U.S. patent number RE47,768 [Application Number 15/344,216] was granted by the patent office on 2019-12-17 for compositions and methods for the production of alpha-herpesviruses.
This patent grant is currently assigned to Sanofi Pasteur Biologics LLC. The grantee listed for this patent is Sanofi Pasteur Biologics LLC. Invention is credited to Simon Delagrave, John Hamberger, Rachid Oubelaid.
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United States Patent |
RE47,768 |
Delagrave , et al. |
December 17, 2019 |
Compositions and methods for the production of
alpha-herpesviruses
Abstract
The present invention relates to virus growth media that improve
the yield of alpha-herpesviruses (e.g., HSV-2) grown in cell
cultures. The growth media of the invention include two additives,
a disaccharide and a lipid mixture, that can be added to serum-free
or serum-enriched growth media to improve the efficiency of virus
production. The invention further provides methods of producing
alpha-herpesviruses (e.g., HSV-2) in such growth media.
Inventors: |
Delagrave; Simon (Stoneham,
MA), Oubelaid; Rachid (Vitry-sur-Seine, FR),
Hamberger; John (Milford, NJ) |
Applicant: |
Name |
City |
State |
Country |
Type |
Sanofi Pasteur Biologics LLC |
Cambridge |
MA |
US |
|
|
Assignee: |
Sanofi Pasteur Biologics LLC
(Cambridge, MA)
|
Family
ID: |
41669238 |
Appl.
No.: |
15/344,216 |
Filed: |
November 4, 2016 |
PCT
Filed: |
August 11, 2009 |
PCT No.: |
PCT/US2009/053407 |
371(c)(1),(2),(4) Date: |
April 28, 2011 |
PCT
Pub. No.: |
WO2010/019572 |
PCT
Pub. Date: |
February 18, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
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61188552 |
Aug 11, 2008 |
|
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Reissue of: |
13057788 |
Aug 11, 2009 |
8877492 |
Nov 4, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
7/00 (20130101); C12N 5/0018 (20130101); C12N
7/00 (20130101); C12N 5/0018 (20130101); C12N
2500/76 (20130101); C12N 2710/16052 (20130101); C12N
2710/16051 (20130101); C12N 2500/34 (20130101); C12N
2710/16052 (20130101); C12N 2710/16051 (20130101); C12N
2500/76 (20130101); C12N 2500/36 (20130101); C12N
2500/34 (20130101); C12N 2500/36 (20130101) |
Current International
Class: |
C12N
15/86 (20060101); C12N 15/63 (20060101); C12N
7/00 (20060101); C12N 5/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 573 107 |
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Dec 1993 |
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EP |
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WO 2007/016239 |
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Feb 2007 |
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WO |
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Other References
Costa et al., "Construction, phenotypic analysis, and
immunogenicity of a UL5/UL29 double deletion mutant of herpes
simplex virus 2," J. Virology, 2000, vol. 74, pp. 7963-7971. cited
by applicant .
Dudek et al., "The MuLV 4070A G541 R Env mutation decreases the
stability and alters the conformation of the TM ectodomain,"
Virology, 2008, vol. 371, pp. 165-175. cited by applicant .
"Immunization with Herpes Simplex Virus Type 2
Replication-Defective Mutant, d15-29, Reduces Latent Viral DNA in
Mouse Trigeminal Ganglia," Vaccine Weekly, Abstract. Feb. 22, 1999.
cited by applicant .
Dudek et al. Virology, Public available on line on Nov. 19, 2027,
vol. 371, pp. 165-175. cited by examiner .
Costa et al. J. Virology, 2000, vol. 74, pp. 7963-7971. cited by
examiner .
International Preliminary Report on Patentability for International
Application No. PCT/US2009/053407, dated Feb. 15, 2011. cited by
applicant .
Extended European Search Report from European Patent Application
No. 09807164.0, dated Dec. 19, 2011 (date of completion of search)
and Dec. 27, 2011 (date of mailing of report). cited by applicant
.
"Immunization with Herpes Simplex Virus Type 2
Replication-Defective Mutant, d15-29, Reduces Latent Viral DNA in
Mouse Trigeminal Ganglia," Vaccine Weekly, Abstract.
<http://www.highbeam.com/doc/1G1-53934235.html>, Feb. 22,
1999. cited by applicant .
International Search Report from International Application No.
PCT/US2009/053407, dated Sep. 18, 2009 (date of completion of
search) and Sep. 28, 2009 (date of mailing of report). cited by
applicant .
Written Opinion from International Application No.
PCT/US2009/053407, dated Sep. 18, 2009 (date of completion of
opinion) and Sep. 28, 2009 (date of mailing of opinion). cited by
applicant.
|
Primary Examiner: Ponnaluri; Padmashri
Attorney, Agent or Firm: McNeill Baur PLLC
Claims
What is claimed is:
1. A composition comprising (i) a growth medium supplemented with
between 5 mM and 500 mM of a disaccharide and a lipid mixture, (ii)
Vero cells that express herpes simplex virus 2 (HSV-2) polypeptides
U.sub.L5 and U.sub.L29, and (iii) an HSV-2 having inactivating
mutations in U.sub.L5 and U.sub.L29.
2. The composition of claim 1, wherein said disaccharide is
sucrose, lactose, maltose, trehalose, or cellobiose, or a mixture
thereof.
3. The composition of claim 1, wherein said disaccharide is present
in said medium at 50 mM or 80 mM, or between 50 mM and 80 mM.
4. The composition of claim 1, wherein said medium further
comprises methyl-.beta.-cyclodextrin and/or serum.
5. The composition of claim 4, wherein said serum is fetal bovine
serum (FBS).
6. The composition of claim 1, wherein said lipid mixture is from a
plant, is synthetic, comprises cholesterol, and/or comprises
stigmastanol.
7. The composition of claim 1, wherein said medium increases
production of said HSV-2 in the cells by at least 10% relative to
HSV-2 production in cells grown in a medium lacking said
disaccharide and said lipid mixture.
