The US anthrax vaccine is predicated on evidence that antibodies directed against protective antigen provide protection against challenge. Work using animal models, including rabbits and guinea pigs, show that while antibodies to PA and in vitro toxin neutralization titers predict survival in an aerosol spore challenge model, this prediction is imperfect [16
]. Since this time, several groups have performed limited characterization of the immune response in individuals receiving the AVA vaccine. These studies have shown that, as with animals, humans vaccinated with AVA produce high titers of antibodies to PA and that antibody titer and in vitro neutralization are correlated [23
]. However, in each of these studies, there were individuals that generated high titers of antibodies to PA but were not able to neutralize toxin. Therefore, it is probable that antibody qualities such as epitope specificity are important factors for effective toxin neutralization. Results from this study provide a biologic explanation for observations of discordance between high concentrations of antibodies but low neutralizing capacities.
In our cohort of AVA immunized subjects, ninety-five percent had measurable antibodies to PA and lower concentrations were associated with longer time since last vaccination and lower total number of doses. Thus, those participants with the highest titers of antibodies received more vaccinations (4-6) and were among those most recently vaccinated (1.1 ± 0.8 years post). These data are consistent with previous reports which show that anti-PA titers decline over time [17
Toxin neutralization is a critical component of protection [17
] and neutralizing antibodies are the best surrogate marker for protective immunity found to date [30
]. Over half of the vaccinated individuals (54%) had little in vitro neutralizing activity above that seen in unvaccinated controls and only twenty-one percent had 50% or greater neutralization at a 1:100 dilution. While it is unclear whether these individuals would be at risk following exposure, a recent report used sera from AVA vaccinated individuals to determine the concentration of PA-specific antibodies necessary for protection. Using a Sterne strain challenge in mice, they found that a toxin neutralization titer of 280 μg/ml or higher (corresponding to 50% neutralization at a 1:112 dilution) provided 79% protection [33
]. These data highlight the need for improved understanding of the variables that determine the quality of vaccine response, potential for different vaccine design and dosing strategies, as well as the desirable properties of specific antibodies used for passive immunization after spore exposure.
Since the highest correlation with in vitro LT neutralization was anti-PA titer, we analyzed the fine specificity of the anti-PA response and identified neutralization-associated humoral epitopes. While there are reports detailing the levels of antigen-specific antibodies following AVA vaccination [34
], the humoral epitope-binding patterns and protective capacity of select specificities have not been well characterized.
We identified 13 unique antibody binding domains after AVA vaccination within all four PA domains [1
]. Three of the antigenic regions were in the PA20
domain and recent evidence indicates that antibodies to this domain can prolong survival of mice challenged with toxin [38
]. An epitope that was identified within sera of participants with high toxin neutralization activity was within the furin cleavage site. Several reports have demonstrated that antibodies or small molecule inhibitors that prevent or slow furin cleavage can provide protection in vivo [39
]. Indeed, Abboud et al
] generated a neutralizing monoclonal antibody in mice that bound to a linear epitope within the furin cleavage site (QKSSN) which is contained within our epitope number 4.
Two epitopes were identified in the ligand-binding domain and mutants in this region have been shown to reduce LF binding and inhibit toxin activity [26
]. The two epitopes found in domain II contain amino acids that are necessary for toxin translocation [28
], as well as sequence homology to the antigenic region (SKNLAPI) bound by a murine monoclonal antibody that provides protection from Sterne strain challenge [39
]. We also identified an antigenic region in the critical receptor-binding domain. Mutations of amino acids within this region result in partial or complete loss of toxicity [29
], and murine monoclonal antibodies to this region neutralize toxin activity in vitro and in vivo [25
]. The antigenic regions within the receptor binding domain that we identified did not overlap with those found by others [25
]. However, Kaur et al
immunized mice with an antigenic region (ID-II), which contained our epitope #12 (LLNIDKDIRK), and demonstrated both in vitro and in vivo protection.
A recent report by Gubbins et al
] demonstrated that AVA-vaccinated individuals generate antibodies to the translocation domain (SFFD), but it is not clear if the antibodies in that study mediate protection. Additionally, others have demonstrated that antibodies from vaccinated individuals directed against the receptor-binding and the ligand-binding regions can provide in vitro protection [47
]. However, the ability of these antibodies to protect against in vivo challenge has not been tested.
We enriched for peptide-specific antibodies against the furin cleavage, ligand-binding, and receptor-binding regions and demonstrated that these antibodies are capable of in vitro neutralization. The neutralizing activity in the un-retained samples is likely due to either low level of antibodies directed against these three regions which were not removed by absorption or to antibodies specific for other peptides since these antibodies account for only three of the antigenic regions identified in the high responders.
Surprisingly, we found that while the antibodies directed to the furin cleavage site mediated the best protection in vitro, the receptor-binding site antibodies resulted in the most robust protection in vivo, despite their apparently equivalent affinity. This may be due in part to rapid furin cleavage and binding of LF to PA before the epitope-specific antibody can bind its target. Pre-incubation of PA with the anti-furin cleavage site antibody probably decreases toxin activity, but this scenario is unlikely to occur naturally. The receptor binding antibody, however, would be able to bind the cellular receptors before the toxin was administered and could result in decreased toxin activity. This is consistent with a previous report showing that monoclonal antibodies to the receptor binding region have neutralizing activity [25
]. Thus, we have demonstrated that epitope-specific antibodies derived from AVA vaccinated individuals can provide protection against lethal toxin challenge.
This is the first report to perform a systematic characterization of the humoral fine-specificity response following human AVA vaccination. We demonstrate that select peptide-specific antibodies enriched from the sera of vaccinated individuals can provide in vivo protection against lethal toxin challenge. The new anthrax vaccination schedule based on quantitative levels of anti-PA following doses may need to be evaluated in the context of antibody specificities that are functionally effective in LT neutralization and critical to survival from challenge. Additionally, the data presented suggest that it may be feasible to create a limited number of monoclonal mixtures that can provide highly effective passive immunity in a post exposure scenario.