US2013095556A1PendingUtilityA1
Novel vesicular stomatitis virus and virus rescue system
Assignee: INT AIDS VACCINE INITIATIVEPriority: Sep 21, 2011Filed: Sep 20, 2012Published: Apr 18, 2013
Est. expirySep 21, 2031(~5.2 yrs left)· nominal 20-yr term from priority
C12N 2760/20221C12N 15/86C12N 2760/20243C12N 2800/22C12N 7/00
58
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Claims
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.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . A vesicular stomatitis virus (VSV) genomic clone comprising:
(a) a VSV genome encoding and expressing a nucleocapsid, phosphoprotein, matrix, glycoprotein and large protein, wherein the VSV genome comprises nucleotide substitutions and amino acid coding changes to improve replicative fitness and genetic stability, (b) a cloning vector, (c) an extended T7 promoter, (d) a hammerhead ribozyme, (e) a hepatitis delta virus ribozyme and T7 terminator (f) unique restriction endonuclease cleavage sites in a VSV genomic sequence (g) a leader and a trailer that are cis-acting sequences controlling mRNA synthesis and replication
2 . The VSV genomic clone of claim 1 , wherein the cloning vector is pSP72 (Genbank X65332.2)
3 . The VSV genomic clone of claim 1 , wherein the extended T7 promoter is PT7-g10.
4 . The VSV genomic clone of claim 1 , wherein the unique restriction endonuclease cleavage sites are 1367 NheI, 2194 SpeI, 2194 BstBI, 4687 PacI, 7532 AvaI, 10190 SalI and 11164 AflII.
5 . The VSV genomic clone of claim 1 , wherein the VSV genomic clone is depicted in FIG. 1 .
6 . The VSV genomic clone of claim 1 , wherein the nucleotide position is according to GenBank Accession Number EF197793 and wherein the nucleotide substitutions are selected from the group consisting of
1371 CA>GC (NheI) After 2195 insert TAG (SpeI) (all genome numbers below adjusted to include +3 bp) 3036 G>T improves match to consensus transcription stop signal 3853 X>A (was an ambiguity in Genbank file) 4691 T>A to generate PacI 7546 C>A silent change in L coding sequence eliminates a BstBI site 1960 TAC>TCC to change Y>S 3247 GTA>ATA to change V>I 3729 AAG>GAG to change K>E 4191 GTA>GAA to change V>E 4386 GGT>GAT to change G>D 4491 ACC>ATC to change T>I 5339 ATT>CTT to change I>L 5834 ACT>GCT to change T>A and 10959 AGA>AAA to change R>K.
7 . The VSV genomic clone of claim 1 , wherein the nucleotide position is according to GenBank Accession Number EF197793 and wherein the nucleotide substitutions are selected from the group consisting of:
Nucleotide position in
Nucleotide position
Nucleotide
EF197793
in rEF197793
Change
Purpose
1
Substitution 1371-2
Substitution 1371-2
CA > GC
Creates a unique NheI cleavage site
between N and P gens
2
Substitution 1960-2
Substitution 1960-2
TAC > TCC
Y > S substitution in P protein amino
acid sequence to agree with consensus.
3
Insert after 2195
3 base insert after
Insert TAG
Creates a unique SpeI site
2195
between P and M genes
4
Substitution 3039
Substitution 3042
G > T
Improves agreement with consensus.
Also improves agreement with
consensus transcription stop signal
5
Substitution 3234-6
Substitution 3237-9
GTA > ATA
V > I substitution in P protein amino
acid sequence to agree with consensus.
6
Substitution 3729-31
Substitution 3732-34
AAG > GAG
K > E substitution in G protein amino
acid sequence to agree with consensus.
7
Substitution 3856
Substitution 3859
N > A
Replace unknown base in Genbank file
with consensus
8
Substitution 4191-93
Substitution 4194-6
GTA > GAA
V > E substitution in G protein amino
acid sequence to agree with consensus.
9
Substitution 4386-88
Substitution 4389-92
GGT > GAT
G > D substitution in G protein amino
acid sequence to agree with consensus.
10
Substitution 4491-93
Substitution 4494-96
ACC > ATC
T > I substitution in G protein amino
acid sequence to agree with consensus.
11
Substitution 4694
Substitution 4697
T > A
Creates unique PacI cleavage site
between G and L genes
12
Substitution 5339-41
Substitution 5342-44
ATT > CTT
I > L substitution in L protein amino
acid sequence to agree with consensus.
13
Substitution 5834-6
Substitution 5837-40
ACT > GCT
T > A substitution in L protein amino
acid sequence to agree with consensus.
14
Substitution 10959-61
Substitution 10962-64
AGA > AAA
R > K substitution in L protein amino
acid sequence to agree with consensus.
15
Substitution 7546
Substitution 7549
C > A
Eliminates a BstBI site in the L gene
sequence making the BstBI site
between the M and G genes unique.
This substitution was silent for amino
acid coding.
8 . The VSV genomic clone of claim 1 , wherein the nucleotide sequences of the VSV genome encoding and expressing a nucleocapsid, phosphoprotein, matrix, glycoprotein and large protein are selected from the group consisting of FIGS. 2B-2G .
9 . The VSV genomic clone of claim 1 , wherein the nucleotide sequences of the VSV genome encoding and expressing a nucleocapsid, phosphoprotein, matrix, glycoprotein and large protein are selected from the group consisting of FIGS. 3A-3G .
10 . A method for rescuing VSV comprising combining a T7 RNA polymerase promoter and a hammerhead ribozyme sequence to increase the efficiency of synthesis and processing of full-length VSV genomic RNA in transfected cells.
11 . The method of claim 10 , wherein the T7 RNA polymerase promoter is a minimal functional sequence designed to initiate transcription very close to or precisely at the 5′ terminus of the genomic clone.
12 . The method of claim 11 , wherein the T7 promoter is a T7 promoter sequence that enhances formation of stable initiation and elongation complexes and a hammerhead ribozyme sequence at the 5′ terminus that catalyzes removal of extra nucleotides restoring the authentic 5′ terminus of the genomic transcript.
13 . The method of claim 10 , wherein the plasmids encoding VSV nucleocapsid, phosphoprotein, matrix, glycoprotein and large protein are optimized to improve expression of the trans-acting proteins to initiate virus rescue.
14 . The method of claim 13 , where the optimization is codon optimization.
15 . The method of claim 14 , wherein the codon optimization comprises replacing a VSV nucleotide sequence with codons used by highly expressed mammalian genes.
16 . The method of claim 14 , 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.
17 . The method of claim 14 , wherein the codon optimization comprises
(a) adding a preferred translational start sequence (the Kozak sequence) and/or (b) adding a preferred translational stop codon.
18 . The method of claim 14 , wherein the codon optimization comprises scanning a sequence for homopolymeric stretches of 5 nucleotides or more and interrupting the sequences by introducing synonymous codons.
19 . The method of claim 14 , wherein the codon optimization comprises scanning a sequence for restriction endonuclease cleavage sites and eliminate any unwanted recognitions signals.
20 . The method of claim 14 , wherein the codon optimization comprises confirming that a modified sequence translates into an expected amino acid sequence.Join the waitlist — get patent alerts
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