U.S. patent application number 13/623437 was filed with the patent office on 2013-04-18 for novel vesicular stomatitis virus and virus rescue system.
This patent application is currently assigned to International AIDS Vaccine Initiative. The applicant listed for this patent is International AIDS Vaccine Initiative. Invention is credited to Christy Jurgens, Christopher L. PARKS, Kevin Wright, Maoli Yuan.
Application Number | 20130095556 13/623437 |
Document ID | / |
Family ID | 48086242 |
Filed Date | 2013-04-18 |
United States Patent
Application |
20130095556 |
Kind Code |
A1 |
PARKS; Christopher L. ; et
al. |
April 18, 2013 |
NOVEL VESICULAR STOMATITIS VIRUS AND VIRUS RESCUE SYSTEM
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.
Inventors: |
PARKS; Christopher L.;
(Boonton, NJ) ; Yuan; Maoli; (Brooklyn, NY)
; Wright; Kevin; (Brooklyn, NY) ; Jurgens;
Christy; (Rahway, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
International AIDS Vaccine Initiative; |
New York |
NY |
US |
|
|
Assignee: |
International AIDS Vaccine
Initiative
New York
NY
|
Family ID: |
48086242 |
Appl. No.: |
13/623437 |
Filed: |
September 20, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61537497 |
Sep 21, 2011 |
|
|
|
Current U.S.
Class: |
435/235.1 |
Current CPC
Class: |
C12N 2760/20221
20130101; C12N 2760/20243 20130101; C12N 15/86 20130101; C12N 7/00
20130101; C12N 2800/22 20130101 |
Class at
Publication: |
435/235.1 |
International
Class: |
C12N 7/00 20060101
C12N007/00 |
Claims
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: TABLE-US-00007 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.
Description
INCORPORATION BY REFERENCE
[0001] This application claims priority to U.S. provisional patent
application Ser. No. 61/537,497 filed Sep. 21, 2011. Reference is
made to U.S. patent application Ser. No. 12/708,940 filed Feb. 19,
2010.
[0002] The foregoing applications, and all documents cited therein
or during their prosecution ("appln cited documents") and all
documents cited or referenced herein ("herein cited documents"),
and all documents cited or referenced in herein cited documents,
together with any manufacturer's instructions, descriptions,
product specifications, and product sheets for any products
mentioned herein or in any document incorporated by reference
herein, are hereby incorporated herein by reference, and may be
employed in the practice of the invention.
FIELD OF THE INVENTION
[0003] The present invention relates to a novel vesicular
stomatitis virus for use in prophylactic and therapeutic
vaccines.
BACKGROUND OF THE INVENTION
[0004] Vesicular stomatitis virus (VSV) is a member of the
Rhabdoviridae family of enveloped viruses that contain a
single-stranded, nonsegmented, negative-sense RNA genome. The VSV
genome is composed of 5 genes arranged sequentially
3'-N-P-M-G-L-S', which each encode a polypeptide found in mature
virions (Rose et al. 2001. Rhabdoviridae: the viruses and their
replication., p. 1221-1244. In D. M. Knipe and P. M. Howley (ed.),
Fields Virology, vol. 1. Lippincott, Williams and Wilkins,
Philadelphia). The virus naturally infects livestock, but is known
to infect humans producing mild illness or no symptoms of infection
(Clarke et al. 2006. Springer seminars in immunopathology
28:239-253 and Letchworth et al. 1999. Vet J 157:239-260).
[0005] VSV is an important technology platform. It is a promising
human vaccine vector candidate for a variety of reasons, notably,
i) as mentioned above, it does not cause serious disease in humans;
ii) genetic systems have been developed for producing recombinant
viruses (Conzelmann. 2004. Curr Top Microbiol Immunol 283:1-41);
iii) it can be modified to express foreign proteins; iv) it
expresses foreign proteins abundantly; v) it elicits immune
responses in infected humans (Reif et al. 1987. Am J Trop Med Hyg
36:177-182); and vi) it has been safely tested as a vaccine vector
in many animal models including nonhuman primates (Clarke et al.
2006. Springer seminars in immunopathology 28:239-253).
[0006] There remains a need to express immunogens in recombinant
vaccines. To do so, it is advantageous to have a vector that is
genetically stable, easily modified, and efficiently
propagated.
[0007] Citation or identification of any document in this
application is not an admission that such document is available as
prior art to the present application.
SUMMARY OF THE INVENTION
[0008] The invention stems, in part, from Applicants wishing to
design a vector with a clearly defined and documented lineage that
was specifically modified without altering amino acid coding or the
function of cis-acting sequences to facilitate subsequent VSV
vector construction. It was also important and practical to start
with a VSV isolate adapted for propagation in primate epithelial
cell lines (rather than commonly-used BHK fibroblastic cells) to
promote greater genetic stability during VSV vector production in
Vero cells used for vaccine manufacturing. Because Applicants'
vaccine development plans included construction of highly modified
VSV vectors that Applicants anticipated to be difficult to rescue,
Applicants designed a cloning plasmid that included strategic
modifications to increase the productivity of Applicants'
recombinant virus rescue system.
[0009] The present invention relates to a vesicular stomatitis
virus (VSV) genomic clone which may comprise: (a) a VSV genome
encoding and expressing a nucleocapsid, phosphoprotein, matrix,
glycoprotein and large protein, wherein the VSV genome may comprise
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
and/or (g) a leader and a trailer that are cis-acting sequences
controlling mRNA synthesis and replication.
[0010] In one embodiment, the cloning vector may be pSP72 (Genbank
X65332.2). In another embodiment, the extended T7 promoter may be
PT7-g10 (Lopez et al. 1997. Journal of molecular biology 269:41-51,
the disclosure of which is incorporated by reference). RNA
polymerase T7 functions first as a DNA binding protein that
recognizes a specific DNA sequence and subsequently transitions its
activity into an elongating RNA polymerase. The nature of the
nucleotide sequence of the region initially transcribed by the
polymerase plays a role in the transition from DNA binding protein
to an elongating polymerase complex. The transcribed region in the
PT7-g10 promoter may include nucleotide sequences that promote more
efficient transition from DNA binding protein to elonagating RNA
polymerase. (Temiakov D, Mentesana P E, Ma K, Mustaev A, Borukhov
S, McAllister W T. The specificity loop of T7 RNA polymerase
interacts first with the promoter and then with the elongating
transcript, suggesting a mechanism for promoter clearance. Proc
Natl Acad Sci USA. 2000 Dec. 19; 97(26):14109-14, the disclosure of
which is incorporated by reference).
[0011] In another embodiment, the unique restriction endonuclease
cleavage sites may be 1367 NheI, 2194 SpeI, 2194 BstBI, 4687 PacI,
7532 AvaI, 10190 SalI and 11164 AflII. In yet another embodiment,
the VSV genomic clones may be depicted in FIG. 1. In another
embodiment, SphI and XhoI may be added for cloning into position 1
between the leader and N gene junction of the VSV genomic clones
depicted in FIG. 1.
[0012] The nucleotide substitutions in the VSV genome may be
selected from the group consisting of: 1371 CA>GC (NheI), after
2195 insert TAG (SpeI) (all genome numbers subsequent to this
insertion have been adjusted to include +3 bp), 3036 G>T
improves match to consensus transcription stop signal, 3853 X>A
(an ambiguity in Genbank file EF197793.1), 4691 T>A to generate
PacI, 7546 C>A silent change in L coding sequence eliminates a
BstBI site. Additionally, amino acids substitutions may be
introduced to increase match with a VSV consensus using nucleotide
substitutions selected from 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/or 10959 AGA>AAA to change
R>K, wherein the nucleotide position is according to GenBank
Accession Number EF197793. The nucleotide substitutions in the VSV
genome (wherein the nucleotide position is according to GenBank
Accession Number EF197793) may be selected from the group
consisting of:
TABLE-US-00001 Nucleotide position Nucleotide position Nucleotide
in 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.
[0013] The VSV genomic clone may comprise the nucleotide sequences
of the VSV genome encoding and expressing a nucleocapsid,
phosphoprotein, matrix, glycoprotein and large protein may be
selected from the group consisting of FIGS. 2B-2G. The VSV genomic
clone may also comprise the nucleotide sequences of the VSV genome
encoding and expressing a nucleocapsid, phosphoprotein, matrix,
glycoprotein and large protein may be selected from the group
consisting of FIGS. 3A-3G.
[0014] The present invention also relates to method for rescuing
VSV, which may comprise 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.
[0015] In one embodiment, the T7 RNA polymerase promoter may be a
minimal functional sequence designed to initiate transcription very
close to or precisely at the 5' terminus of the genomic clone.
Advantageously, the T7 promoter may be 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.
[0016] In another embodiment, the plasmids used to support virus
rescue encoding VSV nucleocapsid, phosphoprotein, matrix,
glycoprotein and large protein may be optimized to improve
expression of the trans-acting proteins to initiate virus rescue.
Advantageously, the optimization is codon optimization. In one
embodiment, the gene optimization may comprise replacing a VSV
nucleotide sequence with codons used by highly expressed mammalian
genes. In another embodiment, the codon optimization may comprise
eliminating potential RNA processing signals in the coding sequence
that might direct unwanted RNA splicing or cleavage/polyadenylation
reaction, wherein the eliminating may comprise: (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. In yet another embodiment, the gene optimization may
comprise (a) adding a preferred translational start sequence (the
Kozak sequence) and/or (b) adding a preferred translational stop
codon. In still another embodiment, the gene optimization may
comprise scanning a sequence for homopolymeric stretches of 5
nucleotides or more and interrupting the sequences by introducing
synonymous codons. In another embodiment, the gene optimization may
comprise scanning a sequence for restriction endonuclease cleavage
sites and eliminate any unwanted recognitions signals. In yet
another embodiment, the gene optimization may comprise confirming
that a modified sequence translates into the expected amino acid
sequence.
[0017] Accordingly, it is an object of the invention to not
encompass within the invention any previously known product,
process of making the product, or method of using the product such
that Applicants reserve the right and hereby disclose a disclaimer
of any previously known product, process, or method. It is further
noted that the invention does not intend to encompass within the
scope of the invention any product, process, or making of the
product or method of using the product, which does not meet the
written description and enablement requirements of the USPTO (35
U.S.C. .sctn.112, first paragraph) or the EPO (Article 83 of the
EPC), such that Applicants reserve the right and hereby disclose a
disclaimer of any previously described product, process of making
the product, or method of using the product.
[0018] It is noted that in this disclosure and particularly in the
claims and/or paragraphs, terms such as "comprises", "comprised",
"comprising" and the like can have the meaning attributed to it in
U.S. patent law; e.g., they can mean "includes", "included",
"including", and the like; and that terms such as "consisting
essentially of" and "consists essentially of" have the meaning
ascribed to them in U.S. patent law, e.g., they allow for elements
not explicitly recited, but exclude elements that are found in the
prior art or that affect a basic or novel characteristic of the
invention.
[0019] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] The following detailed description, given by way of example,
but not intended to limit the invention solely to the specific
embodiments described, may best be understood in conjunction with
the accompanying drawings, in which:
[0021] FIG. 1 depicts a schematic structure of a VSV genomic clone
of the invention. Features include (a) a cloning vector based on
pSP72 (Genbank X65332.2), (b) an extended T7 promoter is PT7-g10
described by Lopez et al. (Lopez et al., 1997. Journal of molecular
biology 269:41-51), (c) a hammerhead ribozyme designed following
the rules for constructing self-cleaving RNA sequences (Inoue et
al. 2003. J Virol Methods 107:229-236 and Ruffner et al. 1990.
Biochemistry 29:10695-10702), (d) a hepatitis delta virus ribozyme
and T7 terminator as described before for the measles virus rescue
system (Radecke et at 1995. The EMBO journal 14:5773-5784, 23 and
Sidhu et al, 1995, Virology 208:800-807), (e) unique restriction
endonuclease cleavage sites indicated above the VSV genomic
sequence (red), (f) leader and trailer as cis-acting sequences in
the termini that control mRNA synthesis and replication and (g) N,
nucleocapsid; P, phosphoprotein; M, matrix; G, glycoprotein; L,
large protein.
[0022] FIG. 2A depicts a schematic VSV genome and cloning fragments
where fragments A and G are combined to produce fragment VSV-AG.
The AG fragment may be cloned first. There are BsmBI sites added to
the termini of AG fragment, which may be used to add ribozyme
sequences to the termini without addition of nucleotides introduced
by the restriction enzyme cleavage site. There may be a polylinker
added between the combined A-G fragments
(NheI-BstBI-PacI-AvaI-SalI-AflII). After cloning the VSV-AG
fragment into the Dual Ribozyme vector, B through F may be inserted
in subsequent cloning steps.
[0023] FIG. 2B depicts a sequence of genome fragment VSV-A/G (1489
bp).
[0024] FIG. 2C depicts a sequence of genome fragment B (1645
bp).
[0025] FIG. 2D depicts a sequence of genome fragment C (1689
bp).
[0026] FIG. 2E depicts a sequence of genome fragment D (2851
bp).
[0027] FIG. 2F depicts a sequence of genome fragment E (2664).
[0028] FIG. 2G depicts a sequence of genome fragment F (930).
[0029] FIG. 3A depicts a schematic VSV annotated rVSV genomic cDNA
and mRNA transcriptional control and processing signals.
[0030] FIGS. 3B-3G depict a sequence of the VSV of FIG. 3A.
DETAILED DESCRIPTION
[0031] Applicants used VSV to develop several types of HIV vaccine
candidate including VSV-SIV and VSV-HIV chimeric viruses in which
the natural VSV attachment protein (G) is functionally replaced
with SIV/HIV Env and EnvG hybrids, vectors designed with Env
epitopes grafted into VSV G and vectors designed to display a
variety of Env immunogens as transmembrane proteins on the surface
of VSV particles and infected cells.
[0032] Applicants used VSV to develop technology platforms for
antibody-based screening and selection procedures that will allow
Applicants to evolve novel Env immunogens. These methods take
advantage of the fact that VSV evolves rapidly when selective
pressure is applied (Novella. 2003. Curr Opin Microbiol 6:399-405).
Methods in development include a procedure that allows Applicants
to select for Env mutants that bind most strongly with monoclonal
antibodies, a method for rapidly producing mutants that escape
neutralizing antibodies that bind HIV Env, and a method for
generating live or inactivated VSV particles displaying Env.
[0033] All of the recombinant VSVs are based on a genomic DNA clone
Applicants designed. Applicants decided to develop Applicants' own
VSV vector for several reasons. First, Applicants wanted to begin
Applicants' vaccine development program with a vector that has a
clearly defined and documented lineage. Second, Applicants planned
to introduce a limited number of strategic nucleotide changes into
the genome that would facilitate subsequent VSV vector construction
without altering amino acid coding or the function of cis-acting
sequences. Third, it was important and practical to start with a
VSV isolate adapted for propagation in primate epithelial cell
lines (rather than commonly-used BHK fibroblastic cells) to promote
greater genetic stability during VSV vector production in Vero
cells used for vaccine manufacturing. Finally, because Applicants'
vaccine development plans included construction of highly modified
VSV vectors that Applicants anticipated to be difficult to rescue,
Applicants designed a cloning plasmid that included strategic
modifications to increase the productivity of Applicants' rescue
system.
[0034] Applicants could have used the VSV vector background
developed in 1995 at Yale University (Lawson et al. 1995.
Proceedings of the National Academy of Sciences of the United
States of America 92:4477-4481) as Applicants' starting material.
Applicants decided against this option because the Yale vector is a
hybrid constructed from sequences originating from multiple VSV
isolates propagated under diverse laboratory conditions (it was
constructed when molecular cloning was considerably more complex
and costly), and for Applicants' purposes, the Yale clone also
needed further modification to introduce unique restriction enzyme
cleavage sites. Thus, Applicants found it simpler to engineer a
vector fitting Applicants' needs by assembling synthetic DNA
fragments based on a virus genomic sequence described in a
manuscript by Remold and colleagues (Remold et al. 2008. Mol Biol
Evol 25:1138-1147). In the end, Applicants' vector nucleotide
sequence differs from circulating wild-type viruses (VSV Indiana)
and the Yale molecular clone by about 1%.
[0035] To construct Applicants' VSV genomic clone (FIG. 1),
Applicants started with the sequence (Genbank Accession EF197793)
of a VSV isolate (Mudd Summers Strain, Indiana Serotype) adapted to
growth in human epithelial cell lines (Remold et al. 2008. Mol Biol
Evol 25:1138-1147). Applicants modified EF197793 nucleotide
sequence to create unique restriction endonuclease cleavage sites
(FIG. 1 and Table 1) that would facilitate subsequent genetic
modification, and Applicants also introduced a number of nucleotide
substitutions and amino acid coding changes that Applicants
anticipated would improve the replicative fitness and genetic
stability of Applicants' recombinant vector based on analysis of
consensus sequences generated by aligning the genomes of
lab-adapted and circulating wild-type viruses. The modified version
of the EF197793 sequence (rEF197793 in Table 1) was then used as a
template to have 6 DNA fragments synthesized, which Applicants
subsequently assembled into the recombinant full-length genomic
clone (FIGS. 2A-2G). An annotated modified VSV genomic sequence is
included in FIGS. 3A-3G.
[0036] Applicants also introduced improvements to the plasmid DNA
cloning vector that enhanced Applicants' ability to rescue
recombinant VSV vectors from transfected cells. Applicants did this
because, as mentioned above, Applicants' vaccine development plans
included construction of highly modified VSV vectors that
Applicants anticipated would be difficult to rescue because
Applicants are adding one or more foreign gene inserts and also
introducing changes expected to decrease replicative fitness.
Negative-strand RNA virus rescue from cloned DNAs is a multistep
process that includes: 1) cotransfection of multiple plasmid DNAs
including the plasmid DNA containing the VSV genomic cDNA, a
plasmid encoding bacteriophage T7 RNA polymerase, and multiple
plasmids expressing viral proteins (i.e. VSV N, P, M, G, and L)
needed to initiate virus replication in transfected cells; 2)
intracellular synthesis of a full-length genomic RNA by
bacteriophage T7 RNA polymerase; 3) precise processing of the
primary genomic transcript to produce requisite termini for
replication; 4) de novo packaging of the genomic RNA by the viral
nucleocapsid protein to generate a functional template for RNA
replication; 5) and finally, initiation of RNA synthesis by the
viral RNA-dependent RNA polymerase (Conzelmann. 2004. Curr Top
Microbiol Immunol 283:1-41 and Neumann et al. 2002. J Gen Virol
83:2635-2662). The rescue process is relatively inefficient and at
times it restricts the ability to rescue the desired recombinant,
although incremental improvements (Ghanem et al. 2011. European
Journal of cell biology, Inoue et al. J Virol Methods 107:229-236,
Parks et al. 1999. J Virol 73:3560-3566, Witko et al. 2010. J Virol
Methods 164:43-50 and Witko et al. 2006. J Virol Methods
135:91-101) in the rescue procedure have made it more efficient
since it was first described (Schnell et al. 1994. Embo J
13:4195-4203). As described herein, to improve Applicants' VSV
rescue system, Applicants used a novel combination of a more
efficient 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.
[0037] The T7 RNA polymerase promoter used in published virus
rescue methods is a minimal functional sequence designed to
initiate transcription very close to or precisely at the 5'
terminus of the genomic clone (Lawson et al. 1995. Proceedings of
the National Academy of Sciences of the United States of America
92:4477-4481, Radecke et al. 1995. The EMBO Journal 14:5773-5784
and Schnell et al. 1994. Embo J 13:4195-4203). Although this
promoter design is effective for forming the 5' end of the genomic
transcript, it is not the most efficient promoter for initiating
productive RNA synthesis. Thus, to improve VSV rescue efficiency,
Applicants developed a modified plasmid that uses a longer T7
promoter sequence known to enhance formation of stable initiation
and elongation complexes (Lopez et al. 1997. Journal of molecular
biology 269:41-51). Because the longer T7 promoter includes
downstream transcribed bacteriophage sequences, extra nucleotides
are added to the primary VSV genomic transcript. To remove these
extra nucleotides, Applicants have incorporated a hammerhead
ribozyme (Inoue et al. J Virol Methods 107:229-236 and Ruffner et
al. 1990. Biochemistry 29:10695-10702) sequence at the 5' that
which catalyzes removal of extra nucleotides restoring the
authentic 5' end of the genomic transcript.
[0038] Finally, the VSV rescue system Applicants developed uses
protocols similar to those described before with modification
(Witko et al. 2006. J Virol Methods 135:91-101). The most
significant change is that Applicants have `optimized` (Examples 3
and 4) Applicants' plasmids encoding N, P, M, G, and L and placed
the optimized genes under control of the human cytomegalovirus
promoter to improve expression of the trans-acting proteins needed
to initiate virus rescue. This modification of the rescue system
was suggested by results showing that codon optimization
significantly enhances expression in transfected cells of
plasmid-encoded viral G proteins from respiratory syncytial virus
and VSV (Ternette et al. 2007. Virol J 4:51 and Witko et al. 2010.
J Virol Methods 164:43-50).
[0039] The present invention also encompasses methods of producing
or eliciting an immune response that may comprise administering to
an animal, advantageously a mammal, any one of the herein disclosed
recombinant VSV vectors.
[0040] The terms "protein", "peptide", "polypeptide", and "amino
acid sequence" are used interchangeably herein to refer to polymers
of amino acid residues of any length. The polymer may be linear or
branched, it may comprise modified amino acids or amino acid
analogs, and it may be interrupted by chemical moieties other than
amino acids. The terms also encompass an amino acid polymer that
has been modified naturally or by intervention; for example
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling or bioactive component.
[0041] As used herein, the terms "antigen" or "immunogen" are used
interchangeably to refer to a substance, typically a protein, which
is capable of inducing an immune response in a subject. The term
also refers to proteins that are immunologically active in the
sense that once administered to a subject (either directly or by
administering to the subject a nucleotide sequence or vector that
encodes the protein) is able to evoke an immune response of the
humoral and/or cellular type directed against that protein.
[0042] The term "antibody" includes intact molecules as well as
fragments thereof, such as Fab, F(ab').sub.2, Fv and scFv which are
capable of binding the epitope determinant. These antibody
fragments retain some ability to selectively bind with its antigen
or receptor and include, for example: [0043] (i) Fab, the fragment
which contains a monovalent antigen-binding fragment of an antibody
molecule may be produced by digestion of whole antibody with the
enzyme papain to yield an intact light chain and a portion of one
heavy chain; [0044] (ii) Fab', the fragment of an antibody molecule
may be obtained by treating whole antibody with pepsin, followed by
reduction, to yield an intact light chain and a portion of the
heavy chain; two Fab' fragments are obtained per antibody molecule;
[0045] (iii) F(ab').sub.2, the fragment of the antibody that may be
obtained by treating whole antibody with the enzyme pepsin without
subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held
together by two disulfide bonds; [0046] (iv) scFv, including a
genetically engineered fragment containing the variable region of a
heavy and a light chain as a fused single chain molecule.
[0047] General methods of making these fragments are known in the
art. (See for example, Harlow and Lane, Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory, New York (1988), which is
incorporated herein by reference).
[0048] It should be understood that the proteins, including the
antibodies and/or antigens of the invention may differ from the
exact sequences illustrated and described herein. Thus, the
invention contemplates deletions, additions and substitutions to
the sequences shown, so long as the sequences function in
accordance with the methods of the invention. In this regard,
particularly preferred substitutions will generally be conservative
in nature, i.e., those substitutions that take place within a
family of amino acids. For example, amino acids are generally
divided into four families: (1) acidic--aspartate and glutamate;
(2) basic--lysine, arginine, histidine; (3) non-polar--alanine,
valine, leucine, isoleucine, proline, phenylalanine, methionine,
tryptophan; and (4) uncharged polar--glycine, asparagine,
glutamine, cysteine, serine threonine, tyrosine. Phenylalanine,
tryptophan, and tyrosine are sometimes classified as aromatic amino
acids. It is reasonably predictable that an isolated replacement of
leucine with isoleucine or valine, or vice versa; an aspartate with
a glutamate or vice versa; a threonine with a serine or vice versa;
or a similar conservative replacement of an amino acid with a
structurally related amino acid, will not have a major effect on
the biological activity. Proteins having substantially the same
amino acid sequence as the sequences illustrated and described but
possessing minor amino acid substitutions that do not substantially
affect the immunogenicity of the protein are, therefore, within the
scope of the invention.
[0049] As used herein the terms "nucleotide sequences" and "nucleic
acid sequences" refer to deoxyribonucleic acid (DNA) or ribonucleic
acid (RNA) sequences, including, without limitation, messenger RNA
(mRNA), DNA/RNA hybrids, or synthetic nucleic acids. The nucleic
acid may be single-stranded, or partially or completely
double-stranded (duplex). Duplex nucleic acids may be homoduplex or
heteroduplex.
[0050] As used herein the term "transgene" may used to refer to
"recombinant" nucleotide sequences that may be derived from any of
the nucleotide sequences encoding the proteins of the present
invention. The term "recombinant" means a nucleotide sequence that
has been manipulated "by man" and which does not occur in nature,
or is linked to another nucleotide sequence or found in a different
arrangement in nature. It is understood that manipulated "by man"
means manipulated by some artificial means, including by use of
machines, codon optimization, restriction enzymes, etc.
[0051] For example, in one embodiment the nucleotide sequences may
be mutated such that the activity of the encoded proteins in vivo
is abrogated. In another embodiment the nucleotide sequences may be
codon optimized, for example the codons may be optimized for human
use. In preferred embodiments the nucleotide sequences of the
invention are both mutated to abrogate the normal in vivo function
of the encoded proteins, and codon optimized for human use. For
example, each of the Gag, Pol, Env, Nef, RT, and IN sequences of
the invention may be altered in these ways.
[0052] As regards codon optimization, the nucleic acid molecules of
the invention have a nucleotide sequence that encodes the antigens
of the invention and may be designed to employ codons that are used
in the genes of the subject in which the antigen is to be produced.
Many viruses, including HIV and other lentiviruses, use a large
number of rare codons and, by altering these codons to correspond
to codons commonly used in the desired subject, enhanced expression
of the antigens may be achieved. In a preferred embodiment, the
codons used are "humanized" codons, i.e., the codons are those that
appear frequently in highly expressed human genes (Andre et al., J.
Virol. 72:1497-1503, 1998) instead of those codons that are
frequently used by HIV. Such codon usage provides for efficient
expression of the transgenic HIV proteins in human cells. Any
suitable method of codon optimization may be used. Such methods,
and the selection of such methods, are well known to those of skill
in the art. In addition, there are several companies that will
optimize codons of sequences, such as Geneart (geneart.com). Thus,
the nucleotide sequences of the invention may readily be codon
optimized.
[0053] The invention further encompasses nucleotide sequences
encoding functionally and/or antigenically equivalent variants and
derivatives of the antigens of the invention and functionally
equivalent fragments thereof. These functionally equivalent
variants, derivatives, and fragments display the ability to retain
antigenic activity. For instance, changes in a DNA sequence that do
not change the encoded amino acid sequence, as well as those that
result in conservative substitutions of amino acid residues, one or
a few amino acid deletions or additions, and substitution of amino
acid residues by amino acid analogs are those which will not
significantly affect properties of the encoded polypeptide.
