Note: Descriptions are shown in the official language in which they were submitted.
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s METI30D FOR MEASURING VIRAL INFECTIVITY
BACKGROUND OF TFiE INVENTION
A particular challenge in the delivery of a gene by
a viral vector for therapeutic purposes is the preparation and
accurate characterization of clinical dosage forms. Total
particle measurement can be made by such techniques as
electron microscopy of viral preparations or measurement of
total DNA by optical density at 260 nm of a sodium dodecyl
sulfate (SDS) treated virus suspension. However, infectivity
of a viral preparation, i.e., the number of infectious viral
particles in a preparation of virus, is more challenging to
accurately measure.
Traditionally, infectivity particles are measured in
culture by a plaque-forming unit assay (pfu) that scores the
number of viral plaques as a function of dilution. An
alternative to the pfu assay is the tissue culture infective
dose procedure (TCIDSo), which estimates infectivity as a
function of intracellular staining for an antigen by direct
immunofluorescence. The methods suffer from limitations
including a high degree of inter-assay variability and are
affected by such factors as virus replication status, vector
characteristics, and virus-cell interactions.
More recently, flow cytometry or FACS (fluorescence-
activated cell sorter) assays have been used to measure the
number of infected cells in cell cultures infected at
relatively high multiplicities of infection. For example,
Saalmuller and Mettenleiter (J. Virol. Methods 44:99-108
(1993)) disclose the identification and quantitation of cells
infected by recombinant pseudorabies virus mutants by the
reaction of intracellular ~i-galactosidase expressed during
infection with recombinant viruses with a fluorogenic
substrate, followed by detection of positive cells in flow
cytometry. Morris et al. (Virolow 197(1):339-48 (1993))
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studied the process of productive and non-productive
recombinant AcMNPV infection in cultured cells by
immunostaining cells to detect the reporter CAT gene product.
The instant invention addresses the need for a more
accurate method of quantitating infectious viral particles in
a population.
SUMMARY OF THE INVENTION
The methods of the instant invention are based on
the unexpected and surprising result that flow cytometry
analysis of cells infected at a low virus to cell ratio yields
a more accurate measurement of infectious virus titer than
traditional titration methods.
One aspect of the invention is a method for
determining the number of infectious virus particles in a
population of virus particles comprising:
i) infecting cells in a cell population at a total
particle to cell ratio of less than about 100:1 to about 0.1:1
to generate infected cells;
ii) reacting a polypeptide expressed by the virus in
infected cells with an antibody labeled with a fluorescent
tag, the antibody having specificity for a polypeptide
expressed by the virus; and
iii) measuring immunofluorescence in the product of
step (ii) by flow cytometry to determine the number of
infected cells, thereby determining the number of infectious
virus particles.
When the virus is a recombinant virus, the viral
polypeptide can be encoded by an exogenous gene, such as a
reporter gene. In some embodiments of the invention, the
exogenous gene is a tumor suppressor gene such as p53 or ,
retinoblastoma (RB). The recombinant virus can be replication
competent or defective, deficient or incompetent.
In some embodiments of the invention, the virus is
adenovirus. Thus, when the infected cells are cultured after
infection to allow expression of a viral polypeptide, the
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viral polypeptide can be an adenovirus polypeptide such as
hexon.
Typically the viral polypeptide is reacted with at
least one antibody, although the antibody can be a mixture of
antibodies. The antibody can be polyclonal or monoclonal.
In preferred embodiments of the invention,~the total
particle to cell ratio is less than about 100:1, typically
less than about 10:1, preferably less than about 5:1, more
preferably less than about 1:1. In some embodiments, the
ratio can be as low as about 0.1:1.
DETAILED DESCRIPTION
' The instant invention provided methods for
quantitating infectious viral particles in a population of
virus particles. The term "infectious" as used herein is
intended to refer to the ability of a virus to enter cells and
direct the synthesis of at least one polypeptide encoded by
the virus. The ability to reproduce the viral nucleic acid is
not required, but is included, by this definition.
