Note: Descriptions are shown in the official language in which they were submitted.
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ONCOLYTIC VIRUS
SUMMARY OF THE INVENTION
This invention provides methods of reducing the viability of a tumor cell,
infecting a neoplasm in a mammal with a virus, or treating a neoplasm in a
mammal, comprising administering a non-naturally occurring virus whereinthe
virus is: a) a reovirus whose mu-2 protein has amino acid residues A, R, M, F,
L, M,
r I, Q, I and S at positions 93, 150, 300, 302, 347, 372, 434, 458, 652 and
726,
respectively; or b) a reassortant of two or more parent strains of a viral
species
selected from the family Reoviridae, or progeny thereof; or c) a virus other
than a
reovirus wherein the virus other than a reovirus is: i) capable of expressing
a
reovirus mu-2 protein having amino acid residues A, R, M, F, L, M, I, Q, I and
S at
positions 93, 150, 300, 302, 34.7, 372, 434, 458, 652 and 726, respectively,
and ii) is
a DNA virus, a positive-sense RNA virus, or a negative-sense RNA virus
selected
from the group~consisting of Orthomyxoviridae, Rhabdoviridae and
Paramyxoviridae. This invention father provides the use of such non-naturally
occurring virus in the manufacture of a medicament for reducing the viability
of a
tumor cell, infecting a neoplasm in a mammal, or treating a neoplasm in a
mammal.
This invention provides a method of identifying a PKR sensitive virus
comprising: a) dividing a sample of a virus to be tested into a first portion
and
second portion; b) contacting PKR +/+ cells with the first portion and
contacting
PKR -/- cells with the second portion, under conditions permitting growth of
the
virus in PKR -/- cells; c) determining the rate of growth of the virus in the
PKR +/+
cells and in the PKR -/- cells; and d) comparing the growth rates from step
c),
wherein a higher rate of growth in the PKR -/- cells than in the PKR +/+ cells
identifies the virus as PKR sensitive. Such PKR sensitive viruses identified
in
accordance with this invention are useful for reducing the viability of a
tumor cell,
infecting a neoplasm in a mammal, or treating a neoplasm in a mammal.
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DESCRIPTION OF THE FIGURES
Figure 1: Virus yield of reovirus strains T1L and T3D in PIER -/- vs. PIER +/+
marine embryo fibroblasts.
Figure 2: Immuno-blot of PKR in MEF Infected with Reo TIL and T3D.
Figure 3: Lungs of mice with ct26 tumors after treatment with reovirus
strains.
T1L, T3D, EB96, EB108 and EB146 relative to untreated control
Iung. The lungs from 2 mice are shown for each treatment.
Figure 4: The weight of BALB-C mouse lungs relative to the presence of
CT26 tumors and reovirus treatment.
Figure 5: Histological sections stained with hematoxylin and eosin showing
lung Iobes of mice with ct26 tumors after treatment with reovirus
strains. T1L, T3D, EB96, EBI08 and EB146 relative to untreated
control lung.
DETAILED DESCRIPTION OF THE INVENTION
Throughout this application amino acids are generally identified using the
standard one-letter abbreviation, but can also be identified by name or
standard
three-letter abbreviations.
T 3D, T1L, T3A and T2J are standard abbreviations for reovirus strains T3
bearing, Tl Lang, T3 Abney, and T2 Jones, respectively. The above-listed names
of strains and their respective abbreviations are used interchangeably.
As used herein "phenotype" refers to the sequence of the expressed proteins
of a virus. In the case of reoviruses the expressed proteins are the gene
products of
the Ll, L2, L3, M1, M2, M3, S1, S2, S3 and S4 genes. Thus, if the amino acid
sequences of the products of these genes are the same in two different
reoviral
strains they are said to have the same phenotype.
As used herein "genotype" refers to the nucleotide sequence of the coding
region of a virus. Thus, for example, if the nucleotide sequences of the L1,
L2, L3,
M1, M~, M3, S1, S2, S3 and S4 genes of two reoviruses are the same in two
different reoviral strains they are said to have the same genotype.
The term "PFU" stands for plaque forming units and is a quantitative
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measure of live virus particles.
Examples of the anti-neoplastic and anti-tumor methods and use of this
invention as described above, include those utilizing a reovirus whose mu-2
protein
has amino acid residues A, R, M, F, L, M, I, Q, I and S at positions 93, 150,
300,
302, 347, 372, 434, 458, 652 and 726, respectively. In a more specific
embodiment
the reeoviral mu-2 protein has the amino acid sequence of the mu-2 protein of
reovirus strain T3 bearing, for example when the mu-2 protein is expressed by
a
gene having the nucleic acid sequence of the M1 gene of reovirus strain T3
bearing.
In a more specific embodiment the reovirus has the same genotype as a reovirus
strain selected from the group consisting of eb86, eb129, eb88, ebl3, and
eb145. In
a more specific embodiment the reovirus has a M1 gene whose sequence is the
same
as the Ml gene of reovirus strain T3 bearing and an L3 gene whose sequence is
the
same as the L3 gene of reovirus strain T1 Lang, for example the virus can have
the
same genotype as a reovirus strain selected from the group consisting of eb28,
eb3l,
eb97, eb123 and g16. In a still more specific embodiment the reovirus has a M1
gene whose sequence is the same as the M1 gene of reovirus strain T3 bearing
and
an L3 gene, Ll gene, and S2 gene whose sequences are the same as the
corresponding genes of reovirus strain T1 Lang, for example reoviruses having
the
same genotype as a reovirus strain selected from eb96, eb146 and eb108. In an
even
more specific embodiment the reovirus has a M1 gene whose sequence is the same
as the M1 gene of reovirus strain T3 bearing and an L3 gene, L1 gene, S2 gene
and
S4 gene whose sequences are the same as the corresponding genes of reovirus
strain
T1 Lang, for example reoviruses having the same genotype as reovirus strain
eb96.
Other examples of the anti-neoplastic and anti-tumor methods and use of this
invention as described above, include those utilizing a virus that is a
reassortant of
two or more parent strains of a viral species selected from the family
Reoviridae, or
progeny thereof. For example, reassortants can be made of two, three or four
of the
reovirus strains T3 bearing, T1 Lang, T3 Abney, and T2 Jones. In a more
specific
embodiment the reassortants are generated from parent strains T3 bearing and
T1
Lang. Examples of such strains include eb118, eb73.1, h17, h15, eb39, and h60
as
well as the other strains shown in Tables 1 and 2.