Conservative amino acid substitutions are glycine/alanine;
valine/isoleucine/leucine; asparagine/glutamine; aspartic
acid/glutamic acid; serine/threonine/methionine; lysine/arginine;
and phenylalanine/tyrosine/tryptophan. In one embodiment, the
variants have at least 50%, at least 55%, at least 60%, at least
65%, at least 70%, at least 75%, at least 80%, at least 85%, at
least 86%, at least 87%, at least 88%, at least 89%, at least 90%,
at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98% or at least 99%
homology or identity to the antigen, epitope, immunogen, peptide or
polypeptide of interest.
[0054] For the purposes of the present invention, sequence identity
or homology is determined by comparing the sequences when aligned
so as to maximize overlap and identity while minimizing sequence
gaps. In particular, sequence identity may be determined using any
of a number of mathematical algorithms. A nonlimiting example of a
mathematical algorithm used for comparison of two sequences is the
algorithm of Karlin & Altschul, Proc. Natl. Acad. Sci. USA
1990; 87: 2264-2268, modified as in Karlin & Altschul, Proc.
Natl. Acad. Sci. USA 1993; 90: 5873-5877.
[0055] Another example of a mathematical algorithm used for
comparison of sequences is the algorithm of Myers & Miller,
CABIOS 1988; 4: 11-17. Such an algorithm is incorporated into the
ALIGN program (version 2.0) which is part of the GCG sequence
alignment software package. When utilizing the ALIGN program for
comparing amino acid sequences, a PAM 120 weight residue table, a
gap length penalty of 12, and a gap penalty of 4 may be used. Yet
another useful algorithm for identifying regions of local sequence
similarity and alignment is the FASTA algorithm as described in
Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85:
2444-2448.
[0056] Advantageous for use according to the present invention is
the WU-BLAST (Washington University BLAST) version 2.0 software.
WU-BLAST version 2.0 executable programs for several UNIX platforms
may be downloaded from ftp://blast.wust1.edu/blast/executables.
This program is based on WU-BLAST version 1.4, which in turn is
based on the public domain NCBI-BLAST version 1.4 (Altschul &
Gish, 1996, Local alignment statistics, Doolittle ed., Methods in
Enzymology 266: 460-480; Altschul et al., Journal of Molecular
Biology 1990; 215: 403-410; Gish & States, 1993; Nature
Genetics 3: 266-272; Karlin & Altschul, 1993; Proc. Natl. Acad.
Sci. USA 90: 5873-5877; all of which are incorporated by reference
herein).
[0057] The various recombinant nucleotide sequences and antibodies
and/or antigens of the invention are made using standard
recombinant DNA and cloning techniques. Such techniques are well
known to those of skill in the art. See for example, "Molecular
Cloning: A Laboratory Manual", second edition (Sambrook et al.
1989).
[0058] The nucleotide sequences of the present invention may be
inserted into "vectors." The term "vector" is widely used and
understood by those of skill in the art, and as used herein the
term "vector" is used consistent with its meaning to those of skill
in the art. For example, the term "vector" is commonly used by
those skilled in the art to refer to a vehicle that allows or
facilitates the transfer of nucleic acid molecules from one
environment to another or that allows or facilitates the
manipulation of a nucleic acid molecule.
[0059] Any vector that allows expression of the antibodies and/or
antigens of the present invention may be used in accordance with
the present invention. In certain embodiments, the antigens and/or
antibodies of the present invention may be used in vitro (such as
using cell-free expression systems) and/or in cultured cells grown
in vitro in order to produce the encoded HIV-antigens and/or
antibodies which may then be used for various applications such as
in the production of proteinaceous vaccines. For such applications,
any vector that allows expression of the antigens and/or antibodies
in vitro and/or in cultured cells may be used.
[0060] For applications where it is desired that the antibodies
and/or antigens be expressed in vivo, for example when the
transgenes of the invention are used in DNA or DNA-containing
vaccines, any vector that allows for the expression of the
antibodies and/or antigens of the present invention and is safe for
use in vivo may be used. In preferred embodiments the vectors used
are safe for use in humans, mammals and/or laboratory animals.
[0061] For the antibodies and/or antigens of the present invention
to be expressed, the protein coding sequence should be "operably
linked" to regulatory or nucleic acid control sequences that direct
transcription and translation of the protein. As used herein, a
coding sequence and a nucleic acid control sequence or promoter are
said to be "operably linked" when they are covalently linked in
such a way as to place the expression or transcription and/or
translation of the coding sequence under the influence or control
of the nucleic acid control sequence. The "nucleic acid control
sequence" may be any nucleic acid element, such as, but not limited
to promoters, enhancers, IRES, introns, and other elements
described herein that direct the expression of a nucleic acid
sequence or coding sequence that is operably linked thereto. The
term "promoter" will be used herein to refer to a group of
transcriptional control modules that are clustered around the
initiation site for RNA polymerase II and that when operationally
linked to the protein coding sequences of the invention lead to the
expression of the encoded protein. The expression of the transgenes
of the present invention may be under the control of a constitutive
promoter or of an inducible promoter, which initiates transcription
only when exposed to some particular external stimulus, such as,
without limitation, antibiotics such as tetracycline, hormones such
as ecdysone, or heavy metals. The promoter may also be specific to
a particular cell-type, tissue or organ. Many suitable promoters
and enhancers are known in the art, and any such suitable promoter
or enhancer may be used for expression of the transgenes of the
invention. For example, suitable promoters and/or enhancers may be
selected from the Eukaryotic Promoter Database (EPDB).
[0062] The present invention relates to a recombinant vesicular
stomatitis virus (VSV) vector expressing a foreign epitope.
Advantageously, the epitope is an HIV epitope. Any HIV epitope may
be expressed in a VSV vector. Advantageously, the HIV epitope is an
HIV antigen, HIV epitope or an HIV immunogen, such as, but not
limited to, the HIV antigens, HIV epitopes or HIV immunogens of
U.S. Pat. Nos. 7,341,731; 7,335,364; 7,329,807; 7,323,553;
7,320,859; 7,311,920; 7,306,798; 7,285,646; 7,285,289; 7,285,271;
7,282,364; 7,273,695; 7,270,997; 7,262,270; 7,244,819; 7,244,575;
7,232,567; 7,232,566; 7,223,844; 7,223,739; 7,223,534; 7,223,368;
7,220,554; 7,214,530; 7,211,659; 7,211,432; 7,205,159; 7,198,934;
7,195,768; 7,192,555; 7,189,826; 7,189,522; 7,186,507; 7,179,645;
7,175,843; 7,172,761; 7,169,550; 7,157,083; 7,153,509; 7,147,862;
7,141,550; 7,129,219; 7,122,188; 7,118,859; 7,118,855; 7,118,751;
7,118,742; 7,105,655; 7,101,552; 7,097,971 7,097,842; 7,094,405;
7,091,049; 7,090,648; 7,087,377; 7,083,787; 7,070,787; 7,070,781;
7,060,273; 7,056,521; 7,056,519; 7,049,136; 7,048,929; 7,033,593;
7,030,094; 7,022,326; 7,009,037; 7,008,622; 7,001,759; 6,997,863;
6,995,008; 6,979,535; 6,974,574; 6,972,126; 6,969,609; 6,964,769;
6,964,762; 6,958,158; 6,956,059; 6,953,689; 6,951,648; 6,946,075;
6,927,031; 6,919,319; 6,919,318; 6,919,077; 6,913,752; 6,911,315;
6,908,617; 6,908,612; 6,902,743; 6,900,010; 6,893,869; 6,884,785;
6,884,435; 6,875,435; 6,867,005; 6,861,234; 6,855,539; 6,841,381
6,841,345; 6,838,477; 6,821,955; 6,818,392; 6,818,222; 6,815,217;
6,815,201; 6,812,026; 6,812,025; 6,812,024; 6,808,923; 6,806,055;
6,803,231; 6,800,613; 6,800,288; 6,797,811; 6,780,967; 6,780,598;
6,773,920; 6,764,682; 6,761,893; 6,753,015; 6,750,005; 6,737,239;
6,737,067; 6,730,304; 6,720,310; 6,716,823; 6,713,301; 6,713,070;
6,706,859; 6,699,722; 6,699,656; 6,696,291; 6,692,745; 6,670,181;
6,670,115; 6,664,406; 6,657,055; 6,657,050; 6,656,471; 6,653,066;
6,649,409; 6,649,372; 6,645,732; 6,641,816; 6,635,469; 6,613,530;
6,605,427; 6,602,709 6,602,705; 6,600,023; 6,596,477; 6,596,172;
6,593,103; 6,593,079; 6,579,673; 6,576,758; 6,573,245; 6,573,040;
6,569,418; 6,569,340; 6,562,800; 6,558,961; 6,551,828; 6,551,824;
6,548,275; 6,544,780; 6,544,752; 6,544,728; 6,534,482; 6,534,312;
6,534,064; 6,531,572; 6,531,313; 6,525,179; 6,525,028; 6,524,582;
6,521,449; 6,518,030; 6,518,015; 6,514,691; 6,514,503; 6,511,845;
6,511,812; 6,511,801; 6,509,313; 6,506,384; 6,503,882; 6,495,676;
6,495,526; 6,495,347; 6,492,123; 6,489,131; 6,489,129; 6,482,614;
6,479,286; 6,479,284; 6,465,634; 6,461,615 6,458,560; 6,458,527;
6,458,370; 6,451,601; 6,451,592; 6,451,323; 6,436,407; 6,432,633;
6,428,970; 6,428,952; 6,428,790; 6,420,139; 6,416,997; 6,410,318;
6,410,028; 6,410,014; 6,407,221; 6,406,710; 6,403,092; 6,399,295;
6,392,013; 6,391,657; 6,384,198; 6,380,170; 6,376,170; 6,372,426;
6,365,187; 6,358,739; 6,355,248; 6,355,247; 6,348,450; 6,342,372;
6,342,228; 6,338,952; 6,337,179; 6,335,183; 6,335,017; 6,331,404;
6,329,202; 6,329,173; 6,328,976; 6,322,964; 6,319,666; 6,319,665;
6,319,500; 6,319,494; 6,316,205; 6,316,003; 6,309,633; 6,306,625
6,296,807; 6,294,322; 6,291,239; 6,291,157; 6,287,568; 6,284,456;
6,284,194; 6,274,337; 6,270,956; 6,270,769; 6,268,484; 6,265,562;
6,265,149; 6,262,029; 6,261,762; 6,261,571; 6,261,569; 6,258,599;
6,258,358; 6,248,332; 6,245,331; 6,242,461; 6,241,986; 6,235,526;
6,235,466; 6,232,120; 6,228,361; 6,221,579; 6,214,862; 6,214,804;
6,210,963; 6,210,873; 6,207,185; 6,203,974; 6,197,755; 6,197,531;
6,197,496; 6,194,142; 6,190,871; 6,190,666; 6,168,923; 6,156,302;
6,153,408; 6,153,393; 6,153,392; 6,153,378; 6,153,377; 6,146,635;
6,146,614; 6,143,876 6,140,059; 6,140,043; 6,139,746; 6,132,992;
6,124,306; 6,124,132; 6,121,006; 6,120,990; 6,114,507; 6,114,143;
6,110,466; 6,107,020; 6,103,521; 6,100,234; 6,099,848; 6,099,847;
6,096,291; 6,093,405; 6,090,392; 6,087,476; 6,083,903; 6,080,846;
6,080,725; 6,074,650; 6,074,646; 6,070,126; 6,063,905; 6,063,564;
6,060,256; 6,060,064; 6,048,530; 6,045,788; 6,043,347; 6,043,248;
6,042,831; 6,037,165; 6,033,672; 6,030,772; 6,030,770; 6,030,618;
6,025,141; 6,025,125; 6,020,468; 6,019,979; 6,017,543; 6,017,537;
6,015,694; 6,015,661; 6,013,484; 6,013,432 6,007,838; 6,004,811;
6,004,807; 6,004,763; 5,998,132; 5,993,819; 5,989,806; 5,985,926;
5,985,641; 5,985,545; 5,981,537; 5,981,505; 5,981,170; 5,976,551;
5,972,339; 5,965,371; 5,962,428; 5,962,318; 5,961,979; 5,961,970;
5,958,765; 5,958,422; 5,955,647; 5,955,342; 5,951,986; 5,951,975;
5,942,237; 5,939,277; 5,939,074; 5,935,580; 5,928,930; 5,928,913;
5,928,644; 5,928,642; 5,925,513; 5,922,550; 5,922,325; 5,919,458;
5,916,806; 5,916,563; 5,914,395; 5,914,109; 5,912,338; 5,912,176;
5,912,170; 5,906,936; 5,895,650; 5,891,623; 5,888,726; 5,885,580
5,885,578; 5,879,685; 5,876,731; 5,876,716; 5,874,226; 5,872,012;
5,871,747; 5,869,058; 5,866,694; 5,866,341; 5,866,320; 5,866,319;
5,866,137; 5,861,290; 5,858,740; 5,858,647; 5,858,646; 5,858,369;
5,858,368; 5,858,366; 5,856,185; 5,854,400; 5,853,736; 5,853,725;
5,853,724; 5,852,186; 5,851,829; 5,851,529; 5,849,475; 5,849,288;
5,843,728; 5,843,723; 5,843,640; 5,843,635; 5,840,480; 5,837,510;
5,837,250; 5,837,242; 5,834,599; 5,834,441; 5,834,429; 5,834,256;
5,830,876; 5,830,641; 5,830,475; 5,830,458; 5,830,457; 5,827,749;
5,827,723; 5,824,497 5,824,304; 5,821,047; 5,817,767; 5,817,754;
5,817,637; 5,817,470; 5,817,318; 5,814,482; 5,807,707; 5,804,604;
5,804,371; 5,800,822; 5,795,955; 5,795,743; 5,795,572; 5,789,388;
5,780,279; 5,780,038; 5,776,703; 5,773,260; 5,770,572; 5,766,844;
5,766,842; 5,766,625; 5,763,574; 5,763,190; 5,762,965; 5,759,769;
5,756,666; 5,753,258; 5,750,373; 5,747,641; 5,747,526; 5,747,028;
5,736,320; 5,736,146; 5,733,760; 5,731,189; 5,728,385; 5,721,095;
5,716,826; 5,716,637; 5,716,613; 5,714,374; 5,709,879; 5,709,860;
5,709,843; 5,705,331; 5,703,057; 5,702,707 5,698,178; 5,688,914;
5,686,078; 5,681,831; 5,679,784; 5,674,984; 5,672,472; 5,667,964;
5,667,783; 5,665,536; 5,665,355; 5,660,990; 5,658,745; 5,658,569;
5,643,756; 5,641,624; 5,639,854; 5,639,598; 5,637,677; 5,637,455;
5,633,234; 5,629,153; 5,627,025; 5,622,705; 5,614,413; 5,610,035;
5,607,831; 5,606,026; 5,601,819; 5,597,688; 5,593,972; 5,591,829;
5,591,823; 5,589,466; 5,587,285; 5,585,254; 5,585,250; 5,580,773;
5,580,739; 5,580,563; 5,573,916; 5,571,667; 5,569,468; 5,558,865;
5,556,745; 5,550,052; 5,543,328; 5,541,100; 5,541,057; 5,534,406
5,529,765; 5,523,232; 5,516,895; 5,514,541; 5,510,264; 5,500,161;
5,480,967; 5,480,966; 5,470,701; 5,468,606; 5,462,852; 5,459,127;
5,449,601; 5,447,838; 5,447,837; 5,439,809; 5,439,792; 5,418,136;
5,399,501; 5,397,695; 5,391,479; 5,384,240; 5,374,519; 5,374,518;
5,374,516; 5,364,933; 5,359,046; 5,356,772; 5,354,654; 5,344,755;
5,335,673; 5,332,567; 5,320,940; 5,317,009; 5,312,902; 5,304,466;
5,296,347; 5,286,852; 5,268,265; 5,264,356; 5,264,342; 5,260,308;
5,256,767; 5,256,561; 5,252,556; 5,230,998; 5,230,887; 5,227,159;
5,225,347; 5,221,610; 5,217,861; 5,208,321; 5,206,136; 5,198,346;
5,185,147; 5,178,865; 5,173,400; 5,173,399; 5,166,050; 5,156,951;
5,135,864; 5,122,446; 5,120,662; 5,103,836; 5,100,777; 5,100,662;
5,093,230; 5,077,284; 5,070,010; 5,068,174; 5,066,782; 5,055,391;
5,043,262; 5,039,604; 5,039,522; 5,030,718; 5,030,555; 5,030,449;
5,019,387; 5,013,556; 5,008,183; 5,004,697; 4,997,772; 4,983,529;
4,983,387; 4,965,069; 4,945,082; 4,921,787; 4,918,166; 4,900,548;
4,888,290; 4,886,742; 4,885,235; 4,870,003; 4,869,903; 4,861,707;
4,853,326; 4,839,288; 4,833,072 and 4,795,739.
[0063] Advantageously, the HIV epitope may be an Env precursor or
gp160 epitope. The Env precursor or gp160 epitope may be recognized
by antibodies PG9, PG16, 2G12, b12, 2F5, 4E10, Z13, or other broad
potent neutralizing antibodies.
[0064] In another embodiment, HN, or immunogenic fragments thereof,
may be utilized as the HIV epitope. For example, the HN nucleotides
of U.S. Pat. Nos. 7,393,949, 7,374,877, 7,306,901, 7,303,754,
7,173,014, 7,122,180, 7,078,516, 7,022,814, 6,974,866, 6,958,211,
6,949,337, 6,946,254, 6,896,900, 6,887,977, 6,870,045, 6,803,187,
6,794,129, 6,773,915, 6,768,004, 6,706,268, 6,696,291, 6,692,955,
6,656,706, 6,649,409, 6,627,442, 6,610,476, 6,602,705, 6,582,920,
6,557,296, 6,531,587, 6,531,137, 6,500,623, 6,448,078, 6,429,306,
6,420,545, 6,410,013, 6,407,077, 6,395,891, 6,355,789, 6,335,158,
6,323,185, 6,316,183, 6,303,293, 6,300,056, 6,277,561, 6,270,975,
6,261,564, 6,225,045, 6,222,024, 6,194,391, 6,194,142, 6,162,631,
6,114,167, 6,114,109, 6,090,392, 6,060,587, 6,057,102, 6,054,565,
6,043,081, 6,037,165, 6,034,233, 6,033,902, 6,030,769, 6,020,123,
6,015,661, 6,010,895, 6,001,555, 5,985,661, 5,980,900, 5,972,596,
5,939,538, 5,912,338, 5,869,339, 5,866,701, 5,866,694, 5,866,320,
5,866,137, 5,864,027, 5,861,242, 5,858,785, 5,858,651, 5,849,475,
5,843,638, 5,840,480, 5,821,046, 5,801,056, 5,786,177, 5,786,145,
5,773,247, 5,770,703, 5,756,674, 5,741,706, 5,705,612, 5,693,752,
5,688,637, 5,688,511, 5,684,147, 5,665,577, 5,585,263, 5,578,715,
5,571,712, 5,567,603, 5,554,528, 5,545,726, 5,527,895, 5,527,894,
5,223,423, 5,204,259, 5,144,019, 5,051,496 and 4,942,122 are useful
for the present invention.
[0065] Any epitope recognized by an HIV antibody may be used in the
present invention. For example, the anti-HIV antibodies of U.S.
Pat. Nos. 6,949,337, 6,900,010, 6,821,744, 6,768,004, 6,613,743,
6,534,312, 6,511,830, 6,489,131, 6,242,197, 6,114,143, 6,074,646,
6,063,564, 6,060,254, 5,919,457, 5,916,806, 5,871,732, 5,824,304,
5,773,247, 5,736,320, 5,637,455, 5,587,285, 5,514,541, 5,317,009,
4,983,529, 4,886,742, 4,870,003 and 4,795,739 are useful for the
present invention. Furthermore, monoclonal anti-HIV antibodies of
U.S. Pat. Nos. 7,074,556, 7,074,554, 7,070,787, 7,060,273,
7,045,130, 7,033,593, RE39,057, 7,008,622, 6,984,721, 6,972,126,
6,949,337, 6,946,465, 6,919,077, 6,916,475, 6,911,315, 6,905,680,
6,900,010, 6,825,217, 6,824,975, 6,818,392, 6,815,201, 6,812,026,
6,812,024, 6,797,811, 6,768,004, 6,703,019, 6,689,118, 6,657,050,
6,608,179, 6,600,023, 6,596,497, 6,589,748, 6,569,143, 6,548,275,
6,525,179, 6,524,582, 6,506,384, 6,498,006, 6,489,131, 6,465,173,
6,461,612, 6,458,933, 6,432,633, 6,410,318, 6,406,701, 6,395,275,
6,391,657, 6,391,635, 6,384,198, 6,376,170, 6,372,217, 6,344,545,
6,337,181, 6,329,202, 6,319,665, 6,319,500, 6,316,003, 6,312,931,
6,309,880, 6,296,807, 6,291,239, 6,261,558, 6,248,514, 6,245,331,
6,242,197, 6,241,986, 6,228,361, 6,221,580, 6,190,871, 6,177,253,
6,146,635, 6,146,627, 6,146,614, 6,143,876, 6,132,992, 6,124,132,
RE36,866, 6,114,143, 6,103,238, 6,060,254, 6,039,684, 6,030,772,
6,020,468, 6,013,484, 6,008,044, 5,998,132, 5,994,515, 5,993,812,
5,985,545, 5,981,278, 5,958,765, 5,939,277, 5,928,930, 5,922,325,
5,919,457, 5,916,806, 5,914,109, 5,911,989, 5,906,936, 5,889,158,
5,876,716, 5,874,226, 5,872,012, 5,871,732, 5,866,694, 5,854,400,
5,849,583, 5,849,288, 5,840,480, 5,840,305, 5,834,599, 5,831,034,
5,827,723, 5,821,047, 5,817,767, 5,817,458, 5,804,440, 5,795,572,
5,783,670, 5,776,703, 5,773,225, 5,766,944, 5,753,503, 5,750,373,
5,747,641, 5,736,341, 5,731,189, 5,707,814, 5,702,707, 5,698,178,
5,695,927, 5,665,536, 5,658,745, 5,652,138, 5,645,836, 5,635,345,
5,618,922, 5,610,035, 5,607,847, 5,604,092, 5,601,819, 5,597,896,
5,597,688, 5,591,829, 5,558,865, 5,514,541, 5,510,264, 5,478,753,
5,374,518, 5,374,516, 5,344,755, 5,332,567, 5,300,433, 5,296,347,
5,286,852, 5,264,221, 5,260,308, 5,256,561, 5,254,457, 5,230,998,
5,227,159, 5,223,408, 5,217,895, 5,180,660, 5,173,399, 5,169,752,
5,166,050, 5,156,951, 5,140,105, 5,135,864, 5,120,640, 5,108,904,
5,104,790, 5,049,389, 5,030,718, 5,030,555, 5,004,697, 4,983,529,
4,888,290, 4,886,742 and 4,853,326, are also useful for the present
invention.
[0066] The vectors used in accordance with the present invention
should typically be chosen such that they contain a suitable gene
regulatory region, such as a promoter or enhancer, such that the
antigens and/or antibodies of the invention may be expressed.
[0067] For example, when the aim is to express the antibodies
and/or antigens of the invention in vitro, or in cultured cells, or
in any prokaryotic or eukaryotic system for the purpose of
producing the protein(s) encoded by that antibody and/or antigen,
then any suitable vector may be used depending on the application.
For example, plasmids, viral vectors, bacterial vectors, protozoan
vectors, insect vectors, baculovirus expression vectors, yeast
vectors, mammalian cell vectors, and the like, may be used.
Suitable vectors may be selected by the skilled artisan taking into
consideration the characteristics of the vector and the
requirements for expressing the antibodies and/or antigens under
the identified circumstances.
[0068] When the aim is to express the antibodies and/or antigens of
the invention in vivo in a subject, for example in order to
generate an immune response against an HIV-1 antigen and/or
protective immunity against HIV-1, expression vectors that are
suitable for expression on that subject, and that are safe for use
in vivo, should be chosen. For example, in some embodiments it may
be desired to express the antibodies and/or antigens of the
invention in a laboratory animal, such as for pre-clinical testing
of the HIV-1 immunogenic compositions and vaccines of the
invention. In other embodiments, it will be desirable to express
the antibodies and/or antigens of the invention in human subjects,
such as in clinical trials and for actual clinical use of the
immunogenic compositions and vaccine of the invention. Any vectors
that are suitable for such uses may be employed, and it is well
within the capabilities of the skilled artisan to select a suitable
vector. In some embodiments it may be preferred that the vectors
used for these in vivo applications are attenuated to vector from
amplifying in the subject. For example, if plasmid vectors are
used, preferably they will lack an origin of replication that
functions in the subject so as to enhance safety for in vivo use in
the subject. If viral vectors are used, preferably they are
attenuated or replication-defective in the subject, again, so as to
enhance safety for in vivo use in the subject.
[0069] In preferred embodiments of the present invention viral
vectors are used. Viral expression vectors are well known to those
skilled in the art and include, for example, viruses such as
adenoviruses, adeno-associated viruses (AAV), alphaviruses,
herpesviruses, retroviruses and poxviruses, including avipox
viruses, attenuated poxviruses, vaccinia viruses, and the modified
vaccinia Ankara virus (MVA; ATCC Accession No. VR-1566). Such
viruses, when used as expression vectors are innately
non-pathogenic in the selected subjects such as humans or have been
modified to render them non-pathogenic in the selected subjects.
For example, replication-defective adenoviruses and alphaviruses
are well known and may be used as gene delivery vectors.
[0070] The present invention relates to recombinant vesicular
stomatitis (VSV) vectors, however, other vectors may be
contemplated in other embodiments of the invention such as, but not
limited to, prime boost administration which may comprise
administration of a recombinant VSV vector in combination with
another recombinant vector expressing one or more HIV epitopes.
[0071] VSV is a very practical, safe, and immunogenic vector for
conducting animal studies, and an attractive candidate for
developing vaccines for use in humans. VSV is a member of the
Rhabdoviridae family of enveloped viruses containing a
nonsegmented, negative-sense RNA genome. The genome is composed of
5 genes arranged sequentially 3'-N-P-M-G-L-5', each encoding a
polypeptide found in mature virions. Notably, the surface
glycoprotein G is a transmembrane polypeptide that is present in
the viral envelope as a homotrimer, and like Env, it mediates cell
attachment and infection.
