ROLE OF THE MUCOSAE-ASSOCIATED EPITHELIAL CHEMOKINE (MEC/CCL28) IN THE MODULATION OF THE IMMUNE RESPONSE AGAINST VIRAL INFECTIONS

Secretory immunity is the main line of defence against mucosal infections as it can provide pathogen blocking secretory IgA at the mucosal surface. CCL28 is a chemokine that binds to CCR3 and CCR10 and potently recruits IgA-secreting plasma cells (IgA-ASCs) in the mucosal lamina propria. Virus-like particles (VLPs) are a novel vaccine approach based on non-pathogenic particles that mimic the structure of authentic virus particles. Immunogenicity of vaccines can be improved by the use of chemokine molecular adjuvants. Here we propose experimental strategies for evaluating CCL28 immunomodulatory effects in mice after vaccination with VLPs of HIV-1IIIB, Influenza A virus (H7N1) and HPV-16 for potential future use of the chemokine as an adjuvant in the development of preventive vaccines against mucosal infections. HIV1IIIB-, H7N1- and HPV16-VLPs were produced in collaboration with the Ruhr-Bochum University, the University of Montpellier and the German Research Cancer Centre of Heidelbergh, respectively. Inbred female Balb/c mice were randomized to receive HIV1IIIB-VLPs or H7N1-VLPs or HPV16-VLPs in the presence or absence of the murine CCL28-expressing plasmid on days 0 and 14. Mice were euthanized on day 28. The murine CCL19 expression vector was used as negative control, as this chemokine binds to CCR7 receptor. HIV1IIIB-VLPs, HPV16-VLPs and CCL28- and CCL19-expressing plasmids were administrated intramuscularly; H7N1-VLPs were administrated intraperitoneally. Blood samples and vaginal secretions were collected by standard methods on days 0, 14 and 28. Saliva samples of H7N1-VLPs treated mice were collected on days 0, 14 and 28 after intraperitoneal injection of carbachol to induce mice drooling. Bronchoalveolar lavages were collected on day 28 by insertion into the trachea of a blunt animal-feeding needle and repeated cycles of injection and aspiration of PBS. Tissues obtained from the spleen, colon, lungs and the uterine cervix were collected on day 28 to obtain the lymphocyte-enriched cell population for cell culture assays and to evaluate IgA-plasma cell distribution at the mucosal level by immunohistochemistry analyses. CCR3 and CCR10 surface receptors were evaluated on circulating splenocytes by flow cytometry. Th1- and Th2-type cytokine production was evaluated in the supernatants from cultivated splenocytes and mucosal T-cells after ex vivo re-stimulation with recombinant HIV-1IIIB gp120 or hemagglutinin (HA) from Influenza virus A H7N1 or HPV-1 L1 protein. Antigen-specific IgG and IgA were measured in sera and mucosal secretions by an ELISA method based on recombinant HIV-1IIIB gp120, HA from Influenza A virus H7N1 and HPV-16 L1 protein. Neutralizing activity of both systemic and mucosal antibodies was assessed as well. Finally, CCL28-mediated recruitment of IgA-ASCs at mucosal sites was evaluated by immunohistochemistry analyses of tissues obtained from the mucosa-associated lymphoid tissue (MALT). Results presented herein show a significant increase in the percentage and in the mean fluorescence intensity (MFI) of CCR3 and CCR10 on CD19+ splenocytes in VLP-CCL28 mice compared to VLP-CCL19 or VLP alone or CCL28 alone or CCL19 alone or saline mice. Antigen-specific production of Th1-type (IFN-gamma) and Th2-type (IL-4 and IL-5) cytokines was significantly augmented in splenocytes and mucosal tissues of VLP-CCL28 mice compared to VLP-CCL19 or VLP alone mice. Total IgA levels were significantly increased compared to baseline values in vaginal secretions of HIV1IIIB-VLP-CCL28 and HPV16-VLP-CCL28 mice, and in saliva samples, BALs and lung homogenates of H7N1-VLP-CCL28 mice on day 28. Antigen-specific IgA were similarly augmented in vaginal secretions of HIV1IIIB-VLP-CCL28 and HPV16-VLP-CCL28 mice and in lungs of H7N1-VLP treated mice. Data were confirmed by the observation that the neutralization ability of both immune sera and mucosal secretions was significantly up-regulated in mice receiving CCL28. Finally, results presented here indicate that immunization on the presence of the CCL28-expressing plasmid significantly increases the quantity of mucosal IgA-ASCs in the rectum of HIV1IIIB-VLP-receiving mice, in the lungs of H7N1-VLP-receiving mice and in the uterine cervix of HPV16-VLP-receiving mice. CCL28 mediates mucosal immunity; the effect is reproducible using this chemokine as an adjuvant in mice immunized with VLPs of mucosally transmitted viruses. CCL28-containing adjuvants should be considered in the development of vaccines against HIV-1, Influenza A virus and HPV to prevent infection of mucosal sites via modulation of mucosal IgA. Nevertheless, the efficacy evaluation of CCL28 adjuvanticity requires further experiments to define immune correlates of mucosal responses to vaccination with VLPs in combination with chemokine adjuvants.