Typically, not every virus particle in a preparation
is infectious. For example, particles can be damaged in
preparation of the virus, thereby not affecting total particle
number but decreasing the number of particles capable of
infection. Furthermore, empty capsids or instability of the
virus extracellularly can also contribute to the decrease in
infectivity. The range of non-infectious particles to
infectious particles in viral preparations can range from 1:1
to greater than 100:1. However, even non-infectious viruses
can cause cytological changes or damage to exposed cells.
Thus, it is advantageous to have an accurate measure of the
number of infectious particles in a population so as to
minimize the number of non-infectious viral particles to which
cells are exposed.
Virtually any virus can be quantitated, or titered,
by the methods of the instant invention, including DNA
viruses, RNA viruses, replication competent viruses,
replication incompetent viruses, recombinant viruses, viruses
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carrying transgenes, etc. Preferably, the virus can infect
cells in culture. Some example of viruses amenable to this
technique include, but are not limited to, adenovirus, adeno-
associated virus, retrovirus, herpes simplex virus,
parvovirus, Epstein Barr virus, rhinotracheitis virus,
parainfluenza virus, bovine diarrhea virus, sindbis virus,
baculovirus, pseudorabies virus, varicella-zoster virus,
cytomegalovirus, HIV, hepatitis A, B, and C viruses, and
vaccinia.
In some embodiments of the invention, infectivity is
measured by antibodies directed against a polypeptide
expressed by the virus. The polypeptide may be a structural
viral polypeptide, a regulatory polypeptide, a polypeptide
such as a polymerase, and so on. In some embodiments of the
invention, the polypeptide is preferably expressed by an
exogenous gene incorporated into the virus, such as a reporter
gene. Some examples of reporter genes include (3-galactosidase
and chloramphenicol transacetylase (CAT). In further
embodiments of the invention, the reporter gene is detected by
antibodies directed against a product of the action of the
reporter gene, such as the action of an enzyme on a substrate.
In other embodiments of the invention, the exogenous gene is a
transgene intended for therapeutic use. Some examples include
but are not limited to tumor suppressor genes, including p53
or retinoblastoma (RB); interleukins, including IL-2, IL-4,
and IL-10; interferons, including alpha-, beta-, and gamma
interferon; other cytokines; thymidine kinase; growth factors,
including GCSF and growth hormone; Factor VIII; adenosine
deaminase, and so on. Typically production of polypeptide
encoded by a transgene will be measured by an antibody
directed against the polypeptide.
Antibodies used for detection can be polyclonal, ,
monoclonal, or include mixtures of such antibodies.
Typically, the detection is done directly by using a ,
fluorescein-conjugated antibody directed against the viral
polypeptide. However, indirect assays are also possible, in
which the antibody directed against the viral polypeptide is
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then reacted with a fluorescein-labeled antibody. Any
fluorescent label compatible with flow cytometry can be used.
To perform the assay of the invention, typically,
the total number of virus particles in a viral preparation is
5 first measured by any of a number of traditional techniques.
For example, an aliquot of a virus preparation can be prepared
in a buffer containing 0.1% sodium dodecyl sulfate (SDS),
after which the optical absorbance is measured at 260 nm
(Maizel et al. yirologv 36:115-125 (1968)). Total particle
counts can also be obtained by preparing a sample of the viral
preparation for electron microscopy, and simply counting the
number of particles. A further technique for particle
enumeration can include the use of anion-exchange
chromatography (Huyghe et al. Oman Gene erarw 6:1403-1416
(1995)).
Cells are then infected with dilutions of the viral
preparations at total particle number to cell number ratios no
higher than about 100:1, typically less than about 10:1,
preferably less than about 5:1, more preferably less than
about 1:1. In some embodiments the ratio is as low as about
0.1:1. Typically, at least one infection will be performed,
although in some embodiments at least two parallel infections
are performed at different particle to cell ratios. The cells
used are typically known to be sensitive to infection by the
virus. It is not required that the cells support replication
by the virus, but the infection is performed under conditions
that allow expression of the viral polypeptide to be detected.