[0072] The VSVs of U.S. Pat. Nos. 7,468,274; 7,419,829; 7,419,674;
7,344,838; 7,332,316; 7,329,807; 7,323,337; 7,259,015; 7,244,818;
7,226,786; 7,211,247; 7,202,079; 7,198,793; 7,198,784; 7,153,510;
7,070,994; 6,969,598; 6,958,226; RE38,824; PPI5,957; 6,890,735;
6,887,377; 6,867,326; 6,867,036; 6,858,205; 6,835,568; 6,830,892;
6,818,209; 5 6,790,641; 6,787,520; 6,743,620; 6,740,764; 6,740,635;
6,740,320; 6,682,907; 6,673,784; 6,673,572; 6,669,936; 6,653,103;
6,607,912; 6,558,923; 6,555,107; 6,533,855; 6,531,123; 6,506,604;
6,500,623; 6,497,873; 6,489,142; 6,410,316; 6,410,313; 6,365,713;
6,348,312; 6,326,487; 6,312,682; 6,303,331; 6,277,633; 6,207,455;
6,200,811; 6,190,650; 6,171,862; 6,143,290; 6,133,027; 6,121,434;
6,103,462; 6,069,134; 6,054,127; 6,034,073; 5,969,211; 10
5,935,822; 5,888,727; 5,883,081; 5,876,727; 5,858,740; 5,843,723;
5,834,256; 5,817,491; 5,792,604; 5,789,229; 5,773,003; 5,763,406;
5,760,184; 5,750,396; 5,739,018; 5,698,446; 5,686,279; 5,670,354;
5,540,923; 5,512,421; 5,090,194; 4,939,176; 4,738,846; 4,622,292;
4,556,556 and 4,396,628 may be contemplated by the present
invention.
[0073] The nucleotide sequences and vectors of the invention may be
delivered to cells, for example if the aim is to express HIV-1
antigens in cells in order to produce and isolate the expressed
proteins, such as from cells grown in culture. For expressing the
antibodies and/or antigens in cells any suitable transfection,
transformation, or gene delivery methods may be used. Such methods
are well known by those skilled in the art, and one of skill in the
art would readily be able to select a suitable method depending on
the nature of the nucleotide sequences, vectors, and cell types
used. For example, transfection, transformation, microinjection,
infection, electroporation, lipofection, or liposome-mediated
delivery could be used. Expression of the antibodies and/or
antigens may be carried out in any suitable type of host cells,
such as bacterial cells, yeast, insect cells, and mammalian cells.
The antibodies and/or antigens of the invention may also be
expressed including using in vitro transcription/translation
systems. All of such methods are well known by those skilled in the
art, and one of skill in the art would readily be able to select a
suitable method depending on the nature of the nucleotide
sequences, vectors, and cell types used.
[0074] In preferred embodiments, the nucleotide sequences,
antibodies and/or antigens of the invention are administered in
vivo, for example where the aim is to produce an immunogenic
response in a subject. A "subject" in the context of the present
invention may be any animal. For example, in some embodiments it
may be desired to express the transgenes of the invention in a
laboratory animal, such as for pre-clinical testing of the HIV-1
immunogenic compositions and vaccines of the invention. In other
embodiments, it will be desirable to express the antibodies and/or
antigens of the invention in human subjects, such as in clinical
trials and for actual clinical use of the immunogenic compositions
and vaccine of the invention. In preferred embodiments the subject
is a human, for example a human that is infected with, or is at
risk of infection with, HIV-1.
[0075] For such in vivo applications the nucleotide sequences,
antibodies and/or antigens of the invention are preferably
administered as a component of an immunogenic composition which may
comprise the nucleotide sequences and/or antigens of the invention
in admixture with a pharmaceutically acceptable carrier. The
immunogenic compositions of the invention are useful to stimulate
an immune response against HIV-1 and may be used as one or more
components of a prophylactic or therapeutic vaccine against HIV-1
for the prevention, amelioration or treatment of AIDS. The nucleic
acids and vectors of the invention are particularly useful for
providing genetic vaccines, i.e. vaccines for delivering the
nucleic acids encoding the antibodies and/or antigens of the
invention to a subject, such as a human, such that the antibodies
and/or antigens are then expressed in the subject to elicit an
immune response.
[0076] The compositions of the invention may be injectable
suspensions, solutions, sprays, lyophilized powders, syrups,
elixirs and the like. Any suitable form of composition may be used.
To prepare such a composition, a nucleic acid or vector of the
invention, having the desired degree of purity, is mixed with one
or more pharmaceutically acceptable carriers and/or excipients. The
carriers and excipients must be "acceptable" in the sense of being
compatible with the other ingredients of the composition.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include,
but are not limited to, water, saline, phosphate buffered saline,
dextrose, glycerol, ethanol, or combinations thereof, buffers such
as phosphate, citrate, and other organic acids; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptide;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.,
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0077] An immunogenic or immunological composition may also be
formulated in the form of an oil-in-water emulsion. The
oil-in-water emulsion may be based, for example, on light liquid
paraffin oil (European Pharmacopea type); isoprenoid oil such as
squalane, squalene, EICOSANE.TM. or tetratetracontane; oil
resulting from the oligomerization of alkene(s), e.g., isobutene or
decene; esters of acids or of alcohols containing a linear alkyl
group, such as plant oils, ethyl oleate, propylene glycol
di(caprylate/caprate), glyceryl tri(caprylate/caprate) or propylene
glycol dioleate; esters of branched fatty acids or alcohols, e.g.,
isostearic acid esters. The oil advantageously is used in
combination with emulsifiers to form the emulsion. The emulsifiers
may be nonionic surfactants, such as esters of sorbitan, mannide
(e.g., anhydromannitol oleate), glycerol, polyglycerol, propylene
glycol, and oleic, isostearic, ricinoleic, or hydroxystearic acid,
which are optionally ethoxylated, and
polyoxypropylene-polyoxyethylene copolymer blocks, such as the
Pluronic.RTM. products, e.g., L121. The adjuvant may be a mixture
of emulsifier(s), micelle-forming agent, and oil such as that which
is commercially available under the name Provax.RTM. (IDEC
Pharmaceuticals, San Diego, Calif.).
[0078] The immunogenic compositions of the invention may contain
additional substances, such as wetting or emulsifying agents,
buffering agents, or adjuvants to enhance the effectiveness of the
vaccines (Remington's Pharmaceutical Sciences, 18th edition, Mack
Publishing Company, (ed.) 1980).
[0079] Adjuvants may also be included. Adjuvants include, but are
not limited to, mineral salts (e.g., AlK(SO.sub.4).sub.2,
AlNa(SO.sub.4).sub.2, AlNH(SO.sub.4).sub.2, silica, alum,
Al(OH).sub.3, Ca3(PO.sub.4).sub.2, kaolin, or carbon),
polynucleotides with or without immune stimulating complexes
(ISCOMs) (e.g., CpG oligonucleotides, such as those described in
Chuang, T. H. et al, (2002) J. Leuk. Biol. 71(3): 538-44;
Ahmad-Nejad, P. et al (2002) Eur. J. Immunol. 32(7): 1958-68; poly
IC or poly AU acids, polyarginine with or without CpG (also known
in the art as IC31; see Schellack, C. et al (2003) Proceedings of
the 34th Annual Meeting of the German Society of Immunology;
Lingnau, K. et al (2002) Vaccine 20(29-30): 3498-508), JuvaVax.TM.
(U.S. Pat. No. 6,693,086), certain natural substances (e.g., wax D
from Mycobacterium tuberculosis, substances found in
Cornyebacterium parvum, Bordetella pertussis, or members of the
genus Brucella), flagellin (Toll-like receptor 5 ligand; see
McSorley, S. J. et al (2002) J. Immunol. 169(7): 3914-9), saponins
such as QS21, QS17, and QS7 (U.S. Pat. Nos. 5,057,540; 5,650,398;
6,524,584; 6,645,495), monophosphoryl lipid A, in particular,
3-de-O-acylated monophosphoryl lipid A (3D-MPL), imiquimod (also
known in the art as IQM and commercially available as Aldara.RTM.;
U.S. Pat. Nos. 4,689,338; 5,238,944; Zuber, A. K. et al (2004)
22(13-14): 1791-8), and the CCR5 inhibitor CMPD167 (see Veazey, R.
S. et al (2003) J. Exp. Med. 198: 1551-1562).
[0080] Aluminum hydroxide or phosphate (alum) are commonly used at
0.05 to 0.1% solution in phosphate buffered saline. Other adjuvants
that may be used, especially with DNA vaccines, are cholera toxin,
especially CTA1-DD/ISCOMs (see Mowat, A. M. et al (2001) J.
Immunol. 167(6): 3398-405), polyphosphazenes (Allcock, H. R. (1998)
App. Organometallic Chem. 12(10-11): 659-666; Payne, L. G. et al
(1995) Pharm. Biotechnol. 6: 473-93), cytokines such as, but not
limited to, IL-2, IL-4, GM-CSF, IL-12, IL-15 IGF-1, IFN-.alpha.,
IFN-.beta., and IFN-.gamma. (Boyer et al., (2002) J. Liposome Res.
121:137-142; WO01/095919), immunoregulatory proteins such as CD4OL
(ADX40; see, for example, WO03/063899), and the CD1a ligand of
natural killer cells (also known as CRONY or .alpha.-galactosyl
ceramide; see Green, T. D. et al, (2003) J. Virol. 77(3):
2046-2055), immunostimulatory fusion proteins such as IL-2 fused to
the Fc fragment of immunoglobulins (Barouch et al., Science
290:486-492, 2000) and co-stimulatory molecules B7.1 and B7.2
(Boyer), all of which may be administered either as proteins or in
the form of DNA, on the same expression vectors as those encoding
the antigens of the invention or on separate expression
vectors.
[0081] In an advantageous embodiment, the adjuvants may be lecithin
is combined with an acrylic polymer (Adjuplex-LAP), lecithin coated
oil droplets in an oil-in-water emulsion (Adjuplex-LE) or lecithin
and acrylic polymer in an oil-in-water emulsion (Adjuplex-LAO)
(Advanced BioAdjuvants (ABA)).
[0082] The immunogenic compositions may be designed to introduce
the nucleic acids or expression vectors to a desired site of action
and release it at an appropriate and controllable rate. Methods of
preparing controlled-release formulations are known in the art. For
example, controlled release preparations may be produced by the use
of polymers to complex or absorb the immunogen and/or immunogenic
composition. A controlled-release formulation may be prepared using
appropriate macromolecules (for example, polyesters, polyamino
acids, polyvinyl, pyrrolidone, ethylenevinylacetate,
methylcellulose, carboxymethylcellulose, or protamine sulfate)
known to provide the desired controlled release characteristics or
release profile. Another possible method to control the duration of
action by a controlled-release preparation is to incorporate the
active ingredients into particles of a polymeric material such as,
for example, polyesters, polyamino acids, hydrogels, polylactic
acid, polyglycolic acid, copolymers of these acids, or ethylene
vinylacetate copolymers. Alternatively, instead of incorporating
these active ingredients into polymeric particles, it is possible
to entrap these materials into microcapsules prepared, for example,
by coacervation techniques or by interfacial polymerization, for
example, hydroxymethylcellulose or gelatin-microcapsule and
poly-(methylmethacrylate) microcapsule, respectively, in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions. Such techniques are disclosed in New Trends and
Developments in Vaccines, Voller et al. (eds.), University Park
Press, Baltimore, Md., 1978 and Remington's Pharmaceutical
Sciences, 16th edition.
[0083] Suitable dosages of the nucleic acids and expression vectors
of the invention (collectively, the immunogens) in the immunogenic
composition of the invention may be readily determined by those of
skill in the art. For example, the dosage of the immunogens may
vary depending on the route of administration and the size of the
subject. Suitable doses may be determined by those of skill in the
art, for example by measuring the immune response of a subject,
such as a laboratory animal, using conventional immunological
techniques, and adjusting the dosages as appropriate. Such
techniques for measuring the immune response of the subject include
but are not limited to, chromium release assays, tetramer binding
assays, IFN-.gamma. ELISPOT assays, IL-2 ELISPOT assays,
intracellular cytokine assays, and other immunological detection
assays, e.g., as detailed in the text "Antibodies: A Laboratory
Manual" by Ed Harlow and David Lane.
[0084] When provided prophylactically, the immunogenic compositions
of the invention are ideally administered to a subject in advance
of HIV infection, or evidence of HIV infection, or in advance of
any symptom due to AIDS, especially in high-risk subjects. The
prophylactic administration of the immunogenic compositions may
serve to provide protective immunity of a subject against HIV-1
infection or to prevent or attenuate the progression of AIDS in a
subject already infected with HIV-1. When provided therapeutically,
the immunogenic compositions may serve to ameliorate and treat AIDS
symptoms and are advantageously used as soon after infection as
possible, preferably before appearance of any symptoms of AIDS but
may also be used at (or after) the onset of the disease
symptoms.
[0085] The immunogenic compositions may be administered using any
suitable delivery method including, but not limited to,
intramuscular, intravenous, intradermal, mucosal, and topical
delivery. Such techniques are well known to those of skill in the
art. More specific examples of delivery methods are intramuscular
injection, intradermal injection, and subcutaneous injection.
However, delivery need not be limited to injection methods.
Further, delivery of DNA to animal tissue has been achieved by
cationic liposomes (Watanabe et al., (1994) Mol. Reprod. Dev.
38:268-274; and WO 96/20013), direct injection of naked DNA into
animal muscle tissue (Robinson et al., (1993) Vaccine 11:957-960;
Hoffman et al., (1994) Vaccine 12: 1529-1533; Xiang et al., (1994)
Virology 199: 132-140; Webster et al., (1994) Vaccine 12:
1495-1498; Davis et al., (1994) Vaccine 12: 1503-1509; and Davis et
al., (1993) Hum. Mol. Gen. 2: 1847-1851), or intradermal injection
of DNA using "gene gun" technology (Johnston et al., (1994) Meth.
Cell Biol. 43:353-365). Alternatively, delivery routes may be oral,
intranasal or by any other suitable route. Delivery may also be
accomplished via a mucosal surface such as the anal, vaginal or
oral mucosa. Immunization schedules (or regimens) are well known
for animals (including humans) and may be readily determined for
the particular subject and immunogenic composition. Hence, the
immunogens may be administered one or more times to the subject.
Preferably, there is a set time interval between separate
administrations of the immunogenic composition. While this interval
varies for every subject, typically it ranges from 10 days to
several weeks, and is often 2, 4, 6 or 8 weeks. For humans, the
interval is typically from 2 to 6 weeks. The immunization regimes
typically have from 1 to 6 administrations of the immunogenic
composition, but may have as few as one or two or four. The methods
of inducing an immune response may also include administration of
an adjuvant with the immunogens. In some instances, annual,
biannual or other long interval (5-10 years) booster immunization
may supplement the initial immunization protocol.
[0086] The present methods also include a variety of prime-boost
regimens, for example DNA prime-VSV boost regimens. In these
methods, one or more priming immunizations are followed by one or
more boosting immunizations. The actual immunogenic composition may
be the same or different for each immunization and the type of
immunogenic composition (e.g., containing protein or expression
vector), the route, and formulation of the immunogens may also be
varied. For example, if an expression vector is used for the
priming and boosting steps, it may either be of the same or
different type (e.g., DNA or bacterial or viral expression vector).
One useful prime-boost regimen provides for two priming
immunizations, four weeks apart, followed by two boosting
immunizations at 4 and 8 weeks after the last priming immunization.
It should also be readily apparent to one of skill in the art that
there are several permutations and combinations that are
encompassed using the DNA, bacterial and viral expression vectors
of the invention to provide priming and boosting regimens.
[0087] The prime-boost regimen may also include VSV vectors that
derive their G protein or G/Stem protein from different serotype
vesicular stomatitis viruses (Rose N F, Roberts A, Buonocore L,
Rose J K. Glycoprotein exchange vectors based on vesicular
stomatitis virus allow effective boosting and generation of
neutralizing antibodies to a primary isolate of human
immunodeficiency virus type 1. J. Virol. 2000 December;
74(23):10903-10). The VSV vectors used in these examples contain a
G or G/Stem protein derived from the Indiana serotype of VSV.
Vectors may also be constructed to express G or G/Stem molecules
derived from other VSV serotypes (i.e. vesicular stomatitis New
Jersey virus or vesicular stomatitis Alagoas virus) or other
vesiculoviruses (i.e. Chandipura virus, Cocal virus, Isfahan
virus). Thus a prime may be delivered in the context of a G or
G/Stem moelcule that is from the Indiana serotype and the immune
system may be boosted with a vector that expresses epitopes in the
context of second serotype like New Jersey. This circumvents anti-G
immunity elicited by the prime, and helps focus the boost response
against the foreign epitope.
[0088] A specific embodiment of the invention provides methods of
inducing an immune response against HIV in a subject by
administering an immunogenic composition of the invention,
preferably which may comprise an VSV vector containing RNA encoding
one or more of the epitopes of the invention, one or more times to
a subject wherein the epitopes are expressed at a level sufficient
to induce a specific immune response in the subject. Such
immunizations may be repeated multiple times at time intervals of
at least 2, 4 or 6 weeks (or more) in accordance with a desired
immunization regime.
[0089] The immunogenic compositions of the invention may be
administered alone, or may be co-administered, or sequentially
administered, with other HIV immunogens and/or HIV immunogenic
compositions, e.g., with "other" immunological, antigenic or
vaccine or therapeutic compositions thereby providing multivalent
or "cocktail" or combination compositions of the invention and
methods of employing them. Again, the ingredients and manner
(sequential or co-administration) of administration, as well as
dosages may be determined taking into consideration such factors as
the age, sex, weight, species and condition of the particular
subject, and the route of administration.
[0090] When used in combination, the other HIV immunogens may be
administered at the same time or at different times as part of an
overall immunization regime, e.g., as part of a prime-boost regimen
or other immunization protocol. In an advantageous embodiment, the
other HIV immunogen is env, preferably the HIV env trimer.
[0091] Many other HIV immunogens are known in the art, one such
preferred immunogen is HIVA (described in WO 01/47955), which may
be administered as a protein, on a plasmid (e.g., pTHr.HIVA) or in
a viral vector (e.g., MVA.HIVA). Another such HIV immunogen is
RENTA (described in PCT/US2004/037699), which may also be
administered as a protein, on a plasmid (e.g., pTHr.RENTA) or in a
viral vector (e.g., MVA.RENTA).
[0092] For example, one method of inducing an immune response
against HIV in a human subject may comprise administering at least
one priming dose of an HIV immunogen and at least one boosting dose
of an HIV immunogen, wherein the immunogen in each dose may be the
same or different, provided that at least one of the immunogens is
an epitope of the present invention, a nucleic acid encoding an
epitope of the invention or an expression vector, preferably a VSV
vector, encoding an epitope of the invention, and wherein the
immunogens are administered in an amount or expressed at a level
sufficient to induce an HIV-specific immune response in the
subject. The HIV-specific immune response may include an
HIV-specific T-cell immune response or an HIV-specific B-cell
immune response. Such immunizations may be done at intervals,
preferably of at least 2-6 or more weeks.
[0093] It is to be understood and expected that variations in the
principles of invention as described above may be made by one
skilled in the art and it is intended that such modifications,
changes, and substitutions are to be included within the scope of
the present invention.
[0094] The invention will now be further described by way of the
following non-limiting examples.
EXAMPLES
Example 1
Recombinant VSV Vector Construction
[0095] Structure of the IAVI VSV genomic clone as depicted in FIG.
1.
Features include: [0096] 1. The cloning vector is based on pSP72
(Genbank X65332.2). [0097] 2. The extended T7 promoter is PT7-g10
described by Lopez et al. (Lopez et al., 1997. Journal of molecular
biology 269:41-51) [0098] 3. The hammerhead ribozyme was designed
following the rules for constructing self-cleaving RNA sequences
(Inoue et al. 2003. J Virol Methods 107:229-236 and Ruffner et al.
1990. Biochemistry 29:10695-10702). [0099] 4. The hepatitis delta
virus ribozyme and T7 RNA polymerase terminator were used as
described before for the measles virus rescue system (Radecke et
al. 1995. The EMBO journal 14:5773-5784, 23 and Sidhu et al, 1995.
Virology 208:800-807) [0100] 5. Unique restriction endonuclease
cleavage sites in the recombinant VSV genomne (red) are indicated
above the genome map. [0101] 6. The Leader and Trailer are
cis-acting sequences in the termini that control mRNA synthesis and
replication. [0102] 7. The viral proteins N, nucleocapsid; P,
phosphoprotein; M, matrix; G, glycoprotein; L, large protein.
[0103] Recombinant VSV Vector Construction [0104] Indiana Serotype
[0105] Based on Genbank EF197793--modified as described below:
[0106] Nucleotide substitutions introduced to generate unique
restriction sites or bring sequence closer to consensus [0107] 1371
CA>GC (NheI) [0108] After 2195 insert TAG (SpeI) (all genome
numbers below adjusted to include +3 by introduced by this
insertion) [0109] 3036 G>T improves match to consensus
transcription stop signal [0110] 3853 X>A (X was an ambiguity in
Genbank file) [0111] 4691 T>A to generate PacI [0112] 7546
C>A silent change in L coding sequence eliminates a BstBI site
[0113] 1960 TAC>TCC to change Y>S [0114] 3247 GTA>ATA to
change V>I [0115] 3729 AAG>GAG to change K>E [0116] 4191
GTA>GAA to change V>E [0117] 4386 GGT>GAT to change G>D
[0118] 4491 ACC>ATC to change T>I [0119] 5339 ATT>CTT to
change I>L [0120] 5834 ACT>GCT to change T>A [0121] 10959
AGA>AAA to change R>K
[0122] A VSV genome and cloning fragments are depicted in FIGS.
2A-G.
TABLE-US-00002 TABLE 1 Modifications introduced into the VSV
genomic sequence (Genbank accession EF197793) are listed. Note that
Line 3 includes a 3 base insertion, which shifts numbering in the
recombinant genomic clone (rEF197793). If nucleotide substitutions
were introduced to change amino acid coding, the base change in the
codon is indicated in red. Nucleotide position Nucleotide position
Nucleotide in 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.
[0123] Genbank X65332.2: Cloning vector pSP72
TABLE-US-00003 LOCUS X65332 2462 bp DNA circular SYN 25 JAN. 2000
DEFINITION Cloning vector pSP72. ACCESSION X65332 VERSION X65332.2
GI:6759494 KEYWORDS beta-lactamase; bla gene; cloning vector;
multiple cloning site; promoter. SOURCE Cloning vector pSP72
ORGANISM Cloning vector pSP72 other sequences; artificial
sequences; vectors. REFERENCE 1 AUTHORS Technical, Services. TITLE
Direct Submission JOURNAL Submitted (23 MAR. 1992) Technical
Services, Promega Corporation, 2800 Woods Hollow Road, Madison, Wi
53711-5399, USA REMARK revised by [2] REFERENCE 2 AUTHORS
Technical, Services. TITLE Direct Submission JOURNAL Submitted (28
MAY 1993) Technical Services, Promega Corporation, 2800 Woods
Hollow Road, Madison, Wi 53711-5399, USA REMARK revised by [3]
REFERENCE 3 (bases 1 to 2462) AUTHORS Technical, Services. TITLE
Direct Submission JOURNAL Submitted (12 JAN. 2000) Technical
Services, Promega Corporation, 2800 Woods Hollow Road, Madison, Wi
53711-5399, USA COMMENT On Jan. 26, 2000 this sequence version
replaced gi:58239. See X65300-X65335 for related vector sequences
This vector can be obtained from Promega Corporation, Madison, WI
Call one of the following numbers for order or technical
information: Order or Technical 800-356-9526 In Wisconsin
800-356-9526 Outside U.S. 608-274-4330. FEATURES
Location/Qualifiers source 1 . . . 2462 /organism="Cloning vector
pSP72" /mol_type="other DNA" /db_xref="taxon:90137" promoter
join(2446 . . . 2462,1 . . . 3) /note="SP6 promoter" misc_feature 1
/note="SP6 transcription initiation site" misc_feature 4 . . . 90
/note="multiple cloning sites" promoter 99 . . . 118 /note="T7
promoter" misc_feature 101 /note="T7 transcription initiation site"
gene complement(1135 . . . 1995) /gene="bla" CDS complement(1135 .
. . 1995) /gene="bla" /codon_start=1 /transl_table=11
/product="Beta-lactamase" /protein_id="CAA46432.1"
/db_xref="GI:58240"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVE
YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
EIGASLIKHW" ORIGIN 1 gaactcgagc agctgaagct tgcatgcctg caggtcgact
ctagaggatc cccgggtacc 61 gagctcgaat tcatcgatga tatcagatct
gccggtctcc ctatagtgag tcgtattaat 121 ttcgataagc caggttaacc
tgcattaatg aatcggccaa cgcgcgggga gaggcggttt 181 gcgtattggg
cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct 241
gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
301 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
gtaaaaaggc 361 cgcgttgctg gcgtttttcc ataggctccg cccccctgac
gagcatcaca aaaatcgacg 421 ctcaagtcag aggtggcgaa acccgacagg
actataaaga taccaggcgt ttccccctgg 481 aagctccctc gtgcgctctc
ctgttccgac cctgccgctt accggatacc tgtccgcctt 541 tctcccttcg
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt 601
gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
661 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
tatcgccact 721 ggcagcagcc actggtaaca ggattagcag agcgaggtat
gtaggcggtg ctacagagtt 781 cttgaagtgg tggcctaact acggctacac
tagaagaaca gtatttggta tctgcgctct 841 gctgaagcca gttaccttcg
gaaaaagagt tggtagctct tgatccggca aacaaaccac 901 cgctggtagc
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 961
tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
1021 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
ttttaaatta 1081 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
acttggtctg acagttacca 1141 atgcttaatc agtgaggcac ctatctcagc
gatctgtcta tttcgttcat ccatagttgc 1201 ctgactcccc gtcgtgtaga
taactacgat acgggagggc ttaccatctg gccccagtgc 1261 tgcaatgata
ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc 1321
agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
1381 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
gcaacgttgt 1441 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
ggtatggctt cattcagctc 1501 cggttcccaa cgatcaaggc gagttacatg
atcccccatg ttgtgcaaaa aagcggttag 1561 ctccttcggt cctccgatcg
ttgtcagaag taagttggcc gcagtgttat cactcatggt 1621 tatggcagca
ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac 1681
tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
1741 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
tgctcatcat 1801 tggaaaacgt tcttcggggc gaaaactctc aaggatctta
ccgctgttga gatccagttc 1861 gatgtaaccc actcgtgcac ccaactgatc
ttcagcatct tttactttca ccagcgtttc 1921 tgggtgagca aaaacaggaa
ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa 1981 atgttgaata
ctcatactct tcctttttca atattattga agcatttatc agggttattg 2041
tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
2101 cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
tgacattaac 2161 ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
cgtttcggtg atgacggtga 2221 aaacctctga cacatgcagc tcccggagac
ggtcacagct tgtctgtaag cggatgccgg 2281 gagcagacaa gcccgtcagg
gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa 2341 ctatgcggca
tcagagcaga ttgtactgag agtgcaccat atggacatat tgtcgttaga 2401
acgcggctac aattaataca taaccttatg tatcatacac atacgattta ggtgacacta
2461 ta//
[0124] Genbank EF197793: Vesicular stomatitis Indiana virus,
complete genome
TABLE-US-00004 LOCUS EF197793 11161 bp cRNA linear VRL 15 APR. 2007
DEFINITION Vesicular stomatitis Indiana virus, complete genome.
