Correction: Nanoformulations of Rilpivirine for Topical Pericoital and Systemic Coitus-Independent Administration Efficiently Prevent HIV Transmission

Vaginal HIV transmission accounts for the majority of new infections worldwide. Currently, multiple efforts to prevent HIV transmission are based on pre-exposure prophylaxis with various antiretroviral drugs. Here, we describe two novel nanoformulations of the reverse transcriptase inhibitor rilpivirine for pericoital and coitus-independent HIV prevention. Topically applied rilpivirine, encapsulated in PLGA nanoparticles, was delivered in a thermosensitive gel, which becomes solid at body temperature. PLGA nanoparticles with encapsulated rilpivirine coated the reproductive tract and offered significant protection to BLT humanized mice from a vaginal high-dose HIV-1 challenge. A different nanosuspension of crystalline rilpivirine (RPV LA), administered intramuscularly, protected BLT mice from a single vaginal high-dose HIV-1 challenge one week after drug administration. Using transmitted/founder viruses, which were previously shown to establish de novo infection in humans, we demonstrated that RPV LA offers significant protection from two consecutive high-dose HIV-1 challenges one and four weeks after drug administration. In this experiment, we also showed that, in certain cases, even in the presence of drug, HIV infection could occur without overt or detectable systemic replication until levels of drug were reduced. We also showed that infection in the presence of drug can result in acquisition of multiple viruses after subsequent exposures. These observations have important implications for the implementation of long-acting antiretroviral formulations for HIV prevention. They provide first evidence that occult infections can occur, despite the presence of sustained levels of antiretroviral drugs. Together, our results demonstrate that topically- or systemically administered rilpivirine offers significant coitus-dependent or coitus-independent protection from HIV infection

<i>In vitro</i> characterization of PLGA/RPV NPs in thermosensitive gel.

<p>(A) Scanning electron microscope image of PLGA/RPV nanoparticles. (B) RPV uptake by HeLa cells. Cells were incubated with 5 μg/ml RPV in solution or in the PLGA/RPV NP formulation. Intracellular RPV and RPV in medium were analyzed by HPLC (n = 3). (C) <i>In vitro</i> analysis of the inhibition of HIV infection by PLGA/ RPV NPs. TZM-bl HIV indicator cells were treated with the indicated concentrations of RPV solution or PLGA/RPV NPs. Cells were challenged with HIV-1_NLX 24 h after RPV treatment. Infection of cells was evaluated by ONE-Glo assay 48 h post infection (n = 3). Data were normalized to luminescence of untreated cells (100%); p = 0.0963.</p

Ability of RPV LA to offer coitus-independent protection from vaginal HIV-1 transmission in BLT mouse.

<p>(A) Longitudinal analysis of RPV levels in plasma of NSG mice injected intramuscularly once with 7.5 mg or 15 mg RPV LA (n = 4 for each group, dotted line indicates IC₉₀). (B) Plasma viral RNA in BLT mice challenged vaginally with HIV-1_CH040 (3.5×10⁵ TCID), a transmitted/founder virus 1-week post administration of RPV LA (15 mg, n = 6) or vehicle (n = 6) intramuscularly. Shown are plasma viral RNA (dotted line-LOQ 400 copies of RNA per ml of plasma). (C) Kaplan-Meier plots representing the percentage of BLT mice protected from HIV transmission by RPV LA as a function of the number of weeks post challenge until the first peripheral blood viral RNA detection. Protected animals were negative for viral RNA in plasma and viral DNA in tissue analyzed after necropsy (P = 0.0047, Log rank/Mantel Cox test). (D). Human CD45 cell levels in peripheral blood (percent of total live cells) and human CD4 levels (percent of human CD3 positive cells) were analyzed by flowcytometry at the indicated times. Solid lines: RPV LA treated mice, dashed lines: control animals.</p

<i>In vitro</i> characterization of PLGA/RPV NPs in thermosensitive gel.

