U.S. patent application number 14/468131 was filed with the patent office on 2014-12-25 for method for production of ph stable enveloped viruses.
The applicant listed for this patent is BAXTER HEALTHCARE SA. Invention is credited to Andrej EGOROV, Brigitte KRENN, Sabine NAKOWITSCH, Julia ROMANOVA, Markus WOLSCHEK.
Application Number | 20140377308 14/468131 |
Document ID | / |
Family ID | 40626824 |
Filed Date | 2014-12-25 |
United States Patent
Application |
20140377308 |
Kind Code |
A1 |
ROMANOVA; Julia ; et
al. |
December 25, 2014 |
METHOD FOR PRODUCTION OF pH STABLE ENVELOPED VIRUSES
Abstract
The present invention provides a method for producing pH-stable
enveloped viruses wherein said viruses are used for infection of
host cells under low pH conditions and for incubation with cell
culture cells under conditions of low pH, as well as influenza
viruses obtainable by this method which exhibit a high growth rate
in cell culture, increased pH and temperature stability and which
have human receptor specificity.
Inventors: |
ROMANOVA; Julia; (Vienna,
AT) ; EGOROV; Andrej; (Vienna, AT) ; KRENN;
Brigitte; (Vienna, AT) ; WOLSCHEK; Markus;
(Vienna, AT) ; NAKOWITSCH; Sabine; (Vienna,
AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BAXTER HEALTHCARE SA |
Glattpark (Opfikon) |
|
CH |
|
|
Family ID: |
40626824 |
Appl. No.: |
14/468131 |
Filed: |
August 25, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13129806 |
May 17, 2011 |
8815252 |
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PCT/EP2009/065812 |
Nov 25, 2009 |
|
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14468131 |
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61117900 |
Nov 25, 2008 |
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Current U.S.
Class: |
424/209.1 ;
435/235.1 |
Current CPC
Class: |
A61K 39/12 20130101;
A61K 39/145 20130101; A61P 31/16 20180101; A61K 2039/5254 20130101;
A61P 37/04 20180101; C12N 7/00 20130101; C12N 2760/16134 20130101;
A61P 31/12 20180101; C12N 2760/16051 20130101; C12N 2760/16021
20130101; C12N 2760/16034 20130101 |
Class at
Publication: |
424/209.1 ;
435/235.1 |
International
Class: |
A61K 39/145 20060101
A61K039/145; C12N 7/00 20060101 C12N007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 25, 2009 |
EP |
09153664.9 |
Claims
1. A method for producing an influenza virus, comprising the steps
of: a) diluting influenza viruses in a solution having a pH of
between 5.2 and 5.9; b) infecting host cells with at least one
infectious virus particle, wherein: i) the virus particle is added
to said cells; and ii) said cells and said virus particle are
incubated at a pH of between 5.2 and 5.9 to provide a virus/cell
complex; c) cultivating infected host cells to propagate viruses;
and d) harvesting the propagated viruses.
2. The method of claim 1, wherein a macrolide polyene antibiotic or
a derivative thereof is added to said cells.
3. The method of claim 1, wherein the cells are tissue culture
cells selected from the group consisting of BSC-1 cells, LLC-MK
cells, CV-1 cells, CHO cells, COS cells, murine cells, human cells,
HeLa cells, 293 cells, VERO cells, MDBK cells, MDCK cells, MDOK
cells, CRFK cells, RAF cells, TCMK cells, LLC-PK cells, PK15 cells,
WI-38 cells, MRC-5 cells, T-FLY cells, BHK cells, SP2/0 cells, NS0
cells, and PerC6 cells.
4. The method of claim 1, wherein the influenza virus is selected
from the group consisting of influenza A, influenza B or and
influenza C.
5. The method of claim 1, wherein the influenza virus is an
attenuated influenza virus.
6. The method of claim 1, wherein the influenza virus comprises a
deletion or modification within the NS1 gene of the virus.
7. The method of claim 1, wherein the influenza virus is a cold
adapted virus.