ACCESSION EF197793 VERSION EF197793.1 GI:144678900 SOURCE Vesicular
stomatitis Indiana virus ORGANISM Vesicular stomatitis Indiana
virus Viruses; ssRNA negative-strand viruses; Mononegavirales;
Rhabdoviridae; Dimarhabdovirus supergroup; Vesiculovirus. REFERENCE
1 (bases 1 to 11161) AUTHORS Remold, S. K., Rambaut, A. and Turner,
P. T. TITLE Evolutionary genomics of host adaptation in Vesicular
stomatitis virus JOURNAL Unpublished REFERENCE 2 (bases 1 to 11161)
AUTHORS Remold, S.K. TITLE Direct Submission JOURNAL Submitted (22
DEC. 2006) Biology, University of Louisville, 139 Life Sciences
Building, Louisville, KY 40292, USA FEATURES Location/Qualifiers
source 1 . . . 11161 /organism="Vesicular stomatitis Indiana virus"
/mol_type="viral cRNA" /isolate="MARMC from S.F. Elena Lab, 2001"
/db_xref="taxon:11277" /country="USA" gene 51 . . . 1376 /gene="N"
CDS 64 . . . 1332 /gene="N" /codon_start=1 /product="nucleoprotein"
/protein_id="ABP01780.1" /db_xref="GI:144678901"
/translation="MSVTVKRIIDNTVIVPKLPANEDPVEYPADYFRKSKEIPLYINT
TKSLSDLRGYVYQGLKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTI
GIFDLVSLKALDGVLPDGVSDASRTSADDKWLPLYLLGLYRVGRTQMPEYRKRLMDGL
TNQCKMINEQFEPLVPEGRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTI
VSRFKDCAALATFGHLCKITGMSTEDVTTWILNREVADEMVQMMLPGQEIDKADSYMP
YLIDFGLSSKSPYSSVKNPAFHFWGQLTALLLRSTRARNARQPDDIEYTSLTTAGLLY
AYAVGSSADLAQQFCVGDSKYTPDDSTGGLTTNAPPQGRDVVEWLGWFEDQNRKPTPD
MMQYAKRAVMSLQGLREKTIGKYAKSEFDK" gene 1386 . . . 2199 /gene="P" CDS
1396 . . . 2193 /gene="P" /codon_start=1 /product="phosphoprotein"
/protein_id="ABP01781.1" /db_xref="GI:144678902"
/translation="MDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDG
VEEHTRPSYFQAADDSDTESEPEIEDNQGLYVPDPEAEQVEGFIQGPLDDYADEDVDV
VFTSDWKQPELESDEHGKTLRLTLPEGLSGEQKSQWLLTIKAVVQSAKHWNLAECTFE
ASGEGVIIKKRQITPDVYKVTPVMNTHPYQSEAVSDVWSLSKTSMTFQPKKASLQPLT
ISLDELFSSRGEFISVGGNGRMSHKEAILLGLRYKKLYNQARVKYSL" gene 2209 . . .
3039 /gene="M" CDS 2250 . . . 2939 /gene="M" /codon_start=1
/product="matrix" /protein_id="ABP01782.1" /db_xref="GI:144678903"
/translation="MSSLKKILGLKGKGKKSKKLGIAPPPYEEDTNMEYAPSAPIDKS
YFGVDEMDTHDPNQLRYEKFFFTVKMTVRSNRPFRTYSDVAAAVSHWDHMYIGMAGKR
PFYKILAFLGSSNLKATPAVLADQGQPEYHAHCEGRAYLPHRMGKTPPMLNVPEHFRR
PFNIGLYKGTIELTMTIYDDESLEAAPMIWDHFNSSKFSDFREKALMFGLIVEKKASG
AWVLDSVSHFK" gene 3049 . . . 4713 /gene="G" CDS 3078 . . . 4613
/gene="G" /codon_start=1 /product="glycoprotein"
/protein_id="ABP01783.1" /db_xref="GI:144678904"
/translation="MKCLLYLAFLFIGVNCKFTIVFPHNQKGNWKNVPSNYHYCPSSS
DLNWHNDLVGTALQVKMPKSHKAIQADGWMCHASKWVTTCDFRWYGPKYITHSIRSFT
PSVEQCKESIEQTKQGTWLNPGFPPQSCGYATVTDAEAAIVQVTPHHVLVDEYTGEWV
DSQFINGKCSNDICPTVHNSTTWHSDYKVKGLCDSNLISMDITFFSEDGELSSLGKKG
TGFRSNYFAYETGDKACKMQYCKHWGVRLPSGVWFEMADKXLFAAARFPECPEGSSIS
APSQTSVDVSLIQDVERILDYSLCQETWSKIRAGLPISPVDLSYLAPKNPGTGPVFTI
INGTLKYFETRYIRVDIAAPILSRMVGMISGTTTERVLWDDWAPYEDVEIGPNGVLRT
SSGYKFPLYMIGHGMLDSDLHLSSKAQVFEHPHIQDAASQLPDGETLFFGDTGLSKNP
IEFVEGWFSSWKSSIASFFFTIGLIIGLFLVLRVGIYLCIKLKHTKKRQIYTDIEMNR LGK"
gene 4723 . . . 11095 /gene="L" CDS 4733 . . . 11062 /gene="L"
/codon start=1 /product="large protein" /protein_id="ABP01784.1"
/db_xref="GI:144678905"
/translation="MEVHDFETDEFNDFNEDDYATREFLNPDERMTYLNHADYNLNSP
LISDDIDNLIRKFNSLPIPSMWDSKNWDGVLEMLTSCQANPISTSQMHKWMGSWLMSD
NHDASQGYSFLHEVDKEAEITFDVVETFIRGWGNKPIEYIKKERWTDSFKILAYLCQK
FLDLHKLTLILNAVSEVELLNLARTFKGKVRRSSHGTNICRIRVPSLGPTFISEGWAY
FKKLDILMDRNFLLMVKDVIIGRMQTVLSMVCRIDNLFSEQDIFSLLNIYRIGDKIVE
RQGNFSYDLIKMVEPICNLKLMKLARESRPLVPQFPHFENHIKTSVDEGAKIDRGIRF
LHDQIMSVKTVDLTLVIYGSFRHWGHPFIDYYTGLEKLHSQVTMKKDIDVSYAKALAS
DLARIVLFQQFNDHKKWFVNGDLLPHDHPFKSHVKENTWPTAAQVQDFGDKWHELPLI
KCFEIPDLLDPSIIYSDKSHSMNRSEVLKHVRMNPNTPIPSKKVLQTMLDTKATNWKE
FLKEIDEKGLDDDDLIIGLKGKERELKLAGRFFSLMSWKLREYFVITEYLIKTHFVPM
FKGLTMADDLTAVIKKMLDSSSGQGLKSYEAICIANHIDYEKWNNHQRKLSNGPVFRV
MGQFLGYPSLIERTHEFFEKSLIYYNGRPDLMRVHNNTLINSTSQRVCWQGQEGGLEG
LRQKGWSILNLLVIQREAKIRNTAVKVLAQGDNQVICTQYKTKKSRNVVELQGALNQM
VSNNEKIMTAIKIGTGKLGLLINDDETMQSADYLNYGKIPIFRGVIRGLETKRWSRVT
CVTNDQIPTCANIMSSVSTNALTVAHFAENPINAMIQYNYFGTFARLLLMMHDPALRQ
SLYEVQDKIPGLHSSTFKYAMLYLDPSIGGVSGMSLSRFLIRAFPDPVTESLSFWRFI
HVHARSEHLKEMSAVFGNPEIAKFRITHIDKLVEDPTSLNIAMGMSPANLLKTEVKKC
LIESRQTIRNQVIKDATIYLYHEEDRLRSFLWSINPLFPRFLSEFKSGTFLGVADGLI
SLFQNSRTIRNSFKKKYHRELDDLIVRSEVSSLTHLGKLHLRRGSCKMWTCSATHADT
LRYKSWGRTVIGTTVPHPLEMLGPQHRKETPCAPCNTSGFNYVSVHCPDGIHDVFSSR
GPLPAYLGSKTSESTSILQPWERESKVPLIKRATRLRDAISWFVEPDSKLAMTILSNI
HSLTGEEWTKRQHGFKRTGSALHRFSTSRMSHGGFASQSTAALTRLMATTDTMRDLGD
QNFDFLFQATLLYAQITTTVARDGWITSCTDHYHIACKSCLRPIEEITLDSSMDYTPP
DVSHVLKTWRNGEGSWGQEIKQIYPLEGNWKNLAPAEQSYQVGRCIGFLYGDLAYRKS
THAEDSSLFPLSIQGRIRGRGFLKGLLDGLMRASCCQVIHRRSLAHLKRPANAVYGGL
IYLIDKLSVSPPFLSLTRSGPIRDELETIPHKIPTSYPTSNRDMGVIVRNYFKYQCRL
IEKGKYRSHYSQLWLFSDVLSIDFIGPFSISTTLLQILYKPFLSGKDKNELRELANLS
SLLRSGEGWEDIHVKFFTKDILLCPEEIRHACKFGIAKDNNKDMSYPPWGRESRGTIT
TIPVYYTTTPYPKMLEMPPRIQNPLLSGIRLGQLPTGAHYKIRSILHGMGIHYRDFLS
CGDGSGGMTAALLRENVHSRGIFNSLLELSGSVMRGASPEPPSALETLGGDKSRCVNG
ETCWEYPSDLCDPRTWDYFLRLKAGLGLQIDLIVMDMEVRDSSTSLKIETNVRNYVHR
ILDEQGVLIYKTYGTYICESEKNAVTILGPMFKTVDLVQTEFSSSQTSEVYMVCKGLK
KLIDEPNPDWSSINESWKNLYAFQSSEQEFARAKKVSTYFTLTGIPSQFIPDPFVNIE
TMLQIFGVPTGVSHAAALKSSDRPADLLTISLFYMAIISYYNINHIRVGPIPPNPPSD
GIAQNVGIAITGISFWLSLMEKDIPLYQQCLAVIQQSFPIRWEAVSVKGGYKQKWSTR
GDGLPKDTRISDSLAPIGNWIRSLELVRNQVRLNPFNEILFNQLCRTVDNHLKWSNLR
RNTGMIEWINRRISKEDRSILMLKSDLHEENSWRD" ORIGIN 1 acgaagacaa acaaaccatt
attatcatta aaaggctcag gagaaacttt aacagtaatc 61 aaaatgtctg
ttacagtcaa gagaatcatt gacaacacag tcatagttcc aaaacttcct 121
gcaaatgagg atccagtgga atacccggca gattacttca gaaaatcaaa ggagattcct
181 ctttacatca atactacaaa aagtttgtca gatctaagag gatatgtcta
ccaaggcctc 241 aaatccggaa atgtatcaat catacatgtc aacagctact
tgtatggagc attgaaggac 301 atccggggta agttggataa agattggtca
agtttcggaa taaacatcgg gaaggcaggg 361 gatacaatcg gaatatttga
ccttgtatcc ttgaaagccc tggacggtgt acttccagat 421 ggagtatcgg
atgcttccag aaccagcgca gatgacaaat ggttgccttt gtatctactt 481
ggcttataca gagtgggcag aacacaaatg cctgaataca gaaaaaggct catggatggg
541 ctgacaaatc aatgcaaaat gatcaatgaa cagtttgaac ctcttgtgcc
agaaggtcgt 601 gacatttttg atgtgtgggg aaatgacagt aattacacaa
aaattgtcgc tgcagtggac 661 atgttcttcc acatgttcaa aaaacatgaa
tgtgcctcgt tcagatacgg aactattgtt 721 tccagattca aagattgtgc
tgcattggca acatttggac acctctgcaa aataaccgga 781 atgtctacag
aagatgtgac gacctggatc ttgaaccgag aagttgcaga tgagatggtc 841
caaatgatgc ttccaggcca agaaattgac aaggctgatt catacatgcc ttatttgatc
901 gactttggat tgtcttctaa gtctccatat tcttccgtca aaaaccctgc
cttccacttc 961 tgggggcaat tgacagctct tctgctcaga tccaccagag
caaggaatgc ccgacagcct 1021 gatgacattg agtatacatc tcttactaca
gcaggtttgt tgtacgctta tgcagtagga 1081 tcctctgctg acttggcaca
acagttttgt gttggagata gcaaatacac tccagatgat 1141 agtaccggag
gattgacgac taatgcaccg ccacaaggca gagatgtggt cgaatggctc 1201
ggatggtttg aagatcaaaa cagaaaaccg actcctgata tgatgcagta tgcgaaacga
1261 gcagtcatgt cactgcaagg cctaagagag aagacaattg gcaagtatgc
taagtcagag 1321 tttgacaaat gaccctataa ttctcagatc acctattata
tattatgcta catatgaaaa 1381 aaactaacag atatcatgga taatctcaca
aaagttcgtg agtatctcaa gtcctattct 1441 cgtctagatc aggcggtagg
agagatagat gagatcgaag cacaacgagc tgaaaagtcc 1501 aattatgagt
tgttccaaga ggacggagtg gaagagcata ctaggccctc ttattttcag 1561
gcagcagatg attctgacac agaatctgaa ccagaaattg aagacaatca aggcttgtat
1621 gtaccagatc cggaagctga gcaagttgaa ggctttatac aggggccttt
agatgactat 1681 gcagatgagg acgtggatgt tgtattcact tcggactgga
aacagcctga gcttgaatcc 1741 gacgagcatg gaaagacctt acggttgaca
ttgccagagg gtttaagtgg agagcagaaa 1801 tcccagtggc ttttgacgat
taaagcagtc gttcaaagtg ccaaacactg gaatctggca 1861 gagtgcacat
ttgaagcatc gggagaaggg gtcatcataa aaaagcgcca gataactccg 1921
gatgtatata aggtcactcc agtgatgaac acacatccgt accaatcaga agccgtatca
1981 gatgtttggt ctctctcaaa gacatccatg actttccaac ccaagaaagc
aagtcttcag 2041 cctctcacca tatccttgga tgaattgttc tcatctagag
gagaattcat ctctgtcgga 2101 ggtaacggac gaatgtctca taaagaggcc
atcctgctcg gtctgaggta caaaaagttg 2161 tacaatcagg cgagagtcaa
atattctctg tagactatga aaaaaagtaa cagatatcac 2221 aatctaagtg
ttatcccaat ccattcatca tgagttcctt aaagaagatt ctcggtctga 2281
aggggaaagg taagaaatct aagaaattag ggatcgcacc acccccttat gaagaggaca
2341 ctaacatgga gtatgctccg agcgctccaa ttgacaaatc ctattttgga
gttgacgaga 2401 tggacactca tgatccgaat caattaagat atgagaaatt
cttctttaca gtgaaaatga 2461 cggttagatc taatcgtccg ttcagaacat
actcagatgt ggcagccgct gtatcccatt 2521 gggatcacat gtacatcgga
atggcaggga aacgtccctt ctacaagatc ttggcttttt 2581 tgggttcttc
taatctaaag gccactccag cggtattggc agatcaaggt caaccagagt 2641
atcatgctca ctgtgaaggc agggcttatt tgccacacag aatggggaag acccctccca
2701 tgctcaatgt accagagcac ttcagaagac cattcaatat aggtctttac
aagggaacga 2761 ttgagctcac aatgaccatc tacgatgatg agtcactgga
agcagctcct atgatctggg 2821 atcatttcaa ttcttccaaa ttttctgatt
tcagagagaa ggccttaatg tttggcctga 2881 ttgtcgagaa aaaggcatct
ggagcttggg tcctggattc tgtcagccac ttcaaatgag 2941 ctagtctagc
ttccagcttc tgaacaatcc ccggtttact cagtctctcc taattccagc 3001
ctttcgaaca actaatatcc tgtcttctct atcccgatga aaaaaactaa cagagatcga
3061 tctgtttcct tgacaccatg aagtgccttt tgtacttagc ttttttattc
atcggggtga 3121 attgcaagtt caccatagtt tttccacaca accaaaaagg
aaactggaaa aatgttcctt 3181 ccaattacca ttattgcccg tcaagctcag
atttaaattg gcataatgac ttagtaggca 3241 cagccttaca agtcaaaatg
cccaagagtc acaaggctat tcaagcagac ggttggatgt 3301 gtcatgcttc
caaatgggtc actacttgtg atttccgctg gtacggaccg aagtatataa 3361
cacattccat ccgatccttc actccatctg tagaacaatg caaggaaagc attgaacaaa
3421 cgaaacaagg aacttggctg aatccaggct tccctcctca aagttgtgga
tatgcaactg 3481 tgacggatgc tgaagcagcg attgtccagg tgactcctca
ccatgtgctt gttgatgaat 3541 acacaggaga atgggttgat tcacagttca
tcaacggaaa atgcagcaat gacatatgcc 3601 ccactgtcca taactccaca
acctggcatt ccgactataa ggtcaaaggg ctatgtgatt 3661 ctaacctcat
ttccatggac atcaccttct tctcagagga cggagagcta tcatccctag 3721
gaaagaaggg cacagggttc agaagtaact actttgctta tgaaactgga gacaaggcct
3781 gcaaaatgca gtactgcaag cattggggag tcagactccc atcaggtgtc
tggttcgaga 3841 tggctgataa ggmtctcttt gctgcagcca gattccctga
atgcccagaa gggtcaagta 3901 tctctgctcc atctcagacc tcagtggatg
taagtctcat tcaggacgtt gagaggatct 3961 tggattattc cctctgccaa
gaaacctgga gcaaaatcag agcgggtctt cccatctctc 4021 cagtggatct
cagctatctt gctcctaaaa acccaggaac cggtcctgtc tttaccataa 4081
tcaatggtac cctaaaatac tttgagacca gatacatcag agtcgatatt gctgctccaa
4141 tcctctcaag aatggtcgga atgatcagtg gaactaccac agaaagggta
ctgtgggatg 4201 actgggctcc atatgaagac gtggaaattg gacccaatgg
agttctgagg accagttcag 4261 gatataagtt tcctttatat atgattggac
atggtatgtt ggactccgat cttcatctta 4321 gctcaaaggc tcaggtgttt
gaacatcctc acattcaaga cgctgcttcg cagcttcctg 4381 atggtgagac
tttatttttt ggtgatactg ggctatccaa aaatccaatc gagtttgtag 4441
aaggttggtt cagtagttgg aagagctcta ttgcctcttt tttctttacc atagggttaa
4501 tcattggact attcttggtt ctccgagttg gtatttatct ttgcattaaa
ttaaagcaca 4561 ccaagaaaag acagatttat acagacatag agatgaaccg
acttggaaag taactcaaat 4621 cctgcacaac agattcttca tgtttgaacc
aaatcaactt gtgatatcat gctcaaagag 4681 gccttaatta tattttaatt
tttaattttt atgaaaaaaa ctaacagcaa tcatggaagt 4741 ccacgatttt
gagaccgacg agttcaatga tttcaatgaa gatgactatg ccacaagaga 4801
attcctgaat cccgatgagc gcatgacgta cttgaatcat gctgattaca atttgaattc
4861 tcctctaatt agtgatgata ttgacaattt gatcaggaaa ttcaattctc
ttccgattcc 4921 ctcgatgtgg gatagtaaga actgggatgg agttcttgag
atgttaacat catgtcaagc 4981 caatcccatc tcaacatctc agatgcataa
atggatggga agttggttaa tgtctgataa 5041 tcatgatgcc agtcaagggt
atagtttttt acatgaagtg gacaaagagg cagaaataac 5101 atttgacgtg
gtggagacct tcatccgcgg ctggggcaac aaaccaattg aatacatcaa 5161
aaaggaaaga tggactgact cattcaaaat tctcgcttat ttgtgtcaaa agtttttgga
5221 cttacacaag ttgacattaa tcttaaatgc tgtctctgag gtggaattgc
tcaacttggc 5281 gaggactttc aaaggcaaag tcagaagaag ttctcatgga
acgaacatat gcaggattag 5341 ggttcccagc ttgggtccta cttttatttc
agaaggatgg gcttacttca agaaacttga 5401 tattctaatg gaccgaaact
ttctgttaat ggtcaaagat gtgattatag ggaggatgca 5461 aacggtgcta
tccatggtat gtagaataga caacctgttc tcagagcaag acatcttctc 5521
ccttctaaat atctacagaa ttggagataa aattgtggag aggcagggaa atttttctta
5581 tgacttgatt aaaatggtgg aaccgatatg caacttgaag ctgatgaaat
tagcaagaga 5641 atcaaggcct ttagtcccac aattccctca ttttgaaaat
catatcaaga cttctgttga 5701 tgaaggggca aaaattgacc gaggtataag
attcctccat gatcagataa tgagtgtgaa 5761 aacagtggat ctcacactgg
tgatttatgg atcgttcaga cattggggtc atccttttat 5821 agattattac
actggactag aaaaattaca ttcccaagta accatgaaga aagatattga 5881
tgtgtcatat gcaaaagcac ttgcaagtga tttagctcgg attgttctat ttcaacagtt
5941 caatgatcat aaaaagtggt tcgtgaatgg agacttgctc cctcatgatc
atccctttaa 6001 aagtcatgtt aaagaaaata catggcctac agctgctcaa
gttcaagatt ttggagataa 6061 atggcatgaa cttccgctga ttaaatgttt
tgaaataccc gacttactag acccatcgat 6121 aatatactct gacaaaagtc
attcaatgaa taggtcagag gtgttgaaac atgtccgaat 6181 gaatccgaac
actcctatcc ctagtaaaaa ggtgttgcag actatgttgg acacaaaggc 6241
taccaattgg aaagaatttc ttaaagagat tgatgagaag ggcttagatg atgatgatct
6301 aattattggt cttaaaggaa aggagaggga actgaagttg gcaggtagat
ttttctccct 6361 aatgtcttgg aaattgcgag aatactttgt aattaccgaa
tatttgataa agactcattt 6421 cgtccctatg tttaaaggcc tgacaatggc
ggacgatcta actgcagtca ttaaaaagat 6481 gttagattcc tcatccggcc
aaggattgaa gtcatatgag gcaatttgca tagccaatca 6541 cattgattac
gaaaaatgga ataaccacca aaggaagtta tcaaacggcc cagtgttccg 6601
agttatgggc cagttcttag gttatccatc cttaatcgag agaactcatg aattttttga
6661 gaaaagtctt atatactaca atggaagacc agacttgatg cgtgttcaca
acaacacact 6721 gatcaattca acctcccaac gagtttgttg gcaaggacaa
gagggtggac tggaaggtct 6781 acggcaaaaa ggatggagta tcctcaatct
actggttatt caaagagagg ctaaaatcag 6841 aaacactgct gtcaaagtct
tggcacaagg tgataatcaa gttatttgca cacagtataa 6901 aacgaagaaa
tcgagaaacg ttgtagaatt acagggtgct ctcaatcaaa tggtttctaa 6961
taatgagaaa attatgactg caatcaaaat agggacaggg aagttaggac ttttgataaa
7021 tgacgatgag actatgcaat ctgcagatta cttgaattat ggaaaaatac
cgattttccg
7081 tggagtgatt agagggttag agaccaagag atggtcacga gtgacttgtg
tcaccaatga 7141 ccaaataccc acttgtgcta atataatgag ctcagtttcc
acaaatgctc tcaccgtagc 7201 tcattttgct gagaacccaa tcaatgccat
gatacagtac aattattttg ggacatttgc 7261 tagactcttg ttgatgatgc
atgatcctgc tcttcgtcaa tcattgtatg aagttcaaga 7321 taagataccg
ggcttgcaca gttctacttt caaatacgcc atgttgtatt tggacccttc 7381
cattggagga gtgtcgggca tgtctttgtc caggtttttg attagagcct tcccagatcc
7441 cgtaacagaa agtctctcat tctggagatt catccatgta catgctcgaa
gtgagcatct 7501 gaaggagatg agtgcagtat ttggaaaccc cgagatagcc
aagtttcgaa taactcacat 7561 agacaagcta gtagaagatc caacctctct
gaacatcgct atgggaatga gtccagcgaa 7621 cttgttaaag actgaggtta
aaaaatgctt aatcgaatca agacaaacca tcaggaacca 7681 ggtgattaag
gatgcaacca tatatttgta tcatgaagag gatcggctca gaagtttctt 7741
atggtcaata aatcctctgt tccctagatt tttaagtgaa ttcaaatcag gcactttttt
7801 gggagtcgca gacgggctca tcagtctatt tcaaaattct cgtactattc
ggaactcctt 7861 taagaaaaag tatcataggg aattggatga tttgattgtg
aggagtgagg tatcctcttt 7921 gacacattta gggaaacttc atttgagaag
gggatcatgt aaaatgtgga catgttcagc 7981 tactcatgct gacacattaa
gatacaaatc ctggggccgt acagttattg ggacaactgt 8041 accccatcca
ttagaaatgt tgggtccaca acatcgaaaa gagactcctt gtgcaccatg 8101
taacacatca gggttcaatt atgtttctgt gcattgtcca gacgggatcc atgacgtctt
8161 tagttcacgg ggaccattgc ctgcttatct agggtctaaa acatctgaat
ctacatctat 8221 tttgcagcct tgggaaaggg aaagcaaagt cccactgatt
aaaagagcta cacgtcttag 8281 agatgctatc tcttggtttg ttgaacccga
ctctaaacta gcaatgacta tactttctaa 8341 catccactct ttaacaggcg
aagaatggac caaaaggcag catgggttca aaagaacagg 8401 gtctgccctt
cataggtttt cgacatctcg gatgagccat ggtgggttcg catctcagag 8461
cactgcagca ttgaccaggt tgatggcaac tacagacacc atgagggatc tgggagatca
8521 gaatttcgac tttttattcc aagcaacgtt gctctatgct caaattacca
ccactgttgc 8581 aagagacgga tggatcacca gttgtacaga tcattatcat
attgcctgta agtcctgttt 8641 gagacccata gaagagatca ccctggactc
aagtatggac tacacgcccc cagatgtatc 8701 ccatgtgctg aagacatgga
ggaatgggga aggttcgtgg ggacaagaga taaaacagat 8761 ctatccttta
gaagggaatt ggaagaattt agcacctgct gagcaatcct atcaagtcgg 8821
cagatgtata ggttttctat atggagactt ggcgtataga aaatctactc atgccgagga
8881 cagttctcta tttcctctat ctatacaagg tcgtattaga ggtcgaggtt
tcttaaaagg 8941 gttgctagac ggattaatga gagcaagttg ctgccaagta
atacaccgga gaagtctggc 9001 tcatttgaag aggccggcca acgcagtgta
cggaggtttg atttacttga ttgataaatt 9061 gagtgtatca cctccattcc
tttctcttac tagatcagga cctattagag acgaattaga 9121 aacgattccc
cacaagatcc caacctccta tccgacaagc aaccgtgata tgggggtgat 9181
tgtcagaaat tacttcaaat accaatgccg tctaattgaa aagggaaaat acagatcaca
9241 ttattcacaa ttatggttat tctcagatgt cttatccata gacttcattg
gaccattctc 9301 tatttccacc accctcttgc aaatcctata caagccattt
ttatctggga aagataagaa 9361 tgagttgaga gagctggcaa atctttcttc
attgctaaga tcaggagagg ggtgggaaga 9421 catacatgtg aaattcttca
ccaaggacat attattgtgt ccagaggaaa tcagacatgc 9481 ttgcaagttc
gggattgcta aggataataa taaagacatg agctatcccc cttggggaag 9541
ggaatccaga gggacaatta caacaatccc tgtttattat acgaccaccc cttacccaaa
9601 gatgctagag atgcctccaa gaatccaaaa tcccctgctg tccggaatca
ggttgggcca 9661 attaccaact ggcgctcatt ataaaattcg gagtatatta
catggaatgg gaatccatta 9721 cagggacttc ttgagttgtg gagacggctc
cggagggatg actgctgcat tactacgaga 9781 aaatgtgcat agcagaggaa
tattcaatag tctgttagaa ttatcagggt cagtcatgcg 9841 aggcgcctct
cctgagcccc ccagtgccct agaaacttta ggaggagata aatcgagatg 9901
tgtaaatggt gaaacatgtt gggaatatcc atctgactta tgtgacccaa ggacttggga
9961 ctatttcctc cgactcaaag caggcttggg gcttcaaatt gatttaattg
taatggatat 10021 ggaagttcgg gattcttcta ctagcctgaa aattgagacg
aatgttagaa attatgtgca 10081 ccggattttg gatgagcaag gagttttaat
ctacaagact tatggaacat atatttgtga 10141 gagcgaaaag aatgcagtaa
caatccttgg tcccatgttc aagacggtcg acttagttca 10201 aacagaattt
agtagttctc aaacgtctga agtatatatg gtatgtaaag gtttgaagaa 10261
attaatcgat gaacccaatc ccgattggtc ttccatcaat gaatcctgga aaaacctgta
10321 cgcattccag tcatcagaac aggaatttgc cagagcaaag aaggttagta
catactttac 10381 cttgacaggt attccctccc aattcattcc tgatcctttt
gtaaacattg agactatgct 10441 acaaatattc ggagtaccca cgggtgtgtc
tcatgcggct gccttaaaat catctgatag 10501 acctgcagat ttattgacca
ttagcctttt ttatatggcg attatatcgt attataacat 10561 caatcatatc
agagtaggac cgatacctcc gaacccccca tcagatggaa ttgcacaaaa 10621
tgtggggatc gctataactg gtataagctt ttggctgagt ttgatggaga aagacattcc
10681 actatatcaa cagtgtttag cagttatcca gcaatcattc ccgattaggt
gggaggctgt 10741 ttcagtaaaa ggaggataca agcagaagtg gagtactaga
ggtgatgggc tcccaaaaga 10801 tacccgaatt tcagactcct tggccccaat
cgggaactgg atcagatctc tggaattggt 10861 ccgaaaccaa gttcgtctaa
atccattcaa tgagatcttg ttcaatcagc tatgtcgtac 10921 agtggataat
catttgaaat ggtcaaattt gcgaagaaac acaggaatga ttgaatggat 10981
caatagacga atttcaaaag aagaccggtc tatactgatg ttgaagagtg acctacacga
11041 ggaaaactct tggagagatt aaaaaatcat gaggagactc caaactttaa
gtatgaaaaa 11101 aactttgatc cttaagaccc tcttgtggtt tttatttttt
atctggtttt gtggtcttcg 11161 t//
Example 2
Vsv Genome and Cloning Fragments
[0125] FIG. 2A depicts a schematic of a VSV genome and cloning
fragments.