8. A method of increasing herpesvirus production in cells, said
method comprising culturing the composition of claim 1, wherein
said composition increases production of the herpesvirus by at
least 10% relative to a herpesvirus cultured in cells grown in a
medium lacking said disaccharide or said lipid mixture, wherein
said herpesvirus is a replication-defective herpesvirus having
inactivating mutations in U.sub.L5 and U.sub.L29, optionally in
combination with an inactivating mutation in U.sub.L41.
9. The method of claim 8, wherein said cells are AV529-19
cells.
10. The method of claim 8, wherein said cells express the
herpesvirus polypeptides U.sub.L5 and/or U.sub.L29.
11. The method of claim 8, wherein said disaccharide is sucrose,
lactose, maltose, trehalose, or cellobiose, or a mixture
thereof.
12. The method of claim 8, wherein said disaccharide is present at
50 mM or 80 mM, or between 50 mM and 80 mM.
13. The method of claim 8, wherein said medium further comprises
methyl-.beta.-cyclodextrin and/or serum.
14. The method of claim 13, wherein said serum is fetal bovine
serum (FBS).
15. The method of claim 8, wherein said lipid mixture is from a
plant, is synthetic, comprises cholesterol, and/or comprises
stigmastanol.
16. The method of claim 8, wherein said method increases production
of a herpesvirus in the cells by at least 10% relative to
herpesvirus production in cells grown in a composition lacking said
disaccharide and said lipid mixture.
17. The composition of claim 1, wherein said HSV-2 has an
inactivating mutation in U.sub.L41.
18. The composition of claim 1, wherein said HSV-2 is strain
dl5-29.
19. The composition of claim 18, wherein said Vero cells are
AV529-19 cells.
.Iadd.20. A composition comprising (i) a growth medium supplemented
with between 5 mM and 500 mM of a disaccharide and a lipid mixture,
(ii) Vero cells engineered to express herpes simplex virus
polypeptides U.sub.L5 and U.sub.L29, and (iii) a herpes simplex
virus 2 (HSV-2) having inactivating mutations in U.sub.L5 and
U.sub.L29..Iaddend.
.Iadd.21. The composition of claim 20, wherein said disaccharide is
sucrose, lactose, maltose, trehalose, or cellobiose, or a mixture
thereof..Iaddend.
.Iadd.22. The composition of claim 20, wherein said disaccharide is
present in said medium at 50 mM or 80 mM, or between 50 mM and 80
mM..Iaddend.
.Iadd.23. The composition of claim 20, wherein said medium further
comprises methyl-.beta.-cyclodextrin and/or serum..Iaddend.
.Iadd.24. The composition of claim 20, wherein said lipid mixture
is from a plant, is synthetic, comprises cholesterol, and/or
comprises stigmastanol..Iaddend.
.Iadd.25. The composition of claim 20, wherein said medium
increases production of said HSV-2 in the cells by at least 10%
relative to HSV-2 production in cells grown in a medium lacking
said disaccharide and said lipid mixture..Iaddend.
.Iadd.26. The composition of claim 20, wherein said HSV-2 is strain
dl5-29..Iaddend.
.Iadd.27. The composition of claim 20, wherein said Vero cells are
AV529-19 cells..Iaddend.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
This application .Iadd.is a reissue application of U.S. Pat. No.
8,877,492, filed as U.S. application Ser. No. 13/057,788 and issued
on Nov. 4, 2014, which .Iaddend.is the U.S. national stage filing
under 35 U.S.C. .sctn.371 of international application
PCT/US2009/053407, filed Aug. 11, 2009, which claims benefit of
U.S. Provisional Application No. 61/188,552, filed Aug. 11,
2008.
FIELD OF THE INVENTION
The invention provides compositions including growth media that
improve the yield of alpha-herpesviruses (e.g., HSV-2) grown in
cell cultures. The growth media of the invention include at least
two additives, a disaccharide and a lipid mixture, which can be
added to serum-free or serum-enriched growth media to improve the
efficiency of virus production. The invention further provides
methods of producing alpha-herpesviruses (e.g., HSV-2) in such
growth media.
BACKGROUND OF THE INVENTION
The alpha-herpesvirus herpes simplex virus type 2 (HSV-2) is the
cause of genital herpes, which can be treated symptomatically but
not cured. Hallmarks of herpes virus infection include the
establishment of lifelong, latent infections that can reactivate to
cause one or more rounds of disease, as well as transmission in the
absence of symptoms. Transmission of HSV-2 to newborns at the time
of delivery may lead to devastating systemic infection with
encephalitis. Further, immunocompromised individuals are at
increased risk of serious disseminated infection. Also, HSV-2 has
been shown to increase significantly the risk of becoming infected
with HIV-1, the virus that causes AIDS. The development of vaccines
to prevent or treat HSV-2 infection is therefore of considerable
importance.
The manufacture of virus-based vaccines (e.g., attenuated or
inactivated virus particles) generally involves the infection of
mammalian cell lines in vitro with a small quantity of a virus,
followed later by harvest of large quantities of progeny virus. The
mammalian cells are propagated in growth media, which are complex
solutions containing a large number of different salts, small
organic molecules such as carbohydrates and lipids, and
macromolecules such as peptides and proteins. Optimization of virus
production methods is important to the development of efficiently
produced, cost-effective herpes virus vaccines.