Other examples of the anti-neoplastic and anti-tumor methods and use of this
invention as described above, include those utilizing a virus other than a
reovirus
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that is: i) capable of expressing a reovirus mu-2 protein having amino acid
residues
A, R, M, F, L, M, I, Q, I and S at positions 93, I50, 300, 302, 347, 372, 434,
458,
652 and 726, respectively, and ii) is a DNA virus, a positive-sense RNA virus,
or a
negative-sense RNA virus selected from the group consisting of the families
Orthomyxoviridae, Rhabdoviridae and Paramyxoviridae. Examples of suitable
DNA viruses include a Herpesvirus, Adenovirus, Parvovirus, Papovavirus,
Iridovirus, Hepadenavirus, Poxvirus, mumps virus, human parainfluenza virus,
measles virus or rubella virus. Examples of suitable a positive-sense RNA
viruses
include a Togavirus, Flavivirus, Picornavirus, or Coronavirus. Examples of
suitable
negative-sense RNA virus selected from the group consisting of
Orthomyxoviridae,
Rhabdoviridae and Paramyxoviridae include an influenza virus or a vesicular
stomatitis virus.
In accordance with the method of identifying a PKR sensitive virus of this
invention as described above, any PKR +/+ and -/- cells can be used, and the
rate of
growth of the virus is determined by any standard technique for monitoring
viral
growth including those that measure the number of virus particles directly or
the
quantity of viral proteins. In a specific embodiment the PKR cells are mouse
embryo fibroblasts. In another specific embodiment the rate of growth of the
virus
is determined by a technique selected from the group consisting of plaque
titer
assay, antibody assay, and Western blot. Each of these techniques is
exemplified
below. Preferably the growth rate of the virus in PKR -/- cells is at least
ten times
higher than the growth rate in PKR +/+ cells.
In all of the anti-neoplastic and anti-tumor methods and use of this invention
as described above, the virus can be a replication competent virus and/or a
clonal
virus. The virus can be administered by any conventional route, including but
not
limited to intranasally, intratracheally, intravenously, intraperitoneally or
intratumorally. In accordance with the method or use of reducing the viability
of a
tumor cell described above, the virus can be administered to the tumor cell
either ih
vivo or ex vivo. When the virus is administered to a mammal, the mammal can be
either a human or a non-human mammal such as a mouse, sheep, cow, pig, dog or
rabbit. While the optimal dose is expected to differ somewhat from patient to
patient and can readily be determined by a skilled clinician, a dosage of from
3 x
10' to 3 x 109 PFU/kg is typical.
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The viruses utilized in accordance with this invention can be produced by
any conventional means, including reassortment among two or more parent virus
strains or the use of standard recombinant genetic techniques. Once produced,
such
viruses can be reproduced by culturing in cells to produce progeny. The
construction of reassortants of viruses is well known and is described, for
example
in Brown, et al., "The L2 Gene of Reovirus Serotype 3 Controls the Capacity to
Interfere, Accumulate Deletions and Establish Persistent Infection" in Double-
Stranded RNA Viruses, Compans, et al. eds. Elsevier (1983). For example,
reassortants can be made of two, three or four of the reovirus strains T3
bearing, T1
Lang, T3 Abney, and T2 Jones. Reassortants of T3 bearing and T1 Lang are
described in Example 2. Preferably the virus is replication competent and/or a
clonal virus.
This invention will be better understood by reference to the following
examples, which illustrate but are not intended to limit the invention
described
herein.
EXPERIMENTS
Experiment 1: Growth of Reovirus Strains T1L and T3D in PKR Knock-Out and
Wild T ae Fibroblast Cells
Viral Growth
The effect of PKR on reovirus infection was examined using PKR knock-out
(PKR -/-) marine embryo fibroblasts (MEF). Both reovirus T1L and T3D grow to
several fold higher titre in PIER -/- relative to PIER +/+ MEF, as measured by
plaque
assay. (Figure 1) This was associated with a higher percentage of antigen
positive
cells detected by fluorescent antibody stainitng described below. Consistent
with
this, infection of PIER -/- MEF resulted in several fold greater amounts of
viral
protein as assayed by western blot described below. Although both T1L and T3D
grew to higher titres in cells lacking the PIER gene T1L virus grew to higher
titres
than T3D in either PKR -/- or PKR +/+ cells. (Figure 1)
Indirect Immunostaining
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Cells were grown on glass coverslips in 35 mm diameter dishes and were
infected with reovirus T1L or T3D at a multiplicity of infection (moi) of 10.
After
48 hours incubation the cells were rinsed in PBS and fixed in prechilled
acetone for
min. After rinsing in PBS (3x5min), 100,1 of an appropriate dilution of type-
specifzc rabbit antivirus antisera was applied and incubated at room
temperature for
30 min. The coverslips were then rinsed in PBS (3x5min) and treated with the
appropriate dilution of Cy3-conjugated donkey anti-rabbit antibody (Jackson
ImmunoResearch Laboratories, Inc.) as the secondary antibody. After another 30
min incubation period at room temperature the coverslips were rinsed in PBS
(3x5min) and mounted on glass slides in Gel/Mount (Biomeda Corp). All antibody
dilutions were done in PBS/3 % BSA.
The samples were examined with a Zeiss microscope equipped with
epifluorescence and a 40X 1.40 NA PlanApo objective. The images were collected
using Image One Metamorph software and a Hamamatsu chilled charge-coupled
digital camera (model C5985). Configuration of the digital images was done
using
Corel Presentations software.