[0126] In the sequences provided in FIGS. 2B-2G, terminal fragments
A and G are combined to produce fragment VS V-AG. [0127] The DNA
fragments are designed for cloning into pSP72 or other similar
cloning vectors. Before adding VSV cDNA sequences, the cloning
plasmid is modified by insertion of the hammerhead and hepatitis
delta virus ribozyme sequences. A BsmBI restriction enzyme cleavage
site is placed between the ribozyme sequences (5'-hammerhead
ribozyme-BsmBI-hepatitis delta virus ribozyme-3') for the purpose
of inserting the VSV-AG fragment. [0128] The AG fragment was
designed with BsmBI sites at the 5' and 3' termini (lower case
nucleotides) for insertion between the ribozyme sequences
introduced in the step above. Because BsmBI cleaves distal to its
recognition sequence (see bullet below), this enzyme may be used to
join the AG fragment directly to the ribozymes while also
eliminating the non-VSV nucleotides added to create the enzyme
cleavage signal. (Ball L A, Pringle C R, Flanagan B, Perepelitsa V
P, Wertz G W. Phenotypic consequences of rearranging the P, M, and
G genes of vesicular stomatitis virus. J. Virol. 1999 June;
73(6):4705-12, the disclosure of which is incorporated by
reference). [0129] Like other restriction endonucleases of this
type (BspMI, Earl, Plel, SfaNI and others), BsmBI cleaves distal to
its recognition sequence:
TABLE-US-00005 [0129] 5'-CGTCTC/N-3' 3'-GCAGAGNNNNN/N-5'
[0130] N is any nucleotide and/indicates cleavage site. [0131] The
VSV-AG fragment also is designed to facilitate subsequent cloning.
Between the fused A and G fragments there is a polylinker sequence
(noted in red nucleotides) that contains restriction endonuclease
cleavage sites needed for sequential cloning of Fragments B-F to
assemble a full-length clone. The polylinker contains
5'-NheI-BstBI-PacI-AvaI-SalI-AflII-3' restriction endonuclease
cleavage sites. Polylinker nucleotides are replaced by VSV genomic
sequence as the full-length clone is assembled.
Example 3
Virus Rescue Support Plasmid Insert Optimization
[0132] Strategy for optimizing gene inserts encoding VSV N, P, M,
G, and L for construction of plasmid DNAs encoding trans-acting
proteins needed to initiate virus rescue. Gene inserts were
optimized using steps in Example 4 then synthesized by a contract
lab and subsequently cloned into a plasmid under the control of the
hCMV promoter and enhancer.
Example 4
Coding Sequence Optimization and Gene Design
[0133] Step 1. Replace VSV sequence with codons used by highly
expressed mammalian genes. Use the CodonJuggle program found in the
GeneDesign Webtool (Richardson et al. 2010. Nucleic Acids Res
38:2603-2606 and Richardson et al. 2006. Genome Res
16:550-556).
[0134] Step 2. Eliminate potential RNA processing signals in the
coding sequence that might direct unwanted RNA splicing or
cleavage/polyadenylation reaction. [0135] a) Identify potential
splice site signals and remove by introducing synonymous codons.
Splice site predictions were made with the webtool at the Berkeley
Drosophila Genome Project website (Reese et al. 1997. J Comput Biol
4:311-323). [0136] b) Scan the insert for consensus
cleavage/polyadenylation signals (AAUAAA) (Zhao et al. 1999. MMBR
63:405-445). Disrupt by introducing synonymous codons.
[0137] Step 3 [0138] a) Add a preferred translational start
sequence (the Kozak sequence) (Kochetov et al. 1998. FEBS letters
440:351-355, Kozak. 1999. Gene 234:187-208, Kozak. 1991. J Biol
Chem 266:19867-19870 and Zhang. 1998. Human molecular genetics
7:919-932). [0139] b) Add a preferred translational stop codon at
the 3' end (Kochetov et al. 1998. FEBS letters 440:351-355, Sun et
al. 2005. J Mol Evol 61:437-444 and Zhang. 1998. Human molecular
genetics 7:919-932).
[0140] Step 4. Scan the sequence for homopolymeric stretches of 5
nucleotides or more. Interrupt these sequences by introducing
synonymous codons.
[0141] Step 5. Scan the sequence for restriction endonuclease
cleavage sites and eliminate any unwanted recognitions signals.
[0142] Step 6. Confirm that the modified sequence translates into
the expected amino acid sequence.
[0143] The invention is further described by the following numbered
paragraphs:
[0144] 1. A vesicular stomatitis virus (VSV) genomic clone
comprising:
[0145] (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,
[0146] (b) a cloning vector,
[0147] (c) an extended T7 promoter,
[0148] (d) a hammerhead ribozyme,
[0149] (e) a hepatitis delta virus ribozyme and T7 terminator
[0150] (f) unique restriction endonuclease cleavage sites in a VSV
genomic sequence
[0151] (g) a leader and a trailer that are cis-acting sequences
controlling mRNA synthesis and replication
[0152] 2. The VSV genomic clone of paragraph 1, wherein the cloning
vector is pSP72 (Genbank X65332.2)
[0153] 3. The VSV genomic clone of paragraph 1 or 2, wherein the
extended T7 promoter is PT7-g10.
[0154] 4. The VSV genomic clone of any one of paragraphs 1 to 3,
wherein the unique restriction endonuclease cleavage sites are 1367
NheI, 2194 SpeI, 2194 BstBI, 4687 PacI, 7532 AvaI, 10190 SalI and
11164 AflII.
[0155] 5. The VSV genomic clone of any one of paragraphs 1 to 4,
wherein the VSV genomic clone is depicted in FIG. 1.
[0156] 6. The VSV genomic clone of any one of paragraphs 1 to 5,
wherein the nucleotide position is according to GenBank Accession
Number EF197793 and wherein the nucleotide substitutions are
selected from the group consisting of [0157] 1371 CA>GC (NheI)
[0158] After 2195 insert TAG (SpeI) (all genome numbers below
adjusted to include +3 bp) [0159] 3036 G>T improves match to
consensus transcription stop signal [0160] 3853 X>A (was an
ambiguity in Genbank file) [0161] 4691 T>A to generate PacI
[0162] 7546 C>A silent change in L coding sequence eliminates a
BstBI site [0163] 1960 TAC>TCC to change Y>S [0164] 3247
GTA>ATA to change V>I [0165] 3729 AAG>GAG to change K>E
[0166] 4191 GTA>GAA to change V>E [0167] 4386 GGT>GAT to
change G>D [0168] 4491 ACC>ATC to change T>I [0169] 5339
ATT>CTT to change I>L [0170] 5834 ACT>GCT to change T>A
and [0171] 10959 AGA>AAA to change R>K.
[0172] 7. The VSV genomic clone of any one of paragraphs 1 to 6,
wherein the nucleotide position is according to GenBank Accession
Number EF197793 and wherein the nucleotide substitutions are
selected from the group consisting of:
TABLE-US-00006 Nucleotide position Nucleotide position Nucleotide
in 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.
[0173] 8. The VSV genomic clone of any one of paragraphs 1 to 7,
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.
[0174] 9. The VSV genomic clone of any one of paragraphs 1 to 7,
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.
[0175] 11. The method of paragraph 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.
[0176] 12. The method of paragraph 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.
[0177] 13. The method of any one of paragraphs 10 to 12, 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.
[0178] 14. The method of paragraph 13, where the optimization is
codon optimization.
[0179] 15. The method of paragraph 14, wherein the codon
optimization comprises replacing a VSV nucleotide sequence with
codons used by highly expressed mammalian genes.
[0180] 16. The method of paragraph 14 or 15, 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: [0181] (a) identifying potential splice site signals and
remove by introducing synonymous codons and/or [0182] (b) scanning
an insert for consensus cleavage/polyadenylation signals (AAUAAA)
and introducing synonymous codons to disrupt the consensus
cleavage/polyadenylation signals.
[0183] 17. The method of any one of paragraphs 14 to 16, wherein
the codon optimization comprises [0184] (a) adding a preferred
translational start sequence (the Kozak sequence) and/or [0185] (b)
adding a preferred translational stop codon.
[0186] 18. The method of any one of paragraphs 14 to 17, wherein
the codon optimization comprises scanning a sequence for
homopolymeric stretches of 5 nucleotides or more and interrupting
the sequences by introducing synonymous codons.
[0187] 19. The method of any one of paragraphs 14 to 18, wherein
the codon optimization comprises scanning a sequence for
restriction endonuclease cleavage sites and eliminate any unwanted
recognitions signals.
[0188] 20. The method of any one of paragraphs 14 to 19, wherein
the codon optimization comprises confirming that a modified
sequence translates into an expected amino acid sequence.
[0189] Having thus described in detail preferred embodiments of the
present invention, it is to be understood that the invention
defined by the above paragraphs is not to be limited to particular
details set forth in the above description as many apparent
variations thereof are possible without departing from the spirit
or scope of the present invention.
Sequence CWU 1
1
181286PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Met Ser Ile Gln His Phe Arg Val Ala Leu Ile
Pro Phe Phe Ala Ala 1 5 10 15 Phe Cys Leu Pro Val Phe Ala His Pro
Glu Thr Leu Val Lys Val Lys 20 25 30 Asp Ala Glu Asp Gln Leu Gly
Ala Arg Val Gly Tyr Ile Glu Leu Asp 35 40 45 Leu Asn Ser Gly Lys
Ile Leu Glu Ser Phe Arg Pro Glu Glu Arg Phe 50 55 60 Pro Met Met
Ser Thr Phe Lys Val Leu Leu Cys Gly Ala Val Leu Ser 65 70 75 80 Arg
Ile Asp Ala Gly Gln Glu Gln Leu Gly Arg Arg Ile His Tyr Ser 85 90
95 Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr Glu Lys His Leu Thr
100 105 110 Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala Ala Ile Thr
Met Ser 115 120 125 Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr Ile
Gly Gly Pro Lys 130 135 140 Glu Leu Thr Ala Phe Leu His Asn Met Gly
Asp His Val Thr Arg Leu 145 150 155 160 Asp Arg Trp Glu Pro Glu Leu
Asn Glu Ala Ile Pro Asn Asp Glu Arg 165 170 175 Asp Thr Thr Met Pro
Val Ala Met Ala Thr Thr Leu Arg Lys Leu Leu 180 185 190 Thr Gly Glu
Leu Leu Thr Leu Ala Ser Arg Gln Gln Leu Ile Asp Trp 195 200 205 Met
Glu Ala Asp Lys Val Ala Gly Pro Leu Leu Arg Ser Ala Leu Pro 210 215
220 Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala Gly Glu Arg Gly Ser
225 230 235 240 Arg Gly Ile Ile Ala Ala Leu Gly Pro Asp Gly Lys Pro
Ser Arg Ile 245 250 255 Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr
Met Asp Glu Arg Asn 260 265 270 Arg Gln Ile Ala Glu Ile Gly Ala Ser
Leu Ile Lys His Trp 275 280 285 22462DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
2gaactcgagc agctgaagct tgcatgcctg caggtcgact ctagaggatc cccgggtacc
60gagctcgaat tcatcgatga tatcagatct gccggtctcc ctatagtgag tcgtattaat
120ttcgataagc caggttaacc tgcattaatg aatcggccaa cgcgcgggga
gaggcggttt 180gcgtattggg cgctcttccg cttcctcgct cactgactcg
ctgcgctcgg tcgttcggct 240gcggcgagcg gtatcagctc actcaaaggc
ggtaatacgg ttatccacag aatcagggga 300taacgcagga aagaacatgt
gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc 360cgcgttgctg
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
420ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
ttccccctgg 480aagctccctc gtgcgctctc ctgttccgac cctgccgctt
accggatacc tgtccgcctt 540tctcccttcg ggaagcgtgg cgctttctca
tagctcacgc tgtaggtatc tcagttcggt 600gtaggtcgtt cgctccaagc
tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg 660cgccttatcc
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
720ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
ctacagagtt 780cttgaagtgg tggcctaact acggctacac tagaagaaca
gtatttggta tctgcgctct 840gctgaagcca gttaccttcg gaaaaagagt
tggtagctct tgatccggca aacaaaccac 900cgctggtagc ggtggttttt
ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc 960tcaagaagat
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
1020ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
ttttaaatta 1080aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
acttggtctg acagttacca 1140atgcttaatc agtgaggcac ctatctcagc
gatctgtcta tttcgttcat ccatagttgc 1200ctgactcccc gtcgtgtaga
taactacgat acgggagggc ttaccatctg gccccagtgc 1260tgcaatgata
ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
1320agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
tccagtctat 1380taattgttgc cgggaagcta gagtaagtag ttcgccagtt
aatagtttgc gcaacgttgt 1440tgccattgct acaggcatcg tggtgtcacg
ctcgtcgttt ggtatggctt cattcagctc 1500cggttcccaa cgatcaaggc
gagttacatg atcccccatg ttgtgcaaaa aagcggttag 1560ctccttcggt
cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
1620tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
tttctgtgac 1680tggtgagtac tcaaccaagt cattctgaga atagtgtatg
cggcgaccga gttgctcttg 1740cccggcgtca atacgggata ataccgcgcc
acatagcaga actttaaaag tgctcatcat 1800tggaaaacgt tcttcggggc
gaaaactctc aaggatctta ccgctgttga gatccagttc 1860gatgtaaccc
actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
1920tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
cgacacggaa 1980atgttgaata ctcatactct tcctttttca atattattga
agcatttatc agggttattg 2040tctcatgagc ggatacatat ttgaatgtat
ttagaaaaat aaacaaatag gggttccgcg 2100cacatttccc cgaaaagtgc
cacctgacgt ctaagaaacc attattatca tgacattaac 2160ctataaaaat
aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga
2220aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
cggatgccgg 2280gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
gggtgtcggg gctggcttaa 2340ctatgcggca tcagagcaga ttgtactgag
agtgcaccat atggacatat tgtcgttaga 2400acgcggctac aattaataca
taaccttatg tatcatacac atacgattta ggtgacacta 2460ta
24623422PRTVesicular stomatitis Indiana virus 3Met Ser Val Thr Val
Lys Arg Ile Ile Asp Asn Thr Val Ile Val Pro 1 5 10 15 Lys Leu Pro
Ala Asn Glu Asp Pro Val Glu Tyr Pro Ala Asp Tyr Phe 20 25 30 Arg
Lys Ser Lys Glu Ile Pro Leu Tyr Ile Asn Thr Thr Lys Ser Leu 35 40
45 Ser Asp Leu Arg Gly Tyr Val Tyr Gln Gly Leu Lys Ser Gly Asn Val
50 55 60 Ser Ile Ile His Val Asn Ser Tyr Leu Tyr Gly Ala Leu Lys
Asp Ile 65 70 75 80 Arg Gly Lys Leu Asp Lys Asp Trp Ser Ser Phe Gly
Ile Asn Ile Gly 85 90 95 Lys Ala Gly Asp Thr Ile Gly Ile Phe Asp
Leu Val Ser Leu Lys Ala 100 105 110 Leu Asp Gly Val Leu Pro Asp Gly
Val Ser Asp Ala Ser Arg Thr Ser 115 120 125 Ala Asp Asp Lys Trp Leu
Pro Leu Tyr Leu Leu Gly Leu Tyr Arg Val 130 135 140 Gly Arg Thr Gln
Met Pro Glu Tyr Arg Lys Arg Leu Met Asp Gly Leu 145 150 155 160 Thr
Asn Gln Cys Lys Met Ile Asn Glu Gln Phe Glu Pro Leu Val Pro 165 170
175 Glu Gly Arg Asp Ile Phe Asp Val Trp Gly Asn Asp Ser Asn Tyr Thr
180 185 190 Lys Ile Val Ala Ala Val Asp Met Phe Phe His Met Phe Lys
Lys His 195 200 205 Glu Cys Ala Ser Phe Arg Tyr Gly Thr Ile Val Ser
Arg Phe Lys Asp 210 215 220 Cys Ala Ala Leu Ala Thr Phe Gly His Leu
Cys Lys Ile Thr Gly Met 225 230 235 240 Ser Thr Glu Asp Val Thr Thr
Trp Ile Leu Asn Arg Glu Val Ala Asp 245 250 255 Glu Met Val Gln Met
Met Leu Pro Gly Gln Glu Ile Asp Lys Ala Asp 260 265 270 Ser Tyr Met
Pro Tyr Leu Ile Asp Phe Gly Leu Ser Ser Lys Ser Pro 275 280 285 Tyr
Ser Ser Val Lys Asn Pro Ala Phe His Phe Trp Gly Gln Leu Thr 290 295
300 Ala Leu Leu Leu Arg Ser Thr Arg Ala Arg Asn Ala Arg Gln Pro Asp
305 310 315 320 Asp Ile Glu Tyr Thr Ser Leu Thr Thr Ala Gly Leu Leu
Tyr Ala Tyr 325 330 335 Ala Val Gly Ser Ser Ala Asp Leu Ala Gln Gln
Phe Cys Val Gly Asp 340 345 350 Ser Lys Tyr Thr Pro Asp Asp Ser Thr
Gly Gly Leu Thr Thr Asn Ala 355 360 365 Pro Pro Gln Gly Arg Asp Val
Val Glu Trp Leu Gly Trp Phe Glu Asp 370 375 380 Gln Asn Arg Lys Pro
Thr Pro Asp Met Met Gln Tyr Ala Lys Arg Ala 385 390 395 400 Val Met
Ser Leu Gln Gly Leu Arg Glu Lys Thr Ile Gly Lys Tyr Ala 405 410 415
Lys Ser Glu Phe Asp Lys 420 4265PRTVesicular stomatitis Indiana
virus 4Met Asp Asn Leu Thr Lys Val Arg Glu Tyr Leu Lys Ser Tyr Ser
Arg 1 5 10 15 Leu Asp Gln Ala Val Gly Glu Ile Asp Glu Ile Glu Ala
Gln Arg Ala 20 25 30 Glu Lys Ser Asn Tyr Glu Leu Phe Gln Glu Asp
Gly Val Glu Glu His 35 40 45 Thr Arg Pro Ser Tyr Phe Gln Ala Ala
Asp Asp Ser Asp Thr Glu Ser 50 55 60 Glu Pro Glu Ile Glu Asp Asn
Gln Gly Leu Tyr Val Pro Asp Pro Glu 65 70 75 80 Ala Glu Gln Val Glu
Gly Phe Ile Gln Gly Pro Leu Asp Asp Tyr Ala 85 90 95 Asp Glu Asp
Val Asp Val Val Phe Thr Ser Asp Trp Lys Gln Pro Glu 100 105 110 Leu
Glu Ser Asp Glu His Gly Lys Thr Leu Arg Leu Thr Leu Pro Glu 115 120
125 Gly Leu Ser Gly Glu Gln Lys Ser Gln Trp Leu Leu Thr Ile Lys Ala
130 135 140 Val Val Gln Ser Ala Lys His Trp Asn Leu Ala Glu Cys Thr
Phe Glu 145 150 155 160 Ala Ser Gly Glu Gly Val Ile Ile Lys Lys Arg
Gln Ile Thr Pro Asp 165 170 175 Val Tyr Lys Val Thr Pro Val Met Asn
Thr His Pro Tyr Gln Ser Glu 180 185 190 Ala Val Ser Asp Val Trp Ser
Leu Ser Lys Thr Ser Met Thr Phe Gln 195 200 205 Pro Lys Lys Ala Ser
Leu Gln Pro Leu Thr Ile Ser Leu Asp Glu Leu 210 215 220 Phe Ser Ser
Arg Gly Glu Phe Ile Ser Val Gly Gly Asn Gly Arg Met 225 230 235 240
Ser His Lys Glu Ala Ile Leu Leu Gly Leu Arg Tyr Lys Lys Leu Tyr 245
250 255 Asn Gln Ala Arg Val Lys Tyr Ser Leu 260 265
5229PRTVesicular stomatitis Indiana virus 5Met Ser Ser Leu Lys Lys
Ile Leu Gly Leu Lys Gly Lys Gly Lys Lys 1 5 10 15 Ser Lys Lys Leu
Gly Ile Ala Pro Pro Pro Tyr Glu Glu Asp Thr Asn 20 25 30 Met Glu
Tyr Ala Pro Ser Ala Pro Ile Asp Lys Ser Tyr Phe Gly Val 35 40 45
Asp Glu Met Asp Thr His Asp Pro Asn Gln Leu Arg Tyr Glu Lys Phe 50
55 60 Phe Phe Thr Val Lys Met Thr Val Arg Ser Asn Arg Pro Phe Arg
Thr 65 70 75 80 Tyr Ser Asp Val Ala Ala Ala Val Ser His Trp Asp His
Met Tyr Ile 85 90 95 Gly Met Ala Gly Lys Arg Pro Phe Tyr Lys Ile
Leu Ala Phe Leu Gly 100 105 110 Ser Ser Asn Leu Lys Ala Thr Pro Ala
Val Leu Ala Asp Gln Gly Gln 115 120 125 Pro Glu Tyr His Ala His Cys
Glu Gly Arg Ala Tyr Leu Pro His Arg 130 135 140 Met Gly Lys Thr Pro
Pro Met Leu Asn Val Pro Glu His Phe Arg Arg 145 150 155 160 Pro Phe
Asn Ile Gly Leu Tyr Lys Gly Thr Ile Glu Leu Thr Met Thr 165 170 175
Ile Tyr Asp Asp Glu Ser Leu Glu Ala Ala Pro Met Ile Trp Asp His 180