The total volume of a virus preparation used to
infect cells in culture is typically determined by the skilled
artisan by taking into account such factors as the total
number of cells to be infected, the particle concentration of
the virus preparation, and the volume of the vessel in which
the infection is performed. Preferably, the particle
concentration of virus used to infect cells in the infection
mixture is at least about 105 particles per ml, more
preferably at least about I06 particles per ml, most
preferably about 10'' particles per ml. The viral preparations
typically are prepared under conditions favorable to stability
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of the virus. Conditions for infection and, optionally,
culture after infection will depend on the particular virus
and the viral or reporter gene used for detection. The term
"culture" as used herein refers to any form of cell culture in
which the minimum requirements are provided to the cells to
enable continued survival for the period of interest. Thus,
for example, culture can refer to preparation of a cell
suspension in a suitable buffer, such as phosphate buffered
saline or an incomplete growth medium, for a period of minutes
or hours, or can refer cells adhering to culture dishes for
minutes to days to weeks in the presence of a suitable
complete growth medium. Typically, sufficient time in culture
is provided for expression of the desired viral polypeptide,
but preferably not enough time is provided for propagation of
the infecting virus which results in further infection of
cells. Thus, it is preferable that only "one round" of
infection occur in these cells. In some embodiments, the
length of time allowed "in culture" will be less than 1 hour
to several hours. In other preferred embodiments, the length
of time will be 1 to 5 days.
Typically, cells are infected under conditions
favoring adsorption of the virus to the cells, although less
optimal conditions can be used in some embodiments.
Typically, viruses are allowed to adsorb to cells for 1-12
hours. In some embodiments, the cells are infected in a
concentrated suspension with concentrated virus, to enhance
the rate of infection or the number of infected cells, then
diluted to a concentration more favorable for cell or viral-
growth. In some embodiments of the invention, it can be
desirable to wash infected cells cultures to remove unabsorbed
virus or components of the medium used for infection, or to
expose the infected cells to media or growth conditions more
favorable to their survival.
After sufficient time has elapsed to allow
expression of the viral polypeptide, the cells are typically
prepared as a suspension of single cells. When the cells are
infected as adherent cells in tissue culture, the cultures are
typically treated with a dissociating agent such as trypsin to
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detach the cells from the substratum. Mechanical means can
also be used to detach cells, such as scraping. CeIIs are
then collected by centrifugation and prepared in a buffer,
such as incomplete or complete growth medium, for reaction
with the detection reagents. Typically cells are "fixed" for
immunostaining by any of a number of standard techniques. A
review of the commonly used fixation techniques is provided by
Bauer and Jacobberger, etg s i~ dell Hiolaav 41:351-376
(1994)),
all purposes. When the polypeptide is detected by its
activity, fluorescent reagents can be introduced into cells to
allow detection of the activity, such as a fluorescein labeled
substrate for an enzyme.
Infected cell populations are then subjected to
analysis by standard flow cytometry, such as by the methods
disclosed by Shapiro, pTa ~ ~;La? Fi oa,L~rtomet~ , 3rd ed. , John
Wiley and Sons (1994?,
The term "FACS" is sometimes used
to refer to flow cytometry, although cell sorting is not
required to practice the instant invention. Typically, a
minimum of about 10,000 events is acquired in the analysis.
Dead cells are typically excluded from the analysis either by
forward/side scatter gating or PI labelling and setting of
electronic windows on the PI negative fraction. A variety of
commercial software packages are available to aid in
preparation and analysis of the data, such as CellQuest'~".
The following example is intended to illustrate but
not limit the invention in any way.
3 0 E7L8M~Ir$
In this example, ACNRH, a recombinant, replicative-
defective adenovirus was titered by TCID~ and by the low
particle number to cell number ratio flow ratio) method of the
present invention. The exemplary virus used essentially
comprised the adenovirus vector backbone disclosed by Wills et
al. (~ns~r~.~ne Therabv Z:I91-197 (1995) ) with full-length
retinoblastoma cDNA inserted into the vector.
