Contribution of immunological memory to protective immunity conferred by a Bacillus anthracis protective antigen-based vaccine

Abstract

Protective antigen (PA)-based vaccination is an effective countermeasure to anthrax infection. While neutralizing anti-PA antibody titers elicited by this vaccine serve as good correlates for protection against anthrax (S. Reuveny, M. D. White, Y. Y. Adar, Y. Kafri, Z. Altboum, Y. Gozes, D. Kobiler, A. Shafferman, and B. Velan, Infect. Immun. 69:2888-2893, 2001), no data are available on the contribution of the immunological memory for PA itself to protection. We therefore developed a guinea pig model in which a primary immunization with threshold levels of PA can induce a long-term T-cell immunological memory response without inducing detectable anti-PA antibodies. A revaccination of primed animals with the same threshold PA levels was effective for memory activation, yielding a robust and rapid secondary response. A challenge with a lethal dose (40 50% lethal doses; 2,000 spores) of spores after the booster vaccinations indicated that animals were not protected at days 2, 4, and 6 postboosting. Protection was achieved only from the 8th day postboosting, concomitant with the detection of protective levels of neutralizing antibody titers in the circulation. The practical implications from the studies reported herein are that, as expected, the protective capacity of memory depends on the PA dose used for the primary immunization and that the effectiveness of booster immunizations for the postexposure treatment of anthrax may be very limited when no detectable antibodies are present in primed animals prior to Bacillus anthracis spore exposure. Therefore, to allow for the establishment of memory-dependent protection prior to the expected onset of disease, booster immunizations should not be used without concomitant antimicrobial treatment in postexposure scenarios.

FIG. 1.

Kinetics of anti-PA Ab generation after primary and secondary immunization. PA-based vaccine doses of 1 μg (solid lines) and 25 μg (dashed lines) were injected (in 0.5 ml s.c.) into 20 guinea pigs. Fourteen weeks after the immunization, the animals were boosted with the same PA-based vaccine dose. The kinetics of Ab generation in these 20 guinea pigs were monitored during the first 14 days after the primary immunization (A and C) and after the booster (B and D), as described in Materials and Methods.

FIG. 2.

Anti-PA Ab avidity measured after primary and secondary immunization. Guinea pigs were immunized with a PA-based vaccine and boosted with the same PA dose 2 months after the first immunization. The anti-PA avidities of individual serum samples were determined 14 days after the first immunization and 14 days after the booster. Relative avidities are expressed as percentages of ELISA optical density values in the absence and presence of 1.5 M thiocyanate. (A) Primary and booster immunizations with 25 μg of PA. (B) Primary and booster immunizations with 1 μg of PA. The anti-PA Ab levels (GMT) in animals immunized with 25 μg of PA were 12,800 after primary vaccination and 25,600 after booster vaccination. In animals vaccinated with 1 μg of PA, a titer of 18,000 was reached after booster vaccination. *, no Ab titers were measured during primary immunization with 1 μg of PA.

FIG. 3.

Cell proliferation index measured before and after booster immunization. The PA-based vaccine was injected (in 0.5 ml s.c.) into guinea pigs, and a booster of the same PA dose was given 8 weeks after the primary immunization. Cell proliferation assays and anti-PA titer ELISAs were performed 14 days after primary and booster immunization. (A) 1-μg dose prebooster; (B) 1-μg dose postbooster; (C) 25-μg dose prebooster (positive control); (D) negative control.