Immunoblotting
Monolayer cultures of MEF were infected at a moi=10 with T1L or T3D
virus as described above. At various times the culture medium was removed and
the cells were rinsed with PBS before solubilizing in 1 ml of sample buffer
(62.SmM Tris-HCl pH6.8, 10% glycerol, 2 % SDS, 0.05% bromophenol blue and 5
2-mercaptoethanol)(Laemmli). Aliquots of 25 u1 volume were subjected to SDS
PAGE and transblotted onto an Immobilon P membrane (Millipore) at 25V
overnight at 4°C. The dried membrane was blocked with 5% (w/v) skim
milk
powder in PBS for lhr at RT. This was followed by the addition of type
specific
rabbit anti-reovirus immune serum as the primary antibody in fresh blocking
solution and incubation for 2hr at 4°C. The membrane was then washed
three times
in PBS and once in TBS (100 mM Tris Hcl pH 7.4, 0.9 % NaCI)to remove
phosphate and incubated in 5% milk in TBS containing 1 uglml protein A
conjugated to alkaline phoshatase obtained from Sigma Chemicals (Oakville,
Ont)
Finally the membrane was washed 4x in TBS before reaction with chromogenic
substrate, vitro blue tetrazolium (NBT) (33 ug/ml) plus 5-bromo-4-chloro-3-
indolyl
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phoshate (BCIP) (3.3 ul/ml), in alkaline phosphatase buffer (100mM NaCI, SmM
MgCl2 and 100mM Tris-HCl pH9.5). The reaction was stopped with PBS
containing 20mM EDTA.
Experiment 2: Reassortants Between Reovirus Strains T1L and T3D
Production of Genetic reassortants between Reovirus Serotype 1 Lang strain and
Serotype 3 bearing strain.
Mouse L929 cells were coinfected with Reovirus Serotype I Lang strain
(TIL) and Serotype 3 bearing strain (T3D) at a multiplicity of infection of S
each.
Virus was harvested 24 hr post infection by 3 cycles of freezing and thawing
before
progeny viruses were isolated by 2 cycles of plaque isolation in L929
monolayers.
Since each of the corresponding genome segments of T1L and T3D is
distinguishable by electrophoretic mobility the genetic composition of each
virus
was determined by polyacrylamide geI electrophoresis of the segmented double
stranded RNA (dsRNA) genome where the mobility of each segment is compared to
the parental strains. Gels prepared as described by Laemmli contained 10%
polyacrylamide and 0.27% methylene bis-acrylamide. Double-stranded RNA was
obtained from L929 cells infected for 3 days and solubilised in buffer
containing
sodium dodecyl sulphate and was detected in gels stained with ethidium bromide
as
described previously (Zou S. and E.G. Brown. (1992) Identification of Sequence
elements containing signals for replication and encapsidation of the reovirus
Ml
genome segment. Virology 186:377-88.. The use of this panel of reassortants
was
first described by E.G. Brown, M. L. Nibert and B.N. Fields (1983) The L2 gene
of
reovirus serotype 3 controls the capacity to interfere, accumulate deletions
and
establish persistent infection. in Double-Stranded RNA Viruses. R.W. Compans
and
D.H.L. Bishop eds. Elsevier Science Publishing Co.
Growth of Reovirus
T1L, T3D and virus stocks from the reassortment procedure described above
were prepared in L929 cells grown in Earl's Minimal Essential Medium (MEM)
supplemented with 5 % fetal bovine serum and penicillin to100 units/ml and
streptomycin to 100 ug/xnl until cytopathic effect was complete. Cells and
culture
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supernatant were subjected to 3 cycles of freezing and thawing before
titration by
plaque assay.
Yields in Mouse Embryo Fibroblasts
Wild type PKR+/+ cells were obtained from Balb-C mice and PKR-/- cells
were obtained from PKR knockout mice. Cell cultures were produced using 15-17
days embryos that had been disaggregated by mincing and trypsin treatment.
Cell
monolayers were grown in 35 mm plastic dishes in MEM supplemented with 10%
FBS and P/S at 37 C in a 5% C02 atmosphere. Cells were infected with titrated
T1L, T3D or reassortant reovirus at a multiplicity of infection (moi) of 10 by
adsorption of stock virus for 0.5 hr with agitation at 15 minute intervals.
LTnadsorbed virus was removed by 3 washes with 2 ml of warm PBS each before
the
addition of 3 ml of MEM supplemented with 5 % fetal bovine serum and
penicillin
to100 units/ml and streptomycin to 100 ug/ml. The yield of T1L and T3D was
assayed at time points over a 4 day period and is shown in Figure 1.
Comparison of
yields of virus from MEF cells infected with reassortant reovirus was done
after 3
days incubation by plaque assay of duplicate cultures. The results are shown
below
in Table 1 (PKR -/-) and Table 2 (PKR +/+).
Plaque assay of reovirus in L929 Cells
Monolayer cultures of L929 cells were decanted of medium and infected in
duplicate with 0.1 ml volumes of serially diluted virus in PBS. Virus was
adsorbed
for 0.5 hr before the application of 3 ml of MEM supplemented with 1 % agax, 5
FBS and P/S. Cultures were incubated at 37 C and supplementary overlays of 2
ml
aliquots of the-same medium was added 3 and 6 days post infection. After 8
days of
infection the monolayers were stained for 24 hr with 2 ml of the same overlay
solution supplemented with neutral red (0.01 % weight/volume) to observe
plaques.
Discussion
The genetic basis for the increased ability of T1L to grow in each cell type
was determined using TIL x T3D reassortants. The difference in yield in wild
type
MEF (PKR +/+) segregated primarily with the M1 gene whereas the difference in
yield in PKR -l- MEF was associated with the L1, L3, M3 and S2 genes and did
not
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involve the Ml gene. The comparison of the genetic basis for replication in
PKR
+/+ relative to PKR -/- MEF cells indicates that the ability of the PKR gene
to
inhibit reovirus infection is dependent on the properties of the M1 gene.
Furthermore the extent of replication and thus exploitation of PKR -/- cells
is
dependent on the nature of the L l, L3, M3 and S2 genes. Thus the reassortant
viruses with the greatest differential ability to replicate in PKR -/-
relative to PKR
+/+ cells possess the T3D M1 gene and the viruses with the greatest ability to
replicate in PKR -l- cells (characteristic of many tumor cells) possess the
L1, L3,
M3 and S2 genes of T1L. Such viruses are restricted in replication of PKR +/+
cells
but replicate to a greater extent than either T1L or T3D in PKR -/- cells and
are
embodied in the properties of the reassortants eb96 and eb108. Statistical
analyses
of the experimental results are shown in Tables 1, 2 and 3.
The amino acid sequences of the T 1 L and T3D mu2 proteins are shown in
Table 4. Each protein is 736 amino acids long and they differ at 10 as
positions.