8. A The method of claim 1, wherein the viruses are diluted in a
buffer is selected from the group consisting of MES
(2-(N-morpholino-ethanesulfonic acid) buffer, citric buffer, and
acetate buffer.
9. A The method of claim 1, wherein in the cultivating step a
cultivation medium is used to cultivate the cells, and wherein the
cultivation medium is SFM opti-pro.TM. medium.
10. The method of claim 1, wherein the viruses are passaged in the
host cells for at least one passage.
11. An influenza virus produced by the method of claim 1, wherein
the virus retains hemagglutination activity at increased
temperature, retains infectivity at a pH range of between 5.5 and
5.8, demonstrates a high growth rate in cell culture, and has human
receptor specificity.
12-13. (canceled)
14. A composition useful for vaccination or therapy of viral
disease comprising an influenza virus produced by the method of
claim 1 and a pharmaceutically acceptable carrier or adjuvant.
15. The method of claim 2, wherein the macrolide polyene antibiotic
is amphotericin B.
16. The method of claim 1, further comprising the step of
inoculating a cell culture using the viruses harvested in step
(d).
17. The method of claim 16, wherein the cell culture comprises a
cell culture medium having a volume of at least 200 liters.
18. The method of claim 1, wherein step (a) comprises diluting
viruses in a solution having a pH of between 5.5 and 5.8.
19. The method of claim 1, wherein the cells and the virus particle
are incubated at a pH of between 5.5 and 5.8.
20. The method of claim 1, wherein the cells and the virus particle
are incubated at a pH of between 5.6.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/129,806, filed May 17, 2011 and entitled
"METHOD FOR PRODUCTION OF pH STABLE ENVELOPED VIRUSES," which is
the U.S. national stage of International Patent Application No.
PCT/EP2009/065812, filed on Nov. 25, 2009 and entitled "METHOD FOR
PRODUCTION OF pH STABLE ENVELOPED VIRUSES", which claims the
benefit of priority under 35 U.S.C. .sctn.119 from European Patent
Application No. 09153664.9, filed Feb. 25, 2009, and under 35
U.S.C. .sctn.120 from U.S. Patent Application No. 61/117,900, filed
Nov. 25, 2008. The disclosures of the foregoing applications are
incorporated herein by reference in their entirety.
SEQUENCE LISTING
[0002] The entire content of a Sequence Listing titled
"Sequence_Listing.txt," created on Aug. 24, 2014 and having a size
of 1 kilobyte, which has been submitted in electronic form in
connection with the present application, is hereby incorporated by
reference herein in its entirety.
FIELD OF THE INVENTION
[0003] The present invention provides a method for producing
pH-stable enveloped viruses wherein said viruses are incubated with
cell culture cells under conditions of low pH. Such viruses include
novel influenza viruses which show high growth rate in cell
culture, increased pH and temperature stability and which have
human receptor specificity.
BACKGROUND
[0004] For preventing disease caused by annual epidemics of viral
infections, vaccination is the most important public health
measure. An effective supply of vaccines is dependent on being able
to quickly produce large quantities of vaccine material (e.g.
virus). The rapid development of vaccines and their abundant
availability is critical in combating many human and animal
diseases. Delays in producing vaccines and shortfalls in their
quantity can cause problems in addressing outbreaks of disease.
[0005] Growth of viruses, especially of influenza virus in
embryonated chicken eggs, has been shown to result in effective
production of influenza virus particles which can be either used
for production of inactivated or live attenuated influenza virus
vaccine strains. Nevertheless during the last few years intensive
efforts have been made in establishing virus production systems
using cell culture because an egg-based method requires a steady
supply of specific pathogen-free eggs which could be problematic in
case of a pandemic. The cell-based technology is an alternative
production process that is independent of eggs suppliers and can be
started as soon as the seed virus is available. Besides this,
inactivated influenza vaccine prepared from the virus grown in
mammalian cells was shown to induce more cross-reactive serum
antibodies and reveals better protection than egg-grown vaccine
(Alymova et al., 1998, J Virol 72, 4472-7). Moreover, according to
previous results receptor specificity and antigenic properties of
human isolates become altered following growth of the virus in
embryonated chicken eggs (Mochalova et al., 2003, Virology 313,
473-80, Romanova et al., 2003, Virology 307, 90-7).
