Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
2002-07-22
2004-03-16
Park, Hankyel T. (Department: 1648)
Chemistry: molecular biology and microbiology
Vector, per se
C435S005000, C435S006120, C536S023720, C424S184100, C424S185100, C424S224100
Reexamination Certificate
active
06706523
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to a vaccine composition and methods of preventing and treating infection in humans and animals therewith. More specifically, the invention relates to a mutant rabies virus wherein the nucleoprotein is mutated at the amino acid wherein phosphorylation occurs. The invention also relates to vectors for delivering a gene to a human or animal, and methods of delivering the gene thereto.
2. Description of the Related Art
Within the Rhabdoviridae family, rabies virus is the prototype of the Lyssavirus genus and vesicular stomatitis virus (VSV) is the prototype of the Vesiculovirus genus (Wagner and Rose, 1996). The genomic RNA is encapsidated with nucleoprotein (N) and this N-RNA complex, together with the phosphoprotein (P, also termed as NS) and RNA-dependent RNA polymerase (L), forms the RNP complex. The N protein of the rhabdoviruses, like the N protein from other members in the order of the mononegavirales, plays vital roles in regulating viral RNA transcription and replication by encapsidating de novo synthesized viral genomic RNA. Although rabies virus N and VSV N do not share a high degree of homology in the primary nucleotide and protein sequences, they do have conserved regions and similar protein characteristics. For example, the N protein of rabies virus has four conserved amino acid stretches homologous with those of VSV (Tordo et al., 1986). In addition, a similar helical structure of N protein exists in both rabies virus and VSV, with an &agr;-helix continuing from the N-terminus through most of the protein, and a &bgr;-turn towards the C-terminus (Barr et al., 1991).
One major structural difference, however, exists between rabies virus N and VSV N. Rabies virus N is phosphorylated while VSV N is not (Sokol and Clark, 1973). The phosphorylation has been mapped to serine residue at position 389 of the rabies virus N (Dietzschold et al., 1987). Previously, it was demonstrated that dephosphorylation of rabies virus N or mutation of the serine 389 to alanine resulted in increased binding to in vitro-synthesized leader RNA (Yang et al., 1999). Furthermore, mutation of the phosphorylated serine to alanine resulted in reduction of viral transcription and replication of a rabies virus minigenomic RNA (Yang et al., 1999). However, in the minigenome system, viral proteins necessary for viral transcription and replication were synthesized by T7 polymerase, and thus their synthesis was not under the control of rabies virus regulatory machinery.
Rabies has always had an aura of tragedy and mystery. Its dramatic clinical expression and almost always fatal outcome guarantee that rabies prevention is given high priority. Despite significant progress in biological research, rabies remains a significant global disease. Annually, more than 70,000 human fatalities are estimated, and millions of others require post-exposure treatment (Meslin et al., 1994; Anonymous, 1993). Although humans are the dead-end host, the disease is epizootic or enzootic in domestic animals as well as in wildlife (Fu, 1997; Rupprecht et al., 1995; Smith et al., 1995). Dogs remain the most important reservoir in Asia, Africa, and Latin America where most human rabies cases occur (Fu, 1997). In countries where dog rabies is controlled through animal vaccination, the number of human cases has been reduced considerably (Smith et al., 1995). However, rabies in wildlife presents a more challenging problem in these countries (Rupprecht et al., 1995; Smith et al., 1995). Fox rabies has been endemic in Europe and North America for many years, although a recent endeavor in oral vaccination has been successful in reducing or even eliminating rabies in many parts of Europe (Brochier et al., 1991). In the United Sates, wildlife rabies accounted for more than 90% of the reported rabies cases (more than 7,000 each year) in the past decade (Rupprecht et al., 1995; Smith et al., 1995; Krebs et al., 2000a; Krebs et al., 2000b) and there are at least five major wildlife rabies reservoirs that maintain concurrent epizootics (Smith et al., 1995). Epizootic raccoon rabies continues to occur in all the states along the eastern seaboard, and it is spreading westwards to Ohio, West Virginia, and Alabama (Krebs et al., 2000b). Skunk rabies remains enzootic in the central states and California (Krebs et al., 2000b). Fox rabies occurs sporadically in Arizona, Alaska, and Texas and also in the eastern states where raccoon rabies is epizootic (Krebs et al., 2000b). Bat rabies is widely distributed throughout the 48 contiguous states (Krebs et al., 2000b). These epizootics of wildlife rabies present a health threat for humans. Therefore, controlling rabies and protecting humans from rabies virus infection requires multi-layered control strategies, particularly vaccination of humans before or after exposure, regular vaccination of pet animals, and vaccination of wildlife.
Vaccination of humans after exposure can be dated back to the time of Pasteur when he injected Joseph Meister with attenuated rabies virus made from neuronal tissue (Pasteur et al., 1996). Since then, human rabies vaccines have gone through successive improvements, particularly the development of human diploid cell culture vaccine (HDCV) by Koprowski and associates at the Wistar Institute (Wiktor et al., 1964). The tissue culture vaccine is not only safe compared with the old brain vaccines because it does not contain neuronal tissues, but it is also more effective. People immunized with HDCV developed high virus neutralizing antibody (VNA) titers as early as 10 days after inoculation, compared with those immunized with the nervous tissue vaccine in whom neutralizing antibody titers do not reach protective levels until 30 days after the immunization (Wiktor et al., 1964). Today, many of the derivatives of tissue culture vaccines are similar to HDCV, and they are both effective and well tolerated. They include the purified chicken embryo cell vaccine (PCEC, Barth et al., 1984; Sehgal et al., 1993), the purified Vero cell rabies vaccine (PVRV, Suntharasamai et al., 1986) and the purified duck embryo cell vaccine (PDRV, Khawplod et al., 1995). A typical post-exposure treatment for an individual bitten by a rabid or a suspected rabid animal consists of the prompt administration of multiple injections of one of the above-mentioned tissue culture vaccines. Depending upon the nature and severity of the bite, it is also recommended that individuals receive antirabies antiserum prepared either in animals (usually equine) or preferably in humans (human rabies immune globulins, or HRIG) (Anonymous, 2000). Less frequently and under special circumstances, humans considered at risk of inapparent rabies exposure, such as animal control officers, veterinarians, and laboratory personnel working with the virus, are immunized against rabies, which is known as pre-exposure vaccination (Anonymous, 2000).
Although the tissue culture vaccines are safe and effective, there are problems. Because all these vaccines are made from inactivated viruses, multiple doses over an extended time period are required to stimulate optimal immune responses (Anonymous, 2000). Failure to complete the whole series of vaccination may result in the development of diseases (Shill et al., 1987; Lumbiganon et al., 1987; Anonymous, 1988). Allergic reactions to proteins contained within cell culture vaccines occur in approximately 6% of the vaccinees given booster injections (CDC, 1984). Indeed, there is some evidence that the most serious adverse reactions are to human albumin denatured by the &bgr;-propiolactone used to inactivate the virus (Warrington et al., 1987; Swanson et al., 1987; Anderson et al., 1987). Furthermore, the high cost of these tissue culture vaccines makes it difficult to effectively utilize in developing countries where it is needed most. Post-exposure treatment may exceed $2,000 to 3,000 dollars (in the United States) per case (Melter, 1996). Most human rabies cases occur in developing countries, where vacci
Burns Doane , Swecker, Mathis LLP
Park Hankyel T.
University of Georgia Research Foundation Inc.
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