Methylglycol chitosan and a synthetic TLR4 agonist enhance immune responses to influenza vaccine administered sublingually

Abstract

Influenza is a vaccine-preventable contagious respiratory illness caused by influenza (flu) viruses which can lead to hospitalization and sometimes even death. Current flu vaccines delivered intramuscularly (IM) or intradermally (ID) are less effective at eliciting protective mucosal immune responses and vaccines delivered intranasally (IN) possess potential safety concerns. Sublingual (SL) vaccination is a promising alternative route for vaccine delivery which has been indicated as safe and effective at inducing protective immune responses in both systemic and mucosal compartments. We evaluated the efficacy of methylglycol chitosan (MGC) and a synthetic toll-like receptor 4 agonist (CRX-601), alone or in combination, for improving systemic and mucosal immune responses to a monovalent detergent-split flu virus vaccine delivered SL. SL vaccination of mice with split-flu vaccine formulated with either MGC or CRX-601 resulted in specific serum IgG and mucosal IgA titers that were significantly greater than titers from non-adjuvanted vaccination and equivalent to or greater than titers in mice vaccinated IM. Our results demonstrate that SL vaccination utilizing MGC or CRX-601 as adjuvants is a viable alternative route of vaccination for flu which can elicit systemic immune responses equivalent to or greater than IM vaccination with the added benefit of stimulating a robust specific mucosal immune response.

Keywords: CRX-601; Chitosan; Influenza; Mucosal vaccination; Sublingual; TLR-4.

Figure 1. Chemical structures of deacetylated chitosan and chitosan derivatives evaluated in this study

The chitosans and derivatives used had > 90% deacetylation as indicated by the manufacturer. Chitosan oligosaccharide lactate is a low molecular weight oligosaccharide available as the lactic acid salt. Glycol chitosan and methylglycol chitosan have the glycol modification with MGC additionally being N-trimethylated.

Figure 2. Colloidal stability of CRX-601 with chitosan derivatives

Photograph of aqueous solution of CRX-601 (1 mg/mL) incubated with the indicated chitosan derivative (5 mg/mL) in 10 mM HEPES for 24h. The picture shows MGC-CRX-601 complexes are colloidally stable whereas visible precipitation is observed with GC and CO. MGC: methylglycol chitosan; GC: glycol chitosan; CO: chitosan oligosaccharide lactate.

Figure 3. MGC-CRX-601 formulations prepared in 10 mM HEPES or HEPES-Saline

Particle size (diameter)/polydispersity (PDI) (A) and zeta-potential (ζ-potential) (B) values as a function of increasing MGC concentration for formulations prepared in 10 mM HEPES (■) vs. 10 mM HEPES-Saline (■) indicating lower polydispersity for formulations prepared in HEPES-Saline. All formulations contained CRX-601 at 1 mg/mL. Particle size is graphically represented by bars while PDI is shown as dot plot. Representative size distribution profiles obtained by dynamic light scattering (DLS) for MGC-CRX-601 complexes at 2 mg/mL MGC prepared in 10 mM HEPES (C) indicating multimodal size distribution vs. those obtained in 10 mM HEPES-Saline (D) indicating a largely unimodal distribution. The data in (A) and (B) represents mean ± standard deviation of three independent experiments. Data were analyzed by t-test (two-tails, equal variance). *, p <0.05, **, p <0.005

Figure 4. MGC and CRX-601 enhance anti-flu IgG titers in mice following SL vaccination

Mice were vaccinated SL with the indicated dose of either flu antigen alone or flu antigen in combination with MGC, CRX-601 or MGC-CRX-601. Serum anti-flu IgG titers (A) 14 days post-secondary SL vaccination (day 35) and (B) 14 days post-tertiary SL vaccination (day 56). Titers obtained from mice vaccinated IM one time (1° IM, collected on day 35) or two times (2° IM, collected on day 56) are included for comparison. The horizontal dashed line represents the lower limit of quantification (LLOQ) for the assay (473.4 ng/mL and 553.1 ng/mL, respectively). Values equal to or less than LLOQ at 1:100 sample dilutions are represented as a value of 1. The number of mice per group that had titers greater than LLOQ, out of the total mice per group, is shown on the X-axis. The mean and standard error are indicated. Data were analyzed by one-way ANOVA with Dunnett post-test. *, p <0.05, **, p <0.005, ***, p <0.0005, ****, p <0.0001 compared to the SL flu antigen only group. Φ, p <0.01, ΦΦ, p <0.05, ΦΦΦ, p <0.005 compared to the indicated IM group.

Figure 5. Functional antibody titers are greater following SL vaccination with formulations containing CRX-601 or MGC

Mice were vaccinated SL with the indicated dose of either flu antigen alone or flu antigen in combination with MGC, CRX-601 or MGC-CRX-601. IM positive control mice were vaccinated two times and are included for comparison. Serum collected on day 56 was analyzed by HAI assay for functional antibody titers. The horizontal dashed line represents the titer necessary for seroconversion (S.C.). Serum samples that did not demonstrate hemagglutination at the lowest dilution (1/20) are represented as a value of 1. The number of mice per group that seroconverted (samples were not diluted), out of the total mice per group, is shown on the X-axis. The mean and standard error are indicated. Data were analyzed by one-way ANOVA with Dunnett post-test. *, p <0.05, **, p <0.005, ***, p <0.0005, ****, p <0.0001 compared to the flu antigen only group.

Figure 6. IgA titers following SL vaccination are enhanced by the addition of CRX-601 or MGC

Mice were vaccinated SL with the indicated dose of either flu antigen alone or flu antigen in combination with MGC, CRX-601 or MGC-CRX-601. Anti-flu IgA titers in vaginal wash samples (VW, black circles) and tracheal wash samples (TW, grey squares) collected on day 56 (14dp3 for SL and 14dp2 for IM) are shown. The horizontal dashed line represents the LLOQ for the VW (black line, ~54.02 ng/mL) and TW (grey line, ~62.18 ng/mL) ELISA assays. Values equal to or less than the LLOQ at 1:40 sample dilution are represented as a value of 1. The number of mice per group that had titers greater than LLOQ, out of the total mice per group, is shown on the X-axis (some TW samples were not assayed due to contamination with blood). The mean and standard error are indicated. Data were analyzed by one-way ANOVA with Dunnett post-test. *, p <0.005, **, p≤0.001, ***, p <0.0001 compared to the flu antigen only group.

Figure 7. Improved IgG titers when MGC is co-formulated with a suboptimal concentration of CRX-601 and flu antigen

Mice were vaccinated SL with the indicated dose of either flu antigen alone or flu antigen in combination with MGC, CRX-601 or MGC-CRX-601. Serum anti-flu IgG titers 14 days post-secondary SL and IM vaccination (day 35) are shown. The horizontal dashed line represents the LLOQ for the ELISA assays (~278.7 ng/mL). Values equal to or less than the LLOQ at 1:100 sample dilutions are represented as a value of 1. The number of mice per group that had titers greater than LLOQ, out of the total mice per group, is shown on the X-axis. The mean and standard error are indicated. Data were analyzed by one-way ANOVA with Dunnett post-test. *, p <0.05, **, p≤0.005, ***, p <0.0001 compared to the indicated group. Φ, p <0.05, ΦΦ, p <0.0001 compared to the IM group. Except where indicated, there were no significant differences between the 2° IM group and groups vaccinated with either MGC or CRX-601.