185 190 Phe Asn Ser Ser Lys Phe Ser Asp Phe Arg Glu Lys Ala Leu Met
Phe 195 200 205 Gly Leu Ile Val Glu Lys Lys Ala Ser Gly Ala Trp Val
Leu Asp Ser 210 215 220 Val Ser His Phe Lys 225 6511PRTVesicular
stomatitis Indiana virusMOD_RES(259)..(259)Any amino acid 6Met Lys
Cys Leu Leu Tyr Leu Ala Phe Leu Phe Ile Gly Val Asn Cys 1 5 10 15
Lys Phe Thr Ile Val Phe Pro His Asn Gln Lys Gly Asn Trp Lys Asn 20
25 30 Val Pro Ser Asn Tyr His Tyr Cys Pro Ser Ser Ser Asp Leu Asn
Trp 35 40 45 His Asn Asp Leu Val Gly Thr Ala Leu Gln Val Lys Met
Pro Lys Ser 50 55 60 His Lys Ala Ile Gln Ala Asp Gly Trp Met Cys
His Ala Ser Lys Trp 65 70 75 80 Val Thr Thr Cys Asp Phe Arg Trp Tyr
Gly Pro Lys Tyr Ile Thr His 85 90 95 Ser Ile Arg Ser Phe Thr Pro
Ser Val Glu Gln Cys Lys Glu Ser Ile 100 105 110 Glu Gln Thr Lys Gln
Gly Thr Trp Leu Asn Pro Gly Phe Pro Pro Gln 115 120 125 Ser Cys Gly
Tyr Ala Thr Val Thr Asp Ala Glu Ala Ala Ile Val Gln 130 135 140 Val
Thr Pro His His Val Leu Val Asp Glu Tyr Thr Gly Glu Trp Val 145 150
155 160 Asp Ser Gln Phe Ile Asn Gly Lys Cys Ser Asn Asp Ile Cys Pro
Thr 165 170 175 Val His Asn Ser Thr Thr Trp His Ser Asp Tyr Lys Val
Lys Gly Leu 180 185 190 Cys Asp Ser Asn Leu Ile Ser Met Asp Ile Thr
Phe Phe Ser Glu Asp 195 200 205 Gly Glu Leu Ser Ser Leu Gly Lys Lys
Gly Thr Gly Phe Arg Ser Asn 210 215 220 Tyr Phe Ala Tyr Glu Thr Gly
Asp Lys Ala Cys Lys Met Gln Tyr Cys 225 230 235 240 Lys His Trp Gly
Val Arg Leu Pro Ser Gly Val Trp Phe Glu Met Ala 245 250 255 Asp Lys
Xaa Leu Phe Ala Ala Ala Arg Phe Pro Glu Cys Pro Glu Gly 260 265 270
Ser Ser Ile Ser Ala Pro Ser Gln Thr Ser Val Asp Val Ser Leu Ile 275
280 285 Gln Asp Val Glu Arg Ile Leu Asp Tyr Ser Leu Cys Gln Glu Thr
Trp 290 295 300 Ser Lys Ile Arg Ala Gly Leu Pro Ile Ser Pro Val Asp
Leu Ser Tyr 305 310 315 320 Leu Ala Pro Lys Asn Pro Gly Thr Gly Pro
Val Phe Thr Ile Ile Asn 325 330 335 Gly Thr Leu Lys Tyr Phe Glu Thr
Arg Tyr Ile Arg Val Asp Ile Ala 340 345 350 Ala Pro Ile Leu Ser Arg
Met Val Gly Met Ile Ser Gly Thr Thr Thr 355 360 365 Glu Arg Val Leu
Trp Asp Asp Trp Ala Pro Tyr Glu Asp Val Glu Ile 370 375 380 Gly Pro
Asn Gly Val Leu Arg Thr Ser Ser Gly Tyr Lys Phe Pro Leu 385 390 395
400 Tyr Met Ile Gly His Gly Met Leu Asp Ser Asp Leu His Leu Ser Ser
405 410 415 Lys Ala Gln Val Phe Glu His Pro His Ile Gln Asp Ala Ala
Ser Gln 420 425 430 Leu Pro Asp Gly Glu Thr Leu Phe Phe Gly Asp Thr
Gly Leu Ser Lys 435 440 445 Asn Pro Ile Glu Phe Val Glu Gly Trp Phe
Ser Ser Trp Lys Ser Ser 450 455 460 Ile Ala Ser Phe Phe Phe Thr Ile
Gly Leu Ile Ile Gly Leu Phe Leu 465 470 475 480 Val Leu Arg Val Gly
Ile Tyr Leu Cys Ile Lys Leu Lys His Thr Lys 485 490 495 Lys Arg Gln
Ile Tyr Thr Asp Ile Glu Met Asn Arg Leu Gly Lys 500 505 510
72109PRTVesicular stomatitis Indiana virus 7Met Glu Val His Asp Phe
Glu Thr Asp Glu Phe Asn Asp Phe Asn Glu 1 5 10 15 Asp Asp Tyr Ala
Thr Arg Glu Phe Leu Asn Pro Asp Glu Arg Met Thr 20 25 30 Tyr Leu
Asn His Ala Asp Tyr Asn Leu Asn Ser Pro Leu Ile Ser Asp 35 40 45
Asp Ile Asp Asn Leu Ile Arg Lys Phe Asn Ser Leu Pro Ile Pro Ser 50
55 60 Met Trp Asp Ser Lys Asn Trp Asp Gly Val Leu Glu Met Leu Thr
Ser 65 70 75 80 Cys Gln Ala Asn Pro Ile Ser Thr Ser Gln Met His Lys
Trp Met Gly 85 90 95 Ser Trp Leu Met Ser Asp Asn His Asp Ala Ser
Gln Gly Tyr Ser Phe 100 105 110 Leu His Glu Val Asp Lys Glu Ala Glu
Ile Thr Phe Asp Val Val Glu 115 120 125 Thr Phe Ile Arg Gly Trp Gly
Asn Lys Pro Ile Glu Tyr Ile Lys Lys 130 135 140 Glu Arg Trp Thr Asp
Ser Phe Lys Ile Leu Ala Tyr
Leu Cys Gln Lys 145 150 155 160 Phe Leu Asp Leu His Lys Leu Thr Leu
Ile Leu Asn Ala Val Ser Glu 165 170 175 Val Glu Leu Leu Asn Leu Ala
Arg Thr Phe Lys Gly Lys Val Arg Arg 180 185 190 Ser Ser His Gly Thr
Asn Ile Cys Arg Ile Arg Val Pro Ser Leu Gly 195 200 205 Pro Thr Phe
Ile Ser Glu Gly Trp Ala Tyr Phe Lys Lys Leu Asp Ile 210 215 220 Leu
Met Asp Arg Asn Phe Leu Leu Met Val Lys Asp Val Ile Ile Gly 225 230
235 240 Arg Met Gln Thr Val Leu Ser Met Val Cys Arg Ile Asp Asn Leu
Phe 245 250 255 Ser Glu Gln Asp Ile Phe Ser Leu Leu Asn Ile Tyr Arg
Ile Gly Asp 260 265 270 Lys Ile Val Glu Arg Gln Gly Asn Phe Ser Tyr
Asp Leu Ile Lys Met 275 280 285 Val Glu Pro Ile Cys Asn Leu Lys Leu
Met Lys Leu Ala Arg Glu Ser 290 295 300 Arg Pro Leu Val Pro Gln Phe
Pro His Phe Glu Asn His Ile Lys Thr 305 310 315 320 Ser Val Asp Glu
Gly Ala Lys Ile Asp Arg Gly Ile Arg Phe Leu His 325 330 335 Asp Gln
Ile Met Ser Val Lys Thr Val Asp Leu Thr Leu Val Ile Tyr 340 345 350
Gly Ser Phe Arg His Trp Gly His Pro Phe Ile Asp Tyr Tyr Thr Gly 355
360 365 Leu Glu Lys Leu His Ser Gln Val Thr Met Lys Lys Asp Ile Asp
Val 370 375 380 Ser Tyr Ala Lys Ala Leu Ala Ser Asp Leu Ala Arg Ile
Val Leu Phe 385 390 395 400 Gln Gln Phe Asn Asp His Lys Lys Trp Phe
Val Asn Gly Asp Leu Leu 405 410 415 Pro His Asp His Pro Phe Lys Ser
His Val Lys Glu Asn Thr Trp Pro 420 425 430 Thr Ala Ala Gln Val Gln
Asp Phe Gly Asp Lys Trp His Glu Leu Pro 435 440 445 Leu Ile Lys Cys
Phe Glu Ile Pro Asp Leu Leu Asp Pro Ser Ile Ile 450 455 460 Tyr Ser
Asp Lys Ser His Ser Met Asn Arg Ser Glu Val Leu Lys His 465 470 475
480 Val Arg Met Asn Pro Asn Thr Pro Ile Pro Ser Lys Lys Val Leu Gln
485 490 495 Thr Met Leu Asp Thr Lys Ala Thr Asn Trp Lys Glu Phe Leu
Lys Glu 500 505 510 Ile Asp Glu Lys Gly Leu Asp Asp Asp Asp Leu Ile
Ile Gly Leu Lys 515 520 525 Gly Lys Glu Arg Glu Leu Lys Leu Ala Gly
Arg Phe Phe Ser Leu Met 530 535 540 Ser Trp Lys Leu Arg Glu Tyr Phe
Val Ile Thr Glu Tyr Leu Ile Lys 545 550 555 560 Thr His Phe Val Pro
Met Phe Lys Gly Leu Thr Met Ala Asp Asp Leu 565 570 575 Thr Ala Val
Ile Lys Lys Met Leu Asp Ser Ser Ser Gly Gln Gly Leu 580 585 590 Lys
Ser Tyr Glu Ala Ile Cys Ile Ala Asn His Ile Asp Tyr Glu Lys 595 600
605 Trp Asn Asn His Gln Arg Lys Leu Ser Asn Gly Pro Val Phe Arg Val
610 615 620 Met Gly Gln Phe Leu Gly Tyr Pro Ser Leu Ile Glu Arg Thr
His Glu 625 630 635 640 Phe Phe Glu Lys Ser Leu Ile Tyr Tyr Asn Gly
Arg Pro Asp Leu Met 645 650 655 Arg Val His Asn Asn Thr Leu Ile Asn
Ser Thr Ser Gln Arg Val Cys 660 665 670 Trp Gln Gly Gln Glu Gly Gly
Leu Glu Gly Leu Arg Gln Lys Gly Trp 675 680 685 Ser Ile Leu Asn Leu
Leu Val Ile Gln Arg Glu Ala Lys Ile Arg Asn 690 695 700 Thr Ala Val
Lys Val Leu Ala Gln Gly Asp Asn Gln Val Ile Cys Thr 705 710 715 720
Gln Tyr Lys Thr Lys Lys Ser Arg Asn Val Val Glu Leu Gln Gly Ala 725
730 735 Leu Asn Gln Met Val Ser Asn Asn Glu Lys Ile Met Thr Ala Ile
Lys 740 745 750 Ile Gly Thr Gly Lys Leu Gly Leu Leu Ile Asn Asp Asp
Glu Thr Met 755 760 765 Gln Ser Ala Asp Tyr Leu Asn Tyr Gly Lys Ile
Pro Ile Phe Arg Gly 770 775 780 Val Ile Arg Gly Leu Glu Thr Lys Arg
Trp Ser Arg Val Thr Cys Val 785 790 795 800 Thr Asn Asp Gln Ile Pro
Thr Cys Ala Asn Ile Met Ser Ser Val Ser 805 810 815 Thr Asn Ala Leu
Thr Val Ala His Phe Ala Glu Asn Pro Ile Asn Ala 820 825 830 Met Ile
Gln Tyr Asn Tyr Phe Gly Thr Phe Ala Arg Leu Leu Leu Met 835 840 845
Met His Asp Pro Ala Leu Arg Gln Ser Leu Tyr Glu Val Gln Asp Lys 850
855 860 Ile Pro Gly Leu His Ser Ser Thr Phe Lys Tyr Ala Met Leu Tyr
Leu 865 870 875 880 Asp Pro Ser Ile Gly Gly Val Ser Gly Met Ser Leu
Ser Arg Phe Leu 885 890 895 Ile Arg Ala Phe Pro Asp Pro Val Thr Glu
Ser Leu Ser Phe Trp Arg 900 905 910 Phe Ile His Val His Ala Arg Ser
Glu His Leu Lys Glu Met Ser Ala 915 920 925 Val Phe Gly Asn Pro Glu
Ile Ala Lys Phe Arg Ile Thr His Ile Asp 930 935 940 Lys Leu Val Glu
Asp Pro Thr Ser Leu Asn Ile Ala Met Gly Met Ser 945 950 955 960 Pro
Ala Asn Leu Leu Lys Thr Glu Val Lys Lys Cys Leu Ile Glu Ser 965 970
975 Arg Gln Thr Ile Arg Asn Gln Val Ile Lys Asp Ala Thr Ile Tyr Leu
980 985 990 Tyr His Glu Glu Asp Arg Leu Arg Ser Phe Leu Trp Ser Ile
Asn Pro 995 1000 1005 Leu Phe Pro Arg Phe Leu Ser Glu Phe Lys Ser
Gly Thr Phe Leu 1010 1015 1020 Gly Val Ala Asp Gly Leu Ile Ser Leu
Phe Gln Asn Ser Arg Thr 1025 1030 1035 Ile Arg Asn Ser Phe Lys Lys
Lys Tyr His Arg Glu Leu Asp Asp 1040 1045 1050 Leu Ile Val Arg Ser
Glu Val Ser Ser Leu Thr His Leu Gly Lys 1055 1060 1065 Leu His Leu
Arg Arg Gly Ser Cys Lys Met Trp Thr Cys Ser Ala 1070 1075 1080 Thr
His Ala Asp Thr Leu Arg Tyr Lys Ser Trp Gly Arg Thr Val 1085 1090
1095 Ile Gly Thr Thr Val Pro His Pro Leu Glu Met Leu Gly Pro Gln
1100 1105 1110 His Arg Lys Glu Thr Pro Cys Ala Pro Cys Asn Thr Ser
Gly Phe 1115 1120 1125 Asn Tyr Val Ser Val His Cys Pro Asp Gly Ile
His Asp Val Phe 1130 1135 1140 Ser Ser Arg Gly Pro Leu Pro Ala Tyr
Leu Gly Ser Lys Thr Ser 1145 1150 1155 Glu Ser Thr Ser Ile Leu Gln
Pro Trp Glu Arg Glu Ser Lys Val 1160 1165 1170 Pro Leu Ile Lys Arg
Ala Thr Arg Leu Arg Asp Ala Ile Ser Trp 1175 1180 1185 Phe Val Glu
Pro Asp Ser Lys Leu Ala Met Thr Ile Leu Ser Asn 1190 1195 1200 Ile
His Ser Leu Thr Gly Glu Glu Trp Thr Lys Arg Gln His Gly 1205 1210
1215 Phe Lys Arg Thr Gly Ser Ala Leu His Arg Phe Ser Thr Ser Arg
1220 1225 1230 Met Ser His Gly Gly Phe Ala Ser Gln Ser Thr Ala Ala
Leu Thr 1235 1240 1245 Arg Leu Met Ala Thr Thr Asp Thr Met Arg Asp
Leu Gly Asp Gln 1250 1255 1260 Asn Phe Asp Phe Leu Phe Gln Ala Thr
Leu Leu Tyr Ala Gln Ile 1265 1270 1275 Thr Thr Thr Val Ala Arg Asp
Gly Trp Ile Thr Ser Cys Thr Asp 1280 1285 1290 His Tyr His Ile Ala
Cys Lys Ser Cys Leu Arg Pro Ile Glu Glu 1295 1300 1305 Ile Thr Leu
Asp Ser Ser Met Asp Tyr Thr Pro Pro Asp Val Ser 1310 1315 1320 His
Val Leu Lys Thr Trp Arg Asn Gly Glu Gly Ser Trp Gly Gln 1325 1330
1335 Glu Ile Lys Gln Ile Tyr Pro Leu Glu Gly Asn Trp Lys Asn Leu
1340 1345 1350 Ala Pro Ala Glu Gln Ser Tyr Gln Val Gly Arg Cys Ile
Gly Phe 1355 1360 1365 Leu Tyr Gly Asp Leu Ala Tyr Arg Lys Ser Thr
His Ala Glu Asp 1370 1375 1380 Ser Ser Leu Phe Pro Leu Ser Ile Gln
Gly Arg Ile Arg Gly Arg 1385 1390 1395 Gly Phe Leu Lys Gly Leu Leu
Asp Gly Leu Met Arg Ala Ser Cys 1400 1405 1410 Cys Gln Val Ile His
Arg Arg Ser Leu Ala His Leu Lys Arg Pro 1415 1420 1425 Ala Asn Ala
Val Tyr Gly Gly Leu Ile Tyr Leu Ile Asp Lys Leu 1430 1435 1440 Ser
Val Ser Pro Pro Phe Leu Ser Leu Thr Arg Ser Gly Pro Ile 1445 1450
1455 Arg Asp Glu Leu Glu Thr Ile Pro His Lys Ile Pro Thr Ser Tyr
1460 1465 1470 Pro Thr Ser Asn Arg Asp Met Gly Val Ile Val Arg Asn
Tyr Phe 1475 1480 1485 Lys Tyr Gln Cys Arg Leu Ile Glu Lys Gly Lys
Tyr Arg Ser His 1490 1495 1500 Tyr Ser Gln Leu Trp Leu Phe Ser Asp
Val Leu Ser Ile Asp Phe 1505 1510 1515 Ile Gly Pro Phe Ser Ile Ser
Thr Thr Leu Leu Gln Ile Leu Tyr 1520 1525 1530 Lys Pro Phe Leu Ser
Gly Lys Asp Lys Asn Glu Leu Arg Glu Leu 1535 1540 1545 Ala Asn Leu
Ser Ser Leu Leu Arg Ser Gly Glu Gly Trp Glu Asp 1550 1555 1560 Ile
His Val Lys Phe Phe Thr Lys Asp Ile Leu Leu Cys Pro Glu 1565 1570
1575 Glu Ile Arg His Ala Cys Lys Phe Gly Ile Ala Lys Asp Asn Asn
1580 1585 1590 Lys Asp Met Ser Tyr Pro Pro Trp Gly Arg Glu Ser Arg
Gly Thr 1595 1600 1605 Ile Thr Thr Ile Pro Val Tyr Tyr Thr Thr Thr
Pro Tyr Pro Lys 1610 1615 1620 Met Leu Glu Met Pro Pro Arg Ile Gln
Asn Pro Leu Leu Ser Gly 1625 1630 1635 Ile Arg Leu Gly Gln Leu Pro
Thr Gly Ala His Tyr Lys Ile Arg 1640 1645 1650 Ser Ile Leu His Gly
Met Gly Ile His Tyr Arg Asp Phe Leu Ser 1655 1660 1665 Cys Gly Asp
Gly Ser Gly Gly Met Thr Ala Ala Leu Leu Arg Glu 1670 1675 1680 Asn
Val His Ser Arg Gly Ile Phe Asn Ser Leu Leu Glu Leu Ser 1685 1690
1695 Gly Ser Val Met Arg Gly Ala Ser Pro Glu Pro Pro Ser Ala Leu
1700 1705 1710 Glu Thr Leu Gly Gly Asp Lys Ser Arg Cys Val Asn Gly
Glu Thr 1715 1720 1725 Cys Trp Glu Tyr Pro Ser Asp Leu Cys Asp Pro
Arg Thr Trp Asp 1730 1735 1740 Tyr Phe Leu Arg Leu Lys Ala Gly Leu
Gly Leu Gln Ile Asp Leu 1745 1750 1755 Ile Val Met Asp Met Glu Val
Arg Asp Ser Ser Thr Ser Leu Lys 1760 1765 1770 Ile Glu Thr Asn Val
Arg Asn Tyr Val His Arg Ile Leu Asp Glu 1775 1780 1785 Gln Gly Val
Leu Ile Tyr Lys Thr Tyr Gly Thr Tyr Ile Cys Glu 1790 1795 1800 Ser
Glu Lys Asn Ala Val Thr Ile Leu Gly Pro Met Phe Lys Thr 1805 1810
1815 Val Asp Leu Val Gln Thr Glu Phe Ser Ser Ser Gln Thr Ser Glu
1820 1825 1830 Val Tyr Met Val Cys Lys Gly Leu Lys Lys Leu Ile Asp
Glu Pro 1835 1840 1845 Asn Pro Asp Trp Ser Ser Ile Asn Glu Ser Trp
Lys Asn Leu Tyr 1850 1855 1860 Ala Phe Gln Ser Ser Glu Gln Glu Phe
Ala Arg Ala Lys Lys Val 1865 1870 1875 Ser Thr Tyr Phe Thr Leu Thr
Gly Ile Pro Ser Gln Phe Ile Pro 1880 1885 1890 Asp Pro Phe Val Asn
Ile Glu Thr Met Leu Gln Ile Phe Gly Val 1895 1900 1905 Pro Thr Gly
Val Ser His Ala Ala Ala Leu Lys Ser Ser Asp Arg 1910 1915 1920 Pro
Ala Asp Leu Leu Thr Ile Ser Leu Phe Tyr Met Ala Ile Ile 1925 1930
1935 Ser Tyr Tyr Asn Ile Asn His Ile Arg Val Gly Pro Ile Pro Pro
1940 1945 1950 Asn Pro Pro Ser Asp Gly Ile Ala Gln Asn Val Gly Ile
Ala Ile 1955 1960 1965 Thr Gly Ile Ser Phe Trp Leu Ser Leu Met Glu
Lys Asp Ile Pro 1970 1975 1980 Leu Tyr Gln Gln Cys Leu Ala Val Ile
Gln Gln Ser Phe Pro Ile 1985 1990 1995 Arg Trp Glu Ala Val Ser Val
Lys Gly Gly Tyr Lys Gln Lys Trp 2000 2005 2010 Ser Thr Arg Gly Asp
Gly Leu Pro Lys Asp Thr Arg Ile Ser Asp 2015 2020 2025 Ser Leu Ala
Pro Ile Gly Asn Trp Ile Arg Ser Leu Glu Leu Val 2030 2035 2040 Arg
Asn Gln Val Arg Leu Asn Pro Phe Asn Glu Ile Leu Phe Asn 2045 2050
2055 Gln Leu Cys Arg Thr Val Asp Asn His Leu Lys Trp Ser Asn Leu
2060 2065 2070 Arg Arg Asn Thr Gly Met Ile Glu Trp Ile Asn Arg Arg
Ile Ser 2075 2080 2085 Lys Glu Asp Arg Ser Ile Leu Met Leu Lys Ser
Asp Leu His Glu 2090 2095 2100 Glu Asn Ser Trp Arg Asp 2105
811161DNAVesicular stomatitis Indiana virus 8acgaagacaa acaaaccatt
attatcatta aaaggctcag gagaaacttt aacagtaatc 60aaaatgtctg ttacagtcaa
gagaatcatt gacaacacag tcatagttcc aaaacttcct 120gcaaatgagg
atccagtgga atacccggca gattacttca gaaaatcaaa ggagattcct
180ctttacatca atactacaaa aagtttgtca gatctaagag gatatgtcta
ccaaggcctc 240aaatccggaa atgtatcaat catacatgtc aacagctact
tgtatggagc attgaaggac 300atccggggta agttggataa agattggtca
agtttcggaa taaacatcgg gaaggcaggg 360gatacaatcg gaatatttga
ccttgtatcc ttgaaagccc tggacggtgt acttccagat 420ggagtatcgg
atgcttccag aaccagcgca gatgacaaat ggttgccttt gtatctactt
480ggcttataca gagtgggcag aacacaaatg cctgaataca gaaaaaggct
catggatggg 540ctgacaaatc aatgcaaaat gatcaatgaa cagtttgaac
ctcttgtgcc agaaggtcgt 600gacatttttg atgtgtgggg aaatgacagt
aattacacaa aaattgtcgc tgcagtggac 660atgttcttcc acatgttcaa
aaaacatgaa tgtgcctcgt tcagatacgg aactattgtt 720tccagattca
aagattgtgc tgcattggca acatttggac acctctgcaa aataaccgga
780atgtctacag aagatgtgac gacctggatc ttgaaccgag aagttgcaga
tgagatggtc 840caaatgatgc ttccaggcca agaaattgac aaggctgatt
catacatgcc ttatttgatc 900gactttggat tgtcttctaa gtctccatat
tcttccgtca aaaaccctgc cttccacttc 960tgggggcaat tgacagctct
tctgctcaga tccaccagag caaggaatgc ccgacagcct 1020gatgacattg
agtatacatc tcttactaca gcaggtttgt tgtacgctta tgcagtagga
1080tcctctgctg acttggcaca acagttttgt gttggagata gcaaatacac
tccagatgat 1140agtaccggag gattgacgac taatgcaccg ccacaaggca
gagatgtggt cgaatggctc 1200ggatggtttg aagatcaaaa cagaaaaccg
actcctgata tgatgcagta tgcgaaacga 1260gcagtcatgt cactgcaagg
cctaagagag aagacaattg gcaagtatgc taagtcagag 1320tttgacaaat
gaccctataa ttctcagatc acctattata tattatgcta catatgaaaa
1380aaactaacag atatcatgga taatctcaca aaagttcgtg agtatctcaa
gtcctattct 1440cgtctagatc aggcggtagg agagatagat gagatcgaag
cacaacgagc tgaaaagtcc 1500aattatgagt tgttccaaga ggacggagtg
gaagagcata ctaggccctc ttattttcag 1560gcagcagatg attctgacac
agaatctgaa ccagaaattg aagacaatca aggcttgtat 1620gtaccagatc
cggaagctga gcaagttgaa ggctttatac aggggccttt agatgactat
1680gcagatgagg acgtggatgt tgtattcact tcggactgga aacagcctga
gcttgaatcc 1740gacgagcatg gaaagacctt acggttgaca ttgccagagg
gtttaagtgg agagcagaaa 1800tcccagtggc ttttgacgat taaagcagtc
gttcaaagtg ccaaacactg gaatctggca
1860gagtgcacat ttgaagcatc gggagaaggg gtcatcataa aaaagcgcca
gataactccg 1920gatgtatata aggtcactcc agtgatgaac acacatccgt
accaatcaga agccgtatca 1980gatgtttggt ctctctcaaa gacatccatg
actttccaac ccaagaaagc aagtcttcag 2040cctctcacca tatccttgga
tgaattgttc tcatctagag gagaattcat ctctgtcgga 2100ggtaacggac
gaatgtctca taaagaggcc atcctgctcg gtctgaggta caaaaagttg
2160tacaatcagg cgagagtcaa atattctctg tagactatga aaaaaagtaa
cagatatcac 2220aatctaagtg ttatcccaat ccattcatca tgagttcctt
aaagaagatt ctcggtctga 2280aggggaaagg taagaaatct aagaaattag
ggatcgcacc acccccttat gaagaggaca 2340ctaacatgga gtatgctccg
agcgctccaa ttgacaaatc ctattttgga gttgacgaga 2400tggacactca
tgatccgaat caattaagat atgagaaatt cttctttaca gtgaaaatga
2460cggttagatc taatcgtccg ttcagaacat actcagatgt ggcagccgct
gtatcccatt 2520gggatcacat gtacatcgga atggcaggga aacgtccctt
ctacaagatc ttggcttttt 2580tgggttcttc taatctaaag gccactccag
cggtattggc agatcaaggt caaccagagt 2640atcatgctca ctgtgaaggc
agggcttatt tgccacacag aatggggaag acccctccca 2700tgctcaatgt
accagagcac ttcagaagac cattcaatat aggtctttac aagggaacga
2760ttgagctcac aatgaccatc tacgatgatg agtcactgga agcagctcct
atgatctggg 2820atcatttcaa ttcttccaaa ttttctgatt tcagagagaa
ggccttaatg tttggcctga 2880ttgtcgagaa aaaggcatct ggagcttggg
tcctggattc tgtcagccac ttcaaatgag 2940ctagtctagc ttccagcttc
tgaacaatcc ccggtttact cagtctctcc taattccagc 3000ctttcgaaca
actaatatcc tgtcttctct atcccgatga aaaaaactaa cagagatcga
3060tctgtttcct tgacaccatg aagtgccttt tgtacttagc ttttttattc
atcggggtga 3120attgcaagtt caccatagtt tttccacaca accaaaaagg
aaactggaaa aatgttcctt 3180ccaattacca ttattgcccg tcaagctcag
atttaaattg gcataatgac ttagtaggca 3240cagccttaca agtcaaaatg
cccaagagtc acaaggctat tcaagcagac ggttggatgt 3300gtcatgcttc
caaatgggtc actacttgtg atttccgctg gtacggaccg aagtatataa
3360cacattccat ccgatccttc actccatctg tagaacaatg caaggaaagc
attgaacaaa 3420cgaaacaagg aacttggctg aatccaggct tccctcctca
aagttgtgga tatgcaactg 3480tgacggatgc tgaagcagcg attgtccagg
tgactcctca ccatgtgctt gttgatgaat 3540acacaggaga atgggttgat
tcacagttca tcaacggaaa atgcagcaat gacatatgcc 3600ccactgtcca
taactccaca acctggcatt ccgactataa ggtcaaaggg ctatgtgatt
3660ctaacctcat ttccatggac atcaccttct tctcagagga cggagagcta
tcatccctag 3720gaaagaaggg cacagggttc agaagtaact actttgctta
tgaaactgga gacaaggcct 3780gcaaaatgca gtactgcaag cattggggag
tcagactccc atcaggtgtc tggttcgaga 3840tggctgataa ggmtctcttt