SUMMARY OF THE INVENTION
In a first aspect, the invention provides compositions useful for
growing cells in culture (e.g., monolayer culture). The
compositions of the invention include growth media including
between 5 mM and 500 mM (e.g., about 50 mM and about 80 mM, or
between about 50 mM and about 80 mM) of a disaccharide (e.g.,
sucrose), and a lipid mixture (e.g., a lipid mixture derived from a
plant or that is synthetically produced, and/or including
cholesterol or stigmastanol). The cells grown in the compositions
of the invention can be, for example, Vero, MRC-5, BHK, CEM, and
LL-1 cells. The disaccharide can be, for example, sucrose, lactose,
maltose, trehalose, or cellobiose, or a mixture thereof. In an
example, the compositions also contain methyl-.beta.-cyclodextrin
or serum, such as fetal bovine serum (FBS). The compositions of the
invention can, for example, increase production of a virus in cells
by at least 10%, relative to cells grown in compositions lacking
the disaccharide and lipid mixture. The virus grown in the media
can be an alpha-herpesvirus, such as herpes simplex virus 1 (HSV-1)
or herpes simplex virus 2 (HSV-2). In one example, the HSV-2 has
one or more inactivating mutations (e.g., deletions) in U.sub.L5
and U.sub.L29 (e.g., virus dl5-29), optionally in combination with
one or more inactivating mutations (e.g., deletions) in U.sub.L41
(e.g., virus dl5-29-41). Further, the compositions of the invention
can include, in addition to media components, as described herein,
cells and/or viruses, as described herein.
In a second aspect, the invention provides methods of increasing
virus production in cells, involving culturing the cells in a
serum-free medium containing between 5 mM and 500 mM (e.g., about
50 mM or about 80 mM, or between about 50 mM and about 80 mM) of a
disaccharide (e.g., sucrose), and a lipid mixture (e.g., a lipid
mixture derived from a plant or that is synthetically produced,
and/or including cholesterol or stigmastanol), where the medium
increases production of a virus in cells by at least 10% relative
to cells grown in a medium lacking the disaccharide and lipid
mixture. In various examples, the cells are mammalian cells, such
as Vero, MRC-5, BHK, CEM, or LL-1 cells. In another example, the
cells (e.g., AV529-19 cells) can support the growth of a
replication defective herpesvirus, such as dl5-29, by expression of
herpesvirus polypeptides U.sub.L5 and/or U.sub.L29. The
disaccharide can be, for example, sucrose, lactose, maltose,
trehalose, or cellobiose, or a mixture thereof. In a further
example, the medium contains methyl-.beta.-cyclodextrin or serum,
such as fetal bovine serum (FBS). The media used in the methods of
the invention can, for example, increase production of a virus in
cells by at least 10% relative to cells grown in a medium lacking a
disaccharide and lipid mixture. The virus grown in the medium can
be an alpha-herpesvirus, such as herpes simplex virus 1 (HSV-1) and
herpes simplex virus 2 (HSV-2). In one example, the HSV-2 has one
or more inactivating mutations (e.g., deletions) in U.sub.L5 and
U.sub.L29 (e.g., virus dl5-29), optionally in combination with one
or more inactivating mutations (e.g., deletions) in U.sub.L41
(e.g., virus dl5-29-41).
The invention provides several advantages. In particular, the
compositions of the invention improve the yield of herpes viruses
grown in cell cultures, which provides benefits with respect to
efficiency and cost of virus production methods.
Other features and advantages of the invention will be apparent
from the following detailed description, the drawings, and the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph showing the average population doubling times of
Vero cells (strain AV529-19) grown in three different cell culture
media (OptiPro SFM, ExCell, and VP SFM AGT), with and without the
addition of sucrose and a lipid mixture.
FIG. 2 is a graph showing the yield of HSV-2 virus dl5-29 (starting
MOI=0.1) produced in AV529-19 cells grown in three different cell
culture media (OptiPro SFM, ExCell, and VP SFM AGT), with and
without the addition of sucrose and a lipid mixture.
FIG. 3 is a graph showing the total yield per flask of HSV-2 virus
dl5-29 (starting MOI=0.1) produced in AV529-19 cells grown in three
different cell culture media (OptiPro SFM, ExCell, and VP SFM AGT),
with and without the addition of sucrose and a lipid mixture.
FIG. 4 is a graph showing the optimization of culture media
additives in OptiPro SFM with 1% FBS. The horizontal line at
.about.13 pfu/cell indicates dl5-29 yield in OptiPro SFM (no FBS)
with 1.times. each of sucrose and a lipid mixture. The horizontal
line at .about.8.5 pfu/cell indicates OptiPro SFM (no FBS) with no
sucrose/lipid mixture additives.
FIG. 5A is a graph showing virus titer from cells grown in three
different culture media (OptiPro, DMEM-F12, and Megavir) in the
presence or absence of sucrose and a lipid mixture.
FIG. 5B is a oneway analysis of the data of FIG. 5A focused on the
viral titer in the presence or absence of additives.
FIG. 5C is a oneway analysis of the data of FIG. 5A focused on the
viral titer in cells incubated in the indicated basal media.
FIG. 6 is a graph showing the PFU/cell production of cells
incubated in three different culture media (OptiPro, DMEM-F12, and
Megavir) with and without sucrose and a lipid mixture.
FIG. 7A is a graph showing viral titer resulting from incubation
with the indicated amounts of sucrose and a lipid mixture in
DMEM-F12.
FIG. 7B is a graph showing the interaction profiles of optimum
amounts of lipid concentrate and sucrose. The upper right quadrant
shows titer as a function of sucrose concentration in the presence
and absence of 1.times. lipid. The lower left quadrant shows titer
as a function of lipid concentration in the presence and absence of
100 mM sucrose.
FIG. 8 is a graph showing PFU/cell production of cells incubated
with the indicated amounts of sucrose and a lipid mixture in
OptiPro.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides compositions and methods for use in virus
production. The compositions of the invention include a cell
culture medium supplemented with disaccharide and lipid mixture
additives. The compositions can be used to culture cells infected
with alpha-herpesviruses and, advantageously, result in high viral
yields of such cultures. As discussed further below, in one
example, the compositions and methods of the invention are used in
the production of a herpes simplex virus, which can be used, e.g.,
as a vaccine or as a delivery vector. The compositions and methods
of the invention are described in further detail as follows.
The present invention is based on the discovery that the addition
of a disaccharide, sucrose, and a lipid mixture to a base cell
culture medium increases the yield of an alpha-herpesvirus (i.e.,
HSV-2 strain dl5-29; see below) by over 100% in static cultures.
