The human antibody response to a large protein antigen such as PA is complex. Exposure to such a molecule activates a large collection of B cells, each of which produces an antibody specific for an individual antigenic epitope associated with the immunogen. Most, if not all, of these antibodies undergo further somatic hypermutation, giving rise to a diverse assemblage of antibody paratopes, each characterized by the three-dimensional structure on the target molecule it recognizes (its epitope) and the strength with which it binds that structure (its affinity). For antibodies that bind determinants physically associated with a pathogen (such as a bacterial cell wall), the functionality of the antibody is often determined by the constant region of the antibody molecule. Complement fixation and opsonization, for example, are facilitated by the constant region and are the primary means by which antibody mediates the destruction of many pathogens. The mechanism by which antibodies neutralize soluble agents such as toxins is not well understood. The findings we present here demonstrate that avid antibody binding alone is insufficient to block PA's role in intoxication and suggest that the antigenic epitope recognized is the primary determining factor in antibody function.
Given the complexity of the role that PA plays in anthrax intoxication, the finding that the majority of PA-specific antibodies in vaccinated individuals do not neutralize LT is unexpected. For PA-mediated cell death to occur in the in vitro assay employed in this study (and presumably in vivo as well), PA must bind to the cell surface receptor and be cleaved by furin to yield cell-associated PA63
. Cell-bound PA63
must then form homoheptamers, the PA63
heptamers must bind LF, and the PA/LF complex must be internalized and released into the cytosol. Our data demonstrate that this complicated chain of events can proceed unimpeded in the presence of antibody bound to each of the participating PA monomers. The predominance of PA20
-specific paratopes in the response may offer a partial explanation of the phenomenon in serum. Antibodies binding the PA20
region of the molecule would be detached from cell-bound PA83
(along with the PA20
fragment) following furin cleavage. Additionally, as free PA20
accumulates in the culture supernatant (or in the serum [13
]), this proteolytic fragment could compete for antibody binding with those PA20
-associated epitopes still associated with intact PA83
. Nine of the 16 PA63
-specific antibodies that we assayed were also nonneutralizing. It remains unclear how PA retains its functionality when complexed with these antibodies.
While we believe the collection of antibodies we isolated to be representative of the overall response, it is not complete, and further investigations of these same donors would undoubtedly uncover additional PA-specific paratopes. Serum from donor 3, for example, exhibited very low, but nevertheless above-background, toxin neutralization activity when tested at low dilutions. None of the antibodies that we isolated from this individual neutralized toxin. This could result either from insufficient sampling or from the fact that the respective B cells were not circulating at the time blood was collected. As a group, the antibody panel represents a wide range of relative avidities, recognizes epitopes distributed throughout the PA monomer, and has both neutralizing and nonneutralizing members. These factors suggest no obvious sampling bias and that the paratope distribution we observe reflects the paratope distribution present during the ongoing immune response in a vaccinated individual.
Establishing the relationship between antibody paratope, antigenic epitope, and antibody function is crucial to the understanding of how toxin-based vaccines give rise to efficacious antibody responses. If binding alone is insufficient, it is a reasonable assumption that antibody binding must disrupt an essential toxin function in order for that antibody to be effective. It has been postulated, for example, that the primary mechanism of action of anti-PA antibodies would be the blockade of binding to the cell surface receptor, and vaccine formulations based solely on the primary receptor binding domain (domain 4) of PA have been proposed (5
). Our findings suggest that these assumptions are premature. Only one of the neutralizing antibodies we isolated (4A12) reacts with the domain 4 region of the molecule, and it is unlikely that any of the PA20
-specific antibodies interfere with receptor binding. Residues associated with heptamer formation, LF/EF binding, or furin cleavage could also give rise to antigenic epitopes, and antibodies recognizing these epitopes might also be effective in neutralizing toxin. We have initiated studies to determine the mechanism by which each of the antibodies we isolated neutralizes PA-mediated toxicity in order to determine if they function through any of these modalities.
Although a subset of PA20-specifc antibodies are capable of neutralizing PA-mediated toxicity, their dominance in the response following vaccination may nevertheless have negative implications for the protective efficacy of PA-based anthrax vaccines. Only 18% of these antibodies neutralize the toxin, and they are less efficient, requiring a higher concentration to achieve neutralization. In addition, in vivo, these antibodies may be effectively blocked by free PA20. In comparison, the population of PA63-specific paratopes contains a higher ratio of neutralizing specificities, and cell-free PA83 or PA63 encountered in serum would not diminish their effectiveness.
Although other anthrax-derived antigens have been shown to elicit protective immune responses and have been proposed for inclusion in new vaccine formulations, the second-generation anthrax vaccines currently under development are based solely on PA (10
). The findings presented here, when considered along with our previous demonstration of a profound domain bias in the PA-specific response toward PA20
-specific epitopes in vaccinated humans (16
), suggest that factors intrinsic to the immunobiology of PA itself may diminish its effectiveness in inducing toxin-neutralizing antibodies. The mechanisms underlying the domain bias of the antibody response to PA remain unknown. We have postulated (16
) that differential antigen processing of free PA20
and cell-bound PA63
may give rise to a preponderance of PA20
-specific antibodies following vaccination, and we have suggested that sequence alterations in the furin recognition sequence that render PA protease resistant might produce a more immunogenic form of the PA monomer. Such a design strategy aimed at shifting epitope dominance toward neutralizing determinants might result in a more efficacious vaccine for the prevention of anthrax infection.