Immunization enhances and accelerates the generation of neutralizing antibodies to a heterologous infecting virus.

<p>Mice were immunized subcutaneously with RV16 VP0 protein plus IFA/CpG or with IFA/CpG adjuvant only and infected intranasally with RV1B, RV29 or sham PBS-challenged as described. Sera were assayed for their ability to prevent cytopathic effect caused by the same RV serotype administered for <i>in vivo</i> infection, using a crystal violet HeLa cell neutalization assay. (a) Neutralization of RV1B cytopathic effect by sera from RV1B-infected or PBS-challenged mice. (b) Neutralization of RV29 cytopathic effect by sera from RV29 infected or PBS challenged mice. Top dotted lines; serum only (uninfected) controls. Bottom dotted lines; virus infected (no serum) control. Open circles are ATCC reference guinea pig anti-sera. Data points represent sera pooled from 4 mice/treatment group. (C) Serum 50% inhibition dilution (ID₅₀) values for RV1B and RV29 neutralization. ND; not detected.</p

Immunization accelerates virus clearance.

<p>Mice were immunized subcutaneously with RV16 VP0 protein plus IFA/CpG or with IFA/CpG adjuvant only and infected intranasally with RV1B or sham PBS-challenged. RV RNA in lung tissue was measured by Taqman qPCR. n = 4 mice/group. n.d., not detected.</p

Immunization enhances lung Th1/Tc1 responses to heterologous RV infection.

<p>Mice were immunized subcutaneously with RV16 VP0 protein plus IFA/CpG, or with IFA/CpG adjuvant only and infected intranasally with RV1B or sham PBS-challenged, as described. (a) Lung tissue IFN-γ, IL-17a and IL-4 mRNA levels measured by Taqman qPCR. (b) T cell cytokine proteins in BAL measured by ELISA. (c) Lung cells harvested 6 days after intranasal challenge were incubated with the indicated stimuli and IFN-γ producing cells were enumerated by ELISPOT assay. n = 4 mice/group. Statistics indicated are for RV-immunized vs RV-adjuvant groups. ***<i>P</i><0.001, **<i>P</i><0.01.</p

Immunization induces systemic, cross-serotype, type I immune responses.

<p>Mice were immunized subcutaneously with RV16 VP0 protein or buffer, with or without IFA/CpG adjuvant, as described. Spleens and serum were harvested 28 days post-immunization. (a) Serum IgG binding to (RV16 VP0 or control polymerase (3′ Pol)) viral proteins were assessed by western blot. (b & c) Splenocytes were stimulated with VP0 or Polymerase (3′ Pol) peptide pools as indicated and (b) IFN-γ and IL-5 producing cells were enumerated by ELISPOT assay and (c) supernatant FN-γ and IL-5 protein levels were measured by cytometric bead array. n = 10 mice/group ***<i>P</i><0.001, **<i>P</i><0.01.</p

Immunization enhances airway lymphocyte responses to heterologous RV infection.

<p>(a) Mice were immunized subcutaneously with RV16 VP0 protein plus IFA/CpG adjuvant, or with IFA/CpG adjuvant only, and infected intranasally with RV1B (RV-Immunized, RV-Adjuvant) or sham PBS-challenged (PBS-Immunized). (b) Lymphocytes in BAL were counted by cytospin assay. (c) BAL and lung CD4+ and CD8+ T cells were enumerated and (d) their expression of the activation marker CD69 was assessed by flow cytometry. (e) CXCL10/IP-10 protein in BAL was measured by ELISA. n = 4 mice/group. Statistics indicated are for RV-immunized vs RV-adjuvant groups. ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05.</p

Immunization enhances effector and memory T cell responses to infection with a more distantly related RV.

<p>Mice were immunized subcutaneously with RV16 VP0 protein plus IFA/CpG or with IFA/CpG adjuvant only and infected intranasally with RV29 or sham PBS-challenged, as described. (a) Lymphocytes in BAL were counted by cytospin assay. (b & c) Total and CD69 expressing CD3+CD4+T cells in BAL (b) and lung (c) were enumerated by flow cytometry. (d & e) Total and CD69 expressing CD3+CD8+T cells in BAL (d) and lung (e) were enumerated by flow cytometry. (f) Lung cells harvested 6 days after intranasal challenge were incubated with the indicated virus, protein, peptide pool or control stimuli and IFN-γ producing cells were measured by ELISPOT assay. (g) Lung cells were stimulated with PMA and ionomycin and intracellular IFN-γ expression in CD4+ and CD8+ T cells was measured by flow cytometry. (h) Graphical data and (i) representative flow cytometry dot plots of CD62L and CD44 memory cell staining of lung CD4+ T cells on day 14 post-infection. n = 4 mice/group. Statistics indicated in a to g are for RV-immunized vs RV-adjuvant groups. ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05.</p

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 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 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