The viral growth media can be formulated with or without fetal
bovine serum (FBS). This result is applicable to other growth
media, including VP-SFM-AGT (Invitrogen/GIBCO) and ExCell
(SAFC)(also see below). The effect of the two additives is due to
increased productivity of virus on a per-cell basis, and not due to
increased cell density per growth vessel. A matrix analysis of
several additive concentrations was performed to ensure that
optimal concentrations of both additives were identified. The
invention was first demonstrated in small culture flasks and later
extended to large 10-layer Nunc cell factories (NCFs). The
compositions and methods of the invention can also be used in
bioreactors for large-scale virus production (e.g., for commercial
viral vaccine production).
As discussed further below, the compositions and methods of the
invention can be used to increase the production efficiency of
alpha-herpesviruses (e.g., HSV-2), such as replication-defective
strains that can be used in therapeutic or prophylactic vaccines or
as delivery vectors.
Viral Growth Media
The invention provides growth media that contain both a
disaccharide (e.g., sucrose) and a lipid mixture that, when used to
produce an alpha-herpesvirus (e.g., HSV-2), is capable of
increasing the viral yield. The disaccharide and lipid mixture can
be added to a base cell culture medium suitable for the culture of
cells supporting virus growth, such as Vero, BHK, and MRC-5 cells.
Exemplary base culture media include OptiPro SFM
(Invitrogen/GIBCO), ExCell (SAFC), VP SFM AGT (Invitrogen/GIBCO)
(FIGS. 1-3), MegaVir (HyClone), DMEM-F12 (Sigma Aldrich), and any
other medium in which the cells and virus can grow, as can be
tested using standard methods (see, e.g., below). A cell culture
medium can be formulated with additional nutrient rich additives
(e.g., with serum, such as fetal bovine serum) or without such
additives (e.g., a "serum-free" medium such as OptiPro SFM).
Exemplary formulations of growth media of the invention are
described below.
Disaccharides
Disaccharides for use in the compositions and methods of the
invention include, for example, sucrose, lactose, maltose,
trehalose, and cellobiose. The disaccharides (e.g., sucrose) can be
added to a suitable growth medium at a concentration of, e.g., 1
mM, 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM,
90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, or more (or at any
amount in ranges between any of the amounts listed above; when
ranges are provided herein, they include both ends of the ranges,
as well as all amounts between the ends, unless otherwise
indicated). Thus, in specific examples, the amount of disaccharide
(e.g., sucrose) can be 50 mM, about 50 mM, 80 mM, about 50 mM, or
between 50 mM and 80 mM. An exemplary method of determining optimal
concentrations of a disaccharide for use with any given virus or
cell line combination is described below. Mixtures of disaccharides
can also be used in any of the compositions or methods of the
invention.
Lipid Mixtures
Lipid mixtures used in the present invention can contain
cholesterol, stigmastanol, other lipids, or combinations of lipids
(including, e.g., cholesterol and/or stigmastanol). The lipid
mixtures can be naturally or synthetically derived. Naturally
derived lipids can be isolated from an animal (e.g., a mammal, such
as a cow) or isolated from a plant (e.g., soybean). Lipid
formulations derived from non-animal sources (e.g., synthetic
lipids or lipids derived from plants) may be preferred in some
instances for use in viral growth media for the production of an
alpha-herpesvirus (e.g., HSV-2) for use in vivo (e.g., in the
treatment of human patients).
A method of determining effective and suitable concentrations of
lipid mixtures for increasing the production of an
alpha-herpesvirus HSV-2, strain dl5-29 is described herein (see,
e.g., FIG. 4), and can be applied in the context of other viruses,
such as those described herein. Prepared lipid mixtures for use in
the compositions and methods of the invention are known in the art
(e.g., Invitrogen/GIBCO 250.times. Cholesterol Lipid Mixture).
Other lipid mixtures are described by Gorfien et al., Biotechnol.
Prog. 16:682-687, 2000. Other examples of such mixtures that can be
used in the invention include adult bovine serum (Sigma Chemical
Co., e.g., Catalog No. L-4646), Ex-Cyte lipid I or Very Low
Endotoxin (VLE) lipid (Miles Inc., Catalog No. 82-004-7, 82-004-8,
and 82-019-1), and soybean lipid extract (Boehringer Mannheim
Biochemicals; e.g., Catalog No. 1074-482; also see Iscove et al.,
J. Expt. Med. 147:923-933, 1979).
Alpha-Herpesviruses
Alpha-herpesviruses are a subfamily of the linear, double-stranded
family of DNA viruses designated Herpesviridae. Alpha-herpesviruses
include the genera Simplexvirus (e.g., ateline herpesvirus 1,
bovine herpesvirus 2, cercopithecine herpesvirus 2, human
herpesvirus 1 (herpes simplex virus-1; HSV-1), human herpesvirus 2
(herpes simplex virus-2; HSV-2), macacine herpesvirus 1 (previously
known as cercopithecine herpesvirus 1), macropodid herpesvirus 1
and 2, papiine herpesvirus 2 (previously known as cercopithecine
herpesvirus 16), and saimiriine herpesvirus 1), Varicellovirus
(e.g., bovine herpesvirus 1, bovine herpesvirus 5, bubaline
herpesvirus 1, canid herpesvirus 1, caprine herpesvirus 1,
cercopithecine herpesvirus 9, cervid herpesvirus 1 and 2, equid
herpesvirus 1, 3, 4, 6 (tentative), 8 and 9, felid herpesvirus 1,
human herpesvirus 3 (varicella zoster virus, VZV), phocid
herpesvirus 1, and suid herpesvirus 1), Mardivirus (e.g., columbid
herpesvirus 1, gallid herpesvirus 2, gallid herpesvirus 3, and
meleagrid herpesvirus 1), and Iltovirus (e.g., gallid herpesvirus
1, psittacid herpesvirus 1, chelonid herpesvirus 5, and chelonid
herpesvirus 6).
