Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
1999-07-09
2002-12-17
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C424S201100, C424S247100, C435S069100, C435S069300, C435S320100, C435S235100, C435S325000, C435S252300, C514S04400A
Reexamination Certificate
active
06495143
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to vaccines for bacterial toxins from
Clostridium botulinum.
INTRODUCTION
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 LD
50
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.
SUMMARY OF THE INVENTION
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.
REFERENCES:
patent: 5792462 (1998-08-01), Johnston et al.
patent: 5814482 (1998-09-01), Dubensky, Jr. et al.
patent: 5919665 (1999-07-01), Williams
patent: 5939070 (1999-08-01), Johnson et al.
patent: WO 96/37616 (1996-11-01), None
patent: WO 98/08540 (1998-03-01), None
Oguma et al. Microbiol. Immunol. 39(3) 161-168, 1995.*
PCT International Search Report for international patent application PCT/US99/15570 (corresponding to US application Ser. No. 09/350,756), dated Jun. 26, 2000 (6 pages).
Anderson et al., “Immune Response in Mice following Immunization with DNA Encoding Fragment C of Tetanus Toxin”, Infection and Immunity, vol. 64, No. 8, document No. XP-002139492, Aug. 1996, pp. 3168-3173.
Pushko, et al., “Replicon-Helper Systems from Attenuated Venezuelan Equine Encephalitis Virus: Expression of Heterologous Genes in Vitro and Immunization against Heterologous Pathogens in Vivo,” Virology:239, pp. 389-401 (1997), document No. XP-0021399493.
Clayton et al., “Protective Vaccination with a Recombinant Fragment of Clostridium botulinum Neurotoxin Serotype A Expressed from a Synthetic Gene inE. coli”, Infection and Immunity, Jul. 1995, vol. 63, No. 7, pp. 2738-2742.
Byrne, et al., “Purification, Potency, and Efficacy of the Botulinum Neurotoxin Type A Binding Domain from Pichia pastoris as a Recombinant Vaccine Candidate”, Infection and Immunity, Oct. 1998, vol. 66, No. 10, pp. 4817-4822.
Pushko, et al., “Replicon-Helper Systems from Attenuated Venezuelan Equine Encephalitis Virus: Expression of Heterologous Genes in Vitro and Immunization against Heterologous Pathogens in Vivo,” Virology:239, pp. 389-401 (1997).
Bavari, et al., “Engineered Bacte
Dertzbaugh Mark T.
Lee John S.
Parker Michael
Pushko Peter
Smith Jonathan F.
Arwine Elizabeth
Harris Charles M.
Mosher Mary E.
The United States of America as represented by the Secretary of
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