Interferon sensitive recombinant poxvirus vaccine

What is described is a recombinant poxvirus, such as vaccinia virus, having enhanced sensitivity to interferon. In one embodiment, the recombinant poxvirus has an open reading frame conferring resistance to interferon deleted therefrom. In another embodiment, the recombinant poxvirus is modified to disrupt K3L gene expression. What is also described is a vaccine containing the recombinant poxvirus having enhanced sensitivity to interferon so that the vaccine has an increased level of safety compared to known recombinant poxvirus vaccines.

FIELD OF THE INVENTION 
The present invention relates to a modified poxvirus and to methods of 
making and using the same. More in particular, the invention relates to 
recombinant poxvirus having enhanced sensitivity to interferon. 
Several publications are referenced in this application by arabic numerals 
within parentheses. Full citation to these references is found at the end 
of the specification immediately preceding the claims. These references 
describe the state-of-the-art to which this invention pertains. 
BACKGROUND OF THE INVENTION 
Vaccinia virus and more recently other poxviruses have been used for the 
insertion and expression of foreign genes. The basic technique of 
inserting foreign genes into live infectious poxvirus involves 
recombination between pox DNA sequences flanking a foreign genetic element 
in a donor plasmid and homologous sequences present in the rescuing 
poxvirus (17). 
Specifically, the recombinant poxviruses are constructed in two steps known 
in the art and analogous to the methods for creating synthetic 
recombinants of the vaccinia virus described in U.S. Pat. No. 4,603,112, 
the disclosure of which patent is incorporated herein by reference. 
First, the DNA gene sequence to be inserted into the virus, particularly an 
open reading frame from a non-pox source, is placed into an E. coli 
plasmid construct into which DNA homologous to a section of DNA of the 
poxvirus has been inserted. Separately, the DNA gene sequence to be 
inserted is ligated to a promoter. The promoter-gene linkage is positioned 
in the plasmid construct so that the promoter-gene linkage is flanked on 
both ends by DNA homologous to a DNA sequence flanking a region of pox DNA 
containing a nonessential locus. The resulting plasmid construct is then 
amplified by growth within E. coli bacteria (20) and isolated (21,22). 
Second, the isolated plasmid containing the DNA gene sequence to be 
inserted is transfected into a cell culture, e.g. chick embryo 
fibroblasts, along with the poxvirus. Recombination between homologous pox 
DNA in the plasmid and the viral genome respectively gives a poxvirus 
modified by the presence, in a nonessential region of its genome, of 
foreign DNA sequences. The term "foreign" DNA designates exogenous DNA, 
particularly DNA from a non-pox source, that codes for gene products not 
ordinarily produced by the genome into which the exogenous DNA is placed. 
Genetic recombination is in general the exchange of homologous sections of 
DNA between two strands of DNA. In certain viruses RNA may replace DNA. 
Homologous sections of nucleic acid are sections of nucleic acid (DNA or 
RNA) which have the same sequence of nucleotide bases. 
Genetic recombination may take place naturally during the replication or 
manufacture of new viral genomes within the infected host cell. Thus, 
genetic recombination between viral genes may occur during the viral 
replication cycle that takes place in a host cell which is co-infected 
with two or more different viruses or other genetic constructs. A section 
of DNA from a first genome is used interchangeably in constructing the 
section of the genome of a second co-infecting virus in which the DNA is 
homologous with that of the first viral genome. 
However, recombination can also take place between sections of DNA in 
different genomes that are not perfectly homologous. If one such section 
is from a first genome homologous with a section of another genome except 
for the presence within the first section of, for example, a genetic 
marker or a gene coding for an antigenic determinant inserted into a 
portion of the homologous DNA, recombination can still take place and the 
products of that recombination are then detectable by the presence of that 
genetic marker or gene in the recombinant viral genome. 
Successful expression of the inserted DNA genetic sequence by the modified 
infectious virus requires two conditions. First, the insertion must be 
into a nonessential region of the virus in order that the modified virus 
remain viable. The second condition for expression of inserted DNA is the 
presence of a promoter in the proper relationship to the inserted DNA. The 
promoter must be placed so that it is located upstream from the DNA 
sequence to be expressed. 
The technology of generating vaccinia virus recombinants has recently been 
extended to other members of the poxvirus family which have a more 
restricted host range. The avipoxvirus, fowlpox, has been engineered as a 
recombinant virus expressing the rabies G gene (23,24). This recombinant 
virus is also described in PCT Publication No. WO89/03429. On inoculation 
of the recombinant into a number of non-avian species an immune response 
to rabies is elicited which in mice, cats and dogs is protective against a 
lethal rabies challenge. 
It is well established that one of the antiviral mechanisms induced by 
interferon (Ifn) is the inhibition of the initiation of protein synthesis 
due to the phosphorylation of eIF-2alpha by the P1 kinase (1,2). Vaccinia 
virus (VV) has been shown to be relatively resistant to Ifn (3,4) and is 
capable of rescuing Ifn-sensitive viruses from the effects of Ifn (5,6), 
by somehow reducing the level of eIF-2alpha phosphorylation. 
VV-based vaccines are useful immunizing agents (14). Recombinant poxvirus 
vaccine candidates, particularly VV vaccine candidates, having enhanced 
sensitivity to interferon, would have an increased level of safety. An 
Ifn-sensitive phenotype would provide a means for drug intervention in the 
event that vaccination leads to vaccinial complications. 
It can thus be appreciated that provision of a recombinant poxvirus, 
particularly recombinant vaccinia virus, having enhanced sensitivity to 
interferon, would be a highly desirable advance over the current state of 
technology. 
OBJECTS OF THE INVENTION 
It is therefore an object of this invention to provide recombinant 
poxviruses, which viruses have enhanced sensitivity to interferon, and to 
provide a method of making such recombinant poxviruses. 
It is an additional object of this invention to provide a recombinant 
poxvirus vaccine having enhanced sensitivity to interferon and 
consequently having an increased level of safety compared to known 
recombinant poxvirus vaccines. 
These and other objects and advantages of the present invention will become 
more readily apparent after consideration of the following. 
STATEMENT OF THE INVENTION 
In one aspect, the present invention relates to a recombinant poxvirus 
having an open reading frame conferring resistance to interferon deleted 
therefrom so that the recombinant poxvirus has enhanced sensitivity to 
interferon. The poxvirus is advantageously a vaccinia virus. 