The observed difference in sensitivity to PKR seen as an ability to replicate
in
pKR+/+ relative to PKR-/- MEF cells is attributed to the difference in amino
acid
sequence between these proteins and thus M1 proteins of reoviruses with these
amino acid changes or other substitutions at these positions are addressed
herein.
The mu2 protein is encoded by the M1 gene. The nucleotide sequences of the T1L
and T3D M1 gene are shown in Table 5. Each genome segment is 2304 nucleotides
long and they differ at 51 nucleotide positions.
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TABLE 2: PIER +/+ (wild type)
VIRUSTITRE L1 L2 L3 M1 M2 M3 S1 S2 S3 S4 RANK
h60 3.96E+08D D L D D D D D L 1
eb392.35E+08L D D L D D D D D D 2
H15 1.78E+08L D D L D D D D D L 3
eb1181.76E+08D D L L D D D D L L 4
eb1461.68E+08L L L D L L L L L D 5
T1 1.50E+08L L L L L L L L L L 6
L
h17 1.46E+08D D !. L D D L D D L 7
eb281.30E+08D D L D D D D L D D 8
eb73.11.23E+08L D L L D D D D D D 9
eb315.20E+07L L L D L I- L D D L 10
eb1234.88E+07D D L D D D D D L D 11
g16 4.03E+07L L L D L L L D L L 12
eb1293.78E+07D D D D D L D L L D 13
eb972.35E+07D D L D D D D D D L 14
eb962.20E+07L D L D L L L L D L 15
eb1081.33E+07L D L D L L L L D D 16
T3D 1.20E+07D D D D D D D D D D 17
eb137.50E+06D D D D D D D D D L 18
eb866.40E+06L D D D D L D D D L 19
eb886.00E+06D D D D L D D D D D 20
eb1452.25E+06D D D D D L L D D D 21
t-test 0.39 0.15 0.056 0.0001 0.68 0.2 0.76 0.56 0.1 0.48
M-W test 0.4 0.35 0.07 0.0009 0.63 0.21 0.8 0.85 0.24 0.42
In Tables 1 and 2, parental origin of genome segments is indicated by L (T1L)
or D
(T3D). Statistical significance was determined using the t-test and the Mann-
Whitney (MVO test.
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TABLE 3: SUSCEPTIBILITY TO PKR SEGREGATES WITH THE M1
GENE
Gene Single Stepwise regression
gene regression
(R2 %) (R2 %)
PKR+/+ PKR-/- PKR+/+ PIER-/-
L1 0 19 (P=.048) 0 L3 + L1
48 (P=.003)
L3 23.8 36 (P=.004) M1+L3 67.0 36 (P=.004)
(P=.025) (P<.001 )
M1 51.6 0 51.6 (P=<.001)L3 + L1+ M1
(P<.001) 56 (P=.0025)
S2 0 16 (P=.073) 0 L3+L1+M3+S
2 63 .4 (P<.001
)
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TABLE 4: Alignment of T1L (GenBank Accession No. CAA42570.1) and T3D
(GenBank Accession No. AAA47256.1) mu2 proteins. These amino acid sequences
were deduced from cDNA. Each protein is 736 nucleotides long and differs at 10
as
positions.
T1L 1
MAYIAVPAVVDSRSSEAIGLLESFGVDAGADANDVSYQDHDYVLDQLQYMLDGYEA
GDVI 60
Consensus
MAYIAVPAVVDSRSSEAIGLLESFGVDAGADANDVSYQDHDYVLDQLQYMLDGYEAGDVI
T3D 1
MAYIAVPAVVDSRSSEAIGLLESFGVDAGADANDVSYQDHDYVLDQLQYMLDGYEA
GDVI 60
T1L 61
DALVHKNWLHHSVYCLLPPKSQLLEYWKSNPSVIPDNVDRRLRKRLMLKKDLRKDD
EYNQ 12 0
Consensus DALVHKNWLHHSVYCLLPPKSQLLEYWKSNPS
IPDNVDRRLRKRLMLKKDLRKDDEYNQ
T3D 61
DALVHKNWLHHSVYCLLPPKSQLLEYWKSNPSAIPDNVDRRLRKRLMLKKDLRKDD
EYNQ 120
T1L 121
LARAFKISDVYAPLISSTTSPMTMIQNLNQGEIVYTTTDRVIGARILLYAPRKYYA
STLS 180
Consensus LARAFKISDVYAPLISSTTSPMTMIQNLN
GEIVYTTTDRVIGARILLYAPRKYYASTLS
T3D 121
LARAFKISDVYAPLISSTTSPMTMIQNLNRGEIVYTTTDRVIGARILLYAPRKYYA
STLS 180
T1L 181
FTMTKCIIPFGKEVGRVPHSRFNVGTFPSIATPKCFVMSGVDIESIPNEFIKLFYQ
RVKS 240
Consensus
FTMTKCIIPFGKEVGRVPHSRFNVGTFPSIATPKCFVMSGVDIESIPNEFIKLFYQRVKS
T3D 181
FTMTKCIIPFGKEVGRVPHSRFNVGTFPSIATPKCFVMSGVDIESIPNEFIKLFYQ
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RVKS 240
T1L 241
VHANILNDISPQIVSDMINRKRLRVHTPSDRRAAQLMHLPYHVKRGASHVDVYKVD
VVDV 300
Consensus
VHANILNDISPQIVSDMINRKRLRVHTPSDRRAAQLMHLPYHVKRGASHVDVYKVDVVD
T3D 241
VHANILNDISPQIVSDMINRKRLRVHTPSDRRAAQLMHLPYHVKRGASHVDVYKVD
VVDM 300
T1L 301
LLEVVDVADGLRNVSRKLTMHTVPVCILEMLGIEIADYCIRQEDGMFTDWFLLLTM
LSDG 360
Consensus L EVVDVADGLRNVSRKLTMHTVPVCILEMLGIEIADYCIRQEDGM
TDWFLLLTMLSDG
T3D 301
LFEVVDVADGLRNVSRKLTMHTVPVCILEMLGIEIADYCIRQEDGMLTDWFLLLTM
LSDG 360
T1L 361
LTDRRTHCQYLINPSSVPPDVILNISITGFINRHTIDVMPDIYDFVKPIGAVLPK
GSFKS 420
consensus LTDRRTHCQYL
NPSSVPPDVILNISITGFINRHTIDVMPDIYDFVKPTGAVLPKGSFKS
T3D 361
LTDRRTHCQYLMNPSSVPPDVILNISITGFINRHTIDVMPDIYDFVKPIGAVLPK
GSFKS 420
T1L 421
TTMRVLDSISILGVQIMPRAHVVDSDEVGEQMEPTFEHAVMEIYKGIAGVDSLDDL
IKWV 480
Consensus TIMRVLDSISILG QIMPRAHVVDSDEVGEQMEPTFE
AVMEIYKGIAGVDSLDDLIKWV
T3D 421
TIMRVLDSISILGIQIMPRAHVVDSDEVGEQMEPTFEQAVMEIYKGIAGVDSLDDL
IKWV 480
T1L 481
LNSDLIPHDDRLGQLFQAFLPLAKDLLAPMARKFYDNSMSEGRLLTFAHADSELLN
ANYF 540
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Consensus
LNSDLIPHDDRLGQLFQAFLPLAKDLLAPMARKFYDNSMSEGRLLTFAHADSELLNANYF
T3D 481
