Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
1997-12-16
2001-07-17
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S218100, C424S093200, C435S235100, C435S236000, C435S320100, C536S023720
Reexamination Certificate
active
06261570
ABSTRACT:
Western equine encephalitis (WEE), eastern equine encephalitis (EEE) and Venezuelan equine encephalitis virus (VEE) are members of the alphavirus genus of the family Togaviridae which is comprised of a large group of mosquito-borne RNA viruses found throughout much of the world. The viruses normally circulate among rodent or avian hosts through the feeding activities of a variety of mosquitoes. Epizootics occur largely as a result of increased-mosquito activity after periods of increased rainfall. Western equine encephalitis virus (WEE) was first recognized in 1930 and causes periodic outbreaks of disease in equines. The virus has been detected over much of the western hemisphere from Argentina north to the more temperate regions of central Canada (For a review, see Reisen and Monath [1988] in
The Arboviruses: Epidemiology and Ecology, Vol. V
. CRC Press, Inc. Boca Raton). Similarly, EEE was first isolated in Virginia and New Jersey in 1933 (Ten Broeck, C. et al. [1935
] J. Exp. Med
. 62:677) and is now known to be focally endemic throughout much of the northern portion of South America, Central America and the eastern part of Mexico and the United States. Venezuelan equine encephalitis virus has six serological subtypes (I-VI). Two of these subtypes, I and III have multiple variants, two of these variants are of particular interest in this application, variant IE,and variant IIIA also called Mucambo virus. A live, attenuated vaccine (TC-83) for VEE IA/B has been used for immunization equines and at-risk laboratory and field personnel (Birge et al. [1961
] Am. J. Hyg
. 73:209-218; Pittman et al. [1996
] Vaccine
14:337-343). The vaccine was credited with helping to limit the northward spread of a serious epizootic of VEE originating in South America in the late 1960's. However, the VEE I/AB vaccines have not yet been licensed by the Food and Drug Administration and have been shown to be effective in preventing disease from VEE IA/B infection only. The current VEE vaccines do not adequately protect against the VEE IE variant or the VEE IIIA variant, as disease has occurred in laboratory workers successfully vaccinated with a vaccine derived from VEE IA/B. In addition, recent unprecedented outbreaks of VEE IE in populations of horses in Mexico indicate a need for a VEE IE vaccine. The lack of adequate cross protection with existing IA/B vaccines documents the need for a VEE IE-specific and a VEE IIIA-specific vaccine.
The vaccines currently in veterinary use for WEE, EEE and VEE IA/B throughout the United States and Canada are formalin-inactivated preparations. Inactivated vaccines for EEE and WEE are also available for use by at-risk laboratory personnel. These inactivated vaccines are poorly immunogenic, require multiple inoculations with frequent boosters and generally result in immunity of short duration. The shortcomings of the available vaccines indicate a need for the development of new vaccines of high immunogenicity which induce a longer lasting immunity for protection against WEE, EEE and VEE subtypes IE and IIIA.
SUMMARY OF THE INVENTION
The present invention satisfies the need mentioned above.
In this application are described live attenuated vaccines for WEE, EEE, VEE IE and VEE IIIA which may provide higher level immunity in humans and equines for many years, and possibly for life. In addition, very large numbers of vaccine doses can be produced from significantly less starting materials than is possible with the existing inactivated products. The vaccine preparations of the present invention comprise full-length cDNA copies of the genomes of WEE or VEE IE which have been altered such that the RNA produced from the cDNA, and the virus produced therefrom is attenuated and useful as a live vaccine for human and veterinary use. The vaccine preparations for VEE IIIA and EEE are novel chimeric viruses which include the newly discovered structural protein genes of VEE IIIA.
The classic methods of deriving live-attenuated vaccines (blind passage in cell cultures) generally result in heterogeneous and undefined products, hence recent attempts to make live vaccines for alphaviruses have relied on genetic engineering procedures.
The alphavirus genome is a single-stranded, positive-stranded RNA approximately 11,400 nucleotides in length. The 5′ two-thirds of the genome consist of a non-coding region of approximately 48 nucleotides followed by a single open reading frame of approximately 7,500 nucleotides which encodes the viral replicase/transcriptase. The 3′ one-third of the genome encodes the viral structural proteins in the order C-E3-E2-6 K-E1, each of which are derived by proteolytic cleavage of the product of a single open reading frame of approximately 3700 nucleotides. The sequences encoding the structural proteins are transcribed as a 26S mRNA from an internal promoter on the negative sense complement of the viral genome. The nucleocapsid (C) protein possesses autoproteolytic activity which cleaves the C protein from the precursor protein soon after the ribosome transits the junction between the C and E3 protein coding sequence. Subsequently, the envelope glycoproteins E2 and E1 are derived by proteolytic cleavage in association with intracellular membranes and form heterodimers. E2 initially appears in the infected cell as a precursor, pE2, which consists of E3 and E2. After extensive glycosylation and transit through the endoplasmic reticulum and the golgi apparatus, E3 is cleaved from E2 by furin-like protease activity at a cleavage site having a consensus sequence of RX(K/R)R, with X being one of many amino acids present in the different viruses, and with the cleavage occuring after the last arginine residue. Subsequently, the E2/E1 complex is transported to the cell surface where it is incorporated into virus budding from the plasma membrane (Strauss and Strauss [1994
] Microbiological Rev
. 58: 491-562). All documents cited herein supra and infra are hereby incorporated in their entirety by reference thereto.
Because the genome of alphavirus is a positive-stranded RNA, and infectious upon transfection of cells in culture, an “infectious clone” approach to vaccine development is particularly suitable for the alphaviruses. In this approach, a full-length cDNA clone of the viral genome is constructed downstream from a RNA polymerase promoter, such that RNA which is equivalent to the viral genome can be transcribed from the DNA clone in vitro. This allows site-directed mutagenesis procedures to be used to insert specific mutations into the DNA clone, which are then reflected in the virus which is recovered by transfection of the RNA.
Previous work with infectious clones of other alphaviruses has demonstrated that disruption of the furin cleavage site results in a virus which incorporates pE2 into the mature virus. Davis et al. (1995, supra) found that disruption of the furin cleavage site in an infectious clone of VEE is a lethal mutation. Transfection of BHK cells with RNA transcribed from this mutant clone resulted in the release of non-infectious particles. However, a low level of infectious virus was produced which contained secondary suppressor mutations such that virus containing pE2 was fully replication competent and subsequently shown to be avirulent but capable of elliciting immunity to lethal virus challenge in a variety of animal species.
The genetic basis for attenuation of the VEE TC-83 vaccine and certain laboratory strains of VEE virus have been studied extensively and has led to the development of improved live, attenuated vaccine candidates (Kinney et al. 1993, supra, Davis et al. 1995, supra). The approach used in this application is similar to that used for VEE, however, following the VEE example exactly did not result in an adequate vaccine for WEE. Changes in the procedure used for VEE were required, none of which could have been predicted from the VEE work, in order to produce the attenuated live WEE virus of the present invention.
Based upon a comparison of the structural p
Crise Bruce J.
Oberste Mark Steven
Parker Michael D.
Schmura Shannon M.
Smith Jonathan F.
Arwine Elizabeth
Harris Charles H.
Mosher Mary E.
The United States of America as represented by the Secretary of
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