According to the present invention, the open reading frame deleted from the 
recombinant poxvirus has homology with eIF-2alpha. 
In another aspect, the present invention relates to a recombinant poxvirus 
modified to disrupt K3L gene expression. The poxvirus is advantageously a 
vaccinia virus. 
In yet another aspect, the present invention relates to a vaccine for 
inducing an immunological response in a host animal inoculated with the 
vaccine, said vaccine including a carrier and a recombinant poxvirus 
having an open reading frame deleted therefrom so that the recombinant 
poxvirus has enhanced sensitivity to interferon and the vaccine has an 
increased level of safety compared to known recombinant poxvirus vaccines. 
The poxvirus used in the vaccine according to the present invention is 
advantageously a vaccinia virus.

DETAILED DESCRIPTION OF THE INVENTION 
A better understanding of the present invention and of its many advantages 
will be had from the following examples, given by way of illustration. 
EXAMPLE 1 
Generation of Vaccina Virus Recombinant vP872 Devoid of the K3L Open 
Reading Frame 
Recent elucidation of the complete nucleotide sequence of the VV genome (7) 
has revealed an open reading frame (ORF), designated as K3L, which has 
27.6% identity to eIF-2alpha over an 87 amino acid region. This example 
describes the generation of a VV mutant, vP872, which was derived by the 
specific deletion of the K3L ORF from the Copenhagen strain of VV (VC-2). 
The amino acid sequence (SEQ ID NO:l) of protein transcribed from K3L ORF 
identified in VC-2 (7) is presented in FIG. 1 and shown in comparison to 
the amino acid sequence (SEQ ID NO: 2) of protein transcribed from 
eIF-2alpha (8). Amino acid homology was obtained using the FASTP (15) 
program of PCGENE against the Swisprot database release 11.0 
(Intelligenetics, Mountain View, Calif.). This alignment has been 
optimized by gap insertions. 
The VV K3L ORF has the potential to encode a 10.5 kDa protein, whereas 
eIF-2alpha has a calculated molecular mass equal to 36.1 kDa. Furthermore, 
the 87 amino acid overlap region spans the amino-terminal portion of 
eIF-2alpha, which contains the serine residue (amino acid 51) which is 
phosphorylated by the interferon-activated P1 kinase (9). It is the 
phosphorylation at this residue which is highly correlated with the rapid 
cessation of protein synthesis in the Ifn-treated system (1,2). 
Referring now to FIG. 2, generation of the VV deletion mutant, vP872, was 
accomplished using the deletion plasmid, pK3Lgpt. Both the upstream (5') 
and downstream (3') sequences relative to the K3L ORF were derived by PCR. 
Oligonucleotides K3L5H2 (SEQ ID NO: 3) (5'-ATCATCAAGCTTGTTAACGGGCTCGTAAAT 
TGG-3'), K3L52 (SEQ ID NO: 4) (5'-ATCGATATTTTTATGCGTGATTGG-3'), K3L3H2 
(SEQ ID NO: 5) (5'-ATCATCAAGCTTTAATTTTTATACCGAAC-3'), and K3L3X2 (SEQ ID 
NO: 3) (5'-ATCATCCTCGAGGCAGGCAATAGCGACATAAAC-3') were used for PCR with 
plasmid, pSD407VC, which contains the VC-2 HindIII K region, as template. 
Oligonucleotides K3L5H2 (SEQ ID NO: 3) and K3L52 (SEQ ID NO: 3) were used 
to generate a 227 bp fragment containing 5' sequences with engineered 
EcoRV and HindIII sites. Oligonucleotides K 3L3H2 (SEQ ID NO: 5) and 
K3L3X2 (SEQ ID NO: 6) were a 239 bp fragment containing 3' sequences with 
engineered XhoI and HindIII sites. The resultant fragments were digested 
with the appropriate restriction enzymes and ligated together into pBS-SK 
(Stratagene, La Jolla, Calif.) vector digested with EcoRV and XhoI. The 
resultant plasmid was designated pK3LA. 
A 1 kb HindIII fragment containing the E. coli gPt gene (ATCC #37145) 
juxtaposed 3' to a 300 bp fragment derived from the upstream region of the 
VC-2 hemorrhagic gene (7,16) was inserted into the unique HindIII site of 
pK3LA, which is situated between the (5') and (3') sequences. The 
resultant plasmid was designated pK3Lgpt and is depicted schematically in 
FIG. 2. 
This plasmid was used in standard in vitro recombination experiments (17) 
with wildtype VC-2 as the rescue virus to generate the K3L-minus mutant, 
vP872. Potential mutants were selected by plating in the presence of 
medium containing mycophenolic acid as described previously (18,19). 
Southern blot analysis of viral DNA derived from the wildtype virus, VC-2, 
and mutant virus, vP872, confirmed the specific deletion of the K3L gene 
and demonstrated no further genomic alterations. 
EXAMPLE 2 
Effect of the K3L Deletion on Protein Synthesis in Ifn-Treated Infected 
Cells 
To assess the effect of the K3L deletion on protein synthesis in 
Ifn-treated infected cells, VC-2 and the deletion mutant, vP872, were 
inoculated onto L929 cell monolayers (ATCC #CCL1) which had been 
pretreated with various concentrations of mouse alpha/beta Ifn. L929 cell 
monolayers were pretreated for 24 hours with either 0, 10, 100, 500, or 
1000 IRU/ml of mouse alpha/beta Ifn (Lee Biomolecular, San Rafael, 
Calif.). Following pretreatment with Ifn, cell monolayers were 
mock-infected, or infected with VC-2 or vP872 at an m.o.i. of 100. After a 
1 hour adsorption period, the inoculum was removed and 2 ml of 
methionine-free medium containing 2% dialysed FBS was applied to the 
monolayers. At 7 hours post infection, the medium was aspirated and 2 ml 
of the above media supplemented with 25 uCi/ml [.sup.35 S]-methionine 
(NEN, Boston, Mass.) was applied to the monolayers. At 8 hours 
postinfection the medium was aspirated from the monolayers and PBS was 
applied. Lysates were prepared by three cycles of freeze-thawing followed 
by clarification on the lysate. Total protein concentrations of the 
lysates were determined using the Bio-Rad Protein Assay kit (Bio-Rad, 
Richmond, Calif.). Aliquots containing equal quantities of total protein 
from each sample were fractionated by SDS-PAGE. The gel was fixed by 
treatment for 45 minutes in a 7.5% acetic acid, 10% methanol, 3% glycerol 
mixture in deionized water (v/v/v). The gel was prepared for fluorography 
by washing the gel for 30 minutes in deionized water followed by treatment 
of the gel for 30 minutes in 1M sodium salicylate. The gel was dried and 
exposed to film for visualization of the protein species. 
