Identification of Linear Epitopes in Bacillus anthracis Protective Antigen Bound by Neutralizing Antibodies

Protective antigen (PA), the binding subunit of anthrax toxin, is the major component in the current anthrax vaccine, but the fine antigenic structure of PA is not well defined. To identify linear neutralizing epitopes of PA, 145 overlapping peptides covering the entire sequence of the protein were synthesized. Six monoclonal antibodies (mAbs) and antisera from mice specific for PA were tested for their reactivity to the peptides by enzyme-linked immunosorbent assays. Three major linear immunodominant B-cell epitopes were mapped to residues Leu¹⁵⁶ to Ser¹⁷⁰, Val¹⁹⁶ to Ile²¹⁰, and Ser³¹² to Asn³²⁶ of the PA protein. Two mAbs with toxin-neutralizing activity recognized two different epitopes in close proximity to the furin cleavage site in domain 1. The three-dimensional complex structure of PA and its neutralizing mAbs 7.5G and 19D9 were modeled using the molecular docking method providing models for the interacting epitope and paratope residues. For both mAbs, LeTx neutralization was associated with interference with furin cleavage, but they differed in effectiveness depending on whether they bound on the N- or C-terminal aspect of the cleaved products. The two peptides containing these epitopes that include amino acids Leu¹⁵⁶–Ser¹⁷⁰ and Val¹⁹⁶–Ile²¹⁰ were immunogenic and elicited neutralizing antibody responses to PA. These results identify the first linear neutralizing epitopes of PA and show that peptides containing epitope sequences can elicit neutralizing antibody responses, a finding that could be exploited for vaccine design.Bacillus anthracis is a Gram-positive, facultatively anaerobic, rod-shaped bacterium that secretes a variety of toxins, including anthrax toxin. This toxin is made up of three proteins as follows: protective antigen (PA),³ edema factor (EF), and lethal factor (LF). Like other binary toxins, anthrax toxin follows the same pattern where distinct subunits are involved in the different steps at which it can act. The B subunit (PA) is involved in receptor binding and cellular internalization into the cytoplasm, whereas the A subunit (EF and/or LF) bears the enzymatic activity (1). Anthrax can occur naturally in animals by spore transmission via ingestion, inhalation, or an open skin wound, but it can also be a result of bioterrorism and biological warfare (2).PA is the component of the currently licensed anthrax vaccine that elicits protective antibodies. Recent studies have demonstrated that a strong humoral response to truncated recombinant PA contributes to a protective immune response to anthrax (3, 4). Accordingly, there is considerable interest in ascertaining the location and immunogenicity of B-cell epitopes on the PA molecule. The development of numerous monoclonal antibodies (mAbs) to different epitopes on the PA molecule that influence PA functions, in conjunction with epitope mapping, has provided an opportunity to study the fine antigenic structure of PA. Most of these epitopes have been defined in mice (5–8), in macaques (9), in rabbits (10), as well as in vaccinated humans (11). Consequently, the epitopes described thus far are localized to three discrete regions within the PA. These regions correspond to the 2β2–2β3 loop of domain 2, the domain 3-domain 4 boundary, and the small loop of domain 4. The 2β2–2β3 loop of domain 2 is responsible for mediating membrane insertion, and many neutralizing murine mAbs target this region (5, 8). The boundary between domains 3 and 4, which does not have a known functional activity, has been suggested as a region recognized by polyclonal antibodies from vaccinated humans and rabbits (6, 12). The cellular receptor binding region is localized to the small loop of domain 4, and this region has been described to be recognized by two neutralizing mAbs (7, 9). With the exception of a neutralizing mAb that bound to PA₂₀ (13), no B-cell epitopes have been reported in domain 1. Therefore, identification of further dominant antigenic epitopes is pivotal for understanding the minimal immunogenic region of PA that will allow for precise direction of potent immune responses.Two mAbs to PA have been reported previously by our laboratory, one known as 7.5G binds to domain 1 and can neutralize the cytotoxic activity of lethal toxin (LeTx) (13). The other, mAb 10F4, binds to domain 4 and has weak neutralizing activity. In addition, we now describe four new anti-PA mAbs, of which only one neutralizes LeTx. The characterization of the B-cell epitopes in PA recognized by protective and nonprotective mAbs is important to better understand the antigenic structure of this toxin, and such information is potentially useful for the design of vaccines and passive immune therapies against B. anthracis. Because PA has been identified as an effective subunit vaccine, we wanted to identify the specific epitopes that provide the protection and use them as immunogens. Using mAbs and 145 overlapping peptides covering the entire sequence of PA, we identify the first linear neutralizing epitopes in domain 1 of PA, and we demonstrate that two peptides containing epitopes in domain 1 were capable of inducing strong LeTx-neutralizing antibody responses.