Architect EIA-4G test in the IPERGAY trial and that only one of them had received TDF/FTC pills but was not adherent. In addition, we tested for HIV infection between two trial visits in six participants who developed signs or symptoms, leading to PrEP (or placebo) withdrawal.

In the six persons who had received TDF/FTC pills, the lack of adherence explains probably the absence of resistance, because of the lack of drug pressure. In some rare samples with detectable drugs, the concentration of tenofovir and FTC were equal or above the mean in-vitro inhibitory concentration 50 (IC50) for HIV-1 (10–60 ng/ml for tenofovir and 4 ng/ml for emtricitabine [23,24]) and the estimated steady state in treated patients (12–91 ng/ml for tenofovir and 47 ng/ml for emtricitabine) [25]. But at the time of resistance testing, tenovovir and FTC were not detectable among four participants. In the later two, high level of drugs was measured in the participant #13 and low in participant #30 without drug-resistance selection. This is in keeping with the lack of resistance selection observed in other randomized trials, where adherence was main factor explaining PrEP failure [3–7,12–17]. Contrary to daily PrEP, on-demand PrEP was not started immediately after the medical consultation, in order to avoid drug pressure in a participant who would later be found infected.

Two limits of our study that might have underestimated the risk of drug resistance are the limited number of participants actually exposed to TDF/FTC, and the frequent UDS failure (32%) despite high viral loads and the absence of new samples to retest the sequencing.

We found no cases of primary resistance to TDF or FTC. In contrast, transmitted viruses resistant to NRTI, zidovudine and NNRTI were detected at low and high levels, respectively, in six participants (19%, 95% CI 7–42). This prevalence of drug resistance observed among drug-naive MSM is in keeping with data reported in France [26,27].

Finally, the absence of minority variants resistant to TDF and FTC both before PrEP initiation and in breakthrough infections confirms the place of TDF/FTC as good candidates for either daily or on-demand PrEP programs.

Acknowledgements

On behalf of the IPERGAY Steering Committee, we acknowledge Professor Mark Wainberg for his scientific input and strong support for the ANRS IPERGAY study.

Author contributions: C.D., M.W. and J.M.M. led the study and wrote the manuscript. C.R., M.L.N. and M.M.D. performed the resistance sequencing. C.C., D.C., I.S. and C.D. analyzed the data. G.P., L.C. and J.M.M. conducted the study at their sites. All the authors critically reviewed and approved the manuscript.

Conflicts of interest

C.D. reports receiving financial support as an adviser for Gilead Sciences, Merck, Bristol-Myers Squib and ViiV healthcare, and research grants from Merck and ViiV Healthcare. G.P. has received consulting fees from Bristol-Myers Squibb, Boehringer Ingelheim, Tibotec, Nephrotek, Gilead Sciences, Roche, MSD, Abbott, and ViiV Healthcare, and research grants from Bristol-Myers Squibb and Gilead Sciences. L.C. has received research grants from ViiV healthcare and Merck, personal fees from Mylan, and nonfinancial support from BMS, Gilead Sciences, Janssen Cilag, MSD, and ViiV healthcare. J.M.M. reports receiving support as an adviser for Gilead Sciences, MSD, Janssen, BMS and ViiV Healthecare and research grant from Gilead Sciences and Merck.

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Keywords:

emtricitabine; on-demand; preexposure prophylaxis; resistance; tenofovir disoproxil fumarate; ultradeep sequencing

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