As expected, uninfected cell controls showed no effect of Ifn on host 
protein synthesis even at high concentrations. Results with the wildtype 
vaccinia virus (VC-2) strain were consistent with previously described 
results (3,4), in that, viral-induced protein synthesis was resistant to 
interferon, although a slight dimunition was noted at high Ifn 
concentrations (greater than 500 IRU/ml). 
Significantly, it was observed that the deletion of K3L from VC-2 resulted 
in an enhanced sensitivity of viral-induced protein synthesis to Ifn. Ifn 
concentrations as low as 10 International Reference Units (IRU)/ml 
significantly reduced the level of virus-induced protein synthesis in 
vP872-infected cells. Viral induced protein synthesis in Ifn treated vP872 
infected L929 cells was almost completely inhibited at Ifn concentrations 
of 100 IRU/ml. It is also noteworthy that the enhanced sensitivity to Ifn 
observed in L929-infected cells cannot merely be attributed to the 
expression of the E. coli gPt gene. Analysis of a VV (Copenhagen strain) 
recombinant not devoid of the K3L ORF and containing the identical Ecogpt 
expression cassette as vP872 displayed an Ifn-resistant phenotype similar 
to wildtype VV. 
Similar results demonstrating an increased sensitivity of VV K3L deletion 
mutants to Ifn were noted in experiments which analyzed the effect of Ifn 
on virus yields from mutant and wildtype virus-infected L929 cells. The 
samples were treated identically as described above except that following 
the adsorption period, 2 ml complete MEM was overlayed, and the samples 
were harvested at 24 hours post infection (rather than 8 hours) in the 
liquid overlay. Lysates were prepared as described above without 
clarification and plated onto monolayers of Veto cells as described 
previously (17). Samples were inoculated in duplicate and plated in 
triplicate. Referring now to FIG. 3, viral yields in the absence of 
interferon are indicated by closed markers on the abscissa. Viral yields 
as a function of interferon concentration are indicated for wildtype VC-2 
infected cells by open diamonds and for vP872 infected cells by open 
circles. Points represent the average of six plates from a representative 
experiment. Plates which were harvested immediately following the 
adsorption period had an average yield of 3.6.times.10.sup.6, considered 
the baseline yield 
It can be seen that low concentrations of Ifn have a small effect on viral 
yield in VC-2-infected L929 cells, whereas the same amount of Ifn reduced 
yield in vP872-infected cells by one log. Concentrations of Ifn greater 
than 10 IRU/ml reduced viral output in vP872-infected L929 cells to levels 
below that of input virus. Conversely, in VC-2-infected L929 cells, no 
concentration of Ifn tested in this experiment was sufficient to reduce 
viral output below this level. 
These results indicate that the VV K3L gene is involved in the 
Ifn-resistant phenotype described previously for VV (3,4). Previously 
reported results have demonstrated that (a) an exogenous source of 
eIF-2alpha could rescue protein synthesis in VSV-infected L cell lysates 
(10), and (b) an exogenous source of eIF-2alpha was able to overcome the 
inhibitory effects of eIF-2alpha phosphorylation and enable the 
replication of a mutant form of adenovirus type 5, which fails to express 
virus-associated RNA (11,12). Of significance, the plasmid-expressed 
exogenous source of eIF-2alpha contained an amino acid substitution of a 
serine to an alanine at position 51, thus preventing the phosphorylation 
at this position, which is an event highly correlated with translational 
repression (10). Interestingly, the VV K3L ORF does not contain a serine 
residue at the equivalent position. 
The VV K3L gene plays an integral role in the resistance to interferon by 
the Copenhagen strain of VV. The WR strain of VV also has a K3L gene (13) 
which shares homology with eIF-2alpha and differs from its Copenhagen 
homolog by three base changes, two of which are conservative at the amino 
acid level. Disruption of K3L gene expression in WR also resulted in an 
increased sensitivity to interferon. 
Thus, recombinant poxvirus containing exogenous DNA coding for an antigen 
and having disrupted or deleted therefrom an open reading frame conferring 
resistance to interferon is useful as vaccines because such poxvirus 
achieves protein synthesis until the levels of interferon are increased as 
in this example; for instance, until exogenous interferon is administered 
to the host to "turn off" the recombinant poxvirus. Nonetheless, such 
recombinant poxvirus will cause the production of sufficient antigen in 
the host cell, unless increased levels of interferon are present, thereby 
providing a useful vaccine which can be "turned off" by administration of 
exogenous interferon. Therefore, with such a recombinant poxvirus vaccine, 
interferon can be used to treat any post-vaccination complication. 
