Attenuation of encephalitogenic alphavirus and uses thereof

Inactive Publication Date: 2010-01-21
FROLOV ILYA V +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0019]FIGS. 5A-5B show effect of the N-terminal deletions in capsid on cellular transcription and cell viability. FIG. 5A is a schematic representation of VEEV genome-based replicons expressing the deleted forms of capsid fused with GFP, and analysis of their ability to establish persistent replication and develop PurR foci. Arrows indicate the positions of the subgenomic promoters. Numbers indicate the first amino acid of CVEE after deletion. In all of the constructs, the initiating capsid AUG was present in its natural position. FIG. 5B compares growth of the cells carrying VEEV replicons expressing GFP or indicated fusions.
[0020]FIGS. 6A-6C compare effect of the expression of CVEE peptides fused with GFP or GFPNLS on cell viability and cellular transcription. FIG

Problems solved by technology

They circulate in the Central, South and North Americas and have an ability to cause fatal disease in humans and horses.
No effective antivirals have been developed against this virus as well.
However, very strong differences in pathogene

Method used

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  • Attenuation of encephalitogenic alphavirus and uses thereof
  • Attenuation of encephalitogenic alphavirus and uses thereof
  • Attenuation of encephalitogenic alphavirus and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cell Cultures

[0061]BHK-21 cells were provided by Dr. Sondra Schlesinger (Washington University, St. Louis, Mo.). NIH 3T3 cells were obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). Both cell lines were maintained at 37° C. in alpha minimum essential medium (aMEM) supplemented with 10% fetal bovine serum (FBS) and vitamins.

example 2

Plasmid Constructs

[0062]VEErepL replicon, having a mutation in the nsP2 gene, Q739→L, is described elsewhere (12, 35). The distinguishing feature of this replicon is in its noncytopathic phenotype and low level of genome RNA replication and transcription of the subgenomic RNA. Such replicons do not overproduce the proteins of interest and generate biologically relevant data. VEErepL / GFP / Pac replicon, used as a noncytopathic control in many experiments, is described elsewhere (11). The genes of tested proteins were cloned into VEErepL under the control of the subgenomic promoter, and the second promoter was driving the expression of puromycin acetyl transferase (Pac) that makes the replicon-containing cells resistant to translational arrest caused by puromycin. All of the tested cassettes expressing capsid protein with different deletions were synthesized by PCR and sequenced before cloning into the vector replicon as GFP fusions. The selection of frame-shift mutant of CVEE is descri...

example 3

RNA Transcriptions

[0063]Plasmids were purified by centrifugation in CsCl gradients. Before being subjected to a transcription reaction, plasmids were linearized using the MluI or NotI restriction sites located downstream of the poly(A) sequence of VEE replicons. RNAs were synthesized by SP6 RNA polymerase in the presence of a cap analog by described conditions (38). The yield and integrity of transcripts were analyzed by gel electrophoresis under non-denaturing conditions. RNA concentration was measured on a Fluor Chem imager (Alpha Innotech), and transcription reactions were used for electroporation without additional purification.

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Abstract

The present invention is drawn to generating attenuated and less cytopathic forms of New World alphaviruses that can be used in immunogenic compositions as vaccines against both Old and New World alphaviruses. In this regard, the present invention discloses that the N-terminal, ˜35-aa-long peptide of VEEV, EEEV and, most likely, of WEEV capsid proteins plays the most critical role in the downregulation of cellular transcription and development of cytopathic effect. The identified, VEEV-specific peptide, CVEE30-68, includes two domains with distinguished functions. The integrity of both domains determines not only the intracellular distribution of CVEE, but is also essential for direct capsid function in the inhibition of transcription. The replacement of the N-terminal fragment of CVEE by its SINV-specific counterpart in VEEV TC-83 genome does not affect virus replication in vitro, but makes it less cytopathic and more attenuated in vivo.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This nonprovisional application claims benefit of priority of U.S. Ser. No. 60 / 964,969, filed Aug. 16, 2007, now abandoned, and is hereby incorporated by reference in its entirety.FEDERAL FUNDING LEGEND[0002]This invention was produced using funds obtained through National Institutes of Health grant AI057156 and Public Health Service grant AI050537. Consequently, the Federal government has certain rights in this invention.BACKGROUND OF THE INVENTION[0003]1. Field of the Invention[0004]The present invention relates generally to the field of virology and vaccine development. More specifically, the present invention provides a method to attenuate encephalitogenic alphaviruses including but not limited to VEEV, EEEV and WEEV. The attenuated phenotype is irreversible and thus, can be effective as human and / or veterinary vaccines in immunogenic composition(s).[0005]2. Description of the Related Art[0006]The Alphavirus genus in the Togaviridae f...

Claims

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Application Information

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IPC IPC(8): A61K39/12C12N7/04A61P31/12
CPCA61K39/12A61K2039/5254C07K14/005C12N2770/36134C12N2770/36122C12N2770/36161C12N7/00A61P31/12A61P31/14A61P37/04
Inventor FROLOV, ILYA V.FROLOVA, ELENAWEAVER, SCOTT C.
Owner FROLOV ILYA V
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