Recombinant nonstructural protein subunit vaccine against...

Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof

Reexamination Certificate

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C424S186100, C424S202100, C435S005000, C435S069100, C435S069300

Reexamination Certificate

active

06416763

ABSTRACT:

TECHNICAL FIELD
This invention relates to protection against and diagnosis of flaviviral infection. More specifically, this invention concerns recombinantly produced subunits of a nonstructural flaviviral protein that is expressed and secreted as a mature polypeptide from eucaryotic cells. Compositions of truncated flaviviral envelope protein in combination with flaviviral nonstructural protein induce a higher degree of protection against flaviviral infection than the truncated protein alone. These compositions may be useful in the prevention, diagnosis or treatment of flaviviral infection. The present invention relates to compositions of matter and methods of making and methods of using said compositions as well as pharmaceutical compositions and methods of treating using said pharmaceutical compositions as well as diagnostic compositions, methods of making and methods of using said diagnostic compositions. The present invention is further useful as a vaccine for immunoprophylaxis.
Several publications are referenced in the present application. Full citation to these references is found at the end of the specification immediately preceding the claims or where the publication is mentioned. Each of these publications is hereby incorporated herein by reference. Said publications relate to the art to which this invention pertains.
BACKGROUND ART
The family Flaviviridae includes the Japanese encephalitis virus (JE), Tick-borne encephalitis virus (TBE), West Nile virus (WN), dengue virus (including the four serotypes of: DEN-1, DEN-2, DEN-3, and DEN4), and the family prototype, yellow fever virus (YF). In the case of dengue, the viruses are transmitted to man by mosquitoes of the genus Aedes, primarily
A. aegypti
and
A. albopictus.
The viruses cause an illness manifested by high fever, headache, aching muscles and joints, and rash. Some cases, typically in children, result in a more severe forms of infection, dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), marked by severe hemorrhage, vascular permeability, or both, leading to shock. Without diagnosis and prompt medical intervention, the sudden onset and rapid progression of DHF/DSS can be fatal.
Flaviviruses are the most significant group of arthropod-transmitted viruses in terms of global morbidity and mortality with an estimated one hundred million cases of dengue fever occurring annually Halstead, S. B. 1988. Pathogenesis of Dengue: Challenges to Molecular Biology
Science
239:476-481. With the global increase in population and urbanization especially throughout the tropics, and the lack of sustained mosquito control measures, the mosquito vectors of flavivirus have distributed throughout the tropics, subtropics, and some temperate areas, bringing the risk of flaviviral infection to over half the world's population. Modern jet travel and human emigration have facilitated global distribution of dengue serotypes, such that now multiple serotypes of dengue are endemic in many regions. Accompanying this in the last 15 years has been an increase in the frequency of dengue epidemics and the incidence of DHF/DSS. For example, in Southeast Asia, DHF/DSS is a leading cause of hospitalization and death among children (Hayes and Gubler, 1992,
Pediatr. Infect. Dis. J.
11:311-317).
Flaviviruses are small, enveloped viruses containing a single, positive-strand, genomic RNA, approximately 10,500 nucleotides in length containing short 5′ and 3′ untranslated regions, a single long open reading frame, a 5′ cap, and a nonpolyadenylated 3′ terminus. The complete nucleotide sequence of numerous flaviviral genomes, including all four DEN serotypes and YF virus have been reported (Fu, J. et al., 1992
Virology
188:953-958; Deubel, V. et al., 1986,
Virology
155:365-377; Hahn, Y. S. et al., 1988,
Virology
162:167-180; Osatomi, K. et al., 1990,
Virology
176:643-647; Zhao, B. E. et al., 1986,
Virology