EXAMPLE 3 
Reinsertion of K3L ORF Into vP872 
To conclusively demonstrate that the increased sensitivity to interferon of 
the K3L.sup.- mutant, vP872, was due to the specific deletion of the K3L 
ORF, the K3L ORF under the control of its endogenous promoter was 
reinserted in vP872 at the ATI locus. To accomplish this, an insertion 
plasmid was engineered as follows: A fragment of 508 bp containing the K3L 
ORF (7) containing its natural promoter was generated by PCR using 
oligonucleotides K3L52 (SEQ ID NO: 4) (5'ATCGATATTTTTATGCGTGATTGG-3') and 
K3LHD (SEQ ID NO: 7) (5'-ATCATCAAGCTTTTATTGATGTCTACACATCC-3') and pSD407 
as template (plasmid pSD407 contains the entire HindIII K genomic fragment 
of vaccinia virus (Copenhagen strain) in the HindIII site of pUCS). This 
fragment was blunt-ended using the Klenow fragment of the E. coli DNA 
polymerase in the presence of 2mM dNTPs and ligated into pSD541 digested 
with SmaI and treated with calf intestine alkaline phosphatase. Plasmid 
pSD541 is a vaccinia insertion plasmid. It is deleted for vaccinia 
sequences, nucleotide 317,812 through 138,976, encompassing the A25L and 
A26L ORFs (7). The deletion junction consists of a polylinker region 
containing XhoI, SmaI and BglII restriction sites, flanked on both sides 
by stop codons and early vaccinia transcriptional terminators (25). pSD541 
was constructed by polymerase chain reaction (PCR) (26) using cloned 
vaccinia SalI E plasmid pSD414 as template. Synthetic oligonucleotides 
MPSYN267 (SEQ ID NO: 8) (5' GGGCTCAAGCTTGCGGCCGCTCATTAGACAAGCGAATGAGGGAC 
3') and MPSYN268 (SEQ ID NO: 8) (5' 
AGATCTCCCGGGCTCGAGTAATTAATTAATTTTTATTACACCAGAAAAGACGGCTTGAGATC 3)' were 
used as primers to generate the left vaccinia arm and synthetic 
oligonucleotides MPSYN269 (SEQ ID NO: 10) (5' 
TAATTACTCGAGCCCGGGAGATCTAATTTAATTTAATTTATATAACTCATTTTTTGAATATACT 3') and 
MPSYN270 (SEQ ID NO: 11) (5' TATCTCGAATTCCCGCGGCTTTAAATGGACGGAACTCTTTTCCCC 
3') were used as primers to generate the right vaccinia arm. PCR products 
consisting of the left and right vaccinia arms were combined, and 
subjected to PCR amplification. The PCR product was digested with EcoRI 
and HindIII and electrophoresed on an agarose gel. The 0.8 kb fragment was 
isolated and ligated into pUC8 cut with EcoRI/HindIII, resulting in 
plasmid pSD541. Potential transformants containing the K3L ORF were 
screened for the insert by colony hybridization using a radiolabeled 
K3L-specific DNA probe. Positives were confirmed by restriction analysis 
and DNA sequence analysis and designated as pK3LGP. 
Plasmid pK3LGP was used in IVR experiments with vP872 as the rescuing 
virus. Recombinants were screened by hybridization with a radiolabeled 
K3L-specific probe. Potential recombinant viruses were purified by three 
rounds of plaque purification. One purified recombinant was amplified and 
confirmed by DNA restriction analysis of the genomic DNA. The vP872 virus 
containing the reinserted K3L ORF was designated as vP1046. 
To determine the effect of reinserting the K3L ORF under the control of its 
endogenous promoter on the interferon sensitive phenotype of vP872, the 
following experiments were performed. Monolayers of mouse L929 cells were 
pretreated with 0, 10, 100, or 1000 units/ml of mouse .alpha.,.beta. 
interferon (Lee Biomolecular, La Jolla, Calif.). These pretreated cell 
monolayers were either mock infected or infected with VC-2 (wildtype 
Copenhagen), vP872, or vP1046 at an m.o.i. of 25 pfu/cell. Virus was 
adsorbed for one hour at 37.degree. C. with rocking every 10 minutes. At 
the end of the adsorption period, the inoculum was aspirated and 2 ml of 
fresh medium was applied to the monolayers. At 7 hour post-infection, the 
medium was aspirated and 2 ml methionine-free medium containing .sup.35 
S-methionine (20 .mu.Ci/ml) was added. The infected monolayers were pulsed 
for one hour, the medium was aspirated, washed 1X with PBS, and then 
harvested by 3 cycles of freeze-thawing in fresh PBS. Total protein 
content was quantitated using the Bio-Rad Protein Assay Kit (Bio-Rad 
Laboratories, Richmond, Calif.) and equal protein amounts were 
fractionated on a 12.5% SDS-polyacrylamide gel (27). The gel was fixed and 
treated for fluorography with 1% Na-Salicylate. 
vP1046 has an interferon-resistant phenotype similar to wildtype VC-2. 
Virus-specific protein synthesis in vP1046 infected cells was not 
inhibited to any significant extent except at interferon concentrations of 
500 units/ml or greater. In contrast, significant reduction in 
virus-induced protein synthesis was observed with vP872 at interferon 
concentrations as low as 10 units/ml. 
Viral yields were also analyzed. The experiment was performed in the same 
way as above except that the infections were harvested at 24 hour 
post-infection by three cycles of freeze-thawing. Virus progeny was 
titrated on Vero cells. As observed in the protein analysis above, vP872 
replication was severely inhibited by as little as 10 units/ml of 
interferon (10% the virus yield observed with no interferon), whereas the 
yields of VC-2 and the K3L restored virus, vP1046, were reduced to these 
levels at interferon concentrations of 500 units/ml. 
EXAMPLE 4 
Delection of K3L ORF From the WR Strain of Vaccinia Virus 
The WR strain of vaccinia virus has a K3L ORF which is 99% homologous at 
the amino acid level to the VC-2 K3L ORF (7,13). To determine whether a 
precise deletion of this K3L ORF from WR has the same phenotypic effect 
with respect to interferon sensitivity, a deletion plasmid to replace the 
K3L ORF with the rabies G gene was engineered. PCR-derived fragments of 
620 bp and 634 bp, consisting of K3L 5' and 3' flanking arms, 
respectively, were generated using pSD407 as template. The 620 bp fragment 
was obtained using oligonucleotides K3LF5 (SEQ ID NO: 12) 
(CCTTATTTTTATGTTCGGTATAAAAATTAAAGCTTCTTGTTAACGGGCTCGTAAATTGG) and K3L5X 
(SEQ ID NO: 13) (ATCATCTCTAGAGAATTAAGAAGATCCGC). The 634 bp fragment was 
derived with oligonucleotides K3LF3 (SEQ ID NO: 14) 
(CCAATTTACGAGCCCGTTAACAAGAAGCTTTAATTTTTATACCGAACATAAAAATAAGG) and K3L3RV 
(SEQ ID NO: 15) (GCGTGTTTTAGTGATATCAAACGG). These fragments were then used 
in equal amounts as template in subsequent PCR fusions using 
oligonucleotide primers K3L5X (SEQ ID NO: 13) and K3L3RV (SEQ ID NO: 15). 
