Efficacy of a vaccine based on protective antigen and killed spores against experimental inhalational anthrax

Abstract

Protective antigen (PA)-based anthrax vaccines acting on toxins are less effective than live attenuated vaccines, suggesting that additional antigens may contribute to protective immunity. Several reports indicate that capsule or spore-associated antigens may enhance the protection afforded by PA. Addition of formaldehyde-inactivated spores (FIS) to PA (PA-FIS) elicits total protection against cutaneous anthrax. Nevertheless, vaccines that are effective against cutaneous anthrax may not be so against inhalational anthrax. The aim of this work was to optimize immunization with PA-FIS and to assess vaccine efficacy against inhalational anthrax. We assessed the immune response to recombinant anthrax PA from Bacillus anthracis (rPA)-FIS administered by various immunization protocols and the protection provided to mice and guinea pigs infected through the respiratory route with spores of a virulent strain of B. anthracis. Combined subcutaneous plus intranasal immunization of mice yielded a mucosal immunoglobulin G response to rPA that was more than 20 times higher than that in lung mucosal secretions after subcutaneous vaccination. The titers of toxin-neutralizing antibody and antispore antibody were also significantly higher: nine and eight times higher, respectively. The optimized immunization elicited total protection of mice intranasally infected with the virulent B. anthracis strain 17JB. Guinea pigs were fully protected, both against an intranasal challenge with 100 50% lethal doses (LD(50)) and against an aerosol with 75 LD(50) of spores of the highly virulent strain 9602. Conversely, immunization with PA alone did not elicit protection. These results demonstrate that the association of PA and spores is very much more effective than PA alone against experimental inhalational anthrax.

FIG. 1.

Efficacy of s.c. vaccination with PA-FIS in the guinea pig and mouse models of respiratory anthrax. (A) Guinea pigs were s.c. immunized twice with PA-FIS (s.c.×2; see Materials and Methods). Three weeks after the booster injection, all animals (six per group) were i.n. infected with 50 LD₅₀ of B. anthracis 9602 spores; 2.6 × 10⁶ spores were recovered from the lungs at time zero. The guinea pigs were observed for 17 days after exposure. The number of survivors in the PA-FIS group was significantly greater than that in the control group (***, P < 0.0001). (B and C) Mice were s.c. immunized twice with either PA-FIS, PA alone, or FIS alone (s.c.×2). Three weeks after the booster injection, all animals (10 per group) were either i.n. infected with 9 LD₅₀ of B. anthracis 9602 spores and observed for 6 days after exposure (B) or i.n. infected with 8 LD₅₀ of B. anthracis 17JB spores and observed for 17 days after exposure (C). The number of mice in the PA-FIS group surviving the 17JB challenge was significantly greater than that in the adjuvant control group (•) (*, P = 0.0102) (C).

FIG. 2.

Efficacy of i.n. vaccination with PA-FIS in the mouse model of respiratory anthrax. Mice were immunized with either PA-FIS or PA alone through the i.n. route (i.n.×2). Three weeks after the booster injection all animals (eight per control group, nine per assay group) were infected i.n. with 9 LD₅₀ of B. anthracis 9602 spores. The mice were observed for 6 days after exposure. The number of mice in the PA-FIS group surviving the challenge was significantly greater than that in either the control group (**, P = 0.0016) or the PA group (*, P = 0.045). The number of mice in the PA group surviving the challenge was significantly greater than that in the control group (*, P = 0.036).

FIG. 3.

Comparison of antibody production in lung mucosal secretions between s.c. and i.n. immunization in mice and guinea pigs. Animals were immunized with PA-FIS either s.c. (s.c.×2) or i.n. (i.n.×2 or i.n.×3). Three weeks after the booster injection, BAL was performed on at least two animals from each experimental group. The titers of anti-PA and antispore total IgG were determined in duplicate by ELISA (left y axis). Anti-PA neutralizing antibody titers were determined by the TNA assay (TNA, right y axis) as described in Materials and Methods. The data reported represent means of two independent experiments, except for the i.n.×3 guinea pig group (one experiment involving four animals). Bars represent means ± the standard deviations of the log₂ values of the titers measured. For each antibody, an unpaired Student t test was used to compare titers obtained after either i.n.×3 or i.n.×2 immunization to those after s.c.×2 immunization. A P value of ≤0.05 was considered to indicate a statistically significant difference between the means. According to the P value, the difference was significant (*), very significant (**), or extremely significant (***).

FIG. 4.

Protection of mice and guinea pigs after a double immunization with PA-FIS. (A) Mice were immunized both s.c. and i.n., (s.c.+i.n.)×3, by the optimized protocol. Three weeks after the second booster injection all animals (10 per group) were i.n. infected with 33 LD₅₀ (8.3 × 10⁷) of B. anthracis 17JB spores. Mice were observed for 23 days after exposure. The number of mice in the PA-FIS group surviving the challenge was statistically greater than that in the adjuvant control group (***, P < 0.0001). Guinea pigs were immunized with either PA-FIS or PA alone both s.c. and i.n.. i.e., (s.c.+i.n.)×3. Three (B) or two (C) weeks after the booster injection, all animals (six per group) were i.n. infected with either 100 LD₅₀ (8 × 10⁶) (B) or 5 LD₅₀ (4 × 10⁵) (C) of B. anthracis 9602 spores. Guinea pigs were observed for 21 days after exposure. In both cases, the number of guinea pigs in the PA-FIS group surviving the challenge was significantly higher than that in the adjuvant control group (***, P < 0.001 [B]; ***, P = 0.0004 [C]). In the experimental results shown in panel C, the number of animals in the PA-FIS group surviving the challenge was significantly higher than that in the PA group (***, P = 0.0005). Also, in panel C the number of survivors in the PA group was significantly higher than that in the adjuvant control group (*, P = 0.0149).

FIG. 5.

Efficacy of PA-FIS against aerosol challenge in the guinea pig model of inhalational anthrax. Guinea pigs were immunized with either PA-FIS or PA alone both s.c. and i.n., i.e., (s.c.+i.n.)×3. Three weeks after the second booster injection all animals (six per group) were exposed to a muzzle-only aerosol challenge with B. anthracis 9602 spores. An aqueous suspension containing 2.9 × 10⁷ spores per ml was aerosolized. The actual inhaled dose was 2.7 × 10⁵ spores, corresponding to approximately 75 LD₅₀, as determined by bacterial counting in the lungs of two control animals at time zero. Guinea pigs were observed for 21 days after exposure. The number of guinea pigs in the PA-FIS group surviving the challenge was significantly higher than those in both the adjuvant control group (***, P = 0.0005) and the PA group (***, P = 0.0008). The number of survivors in the PA group was significantly higher than that in the adjuvant control group (**, P = 0.0013).