In the present study we describe development of a novel approach to develop a vaccine against botulism using inactivated RABV virions as a platform to present the BoNT HC50/A immunogen. Botulism can occur naturally, however, potential use of the toxin as an act of bioterrorism or biological warfare motivated development of this vaccine [4
]. Virus particles present antigens in dense and repetitive manner that maximally stimulate B cell proliferation and antibody production [45
]. Additionally, the virus particles can provide CD4+ T cell epitopes that supply the second signal required for B cell activation [46
]. Subunit vaccines have emerged as a favored candidate vaccine against botulism [42
]. The carboxyterminal half of the botulinum toxin has been the antigen of choice for development of the vaccine since it enables working in a non-toxic environment thereby increasing safety [42
], as well as, decreasing the cost in comparison with working with the fully active toxin [47
]. We generated recombinant RABV particles that display an etiologic agent of botulism, the carboxy-terminal half of botulinum toxin. As previously shown for the incorporation of domain four of the protective antigen (PA) of Anthrax into RABV virions [13
], incorporation of HC50 also required the addition of the RABV G ED, but of a different size. In both case, the recombinant RABV G fusion protein in virions was only detected by Western blotting, indicating that incorporation of such proteins into budding RABV virions is less efficient than that of RABV G protein. We can only speculate about the amount of RABV G-HC50 in the RABV particles, however, RABV G is 24% of the RABV virion mass [48
]. Therefore, the chimeric RABV G HC-50 protein would be 12% of the virion mass if incorporated as efficient as RABV G. Because we neither saw a reduction of RABV G in the recombinant virions (which would be replaced by the RAB-HC50/A-E30 fusion protein) nor we were able to detect RAB-HC50/A-E30 in the coomassie stained gel analyzing the virions, the amount is less, probably around 1-5%, which would result in an estimated amount of 0.1 to 0.5 μg in of 10 ug virions, which were used for the immunizations.
Nevertheless, the fusion protein, RABV G-HC50/A-E30, incorporated into the recombinant virions, induced a strong humoral immune response in vaccinated mice. The magnitude of the RABV-HC50/A induced immune response induced in mice was greater than that induced with recombinant HC50/A admixed with alum. The botulinum toxin is a large protein that has numerous linear and conformational epitopes. There are reported to be a greater numbers of epitopes in the HC50 polypeptide than there are in the rest of the toxin molecule [49
]. Many of these epitopes are potential antibody binding binding sites that can mark the toxin molecule for enhanced clearance from the circulation.
The gold standard for effective neutralization is the demonstration of protection against the lethal effects of the toxin in vaccinated mice. The present study clearly shows the ability of two doses of recombinant RABV-HC50/A virus to evoke immunity against a lethal challenge dose of native botulinum toxin (1× 103 LD50). There was complete immunity and 100% survival of the vaccinated mice in the two independent experiments compared with 80% and 100% survival in mice immunized with the recombinant HC50/A vaccine.
The HC50/A domain of BoNT is already known be an effective vaccine against botulism. However, this study provided further proof for the concept of the potential of RABV recombinant vaccine. Moreover, the RABV vaccine platform seems to work efficiently within a large range of antigen concentration without adjuvant, which is not the case for recombinant HC50 protein, which requires potent adjuvant and high concentration of antigen [41
Many previous reports documented that antibodies raised against the carboxyterminal half of botulinum toxin can neutralize toxin, but few have described the underlying mechanisms. Association of antibodies with the toxin has been shown to enhance toxin clearance from the circulation, as well as to prolong the paralysis times of neuronal tissues [41
]. In this study, we also evaluate mechanism of toxin neutralization and found that our recombinant RABV-HC50/A RABV have induced a rapid and complete neutralization of the toxin from the circulation, and only traces of the toxin were detectable 2 minutes after challenge with the toxin.
In summary, we showed that the display of HC50 by RABV virions is a novel approach for producing a potent BoNT vaccine. Further work will be directed to the inclusion of more serotypes of BoNT (e.g. B and E), larger scale production of the vaccine and clinical testing of this vaccine.