Membrane-bound IL-12 and IL-23 serve as potent mucosal adjuvants when co-presented on whole inactivated influenza vaccines

Abstract

Background: Potent and safe adjuvants are needed to improve the efficacy of parenteral and mucosal vaccines. Cytokines, chemokines and growth factors have all proven to be effective immunomodulatory adjuvants when administered with a variety of antigens. We have previously evaluated the efficacy of membrane-anchored interleukins (IL) such as IL-2 and IL-4 co-presented as Cytokine-bearing Influenza Vaccines (CYT-IVACs) using a mouse model of influenza challenge.

Findings: Here, we describe studies evaluating the parenteral and mucosal adjuvanticity of membrane-bound IL-12 and IL-23 CYT-IVACs in young adult mice. Mucosal immunization using IL-12 and IL-23 bearing whole influenza virus vaccine (WIV) was more effective at eliciting virus-specific nasal IgA and reducing viral lung burden following challenge compared to control WIV vaccinated animals. Both IL-12 and IL-23 bearing WIV elicited the highest anti-viral IgA levels in serum and nasal washes.

Conclusions: This study highlights for the first time the mucosal adjuvant potential of IL-12 and IL-23 CYT-IVAC formulations in eliciting mucosal immune responses and reducing viral lung burden. The co-presentation of immunomodulators in direct context with viral antigen in whole inactivated viral vaccines may provide a means to significantly lower the dose of vaccine required for protection.

Figure 1

Western blot analysis of CYT-IVAC^~IL12 and CYT-IVAC^~IL23. Whole viral lysates were run on 12% SDS-PAGE gel, blotted on PVDF membrane and incubated with IL-12/23p40 antibody followed by anti-species secondary antibodies conjugated to HRP. (A) Dilutions of CYT-IVAC^~IL-12 ranging from 10 μg to 0.5 μg of protein and (B) CYT-IVAC^~IL-23 (lane 2) (5 μg) and PR8 (lane 1) (5 μg) were probed with anti-IL12/23 p40 antibody (eBioscience). (C) Quantitation of virus-incorporated cytokine (pg of cytokine per μg of vaccine) (FlowCytomix™, eBioscience).

Figure 2

Serum anti-viral IgG antibody responses. 8–10 week old female Balb/c mice (Charles River Laboratories, CRL) were lightly anesthetized using isoflurane inhalant and immunized with β-propiolactone-inactivated double gradient purified control WIV (A/PR/8/34), CYT-IVAC^~IL-12 and CYT-IVAC^~IL-23 (n = 5) either intramuscularly (I.M.) in the right hindquarter or intranasally (I.N.) into the nostrils. PBS administered I.M. served as the sham-vaccinated group (n = 5). (A) Blood was collected pre-boost on day 19 (B, C), post-boost on day 35 and serum antibody titers for influenza virus specific IgG were determined by ELISA. Data is displayed as the mean concentration ± SEM. (*p < 0.05 compared to PBS by One way ANOVA, Bonferroni’s multiple comparison test).

Figure 3

CYT-IVAC^~IL12 and CYT-IVAC^~IL23boost mucosal anti-viral responses. (A) Mice were vaccinated with either non-adjuvanted WIV or CYT-IVACs. Blood was collected post-boost on day 35 and serum antibody titers for influenza-specific IgA were determined by ELISA. (B) Mice were challenged with 100LD₅₀ dose of mouse adapted A/PR/8/34 on day 36 following vaccination. At day 4 following challenge, animals (n = 5 mice/group) were sacrificed, nasal washes were collected in PBS (2 ml total volume) and flash frozen. Levels of influenza-specific IgA were quantitated by ELISA, with IgA standard curve extrapolation. Data represents mean concentration ± SE. (A) IM Group *p < 0.05 compared to WIV; IN Group *p < 0.05, ***p < 0.001 compared to WIV by One way ANOVA, (B) ***p < 0.001 compared to WIV, One way ANOVA Bonferroni’s multiple comparison test).

Figure 4

CYT-IVAC immunization reduces viral lung burden following challenge. Animals vaccinated I.M. (A) or I.N. (B) were challenged with a 100LD₅₀ dose of mouse-adapted A/PR/8/34 on day 36 following vaccination. At day 4 following challenge, animals (n = 5) were sacrificed, lung tissues were collected and flash frozen. Viral loads from homogenized lung tissue (n = 5) were determined by tissue culture infectious dose assay. Data is expressed as TCID₅₀ per gram of lung tissue. (*p < 0.05, **p < 0.01, ***p < 0.001 compared to PBS, one way ANOVA, Bonferroni’s multiple comparison test.