A virus grown in the growth medium of the invention can be any
alpha-herpesvirus, or any virus derived therefrom, for example,
HSV-1 or HSV-2 strains. An alpha-herpesvirus produced according to
the methods of the invention can have, for example, the sequence of
an HSV-1 or HSV-2 genome modified by nucleotide mutations,
including deletions and insertions, such as in the viruses
described above and elsewhere herein. In particular, derivatives
that can be used in the practice of the invention include viruses
that have genetic mutations, particularly mutations that result in
viral attenuation. Examples of such viruses include viruses having
deletions in U.sub.L5, U.sub.L29, optionally in combination with
U.sub.L41, such as the HSV-2 strain dl5-29, which is described
further below (see also WO 99/06069; U.S. Patent Publication No.
20020009462; DaCosta et al., J. Virology 74:7963-7971, 2000; and
U.S. Pat. No. 6,841,373), as well as dl5-29-41 (see, e.g., WO
2007/016239).
Additional examples of mutant viruses that can be produced
according to the methods of the invention include strain 1716
(MacLean et al., J. Gen. Virol. 72:631-639, 1991), strains R3616
and R4009 (Chou et al., Proc. Natl. Acad. Sci. USA 89:3266-3270,
1992), and R930 (Chou et al., J. Virol. 68(12):8304-8311, 1994),
all of which have mutations in ICP34.5, strain d120, which has a
deletion in ICP4 (DeLuca et al., J. Virol. 56(2):558-570, 1985),
strain d27-1 (Rice et al., J. Virol. 64(4):1704-1715, 1990), which
has a deletion in ICP27), and strain d92, which has deletions in
both ICP27 and ICP4 (Samaniego et al., J. Virol. 69(9):5705-5715,
1995). Replication competent viruses, such as those that contain
one or more mutations in ICP10, and replication incompetent
viruses, such as those that contain one or more mutations in the
ICP8 gene, can also be produced according to the methods of the
present invention.
In addition, viruses including nucleotide substitutions can also be
used, for example, viruses containing 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 25, 50, 100, 200, 350, 500, or more nucleotide substitutions
(as well as ranges including or between any of these values). The
HSV-1 or HSV-2 genome can alternatively or additionally be modified
by one or more insertions or by an extension at either or both
ends. Viral derivatives that can be produced according to the
methods of the invention also include intertypic recombinants
containing DNA from, e.g., HSV-1 and HSV-2 strains. Derivatives
typically have at least 70% sequence identity to a parent virus
from which it is derived (e.g., HSV-1 or HSV-2), such as, for
example, at least 80%, at least 90%, or at least 95% identity.
In addition to viruses used as vaccines against alpha-herpes virus
infection, the methods of the invention can also be used to produce
viruses that can be used in the delivery of heterologous,
non-herpes virus sequences (see, e.g., WO 01/53507 and U.S. Pat.
No. 7,118,755).
Herpes Simplex Virus 2 Strain dl5-29
Virus dl5-29 is a replication-defective HSV-2 virus, which contains
deletions of the open reading frames for UL5, a component of the
helicase-primase complex and an essential viral protein for viral
DNA synthesis, and UL29, encoding the ICP8 DNA-binding protein,
which is also an essential protein for viral DNA synthesis (see,
e.g., WO 99/06069). Virus dl5-29 replicates only in cells
engineered to complement the two genes, U.sub.L5 and U.sub.L29,
which were deleted from its genome. The Vero cell line, a
transformed cell line derived from African green monkey kidney
cells, is often used as a cell substrate for the production of
various vaccines. After genetic manipulation allows for expression
of herpes viral genes U.sub.L5 and U.sub.L29, engineered Vero cell
strain AV529-19 can be used as a substrate for the production of
virus dl5-29. Use of the methods of the invention in the production
of dl5-29 is described further below. Further, in addition to
dl5-29, as described, for example, in WO 99/06069, the methods of
the invention can be used in connection with other herpes viruses
(e.g., HSV-2) having deletions in U.sub.L5 and U.sub.L29 open
reading frames, or portions thereof, which render the viruses
dependent upon complementation of these proteins for replication.
In addition to dl5-29, as described above, the methods of the
invention can be used in the production of viruses (e.g.,
dl5-29-41), which, in addition to having deletions in U.sub.L5 and
U.sub.L29 sequences, also have an alteration (such as one or more
deletions) of U.sub.L41 nucleic acid sequences, by which the
alteration increases the immunogenicity of the virus. A specific
example of a virus having deletions in U.sub.L5, U.sub.L29, and
U.sub.L41 sequences (dl5-29-41), as well as general teachings of
such viruses, are provided in WO 2007/016239.
Cell Lines
Cell lines used in the invention include cell lines that support
alpha-herpesvirus growth, such as Vero (e.g., strain AV529-19),
MRC-5, BHK, CEM, and LL-1 cells. A suitable cell line is one that
hosts alpha-herpesviruses. Typically, the cell line is a mammalian
cell line, such as a rodent, non-human primate (e.g., monkey), or
human cell line. In order to allow growth of viruses lacking a gene
encoding a protein essential for viral growth, the host cell line
must include a nucleic acid sequence encoding the polypeptide
missing from the virus (e.g., a deleted or mutated polypeptide).
The host cell line can provide more than one polypeptide, in trans,
to support viral growth (e.g., U.sub.L5 and U.sub.L29 are provided
by AV529-19). Such polypeptides include structural and
non-structural (e.g., functional) alpha-herpesvirus polypeptides,
which can complement the growth of another virus in which the gene
for the homologous polypeptide is deleted or otherwise mutated. The
use of a Vero cell line to grow replication-incompetent
herpesviruses is described in, e.g., U.S. Pat. No. 6,841,373, which
is incorporated herein by reference.
