Multicomponent anthrax toxin display and delivery using bacteriophage T4

Abstract

We describe a multicomponent antigen display and delivery system using bacteriophage T4. Two dispensable outer capsid proteins, Hoc (highly antigenic outer capsid protein, 155 copies) and Soc (small outer capsid protein, 810 copies), decorate phage T4 capsid. These proteins bind to the symmetrically localized capsid sites, which appear following prohead assembly and expansion. We hypothesized that multiple antigens fused to Hoc can be displayed on the same capsid and such particles can elicit broad immunological responses. Anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), and their functional domains, were fused to Hoc with an N-terminal hexa-histidine tag and the recombinant proteins were over-expressed in E. coli and purified. Using a defined in vitro assembly system, the anthrax-Hoc fusion proteins were efficiently displayed on T4 capsid, either individually or in combinations. All of the 155 Hoc binding sites can be occupied by one antigen, or they can be split among two or more antigens by varying their molar ratio in the binding reaction. Immunization of mice with T4 phage carrying PA, LF, and EF elicited strong antigen-specific antibodies against all antigens as well as lethal toxin neutralization titers. The triple antigen T4 phage elicited stronger PA-specific immune responses than the phage displaying PA alone. These features offer novel avenues to develop customized multicomponent vaccines against anthrax and other pathogenic diseases.

Structure of the Three N-Terminal Immunoglobulin Domains of the Highly Immunogenic Outer Capsid Protein from a T4-Like Bacteriophage

“…However, when bulky antigens, such as the 88-kDa anthrax protective antigen, were fused to the N terminus of Hoc, there was no change in the affinity of Hoc binding to the capsid, whereas fusion of antigens to the C terminus caused a 13-to 400-fold decrease in affinity (40,41). This is consistent with the notion that the elongated structure of Hoc has its C-terminal domain bound to the capsid.…”

Characterization of a multi-component anthrax vaccine designed to target the initial stages of infection as well as toxaemia

“…Mice have proven historically to be difficult to protect with PA alone against a fully virulent challenge, despite stimulation in mice of high titres of toxin-neutralizing anti-PA antibody (Bielinska et al, 2007;Boyaka et al, 2003;Brossier et al, 2002;Flick-Smith et al, 2005;Hahn et al, 2006;Shivachandra et al, 2007;Welkos et al, 1989;Welkos & Friedlander, 1988;Williamson et al, 2005). In addition, our PA doses were purposefully selected to be suboptimal to increase the chances of revealing any antispore antibody contributions to protection.…”

A Chimeric Protein That Functions as both an Anthrax Dual-Target Antitoxin and a Trivalent Vaccine

“…A recent study has revealed that some DPA mutants are more potent in evoking an anti-PA host immunity response (2,6,51). On the other hand, an earlier study has shown that LFn, native LF, or catalytically inactive LF could augment the immunological response of PA-based vaccines against PA and enhance their immunoprotective efficiency, irrespective of whether immunization was carried out with DNA or protein (12,29,36,43,49). In this study, the chimera LFn-DPA still retained the function of each moiety.…”