It has been reported previously that sera from rhesus macaques immunized with AVA, rPA, or the United Kingdom licensed anthrax vaccine precipitated (AVP) contain toxin-neutralizing antibodies that recognize all four of the domains of PA (47
). While antibody recognition of the N terminus of PA was predominant in macaques immunized with the existing vaccines (AVP and AVA), macaques immunized with rPA recognized both the N- and C-terminal domains of PA (60
). Nonetheless, other studies of the anti-PA responses to AVA have proposed that the PA20 fragment (domain 1a) may carry the immunodominant epitopes in AVA-vaccinated donors. This suggests that removing the immunodominant epitopes found in PA20 from the vaccine would improve the vaccine by targeting RB PA epitopes (47
). Therefore, the PA63 conformer has received some attention as a vaccine component, whether as the recombinant protein, as a truncated gene transcript expressed in Salmonella
spp., or as a component of purified DNA vaccines (8
). The rationale for this approach resides in the concepts that PA63 may present different, more relevant, epitopes to the immunized host than does PA83 and that direct presentation of PA63 eliminates the need for proteolytic processing of the full-length molecule. However, despite some success in animal models, the development of PA63 as a vaccine component is technically challenging because it is difficult to produce and purify (e.g., problems with aggregates and mixture of conformers). Therefore, the focus of recombinant PA vaccine research and development still remains on full-length PA83.
In this study, we demonstrated that LTx-neutralizing responses elicited by AVA vaccination in rhesus macaques can neutralize LTx in vitro
even after it has bound to the cell surface receptor. Receptor binding was confirmed by loss of neutralization by MAb 14B7 while retaining neutralization by MAb 1G3 (26
). The consistent correlations between the NC- and RB-TNA assays indicate that vaccination with AVA generates a repertoire of anti-PA immune responses capable of neutralizing free and RB PA. The results of this study indicate that vaccination by AVA provides protection against multiple targets in LTx intoxication of host cells, perhaps by blocking the PA-LF interaction required for LTx formation, thereby preventing LTx internalization/translocation or by successfully displacing PA from the cell surface receptor. We interpret the high correlation between vaccine-induced and postinfection serum activities in the NC- and RB-TNA assays to mean that the anti-PA response elicited by AVA is qualitatively not different from that following exposure of vaccinated animals to PA during inhalation anthrax.
The neutralizing response in the RB-TNA assay was very consistent across low and high ED50
values, survivors and nonsurvivors, challenge times, and postvaccination and postchallenge, indicating that the anti-PA neutralizing response occurs irrespective of blocking of the interaction between domain 4 of PA and the host cell receptor. It was also determined during these studies that there was a strong positive correlation in both the NC- and RB-TNA assays between the magnitude of the ED50
response and survival. These findings are consistent with results of other studies that employed the traditional R-TNA assay (23
It is important to note that one must be careful when assigning a value to a protective ED50
titer because it depends on the vaccine used and its formulation, the assay from which the ED50
was obtained, the timing of sample collection, and the treatment of the donor prior to sampling (e.g., the number of vaccinations, etc.). For example, the results of this study demonstrated that an ED50
of 2,941 in the RB-TNA assay from the 30-week postvaccination samples was predictive of up to 95% protection. However, this ED50
value may not apply to other vaccines or regimens. Interestingly, observations from the NC- and RB-TNA assays align with data from the R-TNA assay such that the nature of the peak response immediately following the third vaccination with AVA (which, using the original AVA vaccination schedule, was at 4 weeks) gives the best correlation with 95% protection (28
The data presented here demonstrate that there was not a functional bias in the immune response to PA following AVA vaccination, as indicated by the analysis of the pre- and postchallenge samples. In fact, the only clear difference was an increase in the magnitude of the peak anti-PA IgG response, as judged by higher ED50s, to toxin exposure postchallenge. Thus, although the magnitude of the ED50 response was greater after aerosol infection, the relationship between neutralization in the RB assay and in the NC assay did not change compared to the vaccine-induced response alone. Because the animals were primed by PA from the AVA vaccination, the higher neutralizing response after challenge was an anamnestic response due to common epitopes in AVA PA and infection PA.
Taken together, these data demonstrate that the PA component of AVA is highly immunogenic and the humoral immune response generated by vaccination with AVA is potent in neutralizing multiple mechanistic steps of intoxication by anthrax LTx. Importantly, this study showed that the regions of PA occluded during receptor binding via domain 4 are not the only protective epitopes in a PA-based vaccine. Given that the PA domain 4 epitopes can be modified without affecting toxin activity (49
), we conclude that domain-based recombinant vaccines must be evaluated with great care. The results of this study, specifically, the RB-TNA assay results, found that credence should be given to using vaccines that include the 83-kDa form of PA, as it includes not only domain 4 but also domains 2 and 3 and part of domain 1, which provide epitopes necessary for a comprehensive antigenic repertoire and an effective vaccine. In conclusion, these data provide additional evidence that functionally competent full-length PA83 is an excellent vaccine antigen, and the relative ease of its production and purification, compared to properly folded, conformation-competent PA domain-based vaccines, is justification for continuing to pursue PA83 as the primary vaccine candidate.