Attenuated live triple G protein recombinant rabies virus vaccine for pre- and post-exposure prophylaxis of rabies

#The invention relates to nonpathogenic recombinant rabies virus comprising multiple copies of a modified external surface glycoprotein (G) gene. The recombinant rabies virus can be used as a vaccine to protect against infection from rabies virus and to clear rabies virus from nervous tissues after an infection has occurred.

#Rabies causes an estimated 55,000 human deaths globally each year, 23,750 of which occur in Africa (Knobel et al., 2005, Bull World Health Organ 83:360-368). Moreover, 11 million people undergo rabies postexposure prophylaxis (PEP) worldwide each year. Rabies is a zoonotic disease with dogs remaining the principal host in Asia, parts of America, and large parts of Africa, and rabid dogs are the cause of most human rabies cases (Hampson et al., 2009, PLoS Biol 7:e53). It is believed that between 30% to 60% of the victims of dog bites are children under the age of 15. Inappropriate dog vaccination programs, limited access to vaccination, and postexposure treatment of individuals that have been exposed to rabid dogs are major problems in developing countries.

#Rabies virus (RV), a negative-stranded RNA virus of the rhabdoviridae family, has a relatively simple, modular genome that encodes 5 structural proteins: a RNA-dependent RNA polymerase (L), a nucleoprotein (N), a phosphorylated protein (P), a matrix protein (M), and an external surface glycoprotein (G). The N, P, and L together with the genomic RNA form the ribonucleoprotein complex (RNP). The main feature of rabies virus is neuroinvasiveness, which refers to its unique ability to invade the central nervous system (CNS) from peripheral sites. Virus uptake, axonal transport, transsynaptic spread, and the rate of viral replication are key factors that determine the neuroinvasiveness of a RV (Dietzschold et al., 1983, Proc Natl Acad Sci USA 80:70-74; Dietzschold et al., 1987, Proc Natl Acad Sci USA 84:9165-9169; Morimoto et al., 2000, J Neurovirol 6:373-381; Morimoto, et al., 1999, J Virol 73:510-518). The regulation of viral replication also appears to be one of the important mechanisms contributing to RV pathogenesis. Pathogenic RV strains replicate at a lower rate than attenuated strains, which helps preserve the structure of neurons that is used by the viruses to reach the CNS. In addition, the lower expression levels of viral antigens, in particular the RV G, which is the major viral antigen responsible for the induction of protective immunity, hinders early detection by the host immune system (Morimoto, et al., 1999, J Virol 73:510-518). In contrast to wildlife RVs, most attenuated RV strains replicate very quickly and express large amounts of G, thereby inducing strong adaptive immune responses that result in virus clearance. These properties provide the basis for the use of attenuated RV strains for the pre- and PEP of rabies. A live-attenuated RV vaccine is likely to provide effective immunization with a single dose, which has practical, cost, and logistical advantages over conventional multi-dose vaccines with respect to the worldwide eradication of dog rabies. In addition, because live-attenuated RV vaccines are capable of inducing immune responses that can clear virulent RVs from the CNS (Phares et al., 2006, J Immunol 176:7666-7675; Roy et al., 2008, J Neurovirol 14:401-411), there is the possibility that such vaccines could serve as the foundation for the treatment of early stage human rabies.

#It is to be understood that the following detailed description is exemplary and explanatory only, and are not restrictive of the material methods, devices, and kits. The accompanying drawings, which are incorporated herein by reference, and which constitute a part of this specification, illustrate certain embodiments, and together with the detailed description, and serve to explain the principles of the materials and methods. The drawings are exemplary only, and should not be construed as limiting the materials, methods, and compositions described herein.

#FIG. 1 is a schematic of the construction of recombinant RVs containing 1, 2, or 3 modified G genes. To abolish the pathogenicity, two amino acid substitutions were introduced into RV G (Arg333→Glu333 and Asn194→Ser194) resulting in GAS. In SPBAANGAS-GAS(−)-GAS(−), all ATG codons of the last two GAS genes were scrambled. LS=leader sequence; N=nucleoprotein; M=matrix protein; G=glycoprotein; L=RNA-dependent RNA polymerase; TS=terminal sequence; S=scrambled ATG codons.

#FIG. 2A is single-step growth curve for SPBAANGAS-GAS-GAS and SPBAANGAS-GAS(−)-GAS(−). Neuroblastoma (NA) cells were infected in duplicate at a multiplicity of infection (MOI) of 5, and the titers of virus in the tissue culture supernatants were determined at the indicated time points. Data are the means (±SE) of results from 3 independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001).

#The present invention relates to effective and affordable virus vaccines for humans as well as for animals, to methods of making the same, and to methods of using the same for inducing an immune response, preferably a protective immune response in animals and humans. Suitable viruses include, but are not limited to, rabies virus (RV).

#In one embodiment, invention relates to a recombinant nonpathogenic, live rabies virus that has been modified to be nonpathogenic and eliminate revision of the virus to a pathogenic form. For example, the pathogenicity of RV strains can be completely abolished for immunocompetent mice by introducing single amino acid exchanges in their G gene. RVs containing a G having a mutation of Arg333→Glu333 are nonpathogenic for adult mice after intracranial/intracerebral (i.c.) inoculation, and a mutation of Asn194→Ser194 mutation in the same gene prevents the reversion to pathogenic phenotype. The G gene containing both mutations is designated as GAS. GAS has been described in U.S. application Ser. No. 11/571,842, now U.S. Pat. No. 7,695,724, which is incorporated herein by reference in its entirety.

#The present invention is an improvement to attenuated rabies viruses in the art including variants expressing two GAS genes, in that the RV variants of the invention comprising at least three copies of GAS are nonpathogenic when inoculated into, for example, 5 day-old suckling mice, thereby demonstrating that the RV variants do not exhibit residual pathogenicity. The RV variants of the invention are in improvement to existing attenuated RV because the RV variants comprising at least three copies of GAS are nonpathogenic in immunodeficient mammals. The present invention has solved the problem in the art of associated with residual pathogenicity by generating RV variants, which have a reduced or otherwise eliminated pathogenicity in suckling mice and immmunodeficient mammals.