This created a fusion between the 5' and 3' sequences with an XbaI site at 
the 5' end., an intact EcoRV site at the 3' end, and a HindIII site 
between the arms. The 1.2 kb fragment obtained was blunted using the 
Klenow fragment of the E. coli DNA polymerase in the presence of 2mM dNTPs 
and then digested with XbaI. The plasmid vector, pBSgpt, was digested with 
SmaI and XbaI and the K3L fusion arms were inserted (the plasmid pBSgpt 
contains the E. coli expression cassette described in generation of 
vP872). Clones containing the desired insert were screened by colony 
hybridization using the above PCR product as probe. Clones were confirmed 
by XbaI/PstI restriction analysis and verified by sequencing. The sequence 
verified recombinant was designated pK3LAex. This plasmid was partially 
digested with HindIII and the linear product, consisting of three products 
differing in the location of HindIII cleavage, was obtained. Plasmid 
pRW838 was digested with SmaI to liberate a 1.9 kb fragment containing the 
rabies G gene under control of the H6 promoter. The plasmid pRW838 
contains the rabies G gene in an canarypox virus insertion vector. This 
plasmid was generated in the following manner. Oligonucleotides A through 
E (A: (SEQ ID NO: 16) 
CTGAAATTATTTCATTATCGCGATATCCGTTAAGTTTGTATCGTAATGGTTCCTCAGGCTCTCCT GTTTGT; 
B: (SEQ ID NO: 17) CATTACGATACAAACTTAACGGATATCGCGATAATGAAATAATTTCAG; C: 
(SEQ ID NO: 18) 
ACCCCTTCTGGTTTTTCCGTTGTGTTTTGGGAAATTCCCTATTTACACGATCCCAGACAAGCTTA 
GATCTCAG; D: (SEQ ID NO: 19) 
CTGAGATCTAAGCTTGTCTGGGATCGTGTAAATAGGGAATTTCCCAAAACA; E: (SEQ ID NO: 20) 
CAACGGAAAAACCAGAAGGGGTACAAACAGGAGAGCCTGAGGAAC) were Kinased, annealed 
(95.degree. C. for 5 minutes, then cooled to room temperature), and 
inserted between the PvuII sites of pUC9. The resulting plasmid, pRW737, 
was cut with HindIII and BglII and used as a vector for the 1.6 kbp 
HindIII-BglII fragment of ptg155PRO (28) generating pRW739. The ptg155PRO 
HindIII site is 86 bp downstream of the rabies G translation initiation 
codon. BglII is downstream of the rabies G translation stop codon in 
ptg155PRO. pRW739 was partially cut with NruI, completely cut with BglII, 
and a 1.7 kbp NruI-BglII fragment, containing the 3' end of the H6 
promoter through the entire rabies G gene, was inserted between the NruI 
and BamHI sites of pRW824. The resulting plasmid was designated pRW832. 
Insertion into pRW824 added the H6 promoter 5' of NruI. The pRW824 
sequence (SEQ ID NO: 21) of BamHI followed by SmaI is: GGATCCCCGGG. pRW824 
is a plasmid that contains the infectious bronchitis virus peplomer gene 
linked precisely to the vaccinia H6 promoter. Digestion with NruI and 
BamHI completely excises the peplomer gene. The 1.8 kbp pRW832 SmaI 
fragment, containing the entire H6 promoted rabies G, was inserted into 
the SmaI site of pRW831. pRW831 is the C5 locus deorfed vector which was 
derived as follows. The C50RF is contained within pRW764.5. pRW764.5 is a 
0.9 kbp PvuII canarypox fragment cloned between the PvuII sites of pUC9. 
There are two BglII sites in pRW764.5 and they are both in the C50RF. The 
320 bp ORF was deleted from the T of C5's translation initiation codon to 
30 bp upstream of its stop codon. Replacement of the C5 ORF was achieved 
by insertion of annealed oligonucleotides RW145 (SEQ ID NO: 22) 
(5'-ACTCTCAAAAGCTTCCCGGGAATTCTAGCTAGCTAGTTTTTATAAA-3') and RW146 (SEQ ID 
NO: 23) (5'-GATCTTTATAAAAACTAGCTAGCTAGAATTCCCGGGAAGCTTTTGAGAGT-3') into 
pRW764.5 which was partially cut with RsaI and fully cut with GltII to 
delete 306bp. The resulting plasmid, pRW831, contains the following 
sequence (SEQ ID NO: 24) in place of the C5 ORF: 
GCTTCCCGGGAATTCTAGCTAGCTAGTTT. The inserted sequence is followed by TTAT 
which creates TTTTTAT 3' of rabies G in pRW838. 
The 1.9 kbp H6/rabies G fragment was ligated into linearized pK3LAex 
(described above). Recombinants containing the rabies gene were screened 
by colony hybridization. Clones that contained the rabies G gene were 
screened for proper insertion by restriction endonuclease digestion. The 
recombinant was designated pK3LAR. pK3LAR was used in standard 
recombination assays with the WR strain of vaccinia virus as the rescuing 
virus. Screening of this recombinant was by plaque hybridization using a 
rabies-specific probe. The recombinant generated was confirmed by 
restriction analysis and designated vP1033. 
EXAMPLE 5 
The Ability of vP872 to Rescue Vesicular Stomatitis Virus (VSV) and 
Endomyocarditis Virus (EMC) From the Antiviral Effects of Interferon 
The ability of vaccinia virus to rescue the interferon sensitive viruses, 
VSV and EMC, from the antiviral effects of interferon has been well 
documented (5,6). This is especially interesting since these two viruses 
are believed to be inhibited by different interferon-induced pathways 
(5,6,29). This suggests that vaccinia virus can interfere with the 
interferon-induced antiviral pathways at more than one level. It was of 
interest to determine whether the vaccinia virus K3L deletion mutant, 
vP872, had the capacity to rescue these two viruses to the same extent as 
wildtype virus. To test the rescuing potential of vP872, the following 
experiment was performed. L929 cells pretreated with 0, 10, 100, or 1000 
units/ml of mouse .alpha./.beta. IFN were infected with vaccinia (wildtype 
or the K3L-minus recombinant vP872) at an mol of 1 for 2 hours at 
37.degree. C. with rocking every 10 minutes. After 2 hours, the inoculum 
was aspirated and the monolayers washed with PBS. VSV and EMC were then 
inoculated onto the monolayers at an moi of 10 in the presence of 5 
.mu.g/ml actinomycin D (Sigma Chemicals, St. Louis, Mo.). After an hour 
adsorption period at 37.degree. C. (with rocking every 10 minutes), the 
inoculum was aspirated and 2 ml fresh media added. At 7 hours post 
infection with VSV or EMC, the media was removed and replaced with 
methionine-free MEM containing 20 .mu.Ci/ml .sup.35 S-methionine. The 
monolayers were pulsed for 1 hour then harvested by washing twice in PBS 
and lysing the cells by three cycles of freeze-thawing in 0.5 ml PBS. 
