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 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 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 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 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