LNSDLIPHDDRLGQLFQAFLPLAKDLLAPMARKFYDNSMSEGRLLTFAHADSELLN
ANYF 540
T1L 541
GHLLRLKIPYITEVNLMIRKNREGGELFQLVLSYLYKMYATSAQPKWFGSLLRLLI
CPWL 600
Consensus
GHLLRLKIPYITEVNLMTRKNREGGELFQLVLSYLYKMYATSAQPKWFGSLLRLLICPWL
T3D 541
GHLLRLKIPYITEVNLMIRKNREGGELFQLVLSYLYKMYATSAQPKWFGSLLRLL
ICPWL 600
T1L 601
HMEKLIGEADPASTSAEIGWHIPREQLMQDGWCGCEDGFIPWSIRAPRLVMEELM
EKNW 660
consensus HMEKLIGEADPASTSAEIGWHIPREQLMQDGWCGCEDGFIPWSIRAPRLV
EELMEKNW
T3D 601
HMEKLIGEADPASTSAEIGWHIPREQLMQDGWCGCEDGFIPWSIRAPRLVIEELM
EKNW 660
T1L 661
GQYHAQVIVTDQLWGEPRRVSAKAVIKGNHLPVKLVSRFACFTLTAKYEMRLSCG
HSTG 720
Consensus
GQYHAQVIVTDQLWGEPRRVSAKAVIKGNHLPVKLVSRFACFTLTAKYEMRLSCGHSTG
T3D 661
GQYHAQVIVTDQLWGEPRRVSAKAVIKGNHLPVKLVSRFACFTLTAKYEMRLSCG
HSTG 720
T1L 721 RGAAYNARLAFRSDLA 736
Consensus RGAAY ARLAFRSDLA
T3D 721 RGAAYSARLAFRSDLA 736
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TABLE 5: Alignment of the nucleotide sequences of the T1L (GenBank Accession
No. X59945.1) and T3D (GenBank Accession No M27261.1) M1 cDNA encoding
mu-2 protein. The complete coding sequences are shown. Since reoviruses are
double-stranded RNA viruses, the reoviral genome would contain "u" in place to
"t". Each genome segment shown below is 2304 nucleotides long that differ at
51
nucleotide positions.
T1L 1
gctattcgcggtcatggcttacatcgcagttcctgcggtggtggattcacgttcaa
gtga 60
I~IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIII
T3D 1
gctattcgcggtcatggcttacatcgcagttcctgcggtggtggattcacgttcga
gtga 60
T1L 61
ggctattggactgctagaatcgtttggagtagacgctggggctgatgcgaatgacg
tttc 120
IIIIIIIIillllllllllllllllllllllllllllllllllll
IIIIIIIIIIIIII
T3D 61
ggctattggactgctagaatcgtttggagtagacgctggggctgacgcgaatgacg
tttc 120
T1L 121
atatcaagatcatgactatgtgttggatcagttacagtatatgttagatggatatg
aggc 180
IIIIIIIIIIIIIIIIIII~IIIIIIIII~I~IIIIIII
IIIIIIIIIIIIIIIIIIII
T3D 121
atatcaagatcatgactatgtgttggatcagttacagtacatgttagatggatatg
aggc 180
T1L 181
tggcgacgttatcgatgcactcgtccacaagaattggttacatcactccgtctatt
gctt 240
III IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIII
T3D 181
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tggtgacgttatcgatgcactcgtccacaagaattggttacatcactctgtctatt
gctt 240
T1L 241
gttgccacccaaaagtcaactactagagtattggaaaagtaatccttcagtgatac
cgga 300
Illllllllillllllllllll III111111111111111111111111
IIIIIIIII
T3D 241
gttgccacccaaaagtcaactattagagtattggaaaagtaatccttcagcgatac
cgga 300
T1L 301
caacgttgatcgtcggcttcgtaaacgactaatgctaaagaaagatctcagaaaag
atga 360
IIIIII111111111111111111111111111111111111111111111
1111111
T3D 301
caacgttgatcgtcggcttcgtaaaCgactaatgctaaagaaagatctcaggaaag
atga 360
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T1L 361
tgaatacaatcaactagcgcgtgctttcaagatatcggatgtctacgcacctctca
tctc 420
IIIIIII~IIII
IIIIIII~IIIII~IIIIIIII~IIIIIIIIIIIIII~IIIIIIIII
T3D 361
tgaatacaatcagctagcgcgtgctttcaagatatcggatgtctacgcacctctca
tctc 420
T1L 421
atccacgacgtcaccgatgacaatgatccagaacttgaatcaaggcgagatcgtgt
acac 480
IIIIIIIIIIIIIIIIIIIIIIIIIII IIII~IIIIIIII
IIIIIIIIIIIIIIIIII
T3D 421
atccacgacgtcaccgatgacaatgatacagaacttgaatcgaggcgagatcgtgt
acac 480
T1L 481
cacgacggacagggtaattggggctagaatcttgttatatgctcctagaaagtact
atgc 540
IIIIIIIIIIIIIIIII~
IIIIIIIIII~IIIIIIIIIIII~IIIIIIII~I~IIIIII
T3D 481
cacgacggacagggtaataggggctagaatcttgttatatgctcctagaaagtact
atgc 540
T1L 541
gtcaactctatcatttactatgactaagtgcatcattccgtttggcaaagaggtgg
gtcg 600
IIIIIIIII IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII~
llllllllllllll
T3D 541
gtcaactctgtcatttactatgactaagtgcatcattccgtttggtaaagaggtgg
gtcg 600
T1L 601
tgttcctcactctagatttaatgttggcacatttccatcaattgctaccccgaaat
gttt 660
IIIIIIIIIII~I IIIIIIIIIII~IIIIIIIIII
IIII~IIIIIIIIIII~IIIIII
Ig
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T3D 601
tgttcctcactctcgatttaatgttggcacatttccgtcaattgctaccccgaaat
gttt 660
T1L 661
tgtcatgagtggggttgatattgagtccatcccaaatgaattcatcaagttgtttt
acca 720
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIiIII
IIIIIIIIIIIIIIIII
T3D 661
tgtcatgagtggggttgatattgagtccatcccaaatgaatttatcaagttgtttt
acca 720
T1L 721
gcgcgtcaagagtgttcacgccaatatactaaatgacatatcacctcagatcgtct
ctga 780
Illlllillllllllllllll II IIIilllllllllllil
lllllllllllllllll
T3D 721
gcgcgtcaagagtgttcacgctaacatactaaatgacatatctcctcagatcgtct
ctga 780
T1L 781
catgataaacagaaagcgtttgcgcgttcatactccatcagatcgtcgagccgcgc
agtt 840
IIIllllllllllllllll
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIilllllllll
T3D 781
catgataaacagaaagcgtctgcgcgttcatactccatcagatcgtcgagccgcgc