Total VSV or EMC-specific protein synthesis was evaluated upon 
fractionation of equal protein quantities by SDS-PAGE (27). Controls 
consisted of uninfected cells and vaccinia-infected controls not treated 
with actinomycin D. To determine the effect of vaccinia coinfection on VSV 
and EMC yields, the infections were performed as above, but they were 
harvested after 24 hours (without a .sup.35 S-methionine pulse) by three 
cycles of freeze-thawing. The virus was titered on L 929 cells on which 
vaccinia virus does not plaque but VSV and EMC do form plaques. 
A rescue experiment with VSV and EMC viruses, respectively, at the level of 
late protein synthesis was performed. VSV-induced protein synthesis was 
markedly inhibited by interferon concentrations as low as 10 units/ml and 
was virtually abolished at an interferon concentration of 1000 units/ml. 
Both VC-2 and vP872 were able to restore VSV-specific protein synthesis at 
all interferon concentrations tested, although VC-2 was more efficient in 
this regard. EMC showed moderate interferon sensitivity at concentrations 
of 10 units/ml and marked sensitivity at interferon concentrations greater 
that 100 units/ml. Only VC-2 was able to restore protein synthesis to EMC, 
although it is not as dramatic as the rescue of VSV. These results are 
shown in FIGS. 4 and 5 for the rescue experiments with VSV and EMC, 
respectively. It can be seen that VC-2 was able to rescue both VSV and EMC 
viruses from the antiviral effects of interferon. vP872, on the other 
hand, was able to rescue only VSV from the antiviral effects of 
interferon, and even then, not to the same extent as VC-2. 
The results for the VSV rescue experiments can be explained from what is 
known in the literature pertaining to vaccinia virus rescue of this 
interferon sensitive virus. Interferon is known to inhibit VSV replication 
in mouse L929 cells largely via translational shutdown (6). This system 
which shuts down VSV-specific translation is induced by interferon in the 
presence of double-stranded RNA synthesized during the vital replicative 
cycle. The presence of these components activates P1 kinase, which itself 
becomes phosphorylated, and this promotes the phosphorylation of 
eIF2-.alpha.. Phosphorylation of eIF2-.alpha. strongly correlates with a 
cessation of protein synthesis. Vaccinia appears to intercede to block 
this pathway at two levels. First, vaccinia infection is known to alter 
the phosphorylation of P1 kinase. Ten times more double-stranded RNA is 
required to obtain equivalent levels of phosphorylated P1 kinase in 
lysates from vaccinia infected cells than lysates from uninfected cells 
(30). This is apparently due to a vaccinia-encoded function designated as 
SKIF (30) which has characteristics consistent with being the 
double-stranded RNA binding protein recently identified (31). Second, the 
K3L gene product apparently affects the downstream portion of this 
mechanism by acting as a pseudosubstrate of the P1 kinase abrogating its 
ability to phosphorylate eIF2-.alpha.. Therefore, VC-2 which encodes both 
these functions is much more capable of rescuing VSV from the effects of 
interferon than vP872. vp872 still retains some capacity to rescue VSV due 
to its expression of the double-stranded RNA binding protein. 
The major antiviral mechanism of interferon upon EMC replication has been 
shown to be due to RNA breakdown mediated by ribonucleases activated by 
the interferon-induced 2'-5' adenylsynthetase (29). Since vaccinia virus 
has the ability to rescue EMC from the effects of interferon, this 
suggests vaccinia also abrogates this interferon-mediated pathway. In this 
light, however, why vP872 does not rescue EMC in the presence of 
interferon is not certain. Perhaps the vaccinia-induced factor that 
abrogates this antiviral modality is not expressed in sufficient 
quantities in the K3L-minus virus infected cell to achieve this function. 
Nonetheless, this example demonstrates that the recombinant poxvirus of the 
invention having an open reading frame for interferon resistance disrupted 
or deleted therefrom can still function with respect to protein synthesis 
in the presence of interferon, e.g., by expression of the double-stranded 
binding protein. Thus, a recombinant poxvirus containing exogenous DNA 
coding for an antigen and having the open reading frame for interferon 
resistance deleted therefrom is a useful vaccine because it can still 
function with respect to protein synthesis (thereby allowing sufficient 
antigen to be produced to stimulate an immune response); and this 
recombinant poxvirus is even of greater utility because it can be 
substantially "turned off" when interferon levels are raised, e.g., by 
administration of exogenous interferon, for instance, in the event of a 
post-vaccination complication. 
EXAMPLE 6 
Effect of Infection by vP872 OR vP1033 on Phosphorylation of Host Cell P1 
Kinase 
If the K3L-specified gene product, which is homologous to eIF2-.alpha., 
acts as a pseudosubstrate for the P1 kinase preventing phosphorylation of 
eIF2-.alpha. and the cessation of protein synthesis caused by the 
phosphorylation of eIF2-.alpha., the phosphorylation of P1 kinase would be 
similar in wildtype (VC-2) and K3L deletion mutant (vP872 and vP1033) 
infected cells. To investigate the phosphorylation of P1 kinase in these 
infected cell systems, the following experiment was performed. L929 cells 
(5.times.10.sup.4) were plated in a 100mm dish in 10 ml Dulbecco's 
Modified Eagle Medium (D-MEM; Gibco Laboratories, Grand Island, N.Y.) plus 
10% fetal bovine serum (Hyclone Laboratories), 2mM L-glutamine (Gibco 
Laboratories, Grand Island, N.Y.), and 1% penicillin-streptomycin (Gibco). 