Cell lines expressing functional herpes virus structural
polypeptides can be produced by standard methods, such as by
co-transfecting mammalian cells, for example, Vero, MRC-5, BHK,
CEM, and LL-1 cells, with one or more vectors, such as a plasmid
vector, including a nucleic acid molecule encoding the
polypeptide(s), and a vector, such as a plasmid vector, encoding a
selectable marker (e.g., the neo gene for neomycin/G418 antibiotic
resistance). Clones possessing the selectable marker are then
screened further to determine which clones also express functional
polypeptide(s) using methods known to those skilled in the art
(see, e.g., Rice et al., J. Virol. 64(4):1704-1715, 1990).
Compositions Including Media, Cells, and, Optionally, Viruses
The invention also includes compositions that include a medium of
the invention, in combination with cells for use in virus
production, according to the invention. These compositions can
also, optionally, include viruses produced in the cells of the
invention. Thus, compositions of the invention can contain media
that includes a disaccharide (e.g., sucrose, lactose, maltose,
trehalose, and cellobiose) at, for example, any of the
concentrations noted above, as well as a lipid mixture (containing,
for example, cholesterol and/or stigmastanol; and being synthetic
or derived from animal or plant (e.g., soybean) sources). Specific
examples of lipid mixtures and their amounts that can be included
in such compositions are provided above. The media of the invention
can include, for example, the base culture medias described above.
Further, the media in the compositions of the invention can also
optionally include components such as serum and/or amino acids, as
described above.
Cells included in the compositions of the invention include those
described above, e.g., Vero (e.g., AV529-19), MRC-5, BHK, CEM, and
LL-1 cells, which may include transgenes encoding polypeptides that
may be mutated or otherwise deficient in viruses produced in the
cells. The cells can be plated, in suspension, intact, or in
disrupted form (e.g., after sonication). Viruses included in the
compositions of the invention include alpha-herpesviruses,
including HSV-2 viruses as well as the other alpha-herpes viruses
listed above. The viruses can include attenuating and/or other
beneficial mutations as described herein, such as are present in
viruses dl5-29 and dl5-29-41. The compositions of the invention can
be used in methods for producing viruses as described herein.
Production of HSV-2 Strain dl5-29 Using Media Additives
The production of an HSV vaccine virus including deletions in
U.sub.L5 and U.sub.L29 (e.g., dl5-29) can be performed using a
complementary Vero cell line such as AV529-19, which is genetically
engineered two express two genes, U.sub.L5 and U.sub.L29, that are
necessary to support virus growth. Typically, viral yields have
been below 10 pfu per cell, and methods were sought to attempt to
boost the yield. The addition of sucrose and a lipid mixture to the
growth and infection media improved yields to approximately 15 pfu
per cell in preliminary studies using T-flasks and a limited number
of NUNC Cell Factories (NCFs). Based upon these studies, optimized
concentrations of sucrose and the lipid mixture were added to
current media formulations on a larger scale and a production run
utilizing four NCFs was performed to mimic a significant portion of
a production run designed to produce viral seed or vaccine product
for preclinical and clinical studies. The cells were seeded onto
NUNC Cell Factories in OptiPro SFM media containing 10% Fetal
Bovine Serum, 4 mM L-Glutamine, G418, and supplemented with 50 mM
sucrose and 0.5.times. of a 250.times. concentrated lipid mixture
(Invitrogen/GIBCO 250.times. cholesterol lipid concentrate) and
grown for approximately 96 hours at 37.degree. C. and 5% CO.sub.2.
Infection media consisted of the same OptiPro media, only with 1%
FBS and without G418. Infections were at an estimated MOI of 0.01,
and following approximately 72 hours incubation at 34.degree. C.
and 5% CO.sub.2, NCFs were harvested. Aliquots were drawn from each
unit for sonication and viral titer by standard plaque assay, and
bulk harvests were centrifuged and pelleted in stabilization buffer
for downstream processing. Plaque assay results indicated total
viral yield for each NCF of about 2.times.10.sup.10 pfu, with an
average of 19 pfu/cell. This is more than a two-fold increase in
viral yield compared to previous runs without the sucrose and
cholesterol lipid concentrate additives. Viral yields between NCFs
were consistent, ranging from 18 to 20 pfu/cell.
Identification of Optimal Media Additive Concentrations
Preliminary studies were performed in T-25 flasks to determine
optimal concentrations of sucrose and a lipid mixture in growth and
infection media during production of dl5-29 virus. This experiment
to optimize additive concentrations consisted of a matrix of
samples in concentrations of 0.2.times., 0.5.times., 1.0.times.,
2.0.times., and 5.0.times. for each additive with 1.times.
equivalent to 50 mM sucrose or 1.times. of a 250.times. cholesterol
lipid concentrate. Raw data plaque counts were compiled onto an
EXCEL spreadsheet, in which raw titers were calculated by
multiplying the average plaque count by its dilution factor and
dividing that quantity by 0.2. The resulting number was then
multiplied by 5 to reflect the original 1/5 dilution of the
sonicated sample to determine the pfu/mL. Standard deviations and %
CV were also calculated to assist in determining the validity of
the data. Data collected from this study are shown in FIG. 4,
plotting calculated pfu/cell versus additive concentrations. All
data shown are from growth and infection media containing serum,
except the two horizontal lines at .about.8.5 pfu/cell and 13
pfu/cell that indicate serum-free media without and with additives,
respectively. The data indicate a substantial drop in titer with
cholesterol concentration above 1.times.. The highest titers were
achieved with 1.times. sucrose, 0.5.times. cholesterol and 2.times.
sucrose, 1.times. cholesterol. Based upon these data, and for ease
of formulation, the optimum concentrations used to go forward were
sucrose at 1.times. (50 mM) and 0.5.times. of a 250.times.
cholesterol lipid concentrate.
Confirmation of Additive Effect at Larger Scale
Based upon optimization study results, media for growth and
infection were prepared for use in NCFs to compare the effects of
these additives on a larger scale format. Two NCFs were prepared,
one using media with additives (50 mM sucrose, 1.times. cholesterol
lipid concentrate), the other without as a control. Results of this
study are shown in Table 1.
