Nasal aluminum (oxy)hydroxide enables adsorbed antigens to induce specific systemic and mucosal immune responses

Abstract

Some insoluble aluminum salts are commonly used in injectable vaccines as adjuvants to accelerate, prolong, or enhance the antigen-specific immune responses. Data from previous studies testing the nasal mucosal vaccine adjuvant activity of aluminum salts are conflicting. The present study is designed to further assess the feasibility of using aluminum salts in injectable vaccines as nasal mucosal vaccine adjuvants. Using Alhydrogel®, the international scientific standard of aluminum (oxy)hydroxide gels, and ovalbumin or 3 × M2e-HA2, a synthetic influenza virus fusion protein, as antigens, we showed in a mouse model that when dosed intranasally Alhydrogel® enables antigens adsorbed on it to induce stronger antigen-specific immune responses in both serum samples (e.g., specific IgG) and nasal and lung mucosal secretions (i.e., specific IgA) in all immunized mice, as compared with nasal immunization with the antigens alone. Rerouting insoluble aluminum salts in injectable vaccines may represent a viable approach for (nasal) mucosal vaccine adjuvant discovery.

Keywords: MPLA; Mucosal vaccine adjuvant; aluminum salts; antibody responses; cytokine release.

Figure 1.

OVA-specific IgA levels in the nasal wash (A) and BAL samples (B) of mice intranasally immunized with OVA-adsorbed Alhydrogel®. Mice (n = 5) were dosed on days 0, 14, and 28 with OVA/Alhydrogel®, OVA/MPLA, OVA alone, or sterile PBS. The dose of OVA was 5 μg per mouse, 5 μg for MPLA, and 20 μg for Alhydrogel®. The anti-OVA IgA levels (OD₄₅₀ values) in nose wash and BAL samples were measured 14 d after the third immunization (*p < 0.05, vs. OVA; #p < 0.05, OVA/Alhydrogel® vs. OVA/MPLA).

Figure 2.

OVA-specific IgG1 (A) and IgG2a (B) levels in the sera of mice intranasally immunized with OVA-adsorbed Alhydrogel®. Mice (n = 5) were dosed on days 0, 14 and 28 with OVA/Alhydrogel®, OVA/MPLA, OVA alone, or sterile PBS. The dose of OVA was 5 μg per mouse, 5 μg for MPLA and 20 μg for Alhydrogel®. The anti-OVA IgG1 and IgG2a levels (OD₄₅₀ values) in serum samples were measured 14 d after the third immunization (*p < 0.05, vs. OVA; #p < 0.05, OVA/Alhydrogel® vs. OVA/MPLA).

Figure 3.

In vitro release of IL-4 and IFN-γ by splenocytes isolated from mice intranasally immunized with OVA-adsorbed Alhydrogel®. Mice (n = 5) were dosed on days 0, 14, and 28 with OVA/Alhydrogel®, OVA/MPLA, OVA alone, or sterile PBS. On day 42, splenocytes (2 × 10⁶ cells/mL) isolated from individual mouse were stimulated with OVA (10 μg/mL) for 48 h. IL-4 and IFN-γ concentrations in cell culture medium were determined using ELISA kits (*p < 0.05, vs. OVA; #p < 0.05, OVA/MPLA vs. OVA/Alhydrogel®).

Figure 4.

Antigen (3 × M2e-HA2)-specific total IgG levels in the serum (A), and IgA levels in the nasal wash (B) and BAL (C) samples of mice intranasally immunized with 3 × M2e-HA2-adsorbed Alhydrogel®. Mice (n = 5) were dosed on days 0, 14 and 28 with 3 × M2e-HA2/Alhydrogel®, 3 × M2e-HA2/MPLA, 3 × M2e-HA2 alone or sterile PBS. The dose of 3 × M2e-HA2 was 5 μg per mouse, 5 μg for MPLA, and 50 μg for Alhydrogel®. The anti-3 × M2e-HA2 IgG (OD₄₅₀ values) in the serum samples and IgA levels in the nose wash and BAL samples were measured 14 d after the third immunization (*p < 0.05, vs. 3 × M2e-HA2).