Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Virus or component thereof
Reexamination Certificate
1999-12-07
2001-10-02
Mosher, Mary E. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Virus or component thereof
C424S205100, C424S199100, C424S820000, C435S236000, C435S235100
Reexamination Certificate
active
06296854
ABSTRACT:
Venezuelan equine encephalitis virus (VEE) is a member 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.
VEE virus has six serological subtypes (I-VI). Two of these subtypes, I and III have multiple variants. Existing vaccines for VEE are derived from VEE IA/B and have been shown to be effective in preventing disease from VEE IA/B infection. The current vaccines of VEE do not adequately protect against the VEE IE variant, as disease has occurred in laboratory workers vaccinated with a vaccine derived from VEE IA/B. Also, recent unprecedented outbreaks of VEE IE in populations of horses in Mexico indicate a need for a VEE IE vaccine.
The alphavirus vaccines currently in use throughout the United States and Canada for veterinary purposes and for human use and use by at-risk laboratory personnel are formalin-inactivated preparations. These inactivated vaccines are poorly immunogenic, require multiple inoculations and frequent boosters and are inadequately protective in the case of an aerosol exposure to the virus.
In order to develop an improved vaccine of higher immunogenicity requiring less frequent immunizations, and protective against aerosol exposure to the virus, effort has been concentrated on the development of a live-attenuated alphavirus vaccine.
The construction of full-length alphavirus clones has proven to be useful in the development of live-attenuated alphavirus vaccines. The current approach to making live-attenuated alphavirus vaccines from a DNA clone of the virus involves 1) construction of a full-length, virulent clone, 2) identification and introduction of an appropriately attenuating mutation, 3) analysis of stability of the attenuated virus with regard to the specific attenuating mutation(s) in a viable virus vaccine candidate. Points 2 and 3 often involve much effort and expense on the part of the individuals and laboratories and it is possible that the altered antigen structures resulting from the attenuating and/or suppressor mutations would render the vaccine unprotective against infection with the native virus.
Therefore, there is a need for an alphavirus vaccine which is attenuated and retains the natural antigenic structures of the native virus.
SUMMARY OF THE INVENTION
The present invention satisfies the need mentioned above.
In this application is described a live attenuated vaccine for VEE which comprises a viral gene rearrangement as a method of viral attenuation. Alphaviruses with rearranged genes are believed to encode and express native proteins without any known mutation altering the protein conformation. Therefore, the immune reaction is raised against native antigens present on the infecting virus. The gene rearrangement process also obviates the need to search for attenuating mutations and to analyze the stability of the attenuating and/or suppressor mutations.
The mutations in most live-attenuated virus vaccines often result in significantly reduced yields during production, and inadequate replication in vivo because the altered gene products are less efficient in directing virus assembly. That is not the case for the live-attenuated virus of the present invention since it replicates to titers which are equivalent to those seen with wild type, fully virulent VEE viruses.
In addition, the attenuated phenotype is likely very stable since at least two independently unlikely recombination events would by required to revert to a wild type sequence.
Animal studies demonstrated complete protection with a very low dose (10
3
plaque forming units, pfu) of the candidate vaccine. This is at least one order of magnitude less than the dose used for the current vaccine TC-83 and V3526 (Davis, N. L. et al., 1995
, Virology
212, 102-110). Lower vaccination doses provide significant advantages in GMP production and potentially increase the safety of the vaccine.
The vaccine preparations of the present invention comprise the cDNA genome of VEE which has been altered such that the order of genes in the viral genome has been reversed. The resulting virus is attenuated and useful as a live vaccine for human and veterinary use.
The genome of VEE virus is a single-stranded, plus-sense 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 encode the viral replicase/transcriptase. The 3′ one-third of the genome encodes the viral structural proteins in the order C-E3-E2-6K-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 consisting of RKRR 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).
Alphavirus gene expression is biphasic in nature. The viral genome, delivered to the cell after viral and cell membrane fusion, is translated directly starting at a translational start codon located near the 5′ end of the genome. The initial translation products are termed nonstructural proteins because they are not incorporated into the virion particle. These nonstructural proteins serve as polymerases that function both in transcription of viral structural genes and replication of both the positive and negative sense viral genomes. An internal promoter, i.e. 26S promoter, on the full-length negative sense viral RNA is recognized by the viral polymerase leading to transcription of the structural genes.
In a wild type alphavirus infection, there is only one structural gene transcript which gives rise to one polyprotein consisting of all the viral structural proteins. The order of the proteins encoded by the structural gene transcript is capsid protein followed by membrane glycoproteins. Upon translation, the autocatalytic activity of capsid cleaves the capsid protein from the growing nascent chain. This proteolytic event allows proper membrane translocation of the glycoproteins. The capsid proteins complex with the positive sense viral genome to form a ribonucleocapsid. It is the concerted action of the ribonucleocapsid and the glycoproteins that leads to assembly of an infectious virus particle at the plasma membrane.
Systems that employ defective alphaviral genomes lacking all structural genes can be complemented by coexpression of the structural proteins to yield infectious viral particles. Complementation can be accomplished by expression of the capsid and membrane glycoproteins from a single transcript similar to that found in a wild type viral infection. Alternatively, complementation can be carried out using two different transcripts, one encoding a self-cleaving capsid protein and a second encoding the membrane glycoprotein transcript with a translational start. Most often, the complementing genes are u
Crise Bruce J.
Parker Michael D.
Pushko Peter
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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