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Total particle number was obtained by the "SDS/ODz6o"
method and anion exchange chromatography methods described
above. In both assays the measured total particle
concentration was 1.0 X 1012/ml.
Infectious particles were titered by TCIDSO assay as
described by Huyghe et al. (Human Gene Theranv 6:1403-1416 '
(1995)). In brief, 293 cells were plated into a 96-well
microtiter plate: 100,u1 of 5x105 cells/ml for each well in
complete MEM (10% bovine calf serum; 1% glutamine) media
(GIBCO BRL). In a separate plate, a 250-fcl aliquot of virus
sample diluted 1:106 was added to the first column and was
serially diluted two-fold across the plate. Seven rows were
used for samples. One row was used for a negative control. A
100-~1 aliquot of each well was transferred to its identical
position in the 293 seeded plate and allowed to incubate a
37°C in a humidified air/7% COZ incubator for 2 days. The
media was then decanted by inversion and the cells fixed with
50% acetone/50% methanol. After washing with PBS, the fixed
cells were incubated for 45 minutes with a FITC-labeled anti-
Ad5 antibody (Chemicon International #5016) prepared according
to the kit instructions. After washing with PBS, the plate
was examined under a fluorescent microscope (490 mm
excitation, 520 mm emission) and scored for the presence of
label. The titer was determined using the Titerprint Analysis
program (Lynn, Biotechnic~ues 12:880-881 (1992)).
The low ratio assay was performed as follows. 1 X
106 293 cells (human embryonic kidney cells, ATCC CRL 1573)
were seeded per well on 4 6-well dishes. The final volume per
well was 1 ml. After about 2 hr, the medium (Dulbecco's
modified Eagle's medium (DME high glucose) containing 4500
mg/ml D-glucose, supplemented with 5% defined, iron-
supplemented bovine calf serum, 2mM L-glutamine, and l mM
sodium pyruvate) in each well was aspirated and replaced with
1.1 ml of medium (without serum) containing diluted virus.
Adsorption was allowed to occur for 60 minutes, after which an
additional 2m1 of virus-free medium was added to each well.
After about 42 hr, the infected cells cultures were processed
for flow cytometry analysis.
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The cells were detached from the plastic substratum
with a trypsin-EDTA solution (GIHCO-BRL). Detached cells were
collected from each well and centrifuged at about 200 x g for
minutes at room temperature. The supernatants were removed
5 and the cells washed in Dulbecco's phosphate buffered saline
(D-PBS) without calcium or magnesium salts. Pelleted cells
were then resuspended in 2 ml cold acetone: methanol (1:1)
fixative, then held on ice for 15 minutes. 7 ml D-PBS without
calcium or magnesium salts was added to each tube, after which
10 the cells were resuspended in D-PBS with 1% (v/v) calf serum.
After repeating these last two steps, cells were resuspended
in 501 D-PBS with 1% calf serum. 70 ul anti-adenovirus
antibody conjugated with FITC (Chemicon #5016) in 2.0 ml D-PBS
was added to each tube. The samples were incubated at 37°C
for about 50 minutes. The samples were then transferred to
flow cytometry analysis tubes, diluted slightly with 0.5 ml D-
PBS, and analyzed by flow cytometry. A Becton-Dickinson
FACScanT" Flow Cytometer System, PN 34011570, 12-00189-01 with
FACStation (MAC QUADR.A 650 computer, monitor, and printer) was
used with CellQuest~ Software.
The results are shown in Table 1. By the
traditional TCIDSO assay, the total particle number to
infectious unit ratio was 63:1. As is evident in the table, as
the total particle number to cell number ratio decreased, the
calculated total particle number: infectious unit ratio also
decreased to as low as 12:1, thereby providing a value for
infectious titer that was about 5-fold higher than the
traditional assay. Thus, this low ratio assay provides an
unexpectedly better (i.e. much more accurate) enumeration of
3o the number of infectious particles in a viral preparation than
traditional methods for titration. The consequences of such
accurate measurements proved by the instant invention are
especially important in calculating the effective doses of
recombinant viruses for therapeutic use.
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