A Heterologous Helper T-Cell Epitope Enhances the Immunogenicity of a Multiple-Antigenic-Peptide Vaccine Targeting the Cryptic Loop-Neutralizing Determinant ofBacillus anthracisProtective Antigen

Abstract

We previously showed that a multiple antigenic peptide (MAP) displaying amino acids (aa) 305 to 319 from the 2␤2-2␤3 loop of protective antigen (PA) can elicit high-titered antibody that neutralizes lethal toxin (LeTx) in vitro and that this loop-neutralizing determinant (LND) specificity is absent in PA-immune rabbits. Some immune rabbits were, however, nonresponders to the MAP. We hypothesized that the immunogen elicited suboptimal major histocompatibility complex (MHC) class II-restricted T-cell help and that introduction of a functional helper T-cell epitope would increase MHC-restricted responsiveness and the magnitude and affinity of the antibody responses. In the current study, we characterized the T-and B-cell responses to LND peptides in mice, then designed second-generation MAP immunogens for eliciting LND-specific immunity, and tested them in rabbits. Bacillus anthracis is a gram-positive, spore-forming bacterium that naturally infects wildlife and livestock and, less frequently, humans. Since 2001, when spores of B. anthracis sent through the U.S mail resulted in infection in 22 individuals, including five fatal cases of inhalation anthrax, considerable effort has been directed toward reevaluating our preparedness for possible bioterrorist threats, including weaponized anthrax.The morbidity and mortality associated with inhalation anthrax are largely a result of the elaboration of two toxins, lethal toxin (LeTx) and edema toxin. These toxins are classic A-B toxins, where lethal factor and edema factor represent the active moieties and protective antigen (PA) represents the cell-binding moiety (5,6,20). Humoral immunity to PA can successfully mediate protection from lethal challenges in animal models of inhalation anthrax, and the protection is correlated with the ability of PA-specific antibodies to neutralize LeTx in vitro in the toxin neutralization assay (TNA) (17,21,22,35,36,41).While PA-specific antibody titer has been shown to correlate with TNA titers in vitro, this relationship demonstrates variability among different studies and in different species (19,37,40,43,48,50). Most PA-specific neutralizing monoclonal antibodies (MAbs) show a more invariable linear relationship between concentration and toxin neutralization (4, 46). These findings can be reconciled, in part, by the fact that only a fraction of the polyclonal antibody produced in response to vaccination with PA contributes to LeTx neutralization (4,39,40). Indeed, analyses of human and murine MAbs suggest that whereas immunization with whole PA elicits antibodies to a wide range of sequences within the protein, the repertoire of neutralizing antibodies is considerably more focused, limited perhaps to only a couple of major regions in PA, including the anthrax toxin receptor binding region in domain 4 and the lethal factor-and edema factor-binding regions in domain 1 (4,24,25,40). Though PA-specific antibody may contribute to protection through mechanisms other than neutralization, for example, by facilitating opsonization of spores (...

Recombinant Vaccine Displaying the Loop-Neutralizing Determinant from Protective Antigen Completely Protects Rabbits from Experimental Inhalation Anthrax

“…3, T cells from C3H mice immunized with Rec-LND demonstrated strong proliferation to the p38/P4 peptide at all four test antigen doses. T cells specific for the MBP component of Rec-LND exhibited strong restimulation at only the highest test antigen dose, and consistent with previous studies, the LND peptide sequence did not stimulate T cells in mice (7,9).…”

“…7E and F), operationally defined as the quotient of the ED 50 TNA titers and the EC 50 antibody titers ϫ 100. This is functionally equivalent to the proportion of PA-specific antibody participating in neutralization (7). The specific activity of the PA group declined significantly from a mean of 2.47% prechallenge to a mean of 1.27% postchallenge (P ϭ 0.016), whereas specific activity for the Rec-LND group increased significantly following challenge from a mean of 11.09% prechallenge to 38.36% postchallenge (P ϭ 0.031).…”

“…To evaluate whether the significant increases in the neutralization observed in the postchallenge sera of the Rec-LND rabbits might be explained through increases in the LND-specific antibody affinity, we used peptide inhibition to determine the peptide concentration resulting in 50% inhibition (IC 50 s), since we have previously shown that the LND-specific antibody is completely inhibitable with peptide (7,8). The IC 50 s, in turn, are good approximations of the aggregate affinity (K d ) of the LND-specific antibodies (34).…”

“…While MBP can be cleaved from the recombinant protein following purification, it effectively stimulates helper T cell epitopes across multiple major histocompatibility complex (MHC)-disparate strains of inbred mice and, therefore, when retained, can be an effective source of cognate T cell help (28). Such T cell stimulation is particularly critical for the induction of antibody responses against discrete peptide targets, like the LND, since these short sequences are often devoid of intrinsic helper T cell epitopes (7).…”

“…This has included renewed efforts to more critically evaluate the anthrax vaccine currently approved in the United States, BioThrax, as well as continued development of new, alternative vaccines for anthrax (1)(2)(3)(4)(5)(6). We previously showed that immunization of rabbits with a multiple antigenic peptide (MAP), which display multiple copies of a target sequence extending from a branched lysine core, was capable of eliciting antibody specific for a linear determinant in the 2␤2-2␤3 loop, which mediated high-titer neutralization of lethal toxin (LeTx) in vitro (7,8) and protection of rabbits from a targeted aerosol challenge of 200 50% lethal doses (LD 50 ) of B. anthracis Ames strain in vivo (9). The target of the antibody, referred to as the loop-neutralizing determinant (LND), is a critical molecular structure of PA involved in the translocation of edema and lethal factors (LFs) (10)(11)(12).…”