[0006] On the other hand, multiple propagation of viruses in tissue
culture often results in HA mutants that have elevated pH of fusion
(Lin et al., 1997, Virology 233, 402-10) which is correlated to
decreased stability to thermal denaturation of viruses (Ruigrok et
al., 1986). The structure of any protein and its stability are
based on noncovalent interactions like hydrophobic forces, van der
Waal interactions, hydrogen bonds, and ionic interactions.
Mutations which appear upon adaptation of viruses to cell cultures
are known to elevate the threshold of pH of fusion induced by
reduced protein stability because of changed ionic interactions and
salt bridges in HA molecule (Rachakonda et al., 2007, Faseb J21,
995-1002). Destabilizing mutations usually found either at the
interface HA1-HA2 or HA2-HA2 regions or in the N terminus of HA2 in
turn could lead to reduced binding to cell-surface receptors (Korte
et al., 2007, Rachakonda et al., 2007, Faseb J 21, 995-1002,
Shental-Bechor et al., 2002, Biochim Biophys Acta 1565, 81-9),
which leads to decreased virus infectivity and subsequently reduced
immunogenicity of live virus preparations.
[0007] Massaab (Massaab H. F., Journal of Immunology. 1969, 102,
pp. 728-732) tested the biologic and immunologic characteristics of
cold-adapted influenza virus using different genetic markers before
and after adaptation to growth in primary chick kidney tissue
culture and embryonated eggs. It is stated that these strains are
more sensitive to low pH compared to original "wild type" strains
and showed marked decrease in infectivity and hemagglutination
yields.
[0008] Fiszman et al (Journal of Virology, 1974, 13, pp. 801-808)
examined the effect of low pH (pH6.6) on vesicular stomatitis virus
(VSV) and showed that no viral particles or nucleocapsids were
detected. Ackermann W and Massaab H. F. (Journal of Experimental
Medicine FEB 1954, 99, pp 105-117) disclosed the effect of a viral
inhibitor, alpha-amino-p-methoxy-phenylmethanesulfonic acid) upon
the growth cycle of influenza virus.
[0009] Due to difficulties in obtaining high amounts of vaccine
virus preparations from cell culture which are of high stability
and immunogenicity in order to avoid any safety or supply issues,
it is an object of the present invention to make available
processes which lead to efficacious and stable viruses. The object
is achieved by the provision of the embodiments of the present
application.
[0010] The invention relates to a method for producing pH stable
enveloped viruses in tissue cultures by employing conditions of
decreased pH during dilution of virus suspension and host cell
infection. The method of the invention also provides virus of
increased stability and immunogenicity compared to virus particles
derived from presently used methods.
[0011] The invention relates to a method for producing pH stable
enveloped viruses in tissue cultures by employing conditions of
decreased pH during dilution of virus suspension and host cell
infection. The method of the invention also provides virus of
increased stability and immunogenicity compared to virus particles
derived from presently used methods.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1: Immunogenicity of Wisc.L1NS1 and Wisc.L1NS1_HA2_G75R
compared in ferrets after single intranasal immunization with the
dose of 6.0 log TCID50/animal.
[0013] FIG. 2A. Induction of serum antibodies in mouse model.
[0014] FIG. 2B. Reproduction of challenge virus in the lungs and
nasal turbinates of immunized mice.
[0015] FIG. 3A. Sequence comparison of HA molecule of original and
mutant viruses. Substitutions of two nucleotides (aa) to (tt) by
site directed mutagenesis led to the amino acid change K to I at
position 58 of HA2 subunit.
[0016] FIG. 3B. Fusion activity of VN1203 and VN1203 K581 viruses
with human erythrocytes.
[0017] FIG. 3C. IgA antibody titers in mouse nasal washes after
immunization with VN1203 and VN1203 K581 viruses.