agtt 840
T1L 841
gatgcatttgccctaccatgttaaacgaggagcgtctcacgtcgacgtttacaagg
tgga 900
lillllllilll
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
T3D 841
gatgcatttgccttaccatgttaaacgaggagcgtctcacgtcgacgtttacaagg
tgga 900
T1L 901
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tgttgtagacgtgttgttagaggtagtggatgtggccgatgggttgcgcaacgta
tctag 960
Illlllllll 1111111
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIillllllllll
T3D 901
tgttgtagacatgttgttcgaggtagtggatgtggccgatgggttgcgcaacgta
tctag 960
T1L 961
gaaactaactatgcataccgttccggtatgtattcttgaaatgttgggtattgaga
ttgc 1020
IIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
T3D 961
gaaactaactatgcataccgttcctgtatgtattcttgaaatgttgggtattgaga
ttgc 1020
T1L 1021
ggactattgcattcgtcaagaggatggaatgttcacagattggttcctacttttaa
scat 1080
IIIIIIIIIIIIIIIIIIIIIIIIillllll
IIIIIIIIIIIIIIIIIIIIIIIIIIII
T3D 1021
ggactattgcattcgtcaagaggatggaatgctcacagattggttcctacttttaa
coat 1080
T1L 1081
gctatctgatggcttaactgatagaaggacgcattgtcaatacttgattaatccgt
caag 1140
Illllllllllllll Illlllllllllllllllllllllllllllll
IIIIIIIIIII
T3D 1081
gctatctgatggcttgactgatagaaggacgcattgtcaatacttgatgaatccgt
caag 1140
T1L 1141
tgtgcctcctgatgtgatacttaacatctcaattactggatttataaataggcata
caat 1200
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIII
T3D 1241
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tgtgcctcctgatgtgatacttaacatctcaattactggatttataaatagaeata
caat 1200
T1L 1201
cgatgtcatgcctgatatatatgacttcgttaaacccattggcgctgtgctgccta
aggg 1260
T3D 1201
cgatgtcatgcctgacatatatgacttcgttaaacccattggcgctgtgctgccta
aggg 1260
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T1L 1261
atcatttaaatcaacaattatgagagttcttgattcaatatcaatattaggagtcc
agat 1320
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIII II
T3D 1261
atcatttaaatcaacaattatgagagttcttgattcaatatcaatattaggaatcc
aaat 1320
T1L 1321
catgccgcgcgcgcatgtagttgactcggatgaggtgggcgagcaaatggagccta
cgtt 1380
111111111111111111111111111
IIIIllllllllllllllllllllllllllll
T3D 1321
catgccgcgcgcgcatgtagttgactcagatgaggtgggcgagcaaatggagccta
cgtt 1380
T1L 1381
tgagcatgcggttatggagatatacaaagggattgctggcgttgactcgctggatg
atct 1440
111111
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
T3D 1381
tgagcaggcggttatggagatatacaaagggattgctggcgttgactcgctggatg
atct 1440
T1L 1441
catcaagtgggtgctgaactcggatctcattccgcatgatgacaggcttggccaat
tatt 1500
III1111111111 1111111111111111111111111111111111111
11111111
T3D 1441
catcaagtgggtgttgaactcggatctcattccgcatgatgacaggcttggtcaat
tatt 1500
T1L 1501
tcaagcgtttctgcctctcgcaaaggacttgttagctccaatggccagaaagtttt
atga 1560
1111111111 1111111111111111111
11111111111111111111111111111
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T3D 1501
tcaagcgtttttgcctctcgcaaaggacttattagctccaatggccagaaagtttt
atga 1560
T1L 1561
taactcaatgagtgagggtagattgctgacattcgctcatgccgacagtgagttgc
tgaa 1620
IIIIIIIIIIIIIIIIIIIilllllll
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
T3D 1561
taactcaatgagtgagggtagattgctaacattcgctcatgccgacagtgagttgc
tgaa 1620
T1L 1621
cgcaaattactttggtcatttattgcgactaaaaataccatatattacagaggtta
atct 1680
IIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
T3D 1621
cgcaaattattttggtcatttattgcgactaaaaataccatatattacagaggtta
atct 1680
T1L 1681
gatgattcgcaagaatcgtgagggtggagagctatttcagcttgtgttatcgtatc
tata 1740
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIII
T3D 1681
gatgattcgcaagaatcgtgagggtggagagctatttcagcttgtgttatcttatc
tata 1740
T1L 1741
taaaatgtatgctactagcgcgcagcctaaatggtttggatcattattgcgattgt
taat 1800
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
T3D 1741
taaaatgtatgctactagcgcgcagcctaaatggtttggatcattattgcgattgt
taat 1800
T1L 1801
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atgtccctggttacatatggagaaattaataggagaagcagacccggcatctacgt
cggc 1860
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
T3D 1801
atgtccctggttacatatggagaaattaataggagaagcagacccggcatctacgt
cggc 1860
T1L 1861
tgaaattggatggcatatccctcgtgaacagctgatgcaagatggatggtgtggat
gtga 1920
IIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
T3D 1861
tgaaattgggtggcatatccctcgtgaacagctgatgcaagatggatggtgtggat
gtga 1920
T1L 1921
agatggattcattccctatgttagcatacgtgcgccaagactggttatggaggagt
tgat 1980
III IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIII
T3D 1921
agacggattcattccctatgttagcatacgtgcgccaagactggttatagaggagt
tgat 1980
T1L 1981
ggagaagaactggggccaatatcatgcccaagttattgtcactgatcagcttgtcg
tagg 2040
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
