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Title: Recombinant poxviruses
having foreign DNA expressed under the control of poxvirus regulatory
sequences
United States Patent: 6,998,252
Issued: February 14, 2006
Inventors: Moss; Bernard (Bethesda, MD);
Mackett; Michael (Manchester, GB); Smith; Geoffrey L. (Oxford, GB)
Assignee: The United States of America as
represented by the Department of Health and Human Services (Washington,
DC)
Appl. No.: 470360
Filed: June 6, 1995
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Covidien Pharmaceuticals Outsourcing
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Abstract
Recombinant poxviruses, such as vaccinia,
are provided that comprises a segment comprised of (A) a first DNA
sequence encoding a polypeptide that is foreign to poxvirus and (B) a
poxvirus transcriptional regulatory sequence, wherein (i) said
transcriptional regulatory sequence is adjacent to and exerts
transcriptional control over said first DNA sequence and (ii) said segment
is positioned within a nonessential genomic region of said recombinant
poxvirus. Vaccines, carriers, cells, and media comprising recombinant
poxviruses, and methods of immunization with recombinant poxviruses also
are provided.
DETAILED DESCRIPTION
OF THE INVENTION
The invention requires the steps of:
I. Preparation of vector such as a
plasmid containing poxvirus promoter, sites for insertion of a foreign
gene, and poxvirus DNA flanking sequences.
II. Preparation and insertion of foreign
gene into a plasmid or equivalent vector to form chimeric gene.
III. Transfection of cells with the
vector containing chimeric gene.
IV. Isolation of recombinant poxvirus
and detection of foreign gene product.
V. Infection of susceptible cells or
animals with poxvirus recombinants.
This description exemplifying manipulation of the vaccinia virus to
provide useful recombinants are provided as exemplary of methods readily
applicable to poxvirus. As will be readily apparent to those of ordinary
skill in the art. Changes and modifications practiced by or known to those
in the art are within the scope of the invention.
1. Preparation of plasmid vector containing vaccinia virus promoter, sites
for insertion of a foreign gene, and vaccinia virus DNA flanking
sequences.
The vehicle used to assemble the insertion vector may be any convenient
plasmid, cosmid or phage. Plasmids constructed for use include pBR322,
pBR325, l pBR328, pUC7, pUC8, or pUC9 described herein. The vaccinia virus
DNA segment used to promote transcription of the foreign gene contained
nucleotide sequences according and including the start site of an RNA. The
nucleotide sequence and a precise transcriptional map of this region was
needed for application of this method. When a convenient restriction
endonuclease site preceded the RNA start site and another occurred after
the RNA start site but before the first ATG or translational initiation
colon, the promoter segment was excised by restriction endonuclease
digestion and isolated by standard methods such as agarose gel
electrophoresis. When a convenient restriction endonuclease site was not
available, it was necessary to use other methods such as cleaving beyond
the desired site and removing extra nucleotides with an exonuclease such
as Bal, 31. The promoter segment directly or after modification of its
ends was ligated to a plasmid that had been cleaved with a restriction
endonuclease to provide compatible ligatable termini. Ligation of cohesive
or blunt ends followed standard procedures. Additional restriction
endonuclease sites were placed next to the promoter by inserting the
promoter into a plasmid such as pUC9 that already has multiple insertion
sites, however ligation of synthetic polynucleotides should also be
possible. The plasmid containing the promoter was used to transform
bacteria and then purified. Restriction endonucleases were used to cut out
the promoter with adjacent restriction endonuclease sites and the DNA
fragment was purified using conventional methods.
The DNA used to flank the promoter and added restriction endonuclease
sites was derived from a non-essential region of the vaccinia virus
genome. Examples of such non-essential regions include the thymidine
kinase gene and a region of at least 9,000 base-pairs (bp) that is
proximal to the left inverted terminal repetition. DNA containing the
non-essential region was excised by restriction endonuclease cleavage and
purified by agarose gel electrophoresis or other conventional methods. The
segment was then ligated to plasmid DNA that had been cleaved by a
endonuclease to give complementary ligatable termini. The plasmid
containing the vaccinia virus DNA was used to transform bacteria and then
purified. An appropriate restriction endonuclease was used to cleave the
non-essential segment of the vaccinia virus DNA within the plasmid so that
it could be ligated to the previously isolated promoter fragment. In this
manner or by variations of this procedure, a plasmid was obtained that has
a vaccinia virus promoter with adjacent restriction endonuclease sites
flanked by a non-essential segment of vaccinia virus DNA. Since this
plasmid retained the plasmid origin of replication and antibiotic
resistance gene, it was used to transform bacteria and replicated.