[0018] FIG. 3D. HA1 antibody titers in mouse sera after
immunization with VN1203 and VN1203 K581 viruses.
[0019] FIG. 3E. Infectivity of VN 1203 and VN1203K581 viruses for
mice.
[0020] FIG. 4: Sensitivity of Vienna/28 and Vienna/28_HA2_G75R
viruses towards low pH.
DETAILED DESCRIPTION OF THE INVENTION
[0021] The invention relates to a method for producing enveloped
viruses characterized in that it comprises following steps:
[0022] a) diluting viruses in a solution having a pH between 5.2
and 5.9, preferably between 5.4 and 5.8, most preferably at about
5.6;
[0023] b) infecting host cells with at least one infectious virus
particle wherein i) the virus particle is added to said cells and
ii) said cells and said virus particle are incubated at
[0024] The invention relates to a method for producing enveloped
viruses characterized in that it comprises following steps:
[0025] a) diluting viruses in a solution having a pH between 5.2
and 5.9, preferably between 5.4 and 5.8, most preferably at about
5.6;
[0026] b) infecting host cells with at least one infectious virus
particle wherein i) the virus particle is added to said cells and
ii) said cells and said virus particle are incubated at a pH
between 5.2 to 5.9, preferably between 5.4 and 5.8, most preferably
at about 5.6 to provide a virus/cell complex;
[0027] c) cultivating the infected host cells to propagate
viruses;
[0028] d) harvesting the viruses and optionally;
[0029] e) purifying and/or characterising the viruses.
[0030] The virus obtained by said method can be used for production
of lab scale amounts of virus as well as for large scale production
of vaccine virus.
[0031] "Large scale production" means the production in a minimum
cultivation volume of at least 2001, preferably of at least 5001,
preferably of about 10001:
[0032] Vaccine preparations containing enveloped viruses have to be
immunogenic to provide sufficient vaccination. Especially,
inactivated pandemic influenza vaccines like against avian
influenza can be poorly immunogenic and require high doses to
elicit protective antibody responses in humans. Effective antibody
responses provide crucial immunity against virus infection. The
hemagglutinin (HA) protein is the major target of protective
antibody responses induced by viral infection with influenza virus
and by vaccination with both inactivated and live-attenuated flu
vaccines. The structure integrity of HA antigens is critical for
eliciting protective antibody responses.
[0033] The inventors have shown that the inventive method provides
viruses that comprise pH stability and increased immunogenicity.
The HA protein of the virus particles so produced preferably shows
increased stability at low pH. Advantageously the viruses can also
show increased stability at higher temperatures, specifically at
temperatures up to 60.degree. C. These viruses do not significantly
lose hemagglutination activity of HA even when stored at increased
temperatures like e.g. at 60.degree. C. for several minutes up to
several hours. "Not significant" means that hemagglutination
activity is decreased less than fourfold compared to the source
virus. Even after exposure to increased temperatures, said viruses
keep stability when stored for several weeks up to several months
at a temperature between 0.degree. C. and 12.degree. C., preferably
at 4.degree. C. Therefore the viruses produced according to the
invention are highly advantageous for vaccine preparation as said
viruses comprise a stable HA molecule.
[0034] This method can be used specifically for negative-strand RNA
viruses which are a group of animal viruses that comprise several
important human pathogens, including influenza, measles, mumps,
rabies, respiratory syncytial, Ebola and hanta viruses.
[0035] The genomes of these RNA viruses can be unimolecular or
segmented, single stranded of (-) polarity. Two essential
requirements are shared between these viruses: the genomic RNAs
must be efficiently copied into viral RNA, a form which can be used
for incorporation into progeny virus particles and transcribed into
mRNA which is translated into viral proteins. Eukaryotic host cells
typically do not contain machinery for replicating RNA templates or
for translating polypeptides from a negative stranded RNA template.
Therefore negative strand RNA viruses encode and carry an
RNA-dependent RNA polymerase to catalyze synthesis of new genomic
RNA for assembly into progeny and mRNAs for translation into viral
proteins.
