In this study, we examined the PA domain-specific response after anthrax vaccination using PA-based vaccines in humans as well as rabbits and NHPs—the two animal species proposed for Animal Rule-based anthrax vaccine approval. Information regarding the domain specificity of the antibody response to PA-based vaccines has been limited. Flick-Smith et al. suggested that the epitopes most important for protection within PA are found in domain 4 (9
). Abboud and Casadevall, on the other hand, demonstrated that, upon vaccination with various recombinant domain constructs, mice respond strongly to domain 1 (1
). However, both of these studies immunized mice with the purified, recombinant domains themselves (or combinations thereof), not PA83, which could lead to immune responses that are different from those achieved by vaccination with PA. Previous work indentified antibodies specific for domain 1 or domain 4 in humans immunized with AVA by examining the specificity of isolated monoclonal antibodies or through the use of competitive ELISAs (24
). However, those studies were limited since only recombinant domains 1 and 4 or proteolytic PA fragments (PA20 and PA63) were tested, which does not address antibody specificity for individual domains 2 and 3. Our work expands on previous findings in the following ways: (i) by including domains 2 and 3; (ii) by examining sera generated by immunization with both rPA vaccine and AVA; (iii) by examining the domain specificity of functional (i.e., neutralizing) antibodies generated by vaccination; and (iv) by producing data relevant for Animal Rule-based evaluations through the assessment of the domain-specific antibody levels generated by PA-based vaccines in rabbits and NHP.
In order to determine the role of each PA domain individually in the vaccine-induced immune response, we cloned, expressed, and purified each of the four PA domains separately. We then used the purified recombinant domains as competitors in a competitive ELISA format and in a competitive TNA format, in order to identify the domains recognized by antibodies generated after vaccination with PA-based vaccines. Using a competitive ELISA, we found that each domain plays a role in immunogenicity of PA-based vaccines in that IgG antibodies are generated against all four domains, regardless of species. Using a competitive TNA format, we were able to demonstrate that neutralizing antibodies to domains 1, 3, and 4 are generated by vaccination with PA-based vaccines, regardless of species. We were not able to determine whether neutralizing antibodies specific for domain 2 were present in sera from individuals immunized with PA-based vaccines because of the dominant-negative effect of domain 2. This similarity in response suggests that mechanisms of antibody neutralization of toxin action likely do not differ dramatically between species. This information provides important support for the use of an antibody bridge to link protection in animals to human efficacy under the Animal Rule.
Ideally, we would have liked to compare the domain specificities of total IgG antibodies induced by vaccination in the different species in a more quantitative manner. However, using increasing concentrations of competitor up to 600 nM, the largest amount that was technically feasible to use because of volume and production constraints, we found that percent competition continued to increase; thus, we were not able to ascertain that the maximum level of competition had been achieved. We therefore limited our interpretation to qualitative measures: i.e., whether any competition was generated by the addition of soluble PA domains.
The crystal structure of PA monomers shows that each domain is exposed and is potentially accessible to the host immune system. Therefore, an antibody response to each of the domains upon vaccination with PA-based vaccines is not surprising. After heptamerization of PA, a significant amount of surface becomes buried—mostly parts of domains 1 and 2 (20
); however, PA-based vaccines are composed of PA that would not be expected to have heptamerized since the PA is either in the form that is present in the bacterial culture supernatant (for AVA, see references 2
) or a purified form of PA83 (for rPA, see references 2
, and 29
). Our finding that a subset of the antibodies present in the immune sera specific for either domain 1, 3, or 4 were able to neutralize toxin action is consistent with the fact that each domain plays a critical role in toxin action, whether it be effector binding (domain 1) (20
), participation in oligomer formation (domain 3) (16
), or receptor binding (domain 4) (15
). The fact that neutralizing antibodies to at least three individual PA domains are present in immune sera suggests that each of these domains might participate in generating a protective antibody response in both animals and humans after vaccination.
In developing the competitive ELISA, we observed that complete competition of human IgG antibodies could not be obtained using soluble PA83 as the competitor, even at high concentrations, whereas when combined with either rabbit or NHP sera, PA83 at the same concentration was able to nearly completely abrogate IgG antibody binding to the PA83-coated ELISA plate. The incomplete competition in human serum was not due to vaccine differences, as sera from AVA- and rPA-vaccinated humans had the same pattern. However, the difference in competition levels could be lessened through the use of a sandwich competitive ELISA that selectively detected IgG antibodies specific for native PA83. Also, the biphasic nature of the curve was lost in the sandwich competitive ELISA. These findings suggest that humans have at least two significant populations of anti-PA IgG antibodies: one that reacts with native PA83 and one that reacts only with unfolded or incorrectly folded PA83. It is plausible that rabbits and NHPs also have IgG antibodies specific for unfolded or incorrectly folded forms of the protein; however, in these animals, the population is less apparent than in humans, as evidenced by more complete abrogation of binding to plate-bound antigen by soluble PA83 with rabbit and NHP sera and a lack of a biphasic appearance to the competition curves. It is unclear why humans may have a more significant population of IgG antibodies specific for unfolded or denatured PA than the other species. It is possible that humans respond to a slightly different set of B-cell epitopes than the other species.
The data presented herein highlight the similarities and some differences in the immune responses of animals and humans to PA-based vaccines. The overall immunogenicities of the PA domains were comparable in rabbits, NHPs, and humans in that all domains were recognized by IgG antibodies present in immune sera from each of the species. Moreover, neutralizing antibodies to domains 1, 3, and 4 were generated by immunization with PA-based vaccines in each of the species. We noted one difference between the species in that we were able to detect a more apparent population of IgG antibodies specific for unfolded or incorrectly folded PA in immune sera of humans compared to that from rabbits and NHPs. The presence of similar domain specificities of the IgG antibodies induced by vaccination with PA-based vaccines in the different species strengthens the use of an antibody bridge to link animal protection data to human efficacy. However, our finding that a more noticeable proportion of human IgG antibodies may be specific for unfolded or incorrectly folded PA compared to that found in sera of vaccinated rabbits or NHPs cannot be ignored and adds a cautionary note to making a simple link between animal protection data and human efficacy using total antibody levels. Use of neutralizing antibodies as a bridge might better circumvent this difference between species, since neutralizing antibodies would be expected to bind specifically to the native, active form of the protein. We believe that this information may help in evaluation of new PA-based vaccines using the Animal Rule.