Vesicular stomatitis virus and virus rescue system

Patent metadata

Field	Value
Publication number	US-10544430-B2
Application number	US-201715417256-A
Country	US
Kind code	B2
Filing date	Jan 27, 2017
Priority date	Sep 21, 2011
Publication date	Jan 28, 2020
Grant date	Jan 28, 2020

Abstract

The present relation relates to recombinant vesicular stomatitis virus for use as prophylactic and therapeutic vaccines as well as the preparation and purification of immunogenic compositions which are formulated into the vaccines of the present invention.

First claim

What is claimed is:

1. A method for rescuing VSV comprising combining a PT7-g10 promoter operably linked to a hammerhead ribozyme sequence a hepatitis delta virus ribozyme sequence, a T7 terminator and a restriction enzyme cleavage site between the ribozyme sequences to increase the efficiency of synthesis and processing of full-length VSV genomic RNA in transfected cells,
wherein the genomic sequence of the VSV comprises one or more substitutions at nucleotide positions 1371, 2195, 3039, 7546, and 10959 when aligned with SEQ ID NO: 8.

2. The method of claim 1 , wherein the hammerhead ribozyme sequence catalyzes removal of extra nucleotides restoring the authentic 5′ terminus of the genomic transcript.

3. The method of claim 1 , wherein plasmids to support virus rescue encoding VSV nucleocapsid, phosphoprotein, matrix, glycoprotein and large protein are optimized to improve expression of the trans-acting proteins to initiate virus rescue.

4. The method of claim 3 , where the optimization is codon optimization.

5. The method of claim 4 , wherein the codon optimization comprises replacing a VSV nucleotide sequence with codons used by highly expressed mammalian genes.

6. The method of claim 4 , wherein the codon optimization comprises eliminating potential RNA processing signals in the coding sequence that might direct unwanted RNA splicing or cleavage/polyadenylation reaction, wherein the eliminating comprises:
(a) identifying potential splice site signals and remove by introducing synonymous codons and/or
(b) scanning an insert for consensus cleavage/polyadenylation signals (AAUAAA) and introducing synonymous codons to disrupt the consensus cleavage/polyadenylation signals.

7. The method of claim 4 , wherein the codon optimization comprises
(a) adding a preferred translational start sequence (the Kozak sequence) and/or
(b) adding a preferred translational stop codon.

8. The method of claim 4 , wherein the codon optimization comprises scanning a sequence for homopolymeric stretches of 5 nucleotides or more and interrupting the sequences by introducing synonymous codons.

9. The method of claim 4 , wherein the codon optimization comprises confirming that a modified sequence translates into an expected amino acid sequence.

10. The method of claim 1 , wherein the restriction enzyme cleavage site is a BsmB1 restriction enzyme cleavage site.

11. The method claim 1 , wherein the substitutions comprise one or more of substitutions selected from:
1371 CA>GC
2195 insert TAG
3039 G>T
7546 C>A
and 10959 AGA>AAA.

12. The method of claim 1 , wherein the genomic sequence of the VSV comprises the VSV genomic clone of FIG. 1 .

13. A kit for rescuing VSV with increased efficiency, comprising:
(a) A recombinant VSV vector, comprising
(i) an extended T7 promoter;
(ii) a polynucleotide sequence encoding a hammerhead ribozyme;
(iii) a genomic clone of VSV;
(iv) a polynucleotide sequence encoding a hepatitis delta virus ribozyme;
(v) a T7 terminator;

wherein the genomic clone of VSV comprises one or more nucleotide substitutions at nucleotide positions 1371, 2195, 3039, 7546, and 10959 when aligned with SEQ ID NO: 8;
and
(b) one or more vectors comprising polynucleotide sequences encoding one or more of VSV viral proteins nucleocapsid (N), phosphoprotein (P), matrix (M), glycoprotein (G), and large protein (L).

14. The kit of claim 13 , wherein the substitutions comprise one or more of substitutions selected from:
1371 CA>GC
2195 insert TAG
3039 G>T
7546 C>A
and 10959 AGA>AAA.

15. The kit of claim 13 , wherein the VSV genomic clone comprises a polynucleotide sequence encoding a VSV protein G partially or completely replaced with SIV or HIV Env.

16. The kit of claim 13 , wherein the vectors comprising polynucleotide sequences encoding one or more VSV viral proteins are codon optimized for expression in mammalian cells.

17. The kit of claim 15 , wherein the polynucleotide sequences are codon optimized for expression in mammalian cells.

18. The kit of claim 16 , wherein the polynucleotide sequences are under control of human cytomegalovirus promoter.

Assignees

• Int Aids Vaccine Initiative

Inventors

• Parks Christopher L
• Yuan Maoli
• Wright Kevin
• Jurgens Christy

Classifications

• C12N7/00

  Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00) · CPC title

• C12N15/86 Primary

  Viral vectors · CPC title

• C12N2760/20221

  Viruses as such, e.g. new isolates, mutants or their genomic sequences · CPC title

• C12N2760/20243

  viral genome or elements thereof as genetic vector · CPC title

• C12N2800/22

  Vectors comprising a coding region that has been codon optimised for expression in a respective host · CPC title