[0036] Genomic viral RNA must be packaged into viral particles in
order for the virus to be transmitted. The process by which progeny
viral particles are assembled and the protein/protein interactions
occur during assembly is similar within the RNA viruses. The
formation of virus particles ensures the efficient transmission of
the RNA genome from one host cell to another within a single host
or among different host organisms.
[0037] Virus families containing enveloped single-stranded RNA of
the negative-sense genome are classified into groups having
non-segmented genomes (Paramyxoviridae, Rhabdoviridae, Filoviridae
and Borna Disease Virus, Togaviridae) or those having segmented
genomes (Orthomyxoviridae, Bunyaviridae and Arenaviridae). The
Orthomyxoviridae family includes the viruses of influenza, types A,
B and C viruses, as well as Thogoto and Dhori viruses and
infectious salmon anemia virus.
[0038] Preferred embodiments include but are not limited to
influenza virus, respiratory syncytial virus (RSV), Newcastle
disease virus (NDV), vesicular stomatitis virus (VSV), and
parainfluenza virus (PIV).
[0039] Influenza virions consist of an internal ribonucleoprotein
core (a helical nucleocapsid) containing the single-stranded RNA
genome, and an outer lipoprotein envelope lined inside by a matrix
protein (M1). The segmented genome of influenza A virus consists of
eight molecules of linear, negative polarity, single-stranded RNAs
which encodes eleven (some influenza A strains ten) polypeptides,
including: the RNA-dependent RNA polymerase proteins (PB2, PB1 and
PA) and nucleoprotein (NP) which forms the nucleocapsid; the matrix
membrane proteins (M1, M2); two surface glycoproteins which project
from the lipid containing envelope: hemagglutinin (HA) and
neuraminidase (NA); the nonstructural protein (NS1) and nuclear
export protein (NEP). Most influenza A strains also encode an
eleventh protein (PB1-F2) believed to have proapoptotic
properties.
[0040] Transcription and replication of the genome takes place in
the nucleus and assembly occurs via budding on the plasma membrane.
The viruses can reassort genes during mixed infections. Influenza
virus adsorbs via HA to sialyloligo-saccharides in cell membrane
glycoproteins and glycolipids. Following endocytosis of the virion,
a conformational change in the HA molecule occurs within the
cellular endosome which facilitates membrane fusion, thus
triggering uncoating. The nucleocapsid migrates to the nucleus
where viral mRNA is transcribed. Viral mRNA is transcribed by a
unique mechanism in which viral endonuclease cleaves the capped
5'-terminus from cellular heterologous mRNAs which then serve as
primers for transcription of viral RNA templates by the viral
transcriptase. Transcripts terminate at sites 15 to 22 bases from
the ends of their templates, where oligo(U) sequences act as
signals for the addition of poly(A) tracts. Of the eight viral RNA
molecules so produced, six are monocistronic messages that are
translated directly into the proteins representing HA, NA, NP and
the viral polymerase proteins, PB2, PB1 and PA. The other two
transcripts undergo splicing, each yielding two mRNAs which are
translated in different reading frames to produce M1, M2, NS1 and
NEP. In other words, the eight viral RNA segments code for eleven
proteins: nine structural and two nonstructural (NS1 and the
recently identified PB1-F2) proteins.
[0041] The viruses may be selected from naturally occurring
strains, variants or mutants; mutagenized viruses (e.g. generated
by exposure to mutagens, repeated passages and/or passage in
non-permissive hosts); reassortants (in the case of segmented viral
genomes); and/or genetically engineered viruses (e.g. using the
"reverse genetics" techniques) having the desired phenotype.
[0042] The term "passaged" is defined as inoculating host cells
with a defined virus particle number and harvesting said virus
after a defined number of days, typically 2-3 days. The viruses
will have approx. 2 to 4 replication rounds per day.
