Mucosal immunization with a novel nanoemulsion-based recombinant anthrax protective antigen vaccine protects against Bacillus anthracis spore challenge

Abstract

The currently available commercial human anthrax vaccine requires multiple injections for efficacy and has side effects due to its alum adjuvant. These factors limit its utility when immunizing exposed populations in emergent situations. We evaluated a novel mucosal adjuvant that consists of a nontoxic, water-in-oil nanoemulsion (NE). This material does not contain a proinflammatory component but penetrates mucosal surfaces to load antigens into dendritic cells. Mice and guinea pigs were intranasally immunized with recombinant Bacillus anthracis protective antigen (rPA) mixed in NE as an adjuvant. rPA-NE immunization was effective in inducing both serum anti-PA immunoglobulin G (IgG) and bronchial anti-PA IgA and IgG antibodies after either one or two mucosal administrations. Serum anti-PA IgG2a and IgG2b antibodies and PA-specific cytokine induction after immunization indicate a Th1-polarized immune response. rPA-NE immunization also produced high titers of lethal-toxin-neutralizing serum antibodies in both mice and guinea pigs. Guinea pigs nasally immunized with rPA-NE vaccine were protected against an intradermal challenge with approximately 1,000 times the 50% lethal dose ( approximately 1,000x LD(50)) of B. anthracis Ames strain spores (1.38 x 10(3) spores), which killed control animals within 96 h. Nasal immunization also resulted in 70% and 40% survival rates against intranasal challenge with 10x LD(50) and 100x LD(50) (1.2 x 10(6) and 1.2 x 10(7)) Ames strain spores. Our results indicate that NE can effectively adjuvant rPA for intranasal immunization. This potentially could lead to a needle-free anthrax vaccine requiring fewer doses and having fewer side effects than the currently available human vaccine.

FIG. 1.

Time course of serum anti-PA IgG in mice. Mice were intranasally immunized with two doses of vaccine (arrows). (A) Induction of anti-PA IgG in CBA/J mice vaccinated with 20 μg rPA and increasing concentrations of NE. Inset, anti-PA IgG subtypes in CBA/J mice immunized with rPA-NE. Data are presented as ratios of individual IgG2a, IgG2b, and IgG3 titers versus IgG1 titer. (B) Anti-PA IgG in BALB/c mice vaccinated with various formulations of rPA vaccine. The results are presented as the mean ± SEM of individual serum anti-PA IgG end point titers (n = 5 mice per group). *, statistical difference between the titer achieved with rPA-NE vaccination and the antibody titers in the other groups (P < 0.05). Inset, presence of anti-PA IgE in mice immunized with rPA-Alu, showing immuno-dot blots of 1:10 to 1:80 dilutions of pooled sera from mice immunized with rPA-NE (row 1), rPA-MPL A (row 2), rPA-CpG (row 3), control (row 4), and rPA-Alu (row 5).

FIG. 2.

Anti-PA IgA and IgG antibodies in BAL fluid. The anti-PA IgA (A) and anti-PA IgG (B) levels determined by ELISA of BAL fluid from BALB/c mice intranasally vaccinated with various formulations of vaccine are shown. Anti-PA IgA and anti-PA IgG antibodies are expressed as the mean ± SEM of antibody concentrations (n = 5). *, statistical difference between rPA-NE vaccination and other immunization groups (P < 0.05).

FIG. 3.

LeTx neutralization in vitro. RAW264.7 cells were treated with the anthrax LeTx that had been preincubated with a serial dilution of immune, pooled BALB/c sera. Bars represent the antibody dilution in which cells retain 50% viability (NC₅₀). Error bars indicate SEMs.

FIG. 4.

PA-specific induction of splenocyte proliferation in vitro. Splenocytes isolated from immunized mice were stimulated with rPA (5 μg/ml) for 72 h. Proliferation indexes were calculated as a ratio of the activity in rPA-stimulated cells to the activity in resting splenocytes. *, statistical difference between groups (P < 0.05). Error bars indicate SEMs.

FIG. 5.

Immune response and survival of guinea pigs intranasally immunized with rPA-NE vaccine. Hartley guinea pigs were vaccinated with 2 doses of vaccine (at 1 day and 4 weeks, as indicated by arrows). (A) Anti-PA IgG in guinea pig serum. Antibody titers were determined at 3- to 4-week intervals with serum anti-PA IgG measured by ELISA (mean end point titers ± SEM). (B) Intradermal challenge. At 6 months, guinea pigs were intradermally injected with 1,000× LD₅₀ of Ames spores, and mortality was monitored for 14 days. For vaccinated and control animals (n = 9; inset), LeTx neutralization was performed at 22 weeks before the challenge. The antibody titer in which RAW264.7 cells retained 50% viability (NC₅₀) was determined from the cell viability obtained in at least two assays, each performed in triplicate. *, statistically significant difference compared to unvaccinated animals (P < 0.001).

FIG. 6.

Immune response and intranasal challenge of guinea pigs intranasally vaccinated with rPA-NE vaccine. Hartley guinea pigs (n = 10 per group) were vaccinated at 1 day and 4 weeks. (A) Anti-PA IgG and LeTx-neutralizing antibody titers in serum. Antibody titers were determined at 3 and 6 weeks and are presented as the mean ± SEM of individual serum anti-PA IgG end point titers. The LeTx neutralization assay cell was performed before the challenge, with values representing mean titers at which RAW264.7 cells retained 50% viability (NC₅₀). (B and C) Survival curves after intranasal challenge. At 7 weeks, guinea pigs were infected by intranasal instillation of 10× LD₅₀ (B) and 100× LD₅₀ (C) of Ames spores, and animals were monitored up to 16 days. P was <0.05 between all vaccinated groups compared to unvaccinated animals.