The medium was aspirated the next day and 2 ml medium containing 0, 10, 
100, or 1000 units/ml mouse .alpha./.beta. interferon (Lee Biomedical 
Research, Inc., La Jolla, Calif.) was added. After 24 hours, the medium 
was aspirated, the monolayers were washed twice with 5 ml cold PBS, and 
the cells were mock-infected or infected with vaccinia virus VC-2, vP872, 
or vP1033 at an moi of 5 in 0.5 ml D-MEM without additives. Virus was 
adsorbed for 1 hour at 37.degree. C., then the inoculum was aspirated and 
fresh D-MEM containing 5% FBS+2mM L-glutamine+ 1% Penicillin-Streptomycin. 
Monolayers were harvested at 5 hpi by washing once in cold isotonic lysis 
buffer (35 mM Tris, pH 7.0; 146 mM NaC1; 11 mM glucose) followed by 
scraping cells into 5 ml isotonic lysis buffer followed by centrifugation 
for 10 minutes at 1,000 rpm. Supernatant removed and cell pellet lysed in 
100 .mu.l NP-40 lysis buffer (20 mM HEPES, pH 7.6; 120 mM KCl; 5 mM 
MgCl.sub.2 ; 1 mM DTT; 10% glycerol; 0.5% NP-40). Five .mu.l of the above 
lysate from uninfected or virus-infected cells was added to 5 .mu.l lysate 
from IFN-treated uninfected cells in a 1.5 ml Eppendorf tube. Five .mu.l 
P1 kinase assay buffer (60 mM HEPES, pH 7.5; 210 mM KCl; 25 mM MgOAc; 3 mM 
DTT; 2.5 mM ATP), 5 .mu.l dsRNA (Pharmacia LKB Biotechnology, Piscataway, 
N.J.; 0, 0.1, 1, or 10 .mu.g/ml) and 5 .mu.l .sup.32 Pi (2mCi/ml, 
3000Ci/mmol; dupont deNemours, Wilmington, Del.) were then added. 
Reactions were incubated 30 minutes at 30.degree. C. Reactions were 
stopped by the addition of 25 .mu.l 2x Laemmli sample buffer. Samples were 
boiled 3 minutes and fractionated on 12.5% SDS-PAGE. Gels were fixed, and 
an autoradiograph obtained. 
A P1 kinase assay using lysate from uninfected or vaccinia virus-infected 
L929 cells was performed. The results demonstrate that vP872 and vP1033, 
the K3L-minus recombinants derived from VC-2 and WR, respectively, were 
able to inhibit phosphorylation of the P1 kinase to the same extent as 
wild-type VC-2. With uninfected cells, the P1 kinase was phosphorylated in 
the presence of 1 and 10 ug/ml poly(I).multidot.poly(C). With all of the 
vaccinia viruses tested, VC-2, vP872, and vP1033, P1 kinase was not 
phosphorylated except in the presence of 10 ug/ml 
poly(I).multidot.poly(C). It took ten times higher concentrations of 
poly(I).multidot.poly(C) to activate the P1 kinase in cells infected with 
the vaccinia viruses. This corroborates observations made previously for 
the effect of vaccinia virus infection on the phosphorylation of P1 kinase 
(30). The inhibitory effect of P1 kinase phosphorylation is probably due 
to the action of a vaccinia virus encoded double-stranded RNA binding 
protein (31), which has characteristics consistent with the previously 
identified SKIF protein (30). That the phosphorylation of P1 kinase in 
vaccinia infected cells (wildtype or K3L-deletion mutants) was similar is 
consistent with the hypothesis that the presence of K3L probably acts 
mechanistically by preventing the phosphorylation of eIF2-.alpha. by P1 
kinase. 
EXAMPLE 7 
Sensitivity of Fowlpox Virus and Canarypox Virus to chicken Interferon 
Of the avipox viruses, only fowlpox virus has been tested for interferon 
sensitivity and was shown to be resistant to the antiviral effects of 
interferon in chick embryo fibroblasts treated with chicken interferon 
(32). To investigate the sensitivity of canarypox virus to interferon the 
following experiment was performed. Chicken embryo fibroblasts from 11 day 
old chicks (Select Laboratories, Gainesville, Ga.) were plated at 
1.2.times.10.sup.7 cells per 60 mm dish. Thirty minutes after plating, 
chicken interferon (Dr. Philip I. Marcus, University of Connecticut at 
Storrs; 20,000 units/ml) was added to the dishes at a final concentration 
of 0, 10, 100, or 1000 units/mi. After 24 hours, the medium was aspirated 
and the monolayers were infected with fowlpox virus or canarypox virus at 
an moi of 0.1 in 0.2 ml serum-free medium. The virus was adsorbed for 1 
hour at 37.degree. C. with rocking every 10 minutes. At the end of the 
adsorption period, the inoculum was aspirated and 2 ml fresh medium was 
added to the dishes. Virus was harvested at 72 hours post-infection by 
three cycles of freeze-thawing. Virus titrations were performed on CEF 
monolayers. 
FIG. 6 shows the results of a yield reduction experiment. It can be seen 
that fowlpox virus was not inhibited by any of the concentrations of 
interferon tested in this experiment. Canarypox virus, on the other hand, 
was inhibited by interferon concentrations greater than 100 units/ml and 
at an interferon concentration of 1000 units/ml was approximately equal to 
residual input virus of 4.9.times.10.sup.3. The demonstrated sensitivity 
of canarypox virus to interferon (FIG. 6) shows the ability to utilize 
interferon as an antiviral agent in the event of any post-vaccination 
complication induced by a canarypox based recombinant virus vaccine. 
This example illustrates that a poxvirus having an open reading frame for 
interferon resistance disrupted or deleted therefrom is useful as a 
vaccine. Example 22 of U.S. application Ser. No. 07/537,890 filed Jun. 14, 
1990 shows the utility of a recombinant canarypox virus containing 
exogenous DNA coding for rabies. As shown herein, canarypox virus 
naturally fails to have resistance to the antiviral effects of interferon. 
Thus, a recombinant poxvirus containing exogenous DNA coding for an 
antigen and having the open reading frame for interferon resistance 
deleted therefrom functions as the recombinant canarypox virus of Example 
22 of Ser. No. 07/537,890, filed Jun. 14, 1990; namely, that it will 
express the antigen (and thus elicit an immune response in the host), yet 
be able to be "turned off" by the administration of exogenous interferon. 