[0043] It is well known in the art that the wild-type viruses used
in preparation of the vaccine strains for annual vaccination
against epidemic influenza are recommended annually by the World
Health Organization (WHO). These strains may then be used for the
production of reassortant vaccine strains which generally combine
the NA and/or HA genes of the wild-type viruses with the remaining
gene segments derived from a donor virus (often referred to as a
master donor virus or MDV) which will have certain desirable
characteristics. For example, an MDV strain may be cold-adapted
temperature sensitive, attenuated, and/or has a high growth
rate.
[0044] According to a specific embodiment, the influenza virus is
an attenuated influenza virus. Specifically the influenza virus
comprises deletions or modifications within the pathogenicity
factors inhibiting innate immune response of host cells. The
attenuation can exemplarily be derived from cold-adapted virus
strains or due to a deletion or modification within the NS1 gene
(.DELTA.NS1 virus) as described in WO99/64571 and WO99/64068 which
are incorporated herein in total by reference. These viruses are
replication deficient as they undergo abortive replication in the
respiratory tract of animals. Alternatively, the viruses can
comprise deletion or modification of the PB1-F2 gene.
[0045] According to the invention the virus can further comprise
modifications within the HA gene which can increase the stability
of the HA molecule. For example, Steinhauer et al. (1991, PNAS. 88:
11525-1152) identified the K58I mutation in the HA.sub.2 of
influenza Rostock virus (H7N1) to be responsible for a decreased pH
value of membrane fusion compared to the non-mutated virus. This
implies that the conformational change of the HA induced by the
acidic pH happens in the mutated form of the HA at 0.7 lower pH
compared to the wildtype virus. By introducing this mutation to the
X-31 influenza virus (H3 subtype) the same effect was shown.
[0046] The term "reassortant," when referring to a virus, indicates
that the virus includes genetic and/or polypeptide components
derived from more than one parental viral strain or source. For
example, a 7:1 reassortant includes 7 viral genomic segments (or
gene segments) derived from a first parental virus, and a single
complementary viral genomic segment, e.g., encoding hemagglutinin
or neuraminidase, from a second parental virus. A 6:2 reassortant
includes 6 genomic segments, most commonly the 6 internal genes
from a first parental virus, and two complementary segments, e.g.,
hemagglutinin and neuraminidase, from a different parental
virus.
[0047] Specifically, the influenza virus vaccines are derived from
interpandemic or pandemic influenza virus strains, for example of
H1, H3 or B strains. It has been shown that these strains show
highly increased immunogenicity when produced according to the
inventive method.
[0048] The cells which can be used in the method according to the
invention for cultivating the viruses can be any desired type of
cells which can be cultured and which can be infected by enveloped
viruses, specifically by influenza viruses. Specifically it can be
BSC-1 cells, LLC-MK cells, CV-1 cells, CHO cells, COS cells, murine
cells, human cells, HeLa cells, 293 cells, VERO cells, MDBK cells,
MDCK cells, CEK (chicken embryo kidney) CEF (chicken embryo
fibroblasts), MDOK cells, CRFK cells, RAF cells, TCMK cells, LLC-PK
cells, PK15 cells, WI-38 cells, MRC-5 cells, T-FLY cells, BHK
cells, 5P2/0 cells, NS0, PerC6 (human retina cells).
[0049] For diluting the viruses any buffer can be used which can
provide the range of pH values, specifically between pH 5.2 and
5.9, specifically between 5.4 and 5.8 and which is physiological
for the cells. For example, it can be MES
(2-(N-morpholino-ethanesulfonic acid) buffer, citric buffer or
acetic buffer, specifically using buffers based on PBS.
Additionally components can also be added to the dilution solution,
for example salts like sodium chloride, di-sodium hydroxy-phosphate
or potassium di-hydroxy-phosphate etc.
[0050] The term dilution means that virus suspension is diluted to
a content of virus particles that is sufficient for productive
infection of cells.
[0051] According to the inventive method appropriate cells are
infected with at least one virus particle. The number of virus
particles necessary for sufficient infection can be easily
determined by the skilled person. The infection of the cells with
the viruses can be specifically carried out at an m.o.i.
(multiplