c.) 50 μg of Qβ-IL-5 or Qβ-Eot into mice (n = 5) at days 0, 21 and 35. selleck kinase inhibitor The generation of anti-IL-5 and anti-eotaxin IgG antibodies was determined by ELISA. As shown in Fig. 2, 21 days after the initial immunization, high antibody titers against either IL-5 or eotaxin were detected. Subsequent
immunization further increased the titers. For each antigen, a statistically significant increase in titer from days 21 to 54 was observed (p < 0.01). Thus, both vaccines can efficiently overcome B cell unresponsiveness and induce high antibody titers against the displayed auto-antigens. The immune response to vaccination with both Qβ-IL-5 and Qβ-Eot injected simultaneously was next examined. Following immunization, high levels of auto-antibodies against both IL-5 and eotaxin were induced. The kinetics and magnitude of the response were similar to those observed for immunization with the corresponding single antigen (Fig. 2A and B). Again the increase in titers from days 21 to 54 was statistically significant (p < 0.01). These data demonstrate that co-immunization with VLP-based vaccines can http://www.selleckchem.com/products/cobimetinib-gdc-0973-rg7420.html simultaneously break tolerance towards more than one self-antigen and induce high antibody responses against the corresponding molecules. We next checked the neutralizing ability of anti-IL-5 serum in a cell (BCL1 cells) proliferation assay cell. As shown in Fig. 2C, anti-IL-5 antiserum inhibited the proliferation of BCL1 cells induced
by IL-5 in a concentration dependant manner. We further investigated the neutralizing ability of the anti-IL-5 antibodies induced
by Qβ-IL-5 by counting blood eosinophils MycoClean Mycoplasma Removal Kit after immunization. Fig. 2D shows that relative to mice immunized with a control Qβ vaccine, the number of peripheral blood eosinophils in Qβ-IL-5 immunized mice was reduced by 87% (p < 0.01). There was no statistically significant difference between unvaccinated animals and those receiving control Qβ vaccine demonstrating anti-Qβ antibodies do not neutralize IL-5. These results show the anti-IL-5 antibodies induced by immunization with Qβ-IL-5 neutralize the activity of IL-5 in vitro and in vivo. The ability of the vaccines either singly or in combination to induce neutralizing antibodies in vivo in an inflammatory setting was assessed by the use of an OVA-based mouse model of allergic airway inflammation. BALB/c mice (n = 5) were either not vaccinated (injected with PBS) or vaccinated with 50 μg of Qβ-IL-5 or Qβ-Eot singly or with both vaccines simultaneously (a total of 100 μg of vaccine corresponding to 50 μg of Qβ-IL-5 and 50 μg of Qβ-Eot) on days 0, 21 and 35. A three-dose regimen was chosen in order to rapidly establish high antibody titers. After anti-IL-5 and eotaxin antibody titers were confirmed by ELISA, airway inflammation was induced by intraperitoneal (i.p.) and intranasal (i.n.) injection of OVA as described. One day after the final i.n.