Furthermore, the techniques of the earlier Paoletti applications 
(mentioned above and incorporated by reference) can be used to prepare 
recombinant poxviruses containing exogenous DNA, and the techniques 
disclosed herein are used on such recombinant poxviruses to delete 
resistance to interferon, thereby yielding the especially useful viruses 
of this invention (containing exogenous DNA and having interferon 
resistance deleted). Likewise, the skilled artisan can employ the 
techniques herein and then the techniques of the earlier Paoletti 
applications to produce recombinant poxviruses containing exogenous DNA 
and having interferon resistance deleted therefrom. 
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SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 24 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 90 amino acids 
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(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
MetLeuAlaPheCy sTyrSerLeuProAsnAlaGlyAspValIleLys 
151015 
GlyArgValTyrGluXaaLysAspTyrAlaLeuTyrIleTyrLeuPhe 
202530 
AspTyrProHisSerGluAlaXaaIleLeuAlaGluSerValLysMet 
354045 
HisMetAspArgTyrVa lGluTyrArgAspLysLeuValGlyLysThr 
505560 
ValLysValLysValIleArgValAspTyrThrLysGlyTyrIleAsp 
6570 7580 
ValAsnTyrLysArgMetCysArgHisGln 
8590 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 316 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
MetProGlyLeuSerCysArgPheTyrGlnHisLysPheProGluVal 
151015 
GluAspValValMe tValAsnValArgSerIleAlaGluMetGlyAla 
202530 
TyrValSerLeuLeuGluTyrAsnAsnIleGluGlyMetIleLeuLeu 
35 4045 
SerGluLeuSerArgArgArgIleArgSerIleAsnXaaLysLeuIle 
505560 
ArgIleGlyArgAsnGluCysValVa lValIleArgValAspLysGlu 
65707580 
LysGlyTyrIleAspLeuSerLysArgArgValSerProGluGluAla 
85 9095 
IleLysCysGluAspLysPheThrLysSerLysThrValTyrSerIle 
100105110 
LeuArgHisValAlaGluV alLeuGluTyrThrLysAspGluGlnLeu 
115120125 
GluSerLeuPheGlnArgThrAlaTrpValPheAspAspLysTyrLys 
13013 5140 
ArgProGlyTyrGlyAlaTyrAspAlaPheLysHisAlaValSerAsp 
145150155160 
ProSerIleLeuAspSerLeuAs pLeuAsnGluAspGluArgGluVal 
165170175 
LeuIleAsnAsnIleAsnArgArgLeuThrProGlnAlaValLysIle 
180 185190 
ArgAlaAspIleGluValAlaCysTyrGlyTyrGluGlyIleAspAla 
195200205 
ValLysGluAlaLeuArgAlaGly LeuAsnCysSerThrGluThrMet 
210215220 
ProIleLysIleAsnLeuIleAlaProProArgTyrValMetThrThr 
225230 235240 
ThrThrLeuGluArgThrGluGlyLeuSerValLeuAsnGlnAlaMet 
245250255 
AlaValIleLysGluLysIle GluGluLysArgGlyValPheAsnVal 
260265270 
GlnMetGluProLysValValThrAspThrAspGluThrGluLeuAla 
275 280285 
ArgGlnLeuGluArgLeuGluArgGluAsnAlaGluValAspGlyAsp 
290295300 
AspAspAlaGluGluMetGluAlaLysAlaG luAsp 
305310315 
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ATCATCAAGCTTGTTAACGGGCTCGTA AATTGG33 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
ATCGATATTTTTATGCGTGATTGG 24 
(2) INFORMATION FOR SEQ ID NO:5: 
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ATCATCAAGCTTTAATTTTTATACCGAAC 29 
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ATCATCCTCGAGGCAGGCAATAGCGACATAAAC 33 
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ATCATCAAGCTTTTATTGATGTCTACACATCC32 
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(A) LENGTH: 44 base pairs 
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
GGGCTCAAGCTTGCGGCCGCTCATTAGACAAGCGAATGAGGGAC44 
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(i ) SEQUENCE CHARACTERISTICS: 
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AGATCTCCCGGGCTCGAGTAATTAATTAATTTTTATTACACCAGAAAAGACGGCTTGAGA60 
TC 62 
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TAATTACTCGAGCCCGGGAGATCTAATTTAATTTAATTTAT ATAACTCATTTTTTGAATA60 
TACT64 
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TATCTCGAATTCCCGCGGCTTTAAATGGACGGAACTCTTTTCCCC45 
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CCTTATTTTTATGTTCGGTATAAAAATTAAAGCTTCTTGTTAACGGGCTCGTAAATTGG59 
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ATCATCTCT AGAGAATTAAGAAGATCCGC29 
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CCAATTTACGAGCCCGTTAA CAAGAAGCTTTAATTTTTATACCGAACATAAAAATAAGG59 
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(A) LENGTH: 24 base pairs 
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GCGTGTTTTAGTGATATCAAACGG 24 
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(A) LENGTH: 71 base pairs 
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CTGAAATTATTTCATTATCGCGATATCCGTTAAGTTTGTATCGT AATGGTTCCTCAGGCT60 
CTCCTGTTTGT71 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
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CATTACGATACAAACTTAACGGATATCGCGATAATGAAATAATTTCAG48 
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(A) LENGTH: 73 base pairs 
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ACCCCTTCTGGTTTTTCCGTTGTGTTTTGGGAAATTCCCTATTTACACGATCCCAGACAA60 
GCTTAGATCTCAG73 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
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CTGAGATCTAAGCTTGTCTGGGATCGTGTAAATAGGGAATTTCCCAAAACA51 
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(A) LENGTH: 45 base pairs 
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CAACGGAAAAACCAGAAGGGGTACAAACAGGAGAGCCTGAGGAAC45 
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GGATCCCCGGG11 
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(i) SEQUENCE CHARACTERISTICS: 
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ACTCTCAAAAGCTTCCCGGGAATTCTAGCTAGCTAGTTTTTATAAA46 
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GATCTTTATAAAAACTAGCTAGCTAGAATTCCCGGGAAGCTTTTGAGAGT50 
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(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
GC TTCCCGGGAATTCTAGCTAGCTAGTTT29