T3D 1981
ggagaagaactggggccaatatcatgcccaagttattgtcactgatcagcttgtcg
tagg 2040
T1L 2041
cgaaccgcggagggtatctgccaaggctgtgatcaagggtaatcacttaccagtta
agtt 2100
IIIIIIIIIIIIIIIIIIiII IIIIIIIIIIIIIIIIIIII
IIIIIIIIIIIIIIIII
T3D 2041
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cgaaccgcggagggtatctgctaaggctgtgatcaagggtaaccacttacc°agtta
agtt 2100
T1L 2101
agtttcacgatttgcatgtttcacattgacggcgaagtatgagatgaggctctcgt
gcgg 2160
T3D 2101
agtttcacgatttgcatgtttcacattgacggcgaagtatgagatgaggctttcgt
gcgg 2160
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T1L 2161
ccatagcactggacggggggctgcatacaatgcgagactagctttccgatctgact
tggc 2220
IIIIIIIIIIIIIII II IIIIIIIIII
IIIIIIIIIIIIIIIIIIIIIIIIIIIIII
T3D 2161
ccatagcactggacgtggagctgcatacagtgcgagactagctttccgatctgact
tggc 2220
T1L 2221
gtgatccgtgacatgcgtagtgtgacacctgcccctaggtcaatgggggtaggggg
cggg 2280
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII
IIIIIIIIIIillllllllllllllll
T3D 2221
gtgatccgtgacatgcgtagtgtgacacctgctcctaggtcaatgggggtaggggg
cggg 2280
T1L 2281 ctaagactacgtacgcgcttcatc 2304
IIIIIIIIIIIIIIIIIIIIIIII
T3D 2281 ctaagactacgtacgcgcttcatc 2304
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Experiment 3: Assessment of lethal infection in PKR -/- vs. PKR +/+ Mice
Adult Balb-C PIER +/+ or PKR -/- mice were infected with various dosages
of infectious reovirus T1L or T3D via the intraperitoneal (IP) or intranasal
(IN)
route. IP injections involved the administration of 0.1 ml of stock virus or
virus
diluted in PBS. IN infection involved the application of 0.05 ml volumes of
stock
virus or virus diluted in PBS onto the nose-pad of mice anaesthetized with
halothane
(administered at 3% in oxygen). The survival of adult mice was monitored over
a
30 day period. Adult PKR +l+ and PKR -/- mice resisted infection with Se6
infectious T3D virus whereas T1L virus killed PKR -/- mice but not PKR +/+
mice
at this dose. This demonstrates an enhanced ability of T1L to infect the
tissues of
PKR -/- mice. Table 5.
Two day old suckling Balb-C PKR +/+ or PKR -/- mice were infected with
various dosages of infectious reovirus T1L or T3D via the IN route. IN
infectious
involved the application of 0.01 ml volumes of stock virus or virus diluted in
PBS
onto the nose-pad of mice anaesthetized with halothane (administered at 3% in
oxygen). The survival of suckling mice was monitored over an 18 day period.
Suckling PKR +/+ or PKR -/- mice were both susceptible to similar dosages of
T1L
whereas T3D virus killed PKR -/- mice much more effectively than PKR +/+ mice,
killing them at doses more than 100 fold less than those required to kill wild
type
suckling mice. This demonstrates an enhanced ability of T3D to infect the
tissues of
PKR -/- tissues of suckling mice and indicates a difference in the properties
of the
T1L and the T3D strains with respect to differential replication in PKR +/+
versus
PKR -/- mice although both viruses were more restricted in replication of PKR
+/+
mice of different ages (adult versus suckling). Table 5.
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TABLE 5
ADULT T1L virus T3D virus
MICE (S/So) (S/So)
PKR+/+ PKR-/- PKR+/+ PKR-/-
E6 IP ND 100 % (3/3)ND 100 % (3/3)
5 E6 IN 100 % (3/3)0 % (0/3) 100 % (3/3) 100 % (3/3)
5 ES IN ND 100 % (3/3)ND ND
SUCKLING
MICE
3 E6 IN 33 % (2/6) 66 % (2/3) 84 % (5/6) 0 % (0/2)
3 E4 IN 100 % (7/7)ND 100 % (7/7) 0 % ( 0/4)
3 E3 IN ND ND ND 100 % (3/3)
Experiment 4: Reovirus T3D is a stronger inducer of PKR MEF than T1L
Infection of PKR+/+ MEF results in a greater expression of the
phosphorylated form of PKR (Fig. 2). PKR+l+ MEF were infected at a moi of 10
and incubated over a 48 hr period for immunoblot analysis using rabbit anti-
PIER
serum that reacts with the first 100 amino acids of PIER. Proteins were
separated
on a 10% polyacrylamide gel and transferred to IMMOBILON membrane
(Millipore Inc.) before incubation with 1/100 diluted primary antibody in the
presence of casein. After repeated washing the blot was incubated with goat
anti-
rabbit antibody conjugated with alkaline phospatase (1/30,000 dilution) (Sigma
Inc)
for 1 hour before repeated washing and reaction with Attophos substrate for
phosphorescent detection as shown in Figure 2. Activation of PIER results in
an
electrophoretic form of slightly slower mobility indicated as PKR-P. Infection
with
T3D results in a greater production of this form than with infection with T1L.
