Antibody and antiretroviral preexposure prophylaxis prevent cervicovaginal HIV-1 infection in a transgenic mouse model

Abstract

The development of an effective vaccine preventing HIV-1 infection remains elusive. Thus, the development of novel approaches capable of preventing HIV-1 transmission is of paramount importance. However, this is partly hindered by the lack of an easily accessible small-animal model to rapidly measure viral entry. Here, we report the generation of a human CD4- and human CCR5-expressing transgenic luciferase reporter mouse that facilitates measurement of peritoneal and genitomucosal HIV-1 pseudovirus entry in vivo. We show that antibodies and antiretrovirals mediate preexposure protection in this mouse model and that the serum antibody concentration required for protection from cervicovaginal infection is comparable to that required to protect macaques. Our results suggest that this system represents a model for the preclinical evaluation of prophylactic or vaccine candidates. It further supports the idea that broadly neutralizing antibodies should be evaluated for use as preexposure prophylaxis in clinical trials.

Fig 1

Intraperitoneal injection of VSVg-Cre (75 ng p24) into ROSA-Stop-Luc mice results in bioluminescence in the omentum. (A) Representative luminescence 6 days after i.p. injection of control medium or VSVg-Cre. The unmarked signal in lower left abdomen depicts one site of injection. (B) Time course of photon flux per second for omental ROI in mice i.p. injected with VSVg-Cre (n = 5) or control medium (n = 4). †, off-scale value (3.27). (C) (Left) Preincubation with anti-VSVg antibody I1 (total n = 7) blocks VSVg-Cre infection measured 4 days (d4) after i.p. injection by an omental ROI compared to the result for isotype control mice (total n = 8) and untreated mice (total n = 7), used to define 100% infection (I1 and isotype controls, respectively; final concentration, 1 μg/ml). Data were pooled from two experiments. For each experiment, 0% infection was defined by luminescence of uninfected mice (n = 1 to 2). Symbols depict different individual experiments. (Right) Photon flux per second for omenta dissected after i.p. injection of d-luciferin 6 days (d6) after VSVg-Cre injection (n = 4 per group). Dashed line, mean of naive mice; †, negative off-scale values (for I1 mice, −944.7; for naive mice; −6,075); p/s/cm²/sr, photon flux per second per square centimeter per steradian; Ab, antibody.

Fig 2

HIV-Luc_TG mice express hCCR5 and hCD4. (A) Construct used to produce transgenic mice expressing hCCR5 and hCD4 under the control of the human ubiquitin C (UbC) promoter. SV40, simian virus 40; BGH, bovine growth hormone. (B) Representative fluorescence-activated cell sorter analysis of hCCR5 and hCD4 expression on PBMCs of an HIV-Luc_TG mouse, a transgene-negative littermate, and a human healthy control.

Fig 3

MEFs produced from HIV-Luc_TG mice can be used for in vitro analysis of HIV-1 pseudovirus infection. (A) hCCR5 and hCD4 expression on MEFs from an HIV-Luc_TG mouse (solid line) and a ROSA-Stop-Luc mouse (tinted region) determined by flow cytometry. (B) Luminescence after infection of HIV-Luc_TG and ROSA-Stop-Luc MEFs with serial dilutions of preparations of YU2-Cre or VSVg-Cre. Infection of HIV-Luc_TG MEFs was performed in quadruplicate; infection of ROSA-Stop-Luc MEFs was performed in duplicate. Results of a representative experiment for different viral preparations are presented. (C) Luminescence after infection of HIV-Luc_TG MEFs with YU2-Cre (13 TCID₅₀s; red) or VSVg-Cre (35 TCID₅₀s; blue) in the presence of different antiretrovirals. Dashed lines, luminescence in the absence of antiretrovirals (red, YU2-Cre; blue, VSVg-Cre) and for uninfected MEFs (black, YU2-Cre experiments; gray, VSVg-Cre experiments). Infections were performed in duplicate. IC₅₀s were calculated using a nonlinear regression curve. n.a., not applicable.

