PMC

(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.

(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) In vitro 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.

(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.

(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.

(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