This
demonstrates that PKR expression is enhanced in T3D infected cells and
indicates
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that this may be responsible for the greater sensitivity of this virus to the
PKR gene.
Experiment 5: Proof of principle for Improved Oncolysis of reovirus T1L x T3D
Reassortants: Demonstration that reovirus reassortants with the M1 gene of T3D
and the remaining_~enes from T1L and T3D have suberior oncol~properties.
Three reassortants were chosen for testing of oncolytic properties relative to
their parental viruses. Each of the reassortants , EB96, EB 108 and EB 146
posessed
the Ml gene of T3D and were expected to preferentially replicate in cells that
were
damaged in their interferon response. These reassortants also possessed their
L1,
L3 and S2 genes of T1L that would-be predicted to provide optimal replication
abilities.
Oncolytic testing was performed by intranasal infection of 10' pfu of each
virus into mice that possessed lung tumors derived form the CT26 colon tumor
cell
line fo Balb-C origin. Adult female BALB-C mice, 4-6 weeks old, were injected
in
the tail vein with 3 x 105 CT 26 on day 0 of the experiment. On day 7 groups
of 3
mice were anaesthetized and infected with 10~ pfu of virus in a 0.050 volume
of
culture medium. Mice were housed for an additional 6 days before euthanization
with 90% COZ/10% OZ. Lungs were removed, weighed, fixed in formalin and
photographed. One set of lungs was examined histopathologically by hematoxylin
and eosin staining after paraffin embedding and sectioning.
The gross appearance of lungs after treatment showed that the untreated
control lungs were heavily tumor laden having a pebbled surface appearance due
to
contiguous tumor nodules (Fig 3). These animals were in the terminal stages of
cancer since one animal died at this time and the others were in respiratory
distress.
These lungs were 3 times heavier than uninfected balb-c lungs indicating the
increased tumor mass approximated twice the mass of the lung tissue (Fig 4).
Histologically these lungs were covered with a contiguous layer of tumor
nodules
and internal tumor masses seen as eosinophilic growths of cells (Fig 4 and 5).
Infection with T1L virus resulted in a partial freeing of surface tumor growth
observable on gross inspection that was also associated with a decrease in
interior
and surface nodules and a 20 % reduction in lung weight relative to untreated
control (Fig 3, 4 and 5). T3D treatment was not as effective as T1L resulting
in
lungs that were only distinguishable form untreated controls by a slight (8 %)
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decrease in size but were similar in gross and microsopic appearance of tumors
(Fig
3, 4 and 5).
In dramatic contrast the EB96 reassortant virus cleared the lung of gross
tumor mass on treatment (Fig 3). The lungs were of approximately normal weight
having been freed of tumor masses (Fig 4). A small number of residual tumor
cells
remained at this time as detected by histological examination (Fig 5). The
lungs
were of normal size and appearance except for some circular patterns and dents
on
the lungs surface that presumably marked the location of prior tumor nodules.
EB 146 virus was not more effective at tumor lysis than the T3D parental virus
(Fig
3, 4 and 5). Reassortant EB108 was partially effective at oncolysis producing
results that were marginally better but similar than the T1L parental strain.
On
comparison of the genotyoes of the reassortants it can be seen that the 3
ressortants
possess 7 genome segments in common and thus differ in their L2, S3 and S4
genome segments indicating that the latter group of genes include important
modulators of oncolysis. The EB96 reassortant is more effective than EB108
soley
due to the nature of the S4 gene since these viruses only differ in the
parental origin
of this gene. This indicates that the T1L S4 gene conferred enhanced oncolytic
properties relative to the T3D S4 gene. Since the S4 gene encodes the dsRNA
binding protein that blocks PIER activation it is possible that the T1L S4
gene
differs in this ability and thus, in concert with other combinations of T1L
and T3D
genome segments, controls oncolytic potential. In conclusion, the dramatic
increase
in effectiveness of the EB96 reassortant at oncolysis, relative to the
parental T1L
and T3D viruses demonstrates the proof of principle that reassortants of
reovirus
with specific genotyoes have enhanced and effective tumor lysis abilities in
metastatic tumors in hosts with active immune responses. Table 6.
Table 6: Ranking of the ability of reovirus reassortants to lyse ct26 lung
tumors. The relative weight of ct26 tumor bearing lungs relative to untreated
control
tzunor bearing lungs are shown. The parental origin of genome segments are
indicated as L for T1L and D for T3D.
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VIRUS TUMOR L1 L2 L3 M1 M2 M3 S1 S2 S3 S4 RANK
J
eb96 41 L D L D L L L L D L 1
eb108 75 L D L D L L L L D D 2
T1 L 80 L L L L L L L L L L 3
eb146 89 L L L D L L L L L D 4
T3D 92 D D D D D D D D D D 5
Experiment 6: Ability of TIL x T3D Reassortants to lyse tumors in vitro
A panel of tumor cell lines obtained fron the NCI tumor panel (SF539, cns;
SKMEL28, melanoma; HT29; NCI H23, nsc-lung; SW620, colon; DU145,
prostate) were infected with the T1L, T3D, or the reassortants , EB96, EB108
and
EB146 at an moi of 10 and were observed for cytopathic effect over a 5 day
period.
The ability to lyse tumor cells was scored visually on a scale of - to +++,
where -
indicates no difference form mock infected cells and +, ++, and +++ indicate
33
cell destruction, 66 % cell destruction and complete lysis respectively.
Although
different tumor cell types differed in their susceptibility to lysis by
different reovirus
parents or reassortants the reassortants viruses were all as effective or more
effective than the T3D parental virus at tumor cell lysis in vitro (Table 7).
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Table 7: Cytopathology of reovirus '1'1L and T 3D and reassortants in
different tumor cell lines
Tumor
cell
line
SF539 SKMEL28 HT29 NCI H23 SW620 DU145
virus Cns melanoma - nsc-lungcolon prostate
T1L ++ +++ ++ +++ - ++
T3D - +++ + ++ - +
EB96 ++ +++ ++ ++ + +
EB108 ++ +++ ++ ++ + +
EB146 ++ +++ ++ +++ + ++
RAS
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