TABLE-US-00001 TABLE 1 Comparison of dl5-29 yields of NCF treated
or not treated with additives. Avg. Titer Total pfu/ Passage
(pfu/mL) pfu/NCF pfu/cm.sup.2 cell dl5-29 NCF with 29 1.26E+07
1.26E+10 2.00E+06 12.63 additives dl5-29 NCF without 29 6.08E+06
6.08E+09 9.63E+05 6.08 additives
Further analysis was performed to calculate the viral yield per
cm.sup.2, per NCF, and per cell. These calculations were based upon
the total cm.sup.2 of the NCF as 6320, and an estimation of a
confluent NCF as having 1.0.times.10.sup.9 total cells. In
addition, passage numbers for the AV529-19 cells are shown at the
time of infection. Results indicate a benefit from the sucrose and
cholesterol additives. Pfu/cell data were consistent with, or
slightly lower than previous studies, however, the passage number
of the cells used was at the higher limit of optimum use.
Results from a pilot study utilizing 4 NCFs are shown on Table 2.
The results show nearly 2.0.times.10.sup.10 total pfu/NCF, with
yield efficiencies of 18-19 pfu/cell. Yields were consistent
throughout all four NCFs evaluated in this pilot study.
TABLE-US-00002 TABLE 2 Calculated average viral titers (pfu/mL),
total pfu/NCF, pfu/cm.sup.2, and pfu/cell of NCF viral harvests.
Avg. Titer Total pfu/ Passage (pfu/mL) pfu/NCF pfu/cm.sup.2 cell
dl5-29 NCF 1 19 1.93E+07 1.93E+10 3.05E+06 19.29 dl5-29 NCF 2 19
1.82E+07 1.82E+10 2.88E+06 18.21 dl5-29 NCF 3 19 1.97E+07 1.97E+10
3.12E+06 19.71 dl5-29 NCF 4 19 1.94E+07 1.94E+10 3.07E+06 19.38
Based on the results provided above, the best upstream yields
obtained so far are about 1.9.times.10.sup.10 pfu/NCF,
corresponding to an average of 19 pfu/cell. This is more than a
two-fold increase in viral yield as compared to previous runs
without sucrose and cholesterol lipid concentrate. In the four-NCF
pilot study, viral yields between NCFs were very consistent,
ranging from 18 to 20 pfu/cell. The optimal conditions described
here yield useful amounts of virus.
Additional studies were carried out using OptiPro, DMEM-F12, and
Megavir media (see FIGS. 5A-8). Oneway analysis of the titer
production by basal media type shows that Megavir and DMEM-F12
titers have a larger improvement on virus production with sucrose
and cholesterol than OptiPro. This analysis also suggests that the
titer improvement in OptiPro, under certain conditions, may not be
statistically significant (FIGS. 5A-5C and FIG. 6).
Sucrose, as a single factor, has an impact on virus production, but
cholesterol by itself does not improve the virus production (FIGS.
7A and 7B). The simultaneous presence of sucrose and cholesterol
has a significant impact on virus production. To maximize the
titer, the data suggest 1.times. cholesterol and 80 mM sucrose in
DMEM-F 12 to be the optimal condition for viral production.
Observed increases in viral production are partially due to an
increase in the specific production rate of the cells, showing an
increase in the presence of the sucrose and cholesterol from 57.9
PFU/cell to 85.3 PFU/cell (FIG. 8).
Methods
Design of the 4 NCF Pilot Study (Table 2)
The purpose of this study was to scale-up production of herpes
simplex virus strain dl5-29 from infection of Vero (AV529-19) cells
in four Nunc cell factories (NCFs; Nalge-Nunc). The cells can be
grown in OptiPro SFM media (Invitrogen/GIBCO, catalog no.
12309-019) containing 10% fetal bovine serum (FBS; JRH, catalog no.
12106-500M), 4 mM L-glutamine (Invitrogen/GIBCO, catalog no.
25030-081), G418 (Invitrogen/GIBCO, catalog no. 10131-027), and
supplemented with 50 mM sucrose (EMD, catalog no. SX1075-1) and
0.5.times. of a 250.times. cholesterol lipid concentrate
(Invitrogen/GIBCO, catalog no. 12531-018). Infection media
consisted of the same OptiPro media containing 1% FBS but lacking
G418. Cell cultures were infected with an approximate MOI of 0.01,
and allowed to incubate approximately 72 hours at 34.degree. C. and
5% CO.sub.2. Following incubation, NCFs were harvested. Aliquots
were drawn for each unit for sonication and viral titer by standard
plaque assay (described below), and bulk harvests were centrifuged
and pelleted in stabilization buffer for downstream processing.
Preparation of 1 M Sucrose Concentrate in Optipro (400 mL)
Mix the following reagents and warm to 37.degree. C.: sucrose
(136.92 g) OptiPro SFM (200 mL) FBS (10%; 40 mL) L-glutamine (8 mL)
G418 (4 mL) cholesterol lipid concentrate (0.8 mL) When dissolved,
QS volume to 400 mL total with OptiPro SFM, then filter sterilize
(0.2 .mu.m). Preparation of Seeding Media for NCFs (Per L)
Mix the following reagents: OptiPro SFM (868 mL) FBS (10%; 100 mL)
L-glutamine (20 mL) G418 (10 mL) cholesterol lipid concentrate (2
mL) When mixed well, remove 50 mL of media mixture and replace with
50 mL of 1 M sucrose concentrate in OptiPro SFM. The final mixture
will have a sucrose concentration of 50 mM. Preparation of 1 M
Sucrose Concentrate in OptiPro (200 mL for Infection Media)
Mix the following reagents and warm to 37.degree. C.: sucrose
(68.46 g) OptiPro SFM (100 mL) FBS (1%; 2 mL) L-glutamine (4 mL)
cholesterol lipid concentrate (0.4 mL) When dissolved, QS volume to
200 mL total with OptiPro SFM, then filter sterilize (0.2 .mu.m).
