Fig 1.
AAV-MEDI4893 expression kinetics in blood and mucosal tissues of transduced mice and conformation of binding to α-toxin.
(A) Schematic diagram of AAV-MEDI4893 genome. Transgene expression is driven by the ubiquitous CASI promotor, followed by the human MEDI4893 variable heavy chain domain and heavy chain constant domain, a furin F2A self-cleaving peptide, the variable light chain domain and light chain constant domain of MEDI4893. This was followed by a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) and 3x miR142 3p target and a simian virus 40 polyadenylation signal (SV40 polyA), flanked by two AAV2 inverted terminal repeat (ITR) sequences. Genome length from ITR to ITR is 4587 base pairs. Schematic created in BioRender. Hommes, J. (2026) https://BioRender.com/ibh05k0. (B) Plasma expression kinetics of human IgG following AAV-MEDI4893 administration in 6-week-old Balb/c mice (n = 4/group). Blood was collected up to 42 days post-injection, and mAb concentrations were determined by human IgG ELISA. Kruskal-Wallis test; * p < 0.05, ** p < 0.01; mean + SD. (C) Human IgG concentrations measured in bronchoalveolar lavage, peritoneal lavage, intestinal lavage, and vaginal lavage fluids at day 42 post-AAV administration (n = 4 for all groups except n = 3 for vaginal lavage; mean +/- SD). Antibody levels quantified by human IgG ELISA. (D) Functional binding of AAV-expressed MEDI4893 human IgG in mouse plasma to recombinant AT, measured by indirect ELISA at indicated timepoints post-AAV-MEDI4893 administration (n = 4 per group; mean +/- SD).
Fig 2.
AAV-MEDI4893 administration prevents platelet aggregation and mortality following α-toxin infusion.
(A) Survival of untreated control and AAV-MEDI4893-treated mice following AT intoxication. (n = 4 per group); Kaplan-Meier survival analysis with Mantel-Cox test. (B) Quantification of blood platelet levels at baseline (left) and 10 min post-AT infusion (right). (n = 4 per group); unpaired t test, ** p < 0.01; median. (C) Spinning-disk intravital microscopy (SD-IVM images of the liver before and 20 min after AT infusion in control or AAV-MEDI4893 treated C57Bl/6 mice. Images correspond to S1-S2 Videos. Platelets (CD49b; red). Liver resident Kupffer cells (TIM-4; white), and hepatocytes (auto-fluorescent, dull green). (D) Quantification of platelet aggregates (area> than 5-50 µm2) and (E) platelet fluorescent intensity (CD49b) from SD-IVM videos shown in (C). (n = 3 per group); unpaired t-tests; mean + SE. Scale bar 50 µm, (F) Spinning-disk intravital microscopy (SD-IVM images of the liver before and 20 min after AT infusion in AAV-CA45 treated C57Bl/6 mice. Platelets (CD49b; red). Liver resident Kupffer cells (TIM-4; white), and hepatocytes (auto-fluorescent, dull green); Scale bar 100 µm. (G) Quantification of platelet aggregates (area> than 5 µm2) and (H) total area covered by platelets after 20 minutes of AT injection in PBS control mice, AAV-CA45-, or AAV-MEDI4893-treated mice. (n = 5 for PBS, n = 8 for AAV-CA45, and n = 6 for AAV-MEDI4893). Kruskal-Wallis test, * p < 0.05; ** p < 0.01; mean +/- SD. (I) High-magnification SD-IVM images showing colocalization of N-terminally labeled AT (AT-AF647; blue) with platelet aggregates (CD49b; red) 15 minutes post-infusion in control (top) or AAV-MEDI4893-treated mice (bottom). Platelet aggregates (CD49b; red), Kupffer cells (F4/80; white) (J) Quantification of colocalization of platelet aggregates with AT from images in (I) (n = 19 aggregates for control and n = 4 aggregates for AAV-MEDI4893 treated animals). Experiment was repeated twice. Mann-Whitney test, * p < 0.05; mean +/- SD.
Fig 3.
Effect of AAV-MEDI4893 treatment on S. aureus lung infection model.
(A) Survival of mice receiving a lethal dose of S. aureus (5x108 CFU USA300 LAC) in Control or AAV-MEDI4893-treated groups over a period of 4 days post-infection (n = 8 for PBS control mice and n = 7 for AAV-MEDI4893 mice). Survival was tested with Kaplan-Meier with Mantel-Cox test. (B) Sickness score of the same cohorts was assessed over 4 days post-infection using the standardized sepsis scoring system (see S1 Table), Mann-Whitney tests, ** p < 0.01; mean + /- SD. (C) Bacterial burden in lung tissue at 24 hours post intratracheal infection with S. aureus (5x108 CFU USA300 LAC), (n = 5 per group), Mann-Whitney test; median. (D) Total BAL fluid protein concentrations from mice treated with AAV-CA45 or AAV-MEDI4893 (n = 5) Mann-Whitney test, mean + /- SD. (E) Number of total cells collected from BALF of mice treated with AAV-CA45 and AAV-MEDI4893 (n = 5). Mann-Whitney test, mean + /- SD. (F) Representative picture of hemacolor stained cytospins of BALF of mice treated with AAV-CA45 or AAV-MEDI4893. Quantification of (G) neutrophils and (H) monocytes in the bronchoalveolar lavage fluid (BALF) of mice treated with AAV-CA45 and AAV-MEDI4893 after 24 hours of intratracheal infection with a lethal dose of S. aureus (n = 5). Mann-Whitney tests, mean + /- SD.
Fig 4.
Effect of AAV-MEDI4893 treatment on S. aureus skin infection model.
(A) Whole-body bioluminescence imaging of mice bilaterally infected intradermally with S. aureus (2x107 CFU, USA300 LAC-lux per site) at day 0, day 1, day 4, and day 7 post-infection. Control mice (left two) and AAV-MEDI4893-treated mice (right three) were imaged in parallel. (B) Quantification of bioluminescence signals from control mice (n = 14) and AAV-MEDI4893-treated mice (n = 12), Multiple Mann-Whitney tests, *** p < 0.001; mean + SD. (C) Daily photographs of one representative untreated control mouse and one AAV-MEDI4893-treated mouse to monitor dermal necrosis at sites of infection. (D) Quantification of dermal necrosis from panel (C) for control (n = 14 mice) and AAV-MEDI4893-treated mice (n = 12), Multiple Mann-Whitney tests, * p < 0.05, *** p < 0.001; mean + /- SEM. (E) Quantification of bioluminescence signals from mice treated with AAV-CA45 (n = 6) and AAV-MEDI4893 (n = 6), Multiple Mann-Whitney tests, ** p < 0.01; mean + SD. (F) Quantification of dermal necrosis for mice treated with AAV-CA45 (n = 6) and AAV-MEDI4893 (n = 6), Multiple Mann-Whitney tests, ** p < 0.01; mean + /- SEM.