II Preparation and insertion of foreign gene into plasmid vector to form a
chimeric gene.
A segment of DNA containing a foreign gene or a cDNA copy of a foreign
gene was obtained. The DNA segment was cleaved with restriction
endonucleases at a site preceding the translational initiation codon and
distal to the end of the protein coding sequences. When appropriate sites
mare not present, then it was necessary to cleave beyond the desired site
and use an exonuclease such as Bal31 to remove extra nucleotides. For
optimal expression, the first ATG in the segment was used to initiate
translation of the desired gene. Since there is no evidence for splicing
of vaccinia virus RNAs, continuous protein coding sequences was used.
The plasmid constructed in part I of this section was cleaved at a
restriction endonuclease site next to the promoter. The protein coding
segment of the foreign gene was ligated directly to the promoter when it
had complementary termini or after modification of its ends. The plasmid
was used to transform bacteria and then purified. When the foreign gene
was insertable in more than one orientation, it was necessary to analyze
by restriction endonuclease digestion and gel electrophoresis or
nucleotide sequencing to check that the proper one was obtained. The
desired plasmid had the promoter adjacent to the start of the foreign
gene.
III Transfection of cells with plasmid containing chimeric gene.
Plasmids containing chimeric genes flanked by DNA from non-essential
regions of the vaccinia virus genome were used to transfect cells that
were already infected with vaccinia virus. The chimeric gene was inserted
into the vaccinia virus genome by homologous recombination. Typically,
confluent monolayers of BSC-1, TX-143, or other cells in
bottles with a 25 cm2 bottom surface area were infected with
0.01 to 0.05 plaque forming units (pfu) per cell of vaccinia virus.
Approximately 1 μg of plasmid DNA with or without 1 μg of vaccinia virus
DNA and 20 μg of calf thymus DNA or other carrier DNA was mixed in 1 ml of
0.1% dextrose, 0.14 M NaCl, 5 mM KCl, 1 mM Na2HPO4,
20 mM Napes, (pH 7.05) and precipitated by addition of CaCl2 to
a final concentration of 125 mM. The mixture was agitated gently and
allowed to remain at room temperature for about 45 min. Two hr after
infection, 0.8 ml of the fine suspension was added to an Infected
monolayer from which medium had been removed. After 30 min, 8 wt of Eagle
or other tissue culture medium containing 8% fetal bovine serum was added
to each bottle and the incubation was continued at 37° C. for 3.5 more hr.
At 6 hr after infection, fresh medium containing 8% fetal bovine serum was
added and incubation was continued for 48 hr. At this time, the infected
cells were scraped off the bottle, centrifuged, resuspended in tissue
culture medium and homogenized to break the cells and liberate virus.
IV. Isolation of recombinant vaccinia virus and detection of foreign gene
product.
Virus from transfected cells consisted of a population of which only a
small percentage were recombinants. A variety of selective and
non-selective methods were used to isolate these recombinants.
Selective procedures depended on the ability or recombinants to replicate
under conditions that inhibited the original virus. One selective method
involved the inactivation of the vaccinia virus TK gene. This was achieved
by using DNA from the vaccinia virus TK gene to flank the chimeric gene.
When homologous recombination occurred, the chimeric gene was inserted
into the TK gene of virus DNA and the recombinants exhibited a TK negative
(TK-) phenotype. Selective conditions for isolation of TK-
vaccinia virus was achieved by plaquing the virus in monolayers of TK-
negative cells such as TK-143 cells with 25 μg/ml of
5-bromodeoxyuridine (BUdR) in the 1% low melting agar overlay. After 48 to
72 hr at 37° C. in a 5% Co2 humidified atmosphere, plaques were
detected by staining with 0.005% neutral red. Typically more than 30% of
the TK- plaques consisted of recombinants and the remainder
were spontaneous TK- mutants of vaccinia virus.
A second selective method was used when TK- cells were infected
with TK- mutants of vaccinia virus and then transfected with
plasmids that contained a chimeric herpesvirus TK gene. [The TK-
mutants of vaccinia virus were obtained by infecting TK- 143
cells with vaccinia virus in the presence of 25 μg/ml of BUdR. The TK-
negative mutants were then plaqued at least 2 times in succession in TK-143
cells in the presence of BUdR]. Recombinants expressing herpesvirus TK
were selected by plaque assay on TK143 cells with a 1% low melting agar
overlay containing Eagle medium and 8% fetal bovine serum, 100 μM
thymidine, 50 (pH adenosine, 50 (pH guanosine, 10 (pH glycine, 1 (pH
methotrexate. After 48 to 72 hr at 37° C. in a 5% CO2,
humidified atmosphere, the plaques were detected by staining with neutral
red.
