Synthetic Peptide Vaccine Targeting a Cryptic Neutralizing Epitope in Domain 2 ofBacillus anthracisProtective Antigen

Abstract

Current evidence suggests that protective antigen (PA)-based anthrax vaccines may elicit a narrow neutralizing antibody repertoire, and this may represent a vulnerability with PA-based vaccines. In an effort to identify neutralizing specificities which may complement those prevalent in PA antiserum, we evaluated whether sequences within the 2␤2-2␤3 loop of PA, which are apparent in the crystal structure of heptameric but not monomeric PA, might represent a target for an epitope-specific vaccine for anthrax and, further, whether antibodies to these sequences are induced in rabbits immunized with monomeric PA. We evaluated the immunogenicity in rabbits of a multiple antigenic peptide (MAP) displaying copies of amino acids (aa) 305 to 319 of this region. Overall, four out of six rabbits vaccinated with the MAP peptide in Freund's adjuvant developed high-titer, high-avidity antibody responses which cross-reacted with the immobilized peptide sequence comprising aa 305 to 319 and with PA, as determined by an enzyme-linked immunosorbent assay, and which displayed potent and durable neutralization of lethal toxin (LeTx) in vitro, with peak titers which were 452%, 100%, 67%, and 41% of the peak neutralization titers observed in positive-control rabbits immunized with PA. Importantly, analysis of sera from multiple cohorts of rabbits with high-titer immunity to PA demonstrated a virtual absence of this potent antibody specificity, and work by others suggests that this specificity may be present at only low levels in primate PA antiserum. These results highlight the potential importance of this immunologically cryptic neutralizing epitope from PA as a target for alternative and adjunctive vaccines for anthrax.

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

“…Antibody responses were assessed by enzyme-linked immunosorbent assay (ELISA) essentially as described previously (8). For analysis of antibodies specific for PA, wells of microtiter plates (Immulon 2; Thermo Labsystems, Franklin, MA) were coated overnight at 4°C with 100 ng of PA in a 0.05 M carbonate buffer, pH 9.5.…”

“…Mutations or deletions in the linear sequences comprising the LND, especially those involving the F313-F314, have been shown to completely abrogate the cytotoxicity of LeTx in vitro, suggesting that a vaccine which effectively targets the LND would be relatively resistant to circumvention (13)(14)(15)(16). Remarkably, antibodies to the LND appear to be virtually absent in PA-immunized rabbits and may be present only at functionally insignificant levels in human AVA serum (8,17). Thus, the LND appears to be immunologically silent in animals immunized with PA. Development of vaccines capable of eliciting protective immunity against this cryptic epitope, therefore, could be strategic in the event of malicious attempts to circumvent neutralizing specificities elicited by BioThrax or other PA-based vaccines.…”

“…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).…”

Identification of a Continuous Neutralizing Epitope within UL128 of Human Cytomegalovirus

“…In addition, the observation that antibodies in HCMV ϩ individuals react only minimally with peptides containing the 13B5 target sequence may suggest that this sequence is only minimally immunogenic or only rarely recognized by the immune system during natural HCMV infection, which is consistent with characteristics of an immunologically "cryptic" epitope (45). Epitope crypticity is a common pattern of immune evasion and has been described for many other pathogens such as HIV, influenza virus, dengue virus, Plasmodium falciparum, and Bacillus anthracis (45)(46)(47)(48)(49). While it appears contradictory how the 13B5 epitope can be immunologically cryptic and at the same time a target of potent NAb, this could be explained by differences in the accessibility of the 13B5 binding site during natural HCMV infection.…”

Design and Testing of Novel Anthrax Vaccines Utilizing a Tobacco Mosaic Virus Expression System

“…The minimally defined epitopes from these reports formed the majority of the immunogenic PA peptide sequences in this study. Sequences were also selected by analysis of the crystal structure of PA interacting with cellular receptor CMG2 65,72 and from reports of a vulnerable linear epitope located in the 2β2-2β3 region of domain II identified from neutralizing mAbs 57 and peptide focused vaccines 14,20 . The complete list of epitopes selected for this study is described in on the N-terminus, C-terminus, or the surface exposed loop at amino acids 59-65 making genetic manipulation and display of foreign amino acids easier 93 .…”