RV-specific antibody responses in Tbet deficient and wild type mice.

<p>Wild type and Tbet-/- mice were infected intranasally with RV1B or sham infected with PBS. Blood was collected 14 days after infection. (A&B) RV1B-binding IgG2c (A) and IgG1 (B) in sera was measured by ELISA. (C) Neutralisation of RV1B infection of Ohio HeLa cells by pooled sera assessed by crystal violet cell viability staining. ATCC ctl: control reference guinea pig anti-sera. Top dashed line in C, uninfected cells control. Bottom dashed line in C, RV infected cells control. Data represent results from 5–6 pooled sera per treatment group in a single experiment, representative of 3 independent experiments.</p

Helper T cell responses in Tbet deficient and wild type mice.

<p>Wild type and Tbet-/- mice were infected intranasally with RV1B or sham infected with PBS. (A-D) Intranuclear flow cytometry staining for transcription factors Tbet (A), GATA-3 (B), RORγt (C) and FOXP3 (D) in CD3+CD4+ lung T cells, 2 and 7 days post-infection. (E-H) Intracellular flow cytometry staining for cytokines IFN-γ (E), IL-13 (F), IL-17A (G) and IL-10 (H) in lung CD3+CD4+ cells stimulated with PMA and ionomycin, on day 7 post-infection. (I-L) RNA was extracted from lung tissue harvested on day 7 post-infection and expression of IFN-γ (I), IL-13 (J), IL-17a (K) and IL-10 (L) mRNA was quantified by Taqman qPCR. n = 8–9 mice/group.***p<0.001, **p<0.01, *p<0.05, n.s. not significant.</p

Asthma-like airways inflammation is CD4+ T cell dependent.

<p>Tbet-/- mice were infected intranasally with RV1B or sham infected with PBS. In addition, mice were systemically depleted of CD4 expressing cells (anti-CD4), or treated with isotype control antibody (isotype) 3hrs prior to infection. Tissues were harvested at 7 days post-challenge. (A) Lung flow cytometry staining for CD3+CD4+ T cells. (B-D) Levels of cytokines IL-4 (B), IL-13 (C) and IL-17a (D) in lung tissue measured by Taqman qPCR. (E) Total BAL cell counts and (F) eosinophil counts in BAL measured by cytospin assay. (G) MUC5AC levels in BAL measured by ELISA. (H) Representative PAS staining for mucus in lung tissue sections. Scale bars 50μm. n = 12–15 mice/group (mice for which CD4+ cell depletion was not successful were excluded from all analyses (n = 5 of 30)). ***p<0.001, **p<0.01, *p<0.05, n.s. not significant.</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 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 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 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

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

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