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. 2020 Mar 31;94(8):e01912-19.
doi: 10.1128/JVI.01912-19. Print 2020 Mar 31.

Long-Acting Rilpivirine (RPV) Preexposure Prophylaxis Does Not Inhibit Vaginal Transmission of RPV-Resistant HIV-1 or Select for High-Frequency Drug Resistance in Humanized Mice

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Long-Acting Rilpivirine (RPV) Preexposure Prophylaxis Does Not Inhibit Vaginal Transmission of RPV-Resistant HIV-1 or Select for High-Frequency Drug Resistance in Humanized Mice

Kevin Melody et al. J Virol. .
Free PMC article

Abstract

As a long-acting formulation of the nonnucleoside reverse transcriptase inhibitor rilpivirine (RPV LA) has been proposed for use as preexposure prophylaxis (PrEP) and the prevalence of transmitted RPV-resistant viruses can be relatively high, we evaluated the efficacy of RPV LA to inhibit vaginal transmission of RPV-resistant HIV-1 in humanized mice. Vaginal challenges of wild-type (WT), Y181C, and Y181V HIV-1 were performed in mice left untreated or after RPV PrEP. Plasma viremia was measured for 7 to 10 weeks, and single-genome sequencing was performed on plasma HIV-1 RNA in mice infected during PrEP. RPV LA significantly prevented vaginal transmission of WT HIV-1 and Y181C HIV-1, which is 3-fold resistant to RPV. However, it did not prevent transmission of Y181V HIV-1, which has 30-fold RPV resistance in the viruses used for this study. RPV LA did delay WT HIV-1 dissemination in infected animals until genital and plasma RPV concentrations waned. Animals that became infected despite RPV LA PrEP did not acquire new RPV-resistant mutations above frequencies in untreated mice or untreated people living with HIV-1, and the mutations detected conferred low-level resistance. These data suggest that high, sustained concentrations of RPV were required to inhibit vaginal transmission of HIV-1 with little or no resistance to RPV but could not inhibit virus with high resistance. HIV-1 did not develop high-level or high-frequency RPV resistance in the majority of mice infected after RPV LA treatment. However, the impact of low-frequency RPV resistance on virologic outcome during subsequent antiretroviral therapy still is unclear.IMPORTANCE The antiretroviral drug rilpivirine was developed into a long-acting formulation (RPV LA) to improve adherence for preexposure prophylaxis (PrEP) to prevent HIV-1 transmission. A concern is that RPV LA will not inhibit transmission of drug-resistant HIV-1 and may select for drug-resistant virus. In female humanized mice, we found that RPV LA inhibited vaginal transmission of WT or 3-fold RPV-resistant HIV-1 but not virus with 30-fold RPV resistance. In animals that became infected despite RPV LA PrEP, WT HIV-1 dissemination was delayed until genital and plasma RPV concentrations waned. RPV resistance was detected at similar low frequencies in untreated and PrEP-treated mice that became infected. These results indicate the importance of maintaining RPV at a sustained threshold after virus exposure to prevent dissemination of HIV-1 after vaginal infection and low-frequency resistance mutations conferred low-level resistance, suggesting that RPV resistance is difficult to develop after HIV-1 infection during RPV LA PrEP.

Keywords: HIV-1; NNRTI; PrEP; animal model; drug resistance; humanized mice; preexposure prophylaxis; rilpivirine; vaginal transmission.