<p>(A) Scanning electron microscope image of PLGA/RPV nanoparticles. (B) RPV uptake by HeLa cells. Cells were incubated with 5 μg/ml RPV in solution or in the PLGA/RPV NP formulation. Intracellular RPV and RPV in medium were analyzed by HPLC (n = 3). (C) <i>In vitro</i> analysis of the inhibition of HIV infection by PLGA/ RPV NPs. TZM-bl HIV indicator cells were treated with the indicated concentrations of RPV solution or PLGA/RPV NPs. Cells were challenged with HIV-1_NLX 24 h after RPV treatment. Infection of cells was evaluated by ONE-Glo assay 48 h post infection (n = 3). Data were normalized to luminescence of untreated cells (100%); p = 0.0963.</p

Distribution of rhodamine-labeled NPs in thermosensitive gel in the mouse female reproductive tract (FRT) and the ability of PLGA/RPV to offer pericoital protection against HIV-1 transmission.

<p>(A, B) Distribution of rhodamine-labeled PLGA nanoparticles in transverse sections of mouse FRT. Nanoparticles in thermosensitive gel were administered vaginally to humanized BLT mice. FRT was isolated and processed at indicated times and sections were stained for hCD45 (A), hCD3, hCD4, hCD8, hCD11c (B) and DAPI (A, B). Control nanoparticles contain only PLGA. Scale bar = 100 μm. Arrowheads are showing nanoparticles in tissue, arrows are showing nanoparticles on the edge of vaginal epithelium. (C, D) Protection of BLT mice from vaginal HIV-1 infection by topically applied thermosensitive gel containing PLGA/RPV NPs (20 μl of gel, 17.5 μg of RPV per mouse). Controls were treated with vehicle or with thermosensitive gel containing blank NPs. Mice were exposed vaginally to HIV-1_RHPA 1.5 h (n = 4) (C) or 24 h (n = 8, two independent experiments) (D) after vaginal administration of gels. Viral RNA was quantified by real time PCR (RT PCR) with a limit of quantitation (LOQ) of 400 copies of RNA per ml (dotted line); graphs represent means ±SD. (E). Kaplan-Meier plots representing the percentage of BLT mice protected by PLGA/RPV NPs in thermosensitive gel over time until the first peripheral blood viral RNA detection. Protected animals were negative for viral RNA in plasma as well as viral DNA in tissue analyzed after necropsy. Statistical analysis: Log-rank (Mantel-Cox) test; controls vs. 1.5 h p = 0.0084, controls vs. 24 h p = 0.0582</p

Experimental design for the evaluation of efficacy of RPV nanoformulations in prevention of vaginal HIV transmission in humanized BLT mice.

<p>(A) Experimental design for evaluation of efficacy of PLGA nanoparticles loaded with RPV (PLGA/ RPV NPs) in thermosensitive gel as a pericoital PrEP administered as a single topical application 1.5 h or 24 h before HIV-1 challenge. (B) Experimental design for evaluation of efficacy of long acting nanosuspension of RPV administered in a single dose intramuscularly 1 week before HIV-1 challenge, as a coitus-independent approach to prevent vaginal HIV transmission.</p

Analysis of RPV LA protection after exposure to high doses of multiple transmitted/founder viruses and an early passage primary isolate.

<p>(A) Experimental design. BLT mice were challenged with CH040, RHPA or JR-CSF HIV-1 isolates 1 week after RPV LA administration. Four weeks after RPV LA administration, mice were challenged again, but this time with HIV-1_THRO, a transmitted/founder virus. (B) Plasma viral load in RPV LA treated BLT mice (n = 10) and controls (n = 4 for 1^st challenge, n = 6 for 2^nd challenge). Dash line indicates LOQ. Data are presented as mean ± SD. (C) Kaplan-Meier plots representing the percentage of BLT mice protected against HIV transmission by RPV LA intramuscular injection as a function of the number of weeks post 1^st and 2^nd challenges until the first peripheral blood viral RNA detection. Arrows in panels (B) and (C) indicate time of 1^st and 2^nd challenges. RHPA (n = 2) and CH040 (n = 2) were used as controls for the first challenge (Control 1); for the 2^nd challenge, all control animals were exposed to THRO (n = 6; Control 2); RPV LA indicates RPV LA treatment. Statistical analysis: Log-rank (Mantel-Cox) test, 1^st and 2^nd challenge were analyzed separately, controls 1 vs. 1^st challenge p = <0.0001, controls 2 vs. 2^nd challenge p = 0.0038.</p