This invention relates to vaccines for bacterial toxins from Clostridium botulinum. 
Botulism is a disease resulting from the activity of botulinum neurotoxin produced by Clostridium botulinum on the transmission of neuromuscular stimuli. The blockage of stimuli produces neuromuscular weakness and flaccid paralysis which can lead to respiratory failure and death. Food poisoning, infant botulism, and wound botulism are the three ways in which humans are naturally affected by botulinum neurotoxin (BoNT). Ingestion of improperly prepared or canned foods has resulted in numerous cases of botulism. Seven different serotypes of botulinum neurotoxin have been characterized, types A through G, which are antigenically distinct. BoNT are usually expressed in Clostridium botulinum as a single polypeptide chain and then posttranslationally nicked, forming a dichain consisting of a 100-kDa heavy chain and a 50-kDa light chain held together by a single disulfide bond (DasGupta, B. R. 1989, In L. L. simpson (ed.), Botulinum Neurotoxin and Tetanus Toxin. Academic Press, New York, N.Y.). Topologically, these neurotoxins are composed of three domains, a binding domain, a translocation domain, and a catalytic domain, each of which is believed to play a role in intoxication. The carboxy-terminal portion of the heavy chain is responsible for binding nerve cell receptor(s). After toxin binding, it is thought to be internalized into an endosome through receptor-mediated endocytosis (Byrne, M. P. et al., 1998, supra). The product of a gene encoding only the binding domain of BoNT is nontoxic when administered to an organism since it cannot enter the nerve cell without the translocation domain and it lacks the catalytic domain.
The vaccine currently used against botulism is comprised of the complete toxoid (Byrne, M. P. et al., Infect. Immun. 66:4817, 1998). The toxoid vaccine is dangerous and expensive to produce, contains formalin, which is very painful for the recipient, and is incomplete; only five, A-E, of the seven serotypes are represented in the formulation.
Previous work with BoNT serotype A (BoNT/A) demonstrated that the recombinant carboxy terminal of the heavy chain polypeptide (Hc fragment) produced in Escherichia coli only partially protected mice challenged with up to 1,200 LD50 of BoNT/A (LaPenotiere, H. F. et al., 1995, Toxicon 33:1383-1386; Clayton, M. A. et al., 1995, Infect. Immun. 63: 2738-2742). This preparation was difficult to produce due to inclusion bodies and the resulting amount of polypeptide was not large enough to justify large scale production. Furthermore, the product contained E. coli endotoxin.
Therefore, there is a need for an efficacious vaccine against botulism, useful for protecting humans.
The present invention satisfies the need discussed above. The present invention relates to a method and composition for use in inducing an immune response which is protective against intoxication with botulinum neurtoxin (BoNT) serotypes A (BoNTA), B (BoNTB), C (BoNTC), D (BoNTD), E (BoNTE), and F (BoNTF), and G (BoNTG). The invention relates to the use of a replicon vector which results in production of large amounts of a protein encoded by a sequence cloned into the replicon. The protein product is easily purified, available in large quantities, and devoid of endotoxin. Furthermore, immunization with the replicon encoding the desired antigen has the advantage of expressing genes in lymph nodes for a better immune response, and for stimulating mucosal immune responses (Davis et al., 1996, J. Virol. 70, 3781-3787).
The sequences encoding the Hc 50,000 Kd nontoxic fragment of BoNT A-G (see attached sequence and Clayton et al., 1995, Infection and Immunity 63, 2738 -2742) were inserted into the Venezuelan equine encephalitis (VEE) virus replicon described in U.S. Pat. No. 5,792,462 (Hc-replicon). In this vaccine strategy, a gene coding for a protein of interest is cloned in place of the VEE virus structural genes; the result is a self-replicating RNA molecule that encodes its own replicase and transcriptase functions, and in addition makes abundant quantities of mRNA encoding the foreign protein. When replicon RNA is transfected into eukaryotic cells along with two helper RNAs that express the VEE structural proteins (glycoproteins and nucleocapsid), the replicon RNA is packaged into VEE virus-like particles by the VEE virus structural proteins, which are provided in trans. Since the helper RNAs lack packaging signals neccessary for further propagation, the resulting VEE replicon particles (VRPs) which are produced are infectious for one cycle but are defective thereafter. Upon infection of an individual cell with a VRP, an abortive infection occurs in which the infected cell produces the protein of interest in abundance, is ultimately killed by the infection, but does not produce any viral progeny (Pushko et al., 1997, Virology 239, 389-401).
Experiments carried out in cell culture using the Hc-replicon demonstrated that the construct could produce high levels of the Hc polypeptides in eukaryotic cells. Inoculation of VRP containing the Hc-replicon into an inbred mouse strain (BALB/c) or into an outbred mouse strain (SWISS) produced high antibody titers and protected the mice from the effects of botulinum neurotoxin.
Therefore, it is one object of the present invention to provide a VEE virus replicon vector comprising a VEE virus replicon and a DNA fragment encoding any of the botulinum neurotoxin heavy chain fragments of serotypes A, B, C, D, E, F, G, alone or in combination, and fragments thereof such as ASubHc1 or ASubHc2, which define domains within serotype A toxin.
It is another object of the present invention to provide a self replicating RNA comprising the VEE virus replicon and any of the botulinum neurotoxin fragments described above.
It is another object of the present invention to provide infectious VEE virus replicon particles produced from the VEE virus replicon RNA described above.
It is further an object of the invention to provide an immunological composition for the protection of mammals against botulinum intoxication comprising VEE virus replicon particles containing any of the botulinum neurotoxin fragments described above or a combination of different VEE virus replicons each containing a different botulinum neurotoxin fragment.