Preparation of Infection Media for NCFs (Per L)
Mix the following reagents: OptiPro SFM (968 mL) FBS (1%; 10 mL)
L-glutamine (20 mL) cholesterol lipid concentrate (2 mL) When mixed
well, remove 50 mL of media mixture and replace with 50 mL of 1 M
sucrose concentrate in OptiPro. The final mixture will have a
sucrose concentration of 50 mM. Preparation of 10% Sucrose in
1.times. Stabilization Buffer
Mix the following reagents: sucrose (10 g) 10.times. Stabilization
Buffer (10 mL) Add RODI to bring total volume to 100 mL. Mix well
and then place in a 37.degree. C. waterbath for approximately 30
minutes to fully dissolve. Filter-sterilize using a 0.2 .mu.m
sterile filter unit. NUNC Cell Factory Seeding Procedure
Harvest 8 triple flasks of Vero cells (AV529-19) grown to
approximately 90% confluence in OptiPro medium containing 10% FBS
with 4 mM L-glutamine, and G418. Following harvest, draw off a 1 mL
aliquot for cell count using a Vi-Cell instrument. Under aseptic
conditions, seed NCFs at 10.sup.8 cells/NCF using pre-infection
media (OptiPro SFM with 10% FBS+4 mM L-glutamine, G418, 50 mM
sucrose and 0.5.times. cholesterol lipid mixture; 2 L). Grow to
near confluence (approximately 3-4 days) in an incubator at
37.degree. C. and 5% CO.sub.2.
NUNC Cell Factory Infection Procedure
Pour off old media and rinse NCF with 500 mL DPBS and remove.
Infect at full volume infection media (1 L) with dl5-29 at a MOI of
0.01 as follows, assuming 10.sup.9 cells per confluent NCF: NCF
#1-4: 10.sup.7 pfu/NCF of HSV98 (dl5-29) in infection media
(OptiPro SFM with 1% FBS+4 mM L-glutamine, 50 mM sucrose and
0.5.times. cholesterol lipid concentrate). Based on a stock titer
of 1.6.times.10.sup.8 pfu/mL, add 625 .mu.L of virus stock per NCF.
Incubate the NCFs at 34.degree. C. for 72 hours or until
approximately 100% CPE is evident.
NUNC Cell Factory Harvest Procedure
When 100% CPE (nearly 100% of cells rounded up and easily detached)
is evident, tap NCFs several times to dislodge all cells from
monolayer. With 100% CPE, cells should already be mostly detached.
Aseptically pour off infection media from NCF into a sterile
container. Mix well, then draw off 1 mL aliquot and add to sterile
15 mL centrifuge tube containing 3.75 mL of OptiPro SFM with 10%
FBS and 4 mM L-glutamine, and 250 .mu.L of HSA. This is used as a
1:5 dilution of the NCF harvest and is to be sonicated and titered
to determine viral yield. Divide the NCF harvest equally into
4.times.250 mL sterile centrifuge tubes. Spin in centrifuge at 1000
RPM for 10 minutes at 4.degree. C. Decant resulting supernatants
and save in 1 L sterile bottle labeled as conditioned media. Store
bottles at 4.degree. C. Using 20 mL of 10% Sucrose in 1.times.
Stabilization buffer, aseptically resuspend and combine pellets in
BSC. Quick-freeze combined pellet and 1 mL centrifuge tube
containing titer sample in ethanol/dry ice bath until frozen solid.
Then place samples in -80.degree. C. Freezer.
Sonication Procedure
Remove 15 mL centrifuge tubes containing titer samples and thaw
quickly in a 37.degree. C. waterbath. Immediately after thawing,
place on ice. Aseptically sonicate cell suspension sample using
probe sonicator; Outlet level 3, Duty cycle 40% for approximately
15 seconds/mL on ice (total of 75 seconds). Spin down sonicated
suspension samples in centrifuge at 5000 g for 10 minutes at
4.degree. C. to remove cell debris. Aseptically pour off
supernatant into sterile, labeled 15 mL centrifuge tubes. Pellets
are discarded and proceed directly to viral titer procedure.
Virus Infectivity Assay (Plaque A, Assay)
Test samples were tested in 12-well plates that were seeded with
Vero cells (4.times.10.sup.5 cells/well) for virus infectivity and
quantification. They were serially diluted 10-fold in duplicate in
minimum essential medium (MEM) containing 1% FBS supplemented with
100 units/mL penicillin and 100 .mu.g/mL streptomycin, and
L-glutamine (completed cell culture medium). Dilutions were then
plated onto the cells at 200 .mu.L/well in triplicate wells. A
positive control is also serially diluted and plated. The
inoculated plates were incubated at 37.degree. C. with 5% CO.sub.2
for 60 minutes with rocking of the plates every 15 minutes to allow
virus adsorption. One mL of completed medium with 0.8% methyl
cellulose was added to each well and the plates were returned to
incubation at 37.degree. C. with 5% CO.sub.2 for 2 days. Medium was
then removed from all wells and approximately 1 mL of 1% crystal
violet in 70% methanol was added to each well to fix and stain
plates. Plates were left at room temperature for more than 60
minutes to stain cells, then the stain was removed, wells were
washed with tap water, and the plates were air-dried. Viral plaques
were counted using a stereomicroscope with plates held over a light
box. Virus concentrations are expressed as pfu/mL. Further
calculations are then made to determine the total viral yield per
NCF, viral yield/cm.sup.2, and viral yield/cell.
Other Embodiments
All references, publications, patent applications, and patents
cited in this specification are herein incorporated by reference as
if each individual publication or patent were specifically and
individually indicated to be incorporated by reference. Use of
singular forms herein, such as "a" and "the," does not exclude
indication of the corresponding plural form, unless the context
indicates to the contrary. Although the invention has been
described in some detail by way of illustration and example for
purposes of clarity of understanding, it will be readily apparent
to those of ordinary skill in the art in light of the teachings of
the invention that certain changes and modifications may be made
thereto without departing from the spirit or scope of the appended
claims.
Other embodiments are within the following claims.
* * * * *
References