Non-selective methods that depend on identification of virus plaques that
contain the foreign gene were also used. In addition, such methods were
used to confirm the identity of recombinants even after isolation by
selective methods.
DNA—DNA hybridization was used to identify plaques formed by recombinant
virus. One method was referred to as dot blot hybridization. In this
procedure, virus obtained following transfection of infected cells with
chimeric plasmids was plaqued on cell monolayers with a 1% agar overlay.
After 48 to 72 hr, the plaques were detected by staining with neutral red.
Virus within individual plaques were picked using a sterile Pasteur
pipette and used to infect cell monolayers in 16 mm diameter wells of
microtiter dishes. After 48 hr incubation at 37° C., the cells were
scraped, lysed by three freeze-thaw cycles, and collected on
nitrocellulose sheets by filtration using a micro-sample manifold
(Schleicher and Scnuell, N H). The filter was washed with 100 mM NaCl, 50
mM Tris-HCl (pH 7.5), blotted three times on successive Whatman 3 MM
papers saturated with (1) 0.5 M NaOH, (2) 1 M Tris-HCl (pH 7.5), and (3)
2×SSC (SSC is 0.15 M NaCl, 0.015 M sodium citrate), baked at 80° C. for 2
hr and then incubated with 5×Oenhardt's solution [Denhardt, Biochem.
Biophys. Res. Commun., , 23:641-646 (1966)], supplemented with 0.1
mg/ml of denatured salmon sperm DNA in 4×SSC at 65° C. for 4 hr. The
foreign DNA, labeled with 32P by nick translation, and sodium
dodecyl sulfate (SDS) at a final concentration of 0.1% were added and
hybridization continued for 12 hr. The filter was washed twice for 15 min
at 65° C. with 2×SSC/0.1% SDS and then with 0.2×SSC/0.1% SDS. An
autoradiograph was made by placing the filter next to X-ray film and the
presence of dark spots on developed film identified recombinant virus.
Another method of DNA—DNA hybridization used was described by Villarreal
and Berg [Science 196:183-185 (1977)]. In this method, a replica of
virus plaques was made by placing a nitrocellulose filter directly on the
cell monolayer. DNA—DNA hybridization was carried out as above and, after
location of plaques containing recombinant virus, residual virus was
eluted from the agar that originally overlayed the plaques.
Additional methods that depend on expression of the foreign gene were also
used to identify plaques. In one case, 125I-labeled antibodies
to the product of the foreign gene were incubated with the cell monolayer
containing virus plaques. Plaques containing recombinant virus were then
identified by autoradiography. When the herpesvirus thymidine kinase was
expressed, recombinant plaques were detected by incorporation of [125I]deoxycytidine
(1 μC,i/ml) in the presence of 20 μg/ml of tetrahydrouridine. From 14 to
48 hr after infection.
V. Infection of susceptible cells or animals with vaccinia virus
recombinants.
After identification of vaccinia virus recombinants, 2 or more successive
plaque purifications were carried out to obtain pure recombinant virus.
Susceptible cells such as BSC-1, HeLa, MRC-5, or others were infected to
obtain large stocks of recombinant virus. The titers of the stocks were
determined by serial dilution and plaque assay.
To express the foreign gene, cells were infected with 1 to 30 pfu/cell of
crude or purified virus and incubations were carried out at 37° C. for up
to 48 hr. The foreign gene product, depending on its nature was found in
the cell culture medium or within the cells. When present in the cells, it
was liberated by one of a number of methods including sonication,
freeze-thawing, homogenization, or detergent treatment. The foreign
protein was detected by immunological, enzymatic, and electrophoretic
methods.
For infection of animals, recombinant virus was introduced intradermally,
although other routes should be satisfactory. Formation of antibodies to
the product of the foreign gene indicated that the foreign protein was
made and was immunogenic.
Claim 1 of 14 Claims
1. A plasmid that comprises
(A) a segment comprised of (i) a first DNA sequence encoding a polypeptide
that is foreign to poxvirus and (ii) a poxvirus promoter, wherein said
promoter is adjacent to and exerts transcriptional control over said first
DNA sequence; and, flanking said segment, (B) DNA from a nonessential region
of a poxvirus genome.
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