gctgcagcca gattccctga atgcccagaa gggtcaagta 3900tctctgctcc
atctcagacc tcagtggatg taagtctcat tcaggacgtt gagaggatct
3960tggattattc cctctgccaa gaaacctgga gcaaaatcag agcgggtctt
cccatctctc 4020cagtggatct cagctatctt gctcctaaaa acccaggaac
cggtcctgtc tttaccataa 4080tcaatggtac cctaaaatac tttgagacca
gatacatcag agtcgatatt gctgctccaa 4140tcctctcaag aatggtcgga
atgatcagtg gaactaccac agaaagggta ctgtgggatg 4200actgggctcc
atatgaagac gtggaaattg gacccaatgg agttctgagg accagttcag
4260gatataagtt tcctttatat atgattggac atggtatgtt ggactccgat
cttcatctta 4320gctcaaaggc tcaggtgttt gaacatcctc acattcaaga
cgctgcttcg cagcttcctg 4380atggtgagac tttatttttt ggtgatactg
ggctatccaa aaatccaatc gagtttgtag 4440aaggttggtt cagtagttgg
aagagctcta ttgcctcttt tttctttacc atagggttaa 4500tcattggact
attcttggtt ctccgagttg gtatttatct ttgcattaaa ttaaagcaca
4560ccaagaaaag acagatttat acagacatag agatgaaccg acttggaaag
taactcaaat 4620cctgcacaac agattcttca tgtttgaacc aaatcaactt
gtgatatcat gctcaaagag 4680gccttaatta tattttaatt tttaattttt
atgaaaaaaa ctaacagcaa tcatggaagt 4740ccacgatttt gagaccgacg
agttcaatga tttcaatgaa gatgactatg ccacaagaga 4800attcctgaat
cccgatgagc gcatgacgta cttgaatcat gctgattaca atttgaattc
4860tcctctaatt agtgatgata ttgacaattt gatcaggaaa ttcaattctc
ttccgattcc 4920ctcgatgtgg gatagtaaga actgggatgg agttcttgag
atgttaacat catgtcaagc 4980caatcccatc tcaacatctc agatgcataa
atggatggga agttggttaa tgtctgataa 5040tcatgatgcc agtcaagggt
atagtttttt acatgaagtg gacaaagagg cagaaataac 5100atttgacgtg
gtggagacct tcatccgcgg ctggggcaac aaaccaattg aatacatcaa
5160aaaggaaaga tggactgact cattcaaaat tctcgcttat ttgtgtcaaa
agtttttgga 5220cttacacaag ttgacattaa tcttaaatgc tgtctctgag
gtggaattgc tcaacttggc 5280gaggactttc aaaggcaaag tcagaagaag
ttctcatgga acgaacatat gcaggattag 5340ggttcccagc ttgggtccta
cttttatttc agaaggatgg gcttacttca agaaacttga 5400tattctaatg
gaccgaaact ttctgttaat ggtcaaagat gtgattatag ggaggatgca
5460aacggtgcta tccatggtat gtagaataga caacctgttc tcagagcaag
acatcttctc 5520ccttctaaat atctacagaa ttggagataa aattgtggag
aggcagggaa atttttctta 5580tgacttgatt aaaatggtgg aaccgatatg
caacttgaag ctgatgaaat tagcaagaga 5640atcaaggcct ttagtcccac
aattccctca ttttgaaaat catatcaaga cttctgttga 5700tgaaggggca
aaaattgacc gaggtataag attcctccat gatcagataa tgagtgtgaa
5760aacagtggat ctcacactgg tgatttatgg atcgttcaga cattggggtc
atccttttat 5820agattattac actggactag aaaaattaca ttcccaagta
accatgaaga aagatattga 5880tgtgtcatat gcaaaagcac ttgcaagtga
tttagctcgg attgttctat ttcaacagtt 5940caatgatcat aaaaagtggt
tcgtgaatgg agacttgctc cctcatgatc atccctttaa 6000aagtcatgtt
aaagaaaata catggcctac agctgctcaa gttcaagatt ttggagataa
6060atggcatgaa cttccgctga ttaaatgttt tgaaataccc gacttactag
acccatcgat 6120aatatactct gacaaaagtc attcaatgaa taggtcagag
gtgttgaaac atgtccgaat 6180gaatccgaac actcctatcc ctagtaaaaa
ggtgttgcag actatgttgg acacaaaggc 6240taccaattgg aaagaatttc
ttaaagagat tgatgagaag ggcttagatg atgatgatct 6300aattattggt
cttaaaggaa aggagaggga actgaagttg gcaggtagat ttttctccct
6360aatgtcttgg aaattgcgag aatactttgt aattaccgaa tatttgataa
agactcattt 6420cgtccctatg tttaaaggcc tgacaatggc ggacgatcta
actgcagtca ttaaaaagat 6480gttagattcc tcatccggcc aaggattgaa
gtcatatgag gcaatttgca tagccaatca 6540cattgattac gaaaaatgga
ataaccacca aaggaagtta tcaaacggcc cagtgttccg 6600agttatgggc
cagttcttag gttatccatc cttaatcgag agaactcatg aattttttga
6660gaaaagtctt atatactaca atggaagacc agacttgatg cgtgttcaca
acaacacact 6720gatcaattca acctcccaac gagtttgttg gcaaggacaa
gagggtggac tggaaggtct 6780acggcaaaaa ggatggagta tcctcaatct
actggttatt caaagagagg ctaaaatcag 6840aaacactgct gtcaaagtct
tggcacaagg tgataatcaa gttatttgca cacagtataa 6900aacgaagaaa
tcgagaaacg ttgtagaatt acagggtgct ctcaatcaaa tggtttctaa
6960taatgagaaa attatgactg caatcaaaat agggacaggg aagttaggac
ttttgataaa 7020tgacgatgag actatgcaat ctgcagatta cttgaattat
ggaaaaatac cgattttccg 7080tggagtgatt agagggttag agaccaagag
atggtcacga gtgacttgtg tcaccaatga 7140ccaaataccc acttgtgcta
atataatgag ctcagtttcc acaaatgctc tcaccgtagc 7200tcattttgct
gagaacccaa tcaatgccat gatacagtac aattattttg ggacatttgc
7260tagactcttg ttgatgatgc atgatcctgc tcttcgtcaa tcattgtatg
aagttcaaga 7320taagataccg ggcttgcaca gttctacttt caaatacgcc
atgttgtatt tggacccttc 7380cattggagga gtgtcgggca tgtctttgtc
caggtttttg attagagcct tcccagatcc 7440cgtaacagaa agtctctcat
tctggagatt catccatgta catgctcgaa gtgagcatct 7500gaaggagatg
agtgcagtat ttggaaaccc cgagatagcc aagtttcgaa taactcacat
7560agacaagcta gtagaagatc caacctctct gaacatcgct atgggaatga
gtccagcgaa 7620cttgttaaag actgaggtta aaaaatgctt aatcgaatca
agacaaacca tcaggaacca 7680ggtgattaag gatgcaacca tatatttgta
tcatgaagag gatcggctca gaagtttctt 7740atggtcaata aatcctctgt
tccctagatt tttaagtgaa ttcaaatcag gcactttttt 7800gggagtcgca
gacgggctca tcagtctatt tcaaaattct cgtactattc ggaactcctt
7860taagaaaaag tatcataggg aattggatga tttgattgtg aggagtgagg
tatcctcttt 7920gacacattta gggaaacttc atttgagaag gggatcatgt
aaaatgtgga catgttcagc 7980tactcatgct gacacattaa gatacaaatc
ctggggccgt acagttattg ggacaactgt 8040accccatcca ttagaaatgt
tgggtccaca acatcgaaaa gagactcctt gtgcaccatg 8100taacacatca
gggttcaatt atgtttctgt gcattgtcca gacgggatcc atgacgtctt
8160tagttcacgg ggaccattgc ctgcttatct agggtctaaa acatctgaat
ctacatctat 8220tttgcagcct tgggaaaggg aaagcaaagt cccactgatt
aaaagagcta cacgtcttag 8280agatgctatc tcttggtttg ttgaacccga
ctctaaacta gcaatgacta tactttctaa 8340catccactct ttaacaggcg
aagaatggac caaaaggcag catgggttca aaagaacagg 8400gtctgccctt
cataggtttt cgacatctcg gatgagccat ggtgggttcg catctcagag
8460cactgcagca ttgaccaggt tgatggcaac tacagacacc atgagggatc
tgggagatca 8520gaatttcgac tttttattcc aagcaacgtt gctctatgct
caaattacca ccactgttgc 8580aagagacgga tggatcacca gttgtacaga
tcattatcat attgcctgta agtcctgttt 8640gagacccata gaagagatca
ccctggactc aagtatggac tacacgcccc cagatgtatc 8700ccatgtgctg
aagacatgga ggaatgggga aggttcgtgg ggacaagaga taaaacagat
8760ctatccttta gaagggaatt ggaagaattt agcacctgct gagcaatcct
atcaagtcgg 8820cagatgtata ggttttctat atggagactt ggcgtataga
aaatctactc atgccgagga 8880cagttctcta tttcctctat ctatacaagg
tcgtattaga ggtcgaggtt tcttaaaagg 8940gttgctagac ggattaatga
gagcaagttg ctgccaagta atacaccgga gaagtctggc 9000tcatttgaag
aggccggcca acgcagtgta cggaggtttg atttacttga ttgataaatt
9060gagtgtatca cctccattcc tttctcttac tagatcagga cctattagag
acgaattaga 9120aacgattccc cacaagatcc caacctccta tccgacaagc
aaccgtgata tgggggtgat 9180tgtcagaaat tacttcaaat accaatgccg
tctaattgaa aagggaaaat acagatcaca 9240ttattcacaa ttatggttat
tctcagatgt cttatccata gacttcattg gaccattctc 9300tatttccacc
accctcttgc aaatcctata caagccattt ttatctggga aagataagaa
9360tgagttgaga gagctggcaa atctttcttc attgctaaga tcaggagagg
ggtgggaaga 9420catacatgtg aaattcttca ccaaggacat attattgtgt
ccagaggaaa tcagacatgc 9480ttgcaagttc gggattgcta aggataataa
taaagacatg agctatcccc cttggggaag 9540ggaatccaga gggacaatta
caacaatccc tgtttattat acgaccaccc cttacccaaa 9600gatgctagag
atgcctccaa gaatccaaaa tcccctgctg tccggaatca ggttgggcca
9660attaccaact ggcgctcatt ataaaattcg gagtatatta catggaatgg
gaatccatta 9720cagggacttc ttgagttgtg gagacggctc cggagggatg
actgctgcat tactacgaga 9780aaatgtgcat agcagaggaa tattcaatag
tctgttagaa ttatcagggt cagtcatgcg 9840aggcgcctct cctgagcccc
ccagtgccct agaaacttta ggaggagata aatcgagatg 9900tgtaaatggt
gaaacatgtt gggaatatcc atctgactta tgtgacccaa ggacttggga
9960ctatttcctc cgactcaaag caggcttggg gcttcaaatt gatttaattg
taatggatat 10020ggaagttcgg gattcttcta ctagcctgaa aattgagacg
aatgttagaa attatgtgca 10080ccggattttg gatgagcaag gagttttaat
ctacaagact tatggaacat atatttgtga 10140gagcgaaaag aatgcagtaa
caatccttgg tcccatgttc aagacggtcg acttagttca 10200aacagaattt
agtagttctc aaacgtctga agtatatatg gtatgtaaag gtttgaagaa
10260attaatcgat gaacccaatc ccgattggtc ttccatcaat gaatcctgga
aaaacctgta 10320cgcattccag tcatcagaac aggaatttgc cagagcaaag
aaggttagta catactttac 10380cttgacaggt attccctccc aattcattcc
tgatcctttt gtaaacattg agactatgct 10440acaaatattc ggagtaccca
cgggtgtgtc tcatgcggct gccttaaaat catctgatag 10500acctgcagat
ttattgacca ttagcctttt ttatatggcg attatatcgt attataacat
10560caatcatatc agagtaggac cgatacctcc gaacccccca tcagatggaa
ttgcacaaaa 10620tgtggggatc gctataactg gtataagctt ttggctgagt
ttgatggaga aagacattcc 10680actatatcaa cagtgtttag cagttatcca
gcaatcattc ccgattaggt gggaggctgt 10740ttcagtaaaa ggaggataca
agcagaagtg gagtactaga ggtgatgggc tcccaaaaga 10800tacccgaatt
tcagactcct tggccccaat cgggaactgg atcagatctc tggaattggt
10860ccgaaaccaa gttcgtctaa atccattcaa tgagatcttg ttcaatcagc
tatgtcgtac 10920agtggataat catttgaaat ggtcaaattt gcgaagaaac
acaggaatga ttgaatggat 10980caatagacga atttcaaaag aagaccggtc
tatactgatg ttgaagagtg acctacacga 11040ggaaaactct tggagagatt
aaaaaatcat gaggagactc caaactttaa gtatgaaaaa 11100aactttgatc
cttaagaccc tcttgtggtt tttatttttt atctggtttt gtggtcttcg 11160t
1116191489DNAVesicular stomatitis indiana virus 9atagtcgaga
cgacgaagac aaacaaacca ttattatcat taaaaggctc aggagaaact 60ttaacagtaa
tcaaaatgtc tgttacagtc aagagaatca ttgacaacac agtcatagtt
120ccaaaacttc ctgcaaatga ggatccagtg gaatacccgg cagattactt
cagaaaatca 180aaggagattc ctctttacat caatactaca aaaagtttgt
cagatctaag aggatatgtc 240taccaaggcc tcaaatccgg aaatgtatca
atcatacatg tcaacagcta cttgtatgga 300gcattgaagg acatccgggg
taagttggat aaagattggt caagtttcgg aataaacatc 360gggaaggcag
gggatacaat cggaatattt gaccttgtat ccttgaaagc cctggacggt
420gtacttccag atggagtatc ggatgcttcc agaaccagcg cagatgacaa
atggttgcct 480ttgtatctac ttggcttata cagagtgggc agaacacaaa
tgcctgaata cagaaaaagg 540ctcatggatg ggctgacaaa tcaatgcaaa
atgatcaatg aacagtttga acctcttgtg 600ccagaaggtc gtgacatttt
tgatgtgtgg ggaaatgaca gtaattacac aaaaattgtc 660gctgcagtgg
acatgttctt ccacatgttc aaaaaacatg aatgtgcctc gttcagatac
720ggaactattg tttccagatt caaagattgt gctgcattgg caacatttgg
acacctctgc 780aaaataaccg gaatgtctac agaagatgtg acgacctgga
tcttgaaccg agaagttgca 840gatgagatgg tccaaatgat gcttccaggc
caagaaattg acaaggctga ttcatacatg 900ccttatttga tcgactttgg
attgtcttct aagtctccat attcttccgt caaaaaccct 960gccttccact
tctgggggca attgacagct cttctgctca gatccaccag agcaaggaat
1020gcccgacagc ctgatgacat tgagtataca tctcttacta cagcaggttt
gttgtacgct 1080tatgcagtag gatcctctgc tgacttggca caacagtttt
gtgttggaga tagcaaatac 1140actccagatg atagtaccgg aggattgacg
actaatgcac cgccacaagg cagagatgtg 1200gtcgaatggc tcggatggtt
tgaagatcaa aacagaaaac cgactcctga tatgatgcag 1260tatgcgaaac
gagcagtcat gtcactgcaa ggcctaagag agaagacaat tggcaagtat
1320gctaagtcag agtttgacaa atgaccctat aattctcaga tcacctatta
tatattatgc 1380tagcttgttc gaactcttaa ttaacgcccc gagtatgtcg
acgtacttaa gaccctcttg 1440tggtttttat tttttatctg gttttgtggt
cttcgtcgtc tccggccgg 1489101645DNAVesicular stomatitis indiana
virus 10gctagctatg aaaaaaacta acagatatca tggataatct cacaaaagtt
cgtgagtatc 60tcaagtccta ttctcgtcta gatcaggcgg taggagagat agatgagatc
gaagcacaac 120gagctgaaaa gtccaattat gagttgttcc aagaggacgg
agtggaagag catactaggc 180cctcttattt tcaggcagca gatgattctg
acacagaatc tgaaccagaa attgaagaca 240atcaaggctt gtatgtacca
gatccggaag ctgagcaagt tgaaggcttt atacaggggc 300ctttagatga
ctatgcagat gaggacgtgg atgttgtatt cacttcggac tggaaacagc
360ctgagcttga atccgacgag catggaaaga ccttacggtt gacattgcca
gagggtttaa 420gtggagagca gaaatcccag tggcttttga cgattaaagc
agtcgttcaa agtgccaaac 480actggaatct ggcagagtgc acatttgaag
catcgggaga aggggtcatc ataaaaaagc 540gccagataac tccggatgta
tataaggtca ctccagtgat gaacacacat ccgtcccaat 600cagaagccgt
atcagatgtt tggtctctct caaagacatc catgactttc caacccaaga
660aagcaagtct tcagcctctc accatatcct tggatgaatt gttctcatct
agaggagaat 720tcatctctgt cggaggtaac ggacgaatgt ctcataaaga
ggccatcctg ctcggtctga 780ggtacaaaaa gttgtacaat caggcgagag
tcaaatattc tctgtagact agtatgaaaa 840aaagtaacag atatcacaat
ctaagtgtta tcccaatcca ttcatcatga gttccttaaa 900gaagattctc
ggtctgaagg ggaaaggtaa gaaatctaag aaattaggga tcgcaccacc
960cccttatgaa gaggacacta acatggagta tgctccgagc gctccaattg
acaaatccta 1020ttttggagtt gacgagatgg acactcatga tccgaatcaa
ttaagatatg agaaattctt 1080ctttacagtg aaaatgacgg ttagatctaa
tcgtccgttc agaacatact cagatgtggc 1140agccgctgta tcccattggg
atcacatgta catcggaatg gcagggaaac gtcccttcta 1200caagatcttg
gcttttttgg gttcttctaa tctaaaggcc actccagcgg tattggcaga
1260tcaaggtcaa ccagagtatc atgctcactg tgaaggcagg gcttatttgc
cacacagaat 1320ggggaagacc cctcccatgc tcaatgtacc agagcacttc
agaagaccat tcaatatagg 1380tctttacaag ggaacgattg agctcacaat
gaccatctac gatgatgagt cactggaagc 1440agctcctatg atctgggatc
atttcaattc ttccaaattt tctgatttca gagagaaggc 1500cttaatgttt
ggcctgattg tcgagaaaaa ggcatctgga gcttgggtcc tggattctgt
1560cagccacttc aaatgagcta gtctagcttc cagcttctga acaatccccg
gtttactcag 1620tctctcctaa ttccagcctt tcgaa 1645111645DNAVesicular
stomatitis indiana virus 11gctagctatg aaaaaaacta acagatatca
tggataatct cacaaaagtt cgtgagtatc 60tcaagtccta ttctcgtcta gatcaggcgg
taggagagat agatgagatc gaagcacaac 120gagctgaaaa gtccaattat
gagttgttcc aagaggacgg agtggaagag catactaggc 180cctcttattt
tcaggcagca gatgattctg acacagaatc tgaaccagaa attgaagaca
240atcaaggctt gtatgtacca gatccggaag ctgagcaagt tgaaggcttt
atacaggggc 300ctttagatga ctatgcagat gaggacgtgg atgttgtatt
cacttcggac tggaaacagc 360ctgagcttga atccgacgag catggaaaga
ccttacggtt gacattgcca gagggtttaa 420gtggagagca gaaatcccag
tggcttttga cgattaaagc agtcgttcaa agtgccaaac 480actggaatct
ggcagagtgc acatttgaag catcgggaga aggggtcatc ataaaaaagc
540gccagataac tccggatgta tataaggtca ctccagtgat gaacacacat
ccgtcccaat 600cagaagccgt atcagatgtt tggtctctct caaagacatc
catgactttc caacccaaga 660aagcaagtct tcagcctctc accatatcct
tggatgaatt gttctcatct agaggagaat 720tcatctctgt cggaggtaac
ggacgaatgt ctcataaaga ggccatcctg ctcggtctga 780ggtacaaaaa
gttgtacaat caggcgagag tcaaatattc tctgtagact agtatgaaaa
840aaagtaacag atatcacaat ctaagtgtta tcccaatcca ttcatcatga
gttccttaaa 900gaagattctc ggtctgaagg ggaaaggtaa gaaatctaag
aaattaggga tcgcaccacc 960cccttatgaa gaggacacta acatggagta
tgctccgagc gctccaattg acaaatccta 1020ttttggagtt gacgagatgg
acactcatga tccgaatcaa ttaagatatg agaaattctt 1080ctttacagtg
aaaatgacgg ttagatctaa tcgtccgttc agaacatact cagatgtggc
1140agccgctgta tcccattggg atcacatgta catcggaatg gcagggaaac
gtcccttcta 1200caagatcttg gcttttttgg gttcttctaa tctaaaggcc
actccagcgg tattggcaga 1260tcaaggtcaa ccagagtatc atgctcactg
tgaaggcagg gcttatttgc cacacagaat 1320ggggaagacc cctcccatgc
tcaatgtacc agagcacttc agaagaccat tcaatatagg 1380tctttacaag
ggaacgattg agctcacaat gaccatctac gatgatgagt cactggaagc
1440agctcctatg atctgggatc atttcaattc ttccaaattt tctgatttca
gagagaaggc 1500cttaatgttt ggcctgattg tcgagaaaaa ggcatctgga
gcttgggtcc tggattctgt 1560cagccacttc aaatgagcta gtctagcttc
cagcttctga acaatccccg gtttactcag 1620tctctcctaa ttccagcctt tcgaa
1645122851DNAVesicular stomatitis indiana virus 12ttaattaaat
tttaattttt aatttttatg aaaaaaacta acagcaatca tggaagtcca 60cgattttgag
accgacgagt tcaatgattt caatgaagat gactatgcca caagagaatt
120cctgaatccc gatgagcgca tgacgtactt gaatcatgct gattacaatt
tgaattctcc 180tctaattagt gatgatattg acaatttgat caggaaattc
aattctcttc cgattccctc 240gatgtgggat agtaagaact gggatggagt
tcttgagatg ttaacatcat gtcaagccaa 300tcccatctca acatctcaga
tgcataaatg gatgggaagt tggttaatgt ctgataatca 360tgatgccagt
caagggtata gttttttaca tgaagtggac aaagaggcag aaataacatt
420tgacgtggtg gagaccttca tccgcggctg gggcaacaaa ccaattgaat
acatcaaaaa 480ggaaagatgg actgactcat tcaaaattct cgcttatttg
tgtcaaaagt ttttggactt 540acacaagttg acattaatct taaatgctgt
ctctgaggtg gaattgctca acttggcgag 600gactttcaaa ggcaaagtca
gaagaagttc tcatggaacg aacatatgca ggcttagggt 660tcccagcttg
ggtcctactt
ttatttcaga aggatgggct tacttcaaga aacttgatat 720tctaatggac
cgaaactttc tgttaatggt caaagatgtg attataggga ggatgcaaac
780ggtgctatcc atggtatgta gaatagacaa cctgttctca gagcaagaca
tcttctccct 840tctaaatatc tacagaattg gagataaaat tgtggagagg
cagggaaatt tttcttatga 900cttgattaaa atggtggaac cgatatgcaa
cttgaagctg atgaaattag caagagaatc 960aaggccttta gtcccacaat
tccctcattt tgaaaatcat atcaagactt ctgttgatga 1020aggggcaaaa
attgaccgag gtataagatt cctccatgat cagataatga gtgtgaaaac
1080agtggatctc acactggtga tttatggatc gttcagacat tggggtcatc
cttttataga 1140ttattacgct ggactagaaa aattacattc ccaagtaacc
atgaagaaag atattgatgt 1200gtcatatgca aaagcacttg caagtgattt
agctcggatt gttctatttc aacagttcaa 1260tgatcataaa aagtggttcg
tgaatggaga cttgctccct catgatcatc cctttaaaag 1320tcatgttaaa
gaaaatacat ggcctacagc tgctcaagtt caagattttg gagataaatg
1380gcatgaactt ccgctgatta aatgttttga aatacccgac ttactagacc
catcgataat 1440atactctgac aaaagtcatt caatgaatag gtcagaggtg
ttgaaacatg tccgaatgaa 1500tccgaacact cctatcccta gtaaaaaggt
gttgcagact atgttggaca caaaggctac 1560caattggaaa gaatttctta
aagagattga tgagaagggc ttagatgatg atgatctaat 1620tattggtctt
aaaggaaagg agagggaact gaagttggca ggtagatttt tctccctaat
1680gtcttggaaa ttgcgagaat actttgtaat taccgaatat ttgataaaga
ctcatttcgt 1740ccctatgttt aaaggcctga caatggcgga cgatctaact
gcagtcatta aaaagatgtt 1800agattcctca tccggccaag gattgaagtc
atatgaggca atttgcatag ccaatcacat 1860tgattacgaa aaatggaata
accaccaaag gaagttatca aacggcccag tgttccgagt 1920tatgggccag
ttcttaggtt atccatcctt aatcgagaga actcatgaat tttttgagaa
1980aagtcttata tactacaatg gaagaccaga cttgatgcgt gttcacaaca
acacactgat 2040caattcaacc tcccaacgag tttgttggca aggacaagag
ggtggactgg aaggtctacg 2100gcaaaaagga tggagtatcc tcaatctact
ggttattcaa agagaggcta aaatcagaaa 2160cactgctgtc aaagtcttgg
cacaaggtga taatcaagtt atttgcacac agtataaaac 2220gaagaaatcg
agaaacgttg tagaattaca gggtgctctc aatcaaatgg tttctaataa
2280tgagaaaatt atgactgcaa tcaaaatagg gacagggaag ttaggacttt
tgataaatga 2340cgatgagact atgcaatctg cagattactt gaattatgga
aaaataccga ttttccgtgg 2400agtgattaga gggttagaga ccaagagatg
gtcacgagtg acttgtgtca ccaatgacca 2460aatacccact tgtgctaata
taatgagctc agtttccaca aatgctctca ccgtagctca 2520ttttgctgag
aacccaatca atgccatgat acagtacaat tattttggga catttgctag
2580actcttgttg atgatgcatg atcctgctct tcgtcaatca ttgtatgaag
ttcaagataa 2640gataccgggc ttgcacagtt ctactttcaa atacgccatg
ttgtatttgg acccttccat 2700tggaggagtg tcgggcatgt ctttgtccag
gtttttgatt agagccttcc cagatcccgt 2760aacagaaagt ctctcattct
ggagattcat ccatgtacat gctcgaagtg agcatctgaa 2820ggagatgagt
gcagtatttg gaaaccccga g 2851132664DNAVesicular stomatitis indiana
