In this study we have developed a novel reagent that combines the functions of anthrax antitoxin and vaccine in a single compound. It is based on multivalent display of the ANTXR2 VWA domain on the surface of the icosahedral insect nodavirus FHV. We demonstrate that the recombinant VLPs protect cultured cells and rats from anthrax intoxication as efficiently as the highly potent sANTXR2 receptor decoy and that they induce a potent immune response against LeTx when coated with PA. This immune response was neutralizing in vitro and protected animals against LeTx challenge following a single administration without adjuvant.
The motivation for immunogenicity studies was based on the assumption that polyvalent display of PA would induce a more potent immune response than monomeric, recombinant PA, which is currently being developed as a second-generation anthrax vaccine [15
]. Ordered arrays of antigens are known to permit particularly efficient cross-linking of B cell receptors, which in turn leads to faster and more robust B cell proliferation [24
]. Given the exceptionally tight binding of PA to ANTXR2 under natural conditions (Kd
= 170 pM) [21
], we reasoned that complexes formed between chimera 264 and PA would be sufficiently stable to serve as an immunogen in vivo.
In support of this notion, results from in vitro cell intoxication experiments indicated that the complexes were stable for at least 40 h at 37 °C. Based upon a recent observation that naturally occurring PA neutralizing antibodies do not bind to the receptor-binding surface of PA [27
], we reasoned that PA immobilized on these particles should be able to elicit a protective immune response. Indeed, rats survived LeTx challenge 4 wk after a single injection of the VLP-PA complex, whereas animals injected with an equivalent amount of recombinant PA died. This result suggested rapid production of neutralizing antibodies in the absence of adjuvant, two key goals for the development of third-generation anthrax vaccines. No significant antibody response to ANTXR2 was observed, presumably because there are only two–amino acid differences between human ANTXR2 displayed on the particle and endogenous rat ANTXR2 [11
An essential next step will be to characterize the neutralizing antibody response in individual animals after primary and secondary immunization. An important component of this analysis will be to determine the mechanism by which toxin neutralization occurs. For example, we noticed a slight difference in antibody response after primary and secondary immunization and a wide range of antibody titers between individual animals (). It will be of key interest to establish whether these differences correlate with epitope specificity or are based on other immunologic parameters. In addition, it will be critical to confirm our findings in a B. anthracis spore challenge model, and studies to this end are currently underway.
Because the chimeric particles are expressed from an mRNA that contains only the coding sequence of the modified FHV coat protein while all other FHV sequences are missing, the resulting VLPs are not infectious and thus cannot replicate in mammalian tissues [19
]. Even native FHV particles are unable to initiate infection in mammals, as they do not carry the FHV receptor, and because FHV cannot replicate at temperatures above 31 °C [28
]. We have also demonstrated previously that FHV VLPs expressed from baculovirus vectors in Sf21 cells do not contain baculoviral or cellular DNA [19
], thus ruling out potential integration of foreign DNA into mammalian genomes. Based on these properties, the chimeric particles can be expected to have a desirable safety profile for applications in animals and humans.
The idea of combining the functions of anthrax vaccine and antitoxin in a single reagent has been explored previously. Aulinger et al. [29
] demonstrated that a dominant-negative, inhibitory form of PA, DNI-PA, can elicit an antibody response that protects mice from LeTx challenge. DNI-PA forms mixed heptamers with wt PA and thereby acts as an antitoxin to block toxin translocation both in vitro and in vivo [30
]. However, even after two injections in the presence of adjuvant there was only a weak antibody response to DNI-PA, and a third injection had to be performed to generate a sufficient antibody response to protect against LeTx challenge [29
While the potency of the nanoparticles as a vaccine is most likely due to polyvalent display of PA, polyvalency is less of a factor in the function of the particles as an antitoxin given the extremely high affinity between PA and ANTXR2. Moreover, since PA binds as a monomer to the particles, little, if any, polyvalent effect is to be expected. In fact, we detected no significant difference in IC50
when comparing nanoparticles with soluble ANTXR2 in cell intoxication assays. That polyvalency increases the affinity between a ligand and its target receptor is a well-established phenomenon [31
]. Recently, Rai et al. [32
] reported that “pattern matching” is an important parameter for polyvalency to reach its maximum potential. With this approach, they achieved similar IC50
values in cell intoxication assays for liposomes containing inhibitory peptides that block LF binding to the PA heptamer as we observed for our nanoparticles. However, the functionalized liposomes described in their study are without a vaccine application.
In vivo potency of viral nanoparticles is also significantly determined by their pharmacokinetic parameters. Such parameters have recently been reported for viral nanoparticles derived from the plant virus cowpea mosaic virus [33
]. It will be important to determine whether there are significant differences in the plasma clearance kinetics and biodistribution of soluble ANTXR2 versus ANTXR2-containing nanoparticles.
The VWA domain of ANTXR2 was a particularly appealing candidate for insertion into a loop of the FHV coat protein because the N and C termini are only separated by 4.8 Å in the native structure [34
]. In addition, this domain adopts a compact Rossmann-like α/β-fold that can evidently form independently within the context of a larger protein while not interfering with accurate folding of the carrier protein. This hypothesis was supported by the observation that the high-resolution structure of the VWA domain could be fitted easily into the cryoEM density maps. To our knowledge, hepatitis B virus is the only other virus for which icosahedral surface display of an entire protein in its biologically active conformation has been demonstrated. In that case, genetic insertion of the green fluorescent protein in a surface-exposed loop of the core protein resulted in efficient formation of fluorescent hepatitis B virus capsids [35
In principle, it should be possible to expand the use of the FHV platform to display additional anthrax antigens either in the presence or absence of the ANTXR2 VWA domain. Specifically, direct insertion of peptides or entire domains derived from PA, LF, and EF may be feasible as long as the termini of the domains are in close enough proximity for insertion into the FHV coat protein loops. It is also conceivable that the two insertion sites at positions 206 and 264 could be used in combination to create particles with multiple functionalities. This could greatly enhance the protection afforded by the resulting particles.
Numerous other strategies are being pursued to develop improved anthrax vaccines, including PA-expressing Salmonella
] and B. subtilis
], adenovirus encoding PA domain 4 [38
], rabies virus encoding GP-PA fusion protein [39
], and bacteriophage T4 particles decorated with PA-hoc fusion proteins [40
]. None of these, however, combine the function of vaccine and antitoxin. In those cases where immunized animals were challenged with LeTx or anthrax spores, only the adenovirus construct provided complete protection after a single immunization [38
]. The strategy most comparable to that described in our study involves non-covalent surface display of intact proteins and protein complexes on bacteriophage T4 particles. The prolate lattice of the T4 capsid permits efficient surface presentation of anthrax toxin through in vitro addition of Hoc- and/or Soc protein fusions with PA, LF, or EF to hoc−soc−
phage either separately or in combination [40
]. Mice immunized with phage displaying PA, EF, and LF generated high levels of neutralizing antibodies [41
], but results from toxin or spore challenge experiments have not yet been reported.
In summary, we have developed a reagent that serves a dual purpose in combating B. anthracis infection. It functions as a competitive inhibitor of anthrax toxin in vivo, suggesting that it could be useful as a therapeutic compound, particularly in combination with standard antibiotic therapy. In addition, when complexed with PA, it has significant advantages as an immunogen compared to monomeric PA and thus forms the basis for development of an improved anthrax vaccine.