Selection and evaluation of the immunogenicity of protective antigen mutants as anthrax vaccine candidates

Abstract

Protective antigen (PA) is a central component of anthrax toxin and a major antigen in anthrax vaccines. However, the use of native PA as a vaccine is not optimal. If administered to people who have been freshly exposed to anthrax, PA may actually aid in anthrax toxin formation and thus may pose a serious safety concern for postexposure vaccination applications. A non-functional PA mutant may be much safer alternative. To identify an improved anthrax vaccine antigen, we examined four non-functional mutants of PA, each being impaired in a critical step of the cellular intoxication pathway of PA. These mutants were Rec -(unable to bind PA-receptors), SSSR (resistant to activation by furin), Oligo -(unable to form oligomers), and DNI (unable to form endosomal transmembrane pores). When tested in mice and after three doses of immunization, all four mutants were highly potent in eliciting PA-specific, toxin-neutralizing antibodies, with immunogenicity increasing in the order of PA < Rec -< SSSR < Oligo -< DNI. While the differences between Rec -or SSSR and PA were small and not statistically significant, DNI and Oligo -were significantly more immunogenic than wild-type PA. One year after immunization and compared with PA-immunized mice, DNIimmunized mice maintained significantly higher levels of anti-PA IgG with correspondingly higher titers of toxin-neutralizing activity. In contrast, Oligo --immunized mice had high levels of anti-PA IgG but lower titers of toxin-neutralizing activity, suggesting that Oligo -mutation sites may overlap with critical protective epitopes of PA. Our study demonstrates that PA-based vaccines could be improved both in terms of safety and efficacy by strategic mutations that not only render PA nonfunctional but simultaneously enhance its immunogenic potency. Recombinant PA mutants, particularly DNI, hold great promise as better and safer antigens than wild-type PA for use in postexposure vaccination.

Dermatan Sulfate Interacts with Dead Cells and Regulates CD5+ B-Cell Fate

“…Analysis by 3% to 20% native PAGE showed that all SA formed complexes with DSbt at a ratio of 2:1 (DSbt:SA ratio) (data not shown). To test the adjuvant effect of DS, mouse antibody responses to immunizations with pneumococcal type 14 capsular polysaccharide (Pn14) (10 g) and Pn14ϩDS mixture (10 ϩ 10 g) or to a mutant protein of the anthrax toxin protein PA (DNI) 10,11 (10 g) and DNIϩDS mixture (10 ϩ 10 g) were compared. Pn14 was purchased from the American Tissue Culture Collection and purified as described.…”

“…12 DNI was prepared as previously described. 10,11 ELISA Specific and total IgM or IgG (including IgG isotypes) were measured by ELISA in cell culture supernatants or in immunized mouse sera. The following antigens and concentrations were used for plate coating: SA, 10.0 g/mL; Pn14, 20.0 g/mL; histones (unfractionated from calf thymus; Sigma), 25.0 g/mL; double-stranded DNA (dsDNA; activated calf thymus DNA, Sigma), 20.0 g/mL; single-stranded DNA (ssDNA), 20.0 g/mL; goat F(ab') 2 anti-mouse immunoglobulin, 2.5 g/mL; DS-BSA conjugate, 10.0 g/mL; DNI, 5.0 g/mL; and DS, 20.0 g/mL.…”

Investigation of New Dominant-Negative Inhibitors of Anthrax Protective Antigen Mutants for Use in Therapy and Vaccination

“…Therefore, nonfunctional PA DNI mutants may be an ideal substitute for WPA in a potential vaccine against anthrax. This theory was confirmed by evidence showing that double mutation, K397D and D425K, of PA not only rendered PA mutants nonfunctional but also enhanced their immunogenic potency in mice (1,33,35,44). Phenylalanine-427 of PA is another candidate site for mutation because it is crucial for the transport function of PA, being involved in both pore formation and protein translocation (41).…”

“…This is called the dominant-negative inhibitory (DNI) phenotype. In protection against anthrax, the induction of a DNI phenotype may be advantageous since a stronger immunogenic effect than that with WPA is obtained (1,44). Therefore, postexposure use of PA DNI mutants may provide improved immunogenicity and therapeutic activity simultaneously.…”

Residue Histidine 669 Is Essential for the Catalytic Activity of Bacillus anthracis Lethal Factor

“…Because infections are highly fatal, the organisms are easily produced, and the spores spread easily, B. anthracis has been used as a bioweapon in biological war and biological terrorism (38). If inhaled, the spores are phagocytosed by alveolar macrophages, where they germinate to produce vegetative bacteria (10,24).…”