virus 13cccgagatag ccaagttccg aataactcac atagacaagc tagtagaaga
tccaacctct 60ctgaacatcg ctatgggaat gagtccagcg aacttgttaa agactgaggt
taaaaaatgc 120ttaatcgaat caagacaaac catcaggaac caggtgatta
aggatgcaac catatatttg 180tatcatgaag aggatcggct cagaagtttc
ttatggtcaa taaatcctct gttccctaga 240tttttaagtg aattcaaatc
aggcactttt ttgggagtcg cagacgggct catcagtcta 300tttcaaaatt
ctcgtactat tcggaactcc tttaagaaaa agtatcatag ggaattggat
360gatttgattg tgaggagtga ggtatcctct ttgacacatt tagggaaact
tcatttgaga 420aggggatcat gtaaaatgtg gacatgttca gctactcatg
ctgacacatt aagatacaaa 480tcctggggcc gtacagttat tgggacaact
gtaccccatc cattagaaat gttgggtcca 540caacatcgaa aagagactcc
ttgtgcacca tgtaacacat cagggttcaa ttatgtttct 600gtgcattgtc
cagacgggat ccatgacgtc tttagttcac ggggaccatt gcctgcttat
660ctagggtcta aaacatctga atctacatct attttgcagc cttgggaaag
ggaaagcaaa 720gtcccactga ttaaaagagc tacacgtctt agagatgcta
tctcttggtt tgttgaaccc 780gactctaaac tagcaatgac tatactttct
aacatccact ctttaacagg cgaagaatgg 840accaaaaggc agcatgggtt
caaaagaaca gggtctgccc ttcataggtt ttcgacatct 900cggatgagcc
atggtgggtt cgcatctcag agcactgcag cattgaccag gttgatggca
960actacagaca ccatgaggga tctgggagat cagaatttcg actttttatt
ccaagcaacg 1020ttgctctatg ctcaaattac caccactgtt gcaagagacg
gatggatcac cagttgtaca 1080gatcattatc atattgcctg taagtcctgt
ttgagaccca tagaagagat caccctggac 1140tcaagtatgg actacacgcc
cccagatgta tcccatgtgc tgaagacatg gaggaatggg 1200gaaggttcgt
ggggacaaga gataaaacag atctatcctt tagaagggaa ttggaagaat
1260ttagcacctg ctgagcaatc ctatcaagtc ggcagatgta taggttttct
atatggagac 1320ttggcgtata gaaaatctac tcatgccgag gacagttctc
tatttcctct atctatacaa 1380ggtcgtatta gaggtcgagg tttcttaaaa
gggttgctag acggattaat gagagcaagt 1440tgctgccaag taatacaccg
gagaagtctg gctcatttga agaggccggc caacgcagtg 1500tacggaggtt
tgatttactt gattgataaa ttgagtgtat cacctccatt cctttctctt
1560actagatcag gacctattag agacgaatta gaaacgattc cccacaagat
cccaacctcc 1620tatccgacaa gcaaccgtga tatgggggtg attgtcagaa
attacttcaa ataccaatgc 1680cgtctaattg aaaagggaaa atacagatca
cattattcac aattatggtt attctcagat 1740gtcttatcca tagacttcat
tggaccattc tctatttcca ccaccctctt gcaaatccta 1800tacaagccat
ttttatctgg gaaagataag aatgagttga gagagctggc aaatctttct
1860tcattgctaa gatcaggaga ggggtgggaa gacatacatg tgaaattctt
caccaaggac 1920atattattgt gtccagagga aatcagacat gcttgcaagt
tcgggattgc taaggataat 1980aataaagaca tgagctatcc cccttgggga
agggaatcca gagggacaat tacaacaatc 2040cctgtttatt atacgaccac
cccttaccca aagatgctag agatgcctcc aagaatccaa 2100aatcccctgc
tgtccggaat caggttgggc caattaccaa ctggcgctca ttataaaatt
2160cggagtatat tacatggaat gggaatccat tacagggact tcttgagttg
tggagacggc 2220tccggaggga tgactgctgc attactacga gaaaatgtgc
atagcagagg aatattcaat 2280agtctgttag aattatcagg gtcagtcatg
cgaggcgcct ctcctgagcc ccccagtgcc 2340ctagaaactt taggaggaga
taaatcgaga tgtgtaaatg gtgaaacatg ttgggaatat 2400ccatctgact
tatgtgaccc aaggacttgg gactatttcc tccgactcaa agcaggcttg
2460gggcttcaaa ttgatttaat tgtaatggat atggaagttc gggattcttc
tactagcctg 2520aaaattgaga cgaatgttag aaattatgtg caccggattt
tggatgagca aggagtttta 2580atctacaaga cttatggaac atatatttgt
gagagcgaaa agaatgcagt aacaatcctt 2640ggtcccatgt tcaagacggt cgac
266414930DNAVesicular stomatitis indiana virus 14gtcgacttag
ttcaaacaga atttagtagt tctcaaacgt ctgaagtata tatggtatgt 60aaaggtttga
agaaattaat cgatgaaccc aatcccgatt ggtcttccat caatgaatcc
120tggaaaaacc tgtacgcatt ccagtcatca gaacaggaat ttgccagagc
aaagaaggtt 180agtacatact ttaccttgac aggtattccc tcccaattca
ttcctgatcc ttttgtaaac 240attgagacta tgctacaaat attcggagta
cccacgggtg tgtctcatgc ggctgcctta 300aaatcatctg atagacctgc
agatttattg accattagcc ttttttatat ggcgattata 360tcgtattata
acatcaatca tatcagagta ggaccgatac ctccgaaccc cccatcagat
420ggaattgcac aaaatgtggg gatcgctata actggtataa gcttttggct
gagtttgatg 480gagaaagaca ttccactata tcaacagtgt ttagcagtta
tccagcaatc attcccgatt 540aggtgggagg ctgtttcagt aaaaggagga
tacaagcaga agtggagtac tagaggtgat 600gggctcccaa aagatacccg
aatttcagac tccttggccc caatcgggaa ctggatcaga 660tctctggaat
tggtccgaaa ccaagttcgt ctaaatccat tcaatgagat cttgttcaat
720cagctatgtc gtacagtgga taatcatttg aaatggtcaa atttgcgaaa
aaacacagga 780atgattgaat ggatcaatag acgaatttca aaagaagacc
ggtctatact gatgttgaag 840agtgacctac acgaggaaaa ctcttggaga
gattaaaaaa tcatgaggag actccaaact 900ttaagtatga aaaaaacttt
gatccttaag 9301523DNAVesicular stomatitis indiana virus
15tatgaaaaaa actaacagat atc 231623DNAVesicular stomatitis indiana
virus 16tatgaaaaaa agtaacagcg atc 231711164DNAVesicular stomatitis
indiana virus 17acgaagacaa acaaaccatt attatcatta aaaggctcag
gagaaacttt aacagtaatc 60aaaatgtctg ttacagtcaa gagaatcatt gacaacacag
tcatagttcc aaaacttcct 120gcaaatgagg atccagtgga atacccggca
gattacttca gaaaatcaaa ggagattcct 180ctttacatca atactacaaa
aagtttgtca gatctaagag gatatgtcta ccaaggcctc 240aaatccggaa
atgtatcaat catacatgtc aacagctact tgtatggagc attgaaggac
300atccggggta agttggataa agattggtca agtttcggaa taaacatcgg
gaaggcaggg 360gatacaatcg gaatatttga ccttgtatcc ttgaaagccc
tggacggtgt acttccagat 420ggagtatcgg atgcttccag aaccagcgca
gatgacaaat ggttgccttt gtatctactt 480ggcttataca gagtgggcag
aacacaaatg cctgaataca gaaaaaggct catggatggg 540ctgacaaatc
aatgcaaaat gatcaatgaa cagtttgaac ctcttgtgcc agaaggtcgt
600gacatttttg atgtgtgggg aaatgacagt aattacacaa aaattgtcgc
tgcagtggac 660atgttcttcc acatgttcaa aaaacatgaa tgtgcctcgt
tcagatacgg aactattgtt 720tccagattca aagattgtgc tgcattggca
acatttggac acctctgcaa aataaccgga 780atgtctacag aagatgtgac
gacctggatc ttgaaccgag aagttgcaga tgagatggtc 840caaatgatgc
ttccaggcca agaaattgac aaggctgatt catacatgcc ttatttgatc
900gactttggat tgtcttctaa gtctccatat tcttccgtca aaaaccctgc
cttccacttc 960tgggggcaat tgacagctct tctgctcaga tccaccagag
caaggaatgc ccgacagcct 1020gatgacattg agtatacatc tcttactaca
gcaggtttgt tgtacgctta tgcagtagga 1080tcctctgctg acttggcaca
acagttttgt gttggagata gcaaatacac tccagatgat 1140agtaccggag
gattgacgac taatgcaccg ccacaaggca gagatgtggt cgaatggctc
1200ggatggtttg aagatcaaaa cagaaaaccg actcctgata tgatgcagta
tgcgaaacga 1260gcagtcatgt cactgcaagg cctaagagag aagacaattg
gcaagtatgc taagtcagag 1320tttgacaaat gaccctataa ttctcagatc
acctattata tattatgcta gctatgaaaa 1380aaactaacag atatcatgga
taatctcaca aaagttcgtg agtatctcaa gtcctattct 1440cgtctagatc
aggcggtagg agagatagat gagatcgaag cacaacgagc tgaaaagtcc
1500aattatgagt tgttccaaga ggacggagtg gaagagcata ctaggccctc
ttattttcag 1560gcagcagatg attctgacac agaatctgaa ccagaaattg
aagacaatca aggcttgtat 1620gtaccagatc cggaagctga gcaagttgaa
ggctttatac aggggccttt agatgactat 1680gcagatgagg acgtggatgt
tgtattcact tcggactgga aacagcctga gcttgaatcc 1740gacgagcatg
gaaagacctt acggttgaca ttgccagagg gtttaagtgg agagcagaaa
1800tcccagtggc ttttgacgat taaagcagtc gttcaaagtg ccaaacactg
gaatctggca 1860gagtgcacat ttgaagcatc gggagaaggg gtcatcataa
aaaagcgcca gataactccg 1920gatgtatata aggtcactcc agtgatgaac
acacatccgt cccaatcaga agccgtatca 1980gatgtttggt ctctctcaaa
gacatccatg actttccaac ccaagaaagc aagtcttcag 2040cctctcacca
tatccttgga tgaattgttc tcatctagag gagaattcat ctctgtcgga
2100ggtaacggac gaatgtctca taaagaggcc atcctgctcg gtctgaggta
caaaaagttg 2160tacaatcagg cgagagtcaa atattctctg tagactagta
tgaaaaaaag taacagatat 2220cacaatctaa gtgttatccc aatccattca
tcatgagttc cttaaagaag attctcggtc 2280tgaaggggaa aggtaagaaa
tctaagaaat tagggatcgc accaccccct tatgaagagg 2340acactaacat
ggagtatgct ccgagcgctc caattgacaa atcctatttt ggagttgacg
2400agatggacac tcatgatccg aatcaattaa gatatgagaa attcttcttt
acagtgaaaa 2460tgacggttag atctaatcgt ccgttcagaa catactcaga
tgtggcagcc gctgtatccc 2520attgggatca catgtacatc ggaatggcag
ggaaacgtcc cttctacaag atcttggctt 2580ttttgggttc ttctaatcta
aaggccactc cagcggtatt ggcagatcaa ggtcaaccag 2640agtatcatgc
tcactgtgaa ggcagggctt atttgccaca cagaatgggg aagacccctc
2700ccatgctcaa tgtaccagag cacttcagaa gaccattcaa tataggtctt
tacaagggaa 2760cgattgagct cacaatgacc atctacgatg atgagtcact
ggaagcagct cctatgatct 2820gggatcattt caattcttcc aaattttctg
atttcagaga gaaggcctta atgtttggcc 2880tgattgtcga gaaaaaggca
tctggagctt gggtcctgga ttctgtcagc cacttcaaat 2940gagctagtct
agcttccagc ttctgaacaa tccccggttt actcagtctc tcctaattcc
3000agcctttcga acaactaata tcctgtcttc tctatcccta tgaaaaaaac
taacagagat 3060cgatctgttt ccttgacacc atgaagtgcc ttttgtactt
agctttttta ttcatcgggg 3120tgaattgcaa gttcaccata gtttttccac
acaaccaaaa aggaaactgg aaaaatgttc 3180cttccaatta ccattattgc
ccgtcaagct cagatttaaa ttggcataat gacttaatag 3240gcacagcctt
acaagtcaaa atgcccaaga gtcacaaggc tattcaagca gacggttgga
3300tgtgtcatgc ttccaaatgg gtcactactt gtgatttccg ctggtacgga
ccgaagtata 3360taacacattc catccgatcc ttcactccat ctgtagaaca
atgcaaggaa agcattgaac 3420aaacgaaaca aggaacttgg ctgaatccag
gcttccctcc tcaaagttgt ggatatgcaa 3480ctgtgacgga tgctgaagca
gcgattgtcc aggtgactcc tcaccatgtg cttgttgatg 3540aatacacagg
agaatgggtt gattcacagt tcatcaacgg aaaatgcagc aatgacatat
3600gccccactgt ccataactcc acaacctggc attccgacta taaggtcaaa
gggctatgtg 3660attctaacct catttccatg gacatcacct tcttctcaga
ggacggagag ctatcatccc 3720taggaaagga gggcacaggg ttcagaagta
actactttgc ttatgaaact ggagacaagg 3780cctgcaaaat gcagtactgc
aagcattggg gagtcagact cccatcaggt gtctggttcg 3840agatggctga
taaggatctc tttgctgcag ccagattccc tgaatgccca gaagggtcaa
3900gtatctctgc tccatctcag acctcagtgg atgtaagtct cattcaggac
gttgagagga 3960tcttggatta ttccctctgc caagaaacct ggagcaaaat
cagagcgggt cttcccatct 4020ctccagtgga tctcagctat cttgctccta
aaaacccagg aaccggtcct gtctttacca 4080taatcaatgg taccctaaaa
tactttgaga ccagatacat cagagtcgat attgctgctc 4140caatcctctc
aagaatggtc ggaatgatca gtggaactac cacagaaagg gaactgtggg
4200atgactgggc tccatatgaa gacgtggaaa ttggacccaa tggagttctg
aggaccagtt 4260caggatataa gtttccttta tatatgattg gacatggtat
gttggactcc gatcttcatc 4320ttagctcaaa ggctcaggtg tttgaacatc
ctcacattca agacgctgct tcgcagcttc 4380ctgatgatga gactttattt
tttggtgata ctgggctatc caaaaatcca atcgagtttg 4440tagaaggttg
gttcagtagt tggaagagct ctattgcctc ttttttcttt atcatagggt
4500taatcattgg actattcttg gttctccgag ttggtattta tctttgcatt
aaattaaagc 4560acaccaagaa aagacagatt tatacagaca tagagatgaa
ccgacttgga aagtaactca 4620aatcctgcac aacagattct tcatgtttga
accaaatcaa cttgtgatat catgctcaaa 4680gaggccttaa ttaaatttta
atttttaatt tttatgaaaa aaactaacag caatcatgga 4740agtccacgat
tttgagaccg acgagttcaa tgatttcaat gaagatgact atgccacaag
4800agaattcctg aatcccgatg agcgcatgac gtacttgaat catgctgatt
acaatttgaa 4860ttctcctcta attagtgatg atattgacaa tttgatcagg
aaattcaatt ctcttccgat 4920tccctcgatg tgggatagta agaactggga
tggagttctt gagatgttaa catcatgtca 4980agccaatccc atctcaacat
ctcagatgca taaatggatg ggaagttggt taatgtctga 5040taatcatgat
gccagtcaag ggtatagttt tttacatgaa gtggacaaag aggcagaaat
5100aacatttgac gtggtggaga ccttcatccg cggctggggc aacaaaccaa
ttgaatacat 5160caaaaaggaa agatggactg actcattcaa aattctcgct
tatttgtgtc aaaagttttt 5220ggacttacac aagttgacat taatcttaaa
tgctgtctct gaggtggaat tgctcaactt 5280ggcgaggact ttcaaaggca
aagtcagaag aagttctcat ggaacgaaca tatgcaggct 5340tagggttccc
agcttgggtc ctacttttat ttcagaagga tgggcttact tcaagaaact
5400tgatattcta atggaccgaa actttctgtt aatggtcaaa gatgtgatta
tagggaggat 5460gcaaacggtg ctatccatgg tatgtagaat agacaacctg
ttctcagagc aagacatctt 5520ctcccttcta aatatctaca gaattggaga
taaaattgtg gagaggcagg gaaatttttc 5580ttatgacttg attaaaatgg
tggaaccgat atgcaacttg aagctgatga aattagcaag 5640agaatcaagg
cctttagtcc cacaattccc tcattttgaa aatcatatca agacttctgt
5700tgatgaaggg gcaaaaattg accgaggtat aagattcctc catgatcaga
taatgagtgt 5760gaaaacagtg gatctcacac tggtgattta tggatcgttc
agacattggg gtcatccttt 5820tatagattat tacgctggac tagaaaaatt
acattcccaa gtaaccatga agaaagatat 5880tgatgtgtca tatgcaaaag
cacttgcaag tgatttagct cggattgttc tatttcaaca 5940gttcaatgat
cataaaaagt ggttcgtgaa tggagacttg ctccctcatg atcatccctt
6000taaaagtcat gttaaagaaa atacatggcc tacagctgct caagttcaag
attttggaga 6060taaatggcat gaacttccgc tgattaaatg ttttgaaata
cccgacttac tagacccatc 6120gataatatac tctgacaaaa gtcattcaat
gaataggtca gaggtgttga aacatgtccg 6180aatgaatccg aacactccta
tccctagtaa aaaggtgttg cagactatgt tggacacaaa 6240ggctaccaat
tggaaagaat ttcttaaaga gattgatgag aagggcttag atgatgatga
6300tctaattatt ggtcttaaag gaaaggagag ggaactgaag ttggcaggta
gatttttctc 6360cctaatgtct tggaaattgc gagaatactt tgtaattacc
gaatatttga taaagactca 6420tttcgtccct atgtttaaag gcctgacaat
ggcggacgat ctaactgcag tcattaaaaa 6480gatgttagat tcctcatccg
gccaaggatt gaagtcatat gaggcaattt gcatagccaa 6540tcacattgat
tacgaaaaat ggaataacca ccaaaggaag ttatcaaacg gcccagtgtt
6600ccgagttatg ggccagttct taggttatcc atccttaatc gagagaactc
atgaattttt 6660tgagaaaagt cttatatact acaatggaag accagacttg
atgcgtgttc acaacaacac 6720actgatcaat tcaacctccc aacgagtttg
ttggcaagga caagagggtg gactggaagg 6780tctacggcaa aaaggatgga
gtatcctcaa tctactggtt attcaaagag aggctaaaat 6840cagaaacact
gctgtcaaag tcttggcaca aggtgataat caagttattt gcacacagta
6900taaaacgaag aaatcgagaa acgttgtaga attacagggt gctctcaatc
aaatggtttc 6960taataatgag aaaattatga ctgcaatcaa aatagggaca
gggaagttag gacttttgat 7020aaatgacgat gagactatgc aatctgcaga
ttacttgaat tatggaaaaa taccgatttt 7080ccgtggagtg attagagggt
tagagaccaa gagatggtca cgagtgactt gtgtcaccaa 7140tgaccaaata
cccacttgtg ctaatataat gagctcagtt tccacaaatg ctctcaccgt
7200agctcatttt gctgagaacc caatcaatgc catgatacag tacaattatt
ttgggacatt 7260tgctagactc ttgttgatga tgcatgatcc tgctcttcgt
caatcattgt atgaagttca 7320agataagata ccgggcttgc acagttctac
tttcaaatac gccatgttgt atttggaccc 7380ttccattgga ggagtgtcgg
gcatgtcttt gtccaggttt ttgattagag ccttcccaga 7440tcccgtaaca
gaaagtctct cattctggag attcatccat gtacatgctc gaagtgagca
7500tctgaaggag atgagtgcag tatttggaaa ccccgagata gccaagttcc
gaataactca 7560catagacaag ctagtagaag atccaacctc tctgaacatc
gctatgggaa tgagtccagc 7620gaacttgtta aagactgagg ttaaaaaatg
cttaatcgaa tcaagacaaa ccatcaggaa 7680ccaggtgatt aaggatgcaa
ccatatattt gtatcatgaa gaggatcggc tcagaagttt 7740cttatggtca
ataaatcctc tgttccctag atttttaagt gaattcaaat caggcacttt
7800tttgggagtc gcagacgggc tcatcagtct atttcaaaat tctcgtacta
ttcggaactc 7860ctttaagaaa aagtatcata gggaattgga tgatttgatt
gtgaggagtg aggtatcctc 7920tttgacacat ttagggaaac ttcatttgag
aaggggatca tgtaaaatgt ggacatgttc 7980agctactcat gctgacacat
taagatacaa atcctggggc cgtacagtta ttgggacaac 8040tgtaccccat
ccattagaaa tgttgggtcc acaacatcga aaagagactc cttgtgcacc
8100atgtaacaca tcagggttca attatgtttc tgtgcattgt ccagacggga
tccatgacgt 8160ctttagttca cggggaccat tgcctgctta tctagggtct
aaaacatctg aatctacatc 8220tattttgcag ccttgggaaa gggaaagcaa
agtcccactg attaaaagag ctacacgtct 8280tagagatgct atctcttggt
ttgttgaacc cgactctaaa ctagcaatga ctatactttc 8340taacatccac
tctttaacag gcgaagaatg gaccaaaagg cagcatgggt tcaaaagaac
8400agggtctgcc cttcataggt tttcgacatc tcggatgagc catggtgggt
tcgcatctca 8460gagcactgca gcattgacca ggttgatggc aactacagac
accatgaggg atctgggaga 8520tcagaatttc gactttttat tccaagcaac
gttgctctat gctcaaatta ccaccactgt 8580tgcaagagac ggatggatca
ccagttgtac agatcattat catattgcct gtaagtcctg 8640tttgagaccc
atagaagaga tcaccctgga ctcaagtatg gactacacgc ccccagatgt
8700atcccatgtg ctgaagacat ggaggaatgg ggaaggttcg tggggacaag
agataaaaca 8760gatctatcct ttagaaggga attggaagaa tttagcacct
gctgagcaat cctatcaagt 8820cggcagatgt ataggttttc tatatggaga
cttggcgtat agaaaatcta ctcatgccga 8880ggacagttct
ctatttcctc tatctataca aggtcgtatt agaggtcgag gtttcttaaa
8940agggttgcta gacggattaa tgagagcaag ttgctgccaa gtaatacacc
ggagaagtct 9000ggctcatttg aagaggccgg ccaacgcagt gtacggaggt
ttgatttact tgattgataa 9060attgagtgta tcacctccat tcctttctct
tactagatca ggacctatta gagacgaatt 9120agaaacgatt ccccacaaga
tcccaacctc ctatccgaca agcaaccgtg atatgggggt 9180gattgtcaga
aattacttca aataccaatg ccgtctaatt gaaaagggaa aatacagatc
9240acattattca caattatggt tattctcaga tgtcttatcc atagacttca
ttggaccatt 9300ctctatttcc accaccctct tgcaaatcct atacaagcca
tttttatctg ggaaagataa 9360gaatgagttg agagagctgg caaatctttc
ttcattgcta agatcaggag aggggtggga 9420agacatacat gtgaaattct
tcaccaagga catattattg tgtccagagg aaatcagaca 9480tgcttgcaag
ttcgggattg ctaaggataa taataaagac atgagctatc ccccttgggg
9540aagggaatcc agagggacaa ttacaacaat ccctgtttat tatacgacca
ccccttaccc 9600aaagatgcta gagatgcctc caagaatcca aaatcccctg
ctgtccggaa tcaggttggg 9660ccaattacca actggcgctc attataaaat
tcggagtata ttacatggaa tgggaatcca 9720ttacagggac ttcttgagtt
gtggagacgg ctccggaggg atgactgctg cattactacg 9780agaaaatgtg
catagcagag gaatattcaa tagtctgtta gaattatcag ggtcagtcat
9840gcgaggcgcc tctcctgagc cccccagtgc cctagaaact ttaggaggag
ataaatcgag 9900atgtgtaaat ggtgaaacat gttgggaata tccatctgac
ttatgtgacc caaggacttg 9960ggactatttc ctccgactca aagcaggctt
ggggcttcaa attgatttaa ttgtaatgga 10020tatggaagtt cgggattctt
ctactagcct gaaaattgag acgaatgtta gaaattatgt 10080gcaccggatt
ttggatgagc aaggagtttt aatctacaag acttatggaa catatatttg
10140tgagagcgaa aagaatgcag taacaatcct tggtcccatg ttcaagacgg
tcgacttagt 10200tcaaacagaa tttagtagtt ctcaaacgtc tgaagtatat
atggtatgta aaggtttgaa 10260gaaattaatc gatgaaccca atcccgattg
gtcttccatc aatgaatcct ggaaaaacct 10320gtacgcattc cagtcatcag
aacaggaatt tgccagagca aagaaggtta gtacatactt 10380taccttgaca
ggtattccct cccaattcat tcctgatcct tttgtaaaca ttgagactat
10440gctacaaata ttcggagtac ccacgggtgt gtctcatgcg gctgccttaa
aatcatctga 10500tagacctgca gatttattga ccattagcct tttttatatg
gcgattatat cgtattataa 10560catcaatcat atcagagtag gaccgatacc
tccgaacccc ccatcagatg gaattgcaca 10620aaatgtgggg atcgctataa
ctggtataag cttttggctg agtttgatgg agaaagacat 10680tccactatat
caacagtgtt tagcagttat ccagcaatca ttcccgatta ggtgggaggc
10740tgtttcagta aaaggaggat acaagcagaa gtggagtact agaggtgatg
ggctcccaaa 10800agatacccga atttcagact ccttggcccc aatcgggaac
tggatcagat ctctggaatt 10860ggtccgaaac caagttcgtc taaatccatt
caatgagatc ttgttcaatc agctatgtcg 10920tacagtggat aatcatttga
aatggtcaaa tttgcgaaaa aacacaggaa tgattgaatg 10980gatcaataga
cgaatttcaa aagaagaccg gtctatactg atgttgaaga gtgacctaca
11040cgaggaaaac tcttggagag attaaaaaat catgaggaga ctccaaactt
taagtatgaa 11100aaaaactttg atccttaaga ccctcttgtg gtttttattt
tttatctggt tttgtggtct 11160tcgt 111641812DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 18nnnnnngaga cg 12
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