Effect of systemically dosed 14C11 antibody on HRV16 infection in vivo.

<p>Mice were dosed intravenously with 14C11 24 hours prior to intranasal infection with HRV16 (n = 9 for tg− group; n = 6 for tg+ groups). (A) Total BAL cells, amacrophageslymphocytes and neutrophils were assessed by cytospin 2 days after infection. (B) The chemokines CXCL1, CXCL11 and CXCL10 in BAL were determined by MSD or quantitative ELISA 2 days after infection. Data are expressed as mean (± SEM). Significance was assessed by One-way ANOVA test with Bonferroni's Multiple Comparison test as post-test. **p<0.01 and ***p<0.001 vs HRV16 infected transgenic negative mice; ^#p<0.05, ^##p<0.01 and ^###p<0.001 vs HRV16 infected transgenic positive mice; Data are representative of 3 independent experiments.</p

14C11 antibody inhibits major group HRV16 infection <i>in vivo</i>.

<p>Groups of 7 mice were dosed intranasally with 14C11 or isotype control 2 hours prior to intranasal infection with HRV16. (A) Total BAL cells, macrophages, lymphocytes and neutrophils were assessed with differentially cytospin counts day 2 after infection. (B) Levels of proinflammatory cytokines and chemokines IL-1β, IL-6, CXCL1, IFNλ2/3, CXCL11 and CXCL10 in cell-free BAL were determined by MSD or quantitative ELISA 2 days after infection. (C) Proinflammatory cytokines and chemokines IL-1β, IL-6 and CXCL1 in lung homogenate were assessed with MSD 2 days after infection. (D) Groups of 3–5 mice were dosed intranasally with 14C11 or isotype control 2 hours prior to intranasal infection with HRV16 and vRNA in lung tissue was assessed by qPCR at the timepoints indicated. (E) Inflammatory cells in the lungs prior to infection (uninfected) and at 12 hours post-infection (12 p.i.) of huICAM negative (tg−) and huICAM-expressing (tg+) mice without antibody treatment, or for tg+ mice following isotype control antibody (tg+ iso) or 14C11 antibody treatment (tg+ 14C11) assessed by H&E staining Arrows indicate peribronchial cellular inflammation. Data are expressed as mean (± SEM). Significance was assessed by One-way ANOVA test with Bonferroni's Multiple Comparison test as post-test. ***p<0.001 and **p<0.01 vs HRV16 infected transgenic negative mice; ^#p<0.05, ^##p<0.01 and ^###p<0.001 vs HRV16 infected transgenic positive mice. Data are representative of 3 independent experiments each.</p

14C11 inhibits major group HRV replication <i>in vitro</i>.

<p>Ohio HeLa cells were pre-incubated with serial dilutions of 14C11 or isotype control and infected with (A) HRV16, (B) HRV14 and (C) minor group HRV25. The antiviral effect of 14C11 was determined by CPE reduction assay and expressed as % of control. (D) IC₅₀s for 14C11 were determined for major HRVs by CPE assay as indicated in. Experiment (A), (B) and (C) were performed in sextuplicates and (D) is a pool of two independent experiments. Data are expressed as mean (± SEM).</p

14C11 does not inhibit inflammation induced via mechanisms independent of human ICAM-1.

<p>Mice were dosed intravenously with 14C11 24 hours prior to intranasal dosing with HRV1B, UV-inactivated HRV1B (UV) or HRV16 (n = 4 for tg− UV, tg− 1B, tg+ 16 iso and tg+ 16 14C11 groups; n = 6 for tg+ UV, tg+ 1B, tg+ 1B iso and tg+ 1B 14C11 groups). (A)Total BAL cells, neutrophils (on day 1) and lymphocytes (on day 4) were assessed with differentially stained cytospins. Data expressed as mean (± SEM). Significance was assessed by One-way ANOVA test with Bonferroni's Multiple Comparison test as post-test. ***p<0.001 vs UV-RV1B in transgenic negative mice; ^###p<0.001 vs UV-RV1B in transgenic positive mice; ^§p<0.05 and ^§§§p<0.001 vs isotype treated HRV16 infected transgenic positive mice. Data are representative of 2 independent experiments. (B) Groups of 7 mice were dosed intravenously with 14C11 24 hours prior to intranasal dosing with 1 µg LPS/mouse. Total BAL cells, lymphocytes and neutrophils were assessed with differentially stained cytospins day 1 after infection. Data are expressed as mean (± SEM). Significance was assessed by One-way ANOVA test with Bonferroni's Multiple Comparison test as post-test. *p<0.05, **p<0.01 and ***p<0.001 vs transgenic positive mice without treatment. Data are representative of 2 independent experiments.</p

14C11 binds in domain 1 of human ICAM-1, does not bind to mouse ICAM-1 and does not inhibit human ICAM-1/LFA-1 interaction.

<p>(A)14C11 and (B) 84H10 antibody were tested for binding of antigens (human ICAM-1 (Hu ICAM-1), mouse ICAM-1 (Mu ICAM-1) and a recombinant protein consisting of human ICAM-1 D1 and mouse ICAM-1 D2-5 (Hu1 Mu2-5)). Jurkat E6.1 cells were labelled with Calcein-AM dye. Serial dilutions of the antibodies 14C11, 84H10 and isotype control were added in an ICAM-1-Fc coated plate. (C) PMA and labelled Jurkat E6.1 cells were added into the plate. Binding to LFA-1 was determined by fluorescein detection. Data were analysed by normalising values to no PMA (0% signal) and PMA no antibody (100% signal) controls. Data are a representative of three experiments for (A) and (B) and four to seven experiments for (C). Data are expressed as mean (± SEM).</p

Time course of topically dosed 14C11 antibody-mediated inhibition of major group HRV16 infection.

<p>Mice were dosed intranasally with 14C11 or isotype control 2 hours prior to intranasal infection with HRV16. (A)Total BAL cells, macrophages, lymphocytes and neutrophils were assessed with differentially stained cytospins day 2, 4 and 7 after infection. (B) CXCL1, CXCL10 and CXCL11 in the BAL were determined by MSD or quantitative ELISA. (C) HRV16 specific serum IgG1 and IgG2a on day 7 post-infection. Data are a pool of 2 experiments with n = 4 mice per group each. Data are expressed as mean (± SEM). Significance was assessed by Three-way analysis of variance (A and B) or by One-way ANOVA with Bonferroni's Multiple Comparison test as post-test (C). **p<0.01 and ***p<0.001 vs HRV16 infected transgenic negative mice; ^#p<0.05 and ^###p<0.001 vs HRV16 infected transgenic positive mice.</p