155:77-88; Mackow, E. et al., 1987,
Virology
159:217-228; Rice, C. M. et al., 1985,
Science
229:726-733). All flaviviral proteins are derived from a single long polyprotein through precise processing events mediated by host as well as virally encoded proteases. The ten gene products encoded by the single open reading frame are translated as a polyprotein organized in the order, capsid (C), ‘preMembrane’ (prM, which is processed to ‘Membrane’ (M) just prior to virion release from the cell) and ‘envelope (E)’; following this are the non-structural (NS) proteins: NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5 (reviewed in Chambers, Thomas J., Chang S. Hahn, Ricardo Galler, and Charles M. Rice. 1990. Flavivirus Genome Organization, Expression, and Replication.
Ann. Rev. Microbiol.
44: 649-688; Henchal, Erik A., and J. Robert Putnak. 1990. The Dengue Virus.
Clin. Microbiol. Rev.
3 (4): 376-396). A stretch of hydrophobic residues at the C-terminal end of E serve both as its membrane anchor as well as signal sequence directing NS1 for translocation into the endoplasmic reticular lumen. Thus precise cleavage at the E-NS1 junction is provided by host signal peptidase Falgout, B., R. Chanock, and C.-J. Lai. 1989. Proper Processing of Dengue Virus Nonstructural Glycoprotein NS1 Requires the N-Terminal Hydrophobic Signal Sequence and the Downstream Nonstructural Protein NS2a.
J. Virol.
63: 1852-60), while the virally-encoded protease NS2a is responsible for processing at the NS1 C-terminus Leblois, H., and P. R. Young. 1995. Maturation of the Dengue-2 Virus NS1 Protein in Insect Cells: Effects of Downstream NS2A Sequences on Baculovirus-expressed Gene Constructs.
J. Gen. Virol.
76: 979-984). A role for NS1 in replication of viral RNA is suggested by immunolocalization studies which demonstrate its association with the replicative form dsRNA Mackenzie, J. M., M. K. Jones, and P. R. Young. 1996. Immunolocalization of the Dengue Virus Nonstructural Glycoprotein NS1 Suggests a Role in Viral RNA Replication.
Virol.
220: 232-240) as well as blockage of RNA accumulation by a temperature-sensitive NS1 mutation Muylaert, I. R., R. Galler, and C. M. Rice. 1997. Genetic Analysis of the Yellow Fever Virus NS1 Protein: Identification of a Temperature-Sensitive Mutation which Blocks RNA Accumulation.
J. Virol
71: 291-98). Further studies utilizing gene complementation in order to provide the NS1 functions in trans have more precisely definedits role in RNA replication to be just prior to or at initiation of minus-strand synthesis Lindenbach, B. D. and C. M. Rice. 1997. Trans-Complementation of Yellow Fever Virus NS1 Reveals a Role in Early RNA Replication.
J. Virol
71: 9608-17). Meanwhile, work of others has indicated that the RNA-dependent RNA polymerase activity necessary for viral nucleic acid replication is provided by NS5 Tan, B. H., J. Fu, R. J. Sugrue, E. H. Yap, Y. C. Chan and Y. H. Tan. 1996. Recombinant Dengue Type 1 Virus NS5 Protein Expressed in
Escherichia coli
Exhibits RNA-Dependent RNA Polymerase Activity.
Virology
216: 317-25).
Processing of the encoded polyprotein is initiated cotranslationally, and full maturation requires both host and virally-encoded proteases. The sites of proteolytic cleavage in the YF virus have been determined by comparing the nucleotide sequence and the amino terminal sequences of the viral proteins. Subsequent to initial processing of the polyprotein, prM is converted to M during viral release (Wengler, G. et al., 1989.
J. Virol
63:2521-2526) and anchored C is processed during virus maturation (Nowak et al., 1987.
Virology
156:127-137). The envelope of flaviviruses is derived from the host cell membrane and is decorated with virally-encoded transmembrane proteins membrane (M) and envelope (E). While mature E protein and the precursor to M, prM, are glycosylated, the much smaller mature M protein is not. The E glycoprotein, which is the largest viral structural protein, contains functional domains responsible for cell surface attachment and intraendosomal fusion activities. It is also a major target of the host immune system, inducing virus neutralizing antibodie

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