Figures

FIG 1
FIG 1
In vitro HIV-1NL4-BAL susceptibility to RPV and replication. (A) RPV susceptibility of WT, Y181C, Y181I, and Y181V HIV-1NL4-BAL was measured in TZM-bl cells in the presence of multiple concentrations of RPV. The results are representative of 3 independent experiments, each performed in triplicate. Error bars represent standard deviations (SD). Average fold changes in resistance for each of the mutants compared to that of WT HIV-1NL4-BAL are designated next to each curve. (B) Replication of WT, Y181C, Y181I, and Y181V HIV-1NL4-BAL was measured in human primary PBMCs from 3 individual donors by production of p24 in cell culture supernatants over time. Results are shown as means with standard errors of the means (SEM).
FIG 2
FIG 2
Inhibition of WT HIV-1NL4-BAL by RPV and RPV LA in vitro. Infection of WT HIV-1NL4-BAL was measured in TZM-bl cells in the presence of multiple concentrations of RPV or RPV LA. Error bars represent SD.
FIG 3
FIG 3
Plasma and female genital tract tissue RPV concentrations in female BLT mice after a single RPV LA dose. A single 150-mg/kg dose of RPV LA was administered to 14 female BLT mice. RPV concentrations were measured in plasma and female genital tract at 1, 3, 6, 12, 24, and 48 h and 7 days postdose. Each point represents the means from ≥2 animals, and error bars represent 95% confidence intervals. The dashed lines represent the range of measured concentrations from plasma (gray) or genital tract tissues (blue) from women during 28 days postdose (13). The red line indicates the previously reported protein-adjusted EC90 (12 ng/ml or 33 nM) (15). The arrows denote time points at which mice were challenged after RPV LA dose.
FIG 4
FIG 4
Immunofluorescent staining of human CD4 and CD68 in murine and human female genital tract tissues. Tissues were obtained and imaged from the female genital tract of a NSG mouse prior to reconstitution with human cells, a reconstituted BLT mouse, and cervical tissue from a human woman. Tissues were fixed, sectioned, and stained with anti-human CD4 (green) and anti-human CD68 (red) antibodies and Hoechst stain (nuclei; blue). Representative images are shown.
FIG 5
FIG 5
Plasma viremia and time to infection of RPV LA-treated animals after vaginal HIV-1NL4-BAL challenge. (A, C, and E) Average plasma viral RNA levels in female BLT mice challenged vaginally with WT (A), Y181C (C), or Y181V HIV-1NL4-BAL (E) after no treatment (black) or at day 1 (red) or 7 (blue) after RPV LA dose. Error bars represent the standard errors of the means (SEM). The limit of quantitation was 120 viral RNA copies/ml. P values of <0.05 are shown for time points comparing untreated versus RPV LA d1 groups (*) or untreated versus RPV LA d7 groups (#). (B, D, and F) Kaplan-Meier curves are shown for animals that remained negative over time after challenge with WT (B), Y181C (D), or Y181V (F) HIV-1NL4-BAL. P values between untreated and RPV LA treatment groups are shown.
FIG 6
FIG 6
Comparison of time to infection of animals challenged with WT and mutant HIV-1NL4-BAL either in the absence or in the presence of RPV LA PrEP. Kaplan-Meier curves are shown for all animals that remained negative over time in the absence of treatment (A), 1 day after RPV LA dose (B), or 7 days after RPV dose (C). P values between groups challenged with WT or mutant HIV-1NL4-BAL are shown.
FIG 7
FIG 7
Plasma and female genital tract tissue drug concentrations in BLT mice after a single or multiple doses of TFV and FTC. A single dose of TFV (150 mg/kg) and FTC (100 mg/kg) was administered to 12 female BLT mice for PK measurements. (A) TFV (left) and FTC (right) concentrations were measured in plasma (black) and female genital tract (blue) at 1, 3, 6, 12, 24, and 48 h postdose. The dashed lines represent the range of measured TFV and FTC concentrations from human plasma (gray) or genital tract tissues (blue) (54). (B) TFV and FTC (blue circles) and active, phosphorylated TFV and FTC, TFVdp and FTCtp, respectively (blue triangles), were measured in the female genital tract. The blue dashed lines represent the range of measured TFVdp and FTCtp concentrations from human genital tract tissues (54). In addition, two doses of TFV (150 mg/kg) and FTC (275 mg/kg) were administered 24 h apart to an additional 3 animals, and the drugs and metabolites were measured 12 h after the last dose (red symbols). Each point represents the means from ≥2 animals, and error bars represent the interquartile ranges. The arrows denote the time point at which mice were challenged after TFV/FTC dose.
FIG 8
FIG 8
Plasma viremia and time to infection of TFV/FTC-treated animals after vaginal HIV-1NL4-BAL challenge. (A and C) Average plasma viral RNA levels in female BLT mice challenged vaginally with WT (A) or Y181C (C) HIV-1NL4-BAL after no treatment or during 5 days of TFV/FTC treatment. Error bars represent SEM. The limit of quantitation was 120 HIV-1 RNA copies/ml. P values of <0.05 are shown for time points comparing untreated to TFV/FTC-treated groups (*). (B and D) Kaplan-Meier curves are shown for animals that remained negative over time after challenge with WT (B) or Y181C (D) HIV-1NL4-BAL. P values are shown between treatment groups.
FIG 9
FIG 9
In vitro HIV-1NL4-BAL replication and susceptibility to RPV. (A) Replication of WT HIV-1NL4-BAL and WT HIV-1CH185 was measured in human primary PBMCs from 3 individual donors by production of p24 in cell culture supernatants. Results show means with SEM. (B) RPV susceptibility of WT and Y181V HIV-1NL4-BAL as well as WT and Y181V HIV-1CH185 was measured in TZM-bl cells in the presence of multiple concentrations of RPV. The results are representative of 2 independent experiments, each performed in triplicate. Error bars represent SD. Average fold changes in resistance for each of the mutants compared to levels for WT HIV-1NL4-BAL are designated next to each curve.
FIG 10
FIG 10
Plasma viremia and time to infection of RPV LA-treated animals after vaginal HIV-1CH185 challenge. (A and C) Average plasma viral RNA levels in female BLT mice challenged vaginally with WT (A) or Y181V (C) HIV-1CH185 after no treatment (black) or at day 1 after RPV LA dose (red). Error bars represent the SEM. The limit of quantitation was 120 viral RNA copies/ml. (B and D) Kaplan-Meier curves are shown for animals that remained negative over time after challenge with WT (B) or Y181V (D) HIV-1CH185. (E) Kaplan-Meier curves are shown for animals that remained negative after challenge with WT HIV-1NL4-BAL or WT HIV-1CH185. P values are shown between untreated and RPV LA treatment groups. NS denotes a P value of >0.05 (not significant).
FIG 11
FIG 11
Percentage of HIV-1 sequences with RPV resistance in HIV-infected BLT mice and untreated humans. The frequencies of new RPV resistance mutations identified by SGS in HIV-infected mice in the untreated (black), RPV LA d1-treated (red), and RPV LA d7-treated (blue) groups (A) and RPV resistance mutations identified in a cohort of untreated HIV-infected people (67) (B) were plotted.

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