Fig 4

HIV-Luc_TG mice become infected with HIV-1 pseudovirus after i.p. injection. (A) Representative luminescence 8 days after i.p. injection. (B) Time course of changes in photon flux per second for an omental ROI for HIV-Luc_TG mice and transgene-negative littermates injected i.p. with YU2-Cre (585 TCID₅₀s) compared to the luminescence of uninjected mice (left) and photon flux per second 8 days after injection (right). Pooled data from two experiments depicted by individual symbols are presented (total n starting on day 3, 7 to 8 [one mouse died during the course of the experiment] for HIV-Luc_TG mice, 8 for negative mice, and 2 for uninjected mice). (C) Detection of tdTomato-positive (tdTomato⁺) cells in peritoneal lavage fluid obtained 6 days after i.p. injection of YU2-Cre (585 TCID₅₀s) in hCCR5⁺ hCD4⁺ ROSA26^tdTomato/Luc mice or transgene-negative littermates. Representative tdTomato expression found in different cellular subsets of an hCCR5⁺/hCD4⁺ mouse is shown. (Left) Gating strategy for cells pregated on the basis of forward scatter and sideward scatter (SSC); (right) mean frequency (n = 3 per group) of tdTomato-positive cells in cellular subsets of hCCR5⁺/hCD4⁺ mice and negative littermates. (D) Ex vivo luminescence analysis of dissected omenta after i.p. d-luciferin injection. Analysis was performed 8 days after i.p. YU2-Cre injection (585 TCID₅₀s) in HIV-Luc_TG mice (left) and 6 days after i.p. VSVg-Cre injection (2,190 TCID₅₀s) in ROSA-Stop-Luc mice (right). Mice were pretreated with antiretroviral compounds starting 24 h prior to viral challenge (efavirenz, 0.5 mg twice p.o.; maraviroc, 1.5 mg twice p.o.; enfuvirtide, 40 μg four times s.c.) or untreated. The log differences in the arithmetic means of photon flux per second defining 0% and 100% infection are 1.2 (YU2) and 3.0 (VSVg). (E) Ex vivo luminescence analysis of dissected omenta after i.p. d-luciferin injection. Analysis was performed 8 days after i.p. YU2-Cre (585 TCID₅₀s) injection in HIV-Luc_TG mice. Mice were s.c. pretreated with 3BNC117 or 200 μg mGO53 24 h prior to viral challenge. The log difference in the arithmetic mean of photon flux per second defining 0% and 100% infection is 0.74. For panels D and E, untreated or isotype-treated mice were used to define 100% infection, respectively, and naive mice were used to define 0% infection (n ≥ 4 for virus-injected groups, n = 2 to 3 for naive mice).

Fig 5

HIV-Luc_TG mice become infected with HIV-1 pseudoviruses after intravaginal application. (A) Representative luminescence in HIV-Luc_TG mice and transgene-negative littermates 5 days after i.vag. YU2-Cre application (three times with 160 TCID₅₀s). (B) Timeline for experiments (left; d, day; Tx, treatment) and time course of photon flux per second for vaginal ROI after i.vag. application of YU2-Cre (three times with 160 TCID₅₀s each time) (middle) in PBS-treated HIV-Luc_TG (n = 6) mice and transgene-negative littermates (n = 4). Dashed line, luminescence 5 days after application in ROSA-Stop-Luc mice (n = 3). (Right) Pooled luminescence 5 days (d5) after infection for PBS-treated (total n = 17) or mGO53-treated (200 μg s.c., total n = 10) HIV-Luc_TG mice and transgene-negative littermates (total n = 6). Symbols depict individual experiments (n = 7). (C) Photon flux per second 5 days after i.vag. application of JR-FL-Cre (MEF titer > 1:270; three times with 40 μl each time) and YU2-Cre (three times with 160 TCID₅₀s each time) in mice homozygous for hCCR5 and hCD4 compared to transgene-negative littermates. Pooled data from two experiments are presented (total n for hCCR5⁺/hCD4⁺ mice, ≥5; total n for negative mice, 2). (D) Infection in mice pretreated with maraviroc (1.5 mg twice p.o.) or controls 5 days after i.vag. application of pseudovirus. (Left) YU2-Cre infection (three times with 160 TCID₅₀s each time) in HIV-Luc_TG mice (n = 4). mGO53-pretreated mice (200 μg s.c., n = 4) (red bar) and ROSA-Stop-Luc mice (n = 3; see panel B) from the same experiment were used to define 100% and 0% infection, respectively. (Right) Total photon flux per second in ROSA-Stop-Luc mice after VSVg-Cre application (three times with 175 TCID₅₀s each time). Pooled data from two individual experiments are presented (total n = 6 per group). (E) Infection 5 days after intravaginal application of YU2-Cre (three times with 160 TCID₅₀s each time) in HIV-Luc_TG mice s.c. pretreated with 3BNC117 (for 2 μg, total n = 3; for 20 μg, total n = 7; for 200 μg, total n = 6) or 200 μg mGO53 (total n = 10; see panel B) 24 h prior to viral challenge. Pooled mGO53-treated mice and pooled transgene-negative littermates (total n = 6; see panel B) were used to define 100% and 0% infection, respectively. Pooled data from a total of five experiments depicted by individual symbols are presented. (F) Infection 5 days after intravaginal application of YU2-Cre (three times with 160 TCID₅₀s each time) in HIV-Luc_TG mice s.c. pretreated with 3BC176 or PBS (n = 4 per group) 24 h prior to viral challenge. PBS-treated mice and pooled transgene-negative littermates (total n = 6; see panel B) were used to define 100% and 0% infection, respectively. Numbers show human IgG serum concentration (n = 5 per group) in μg/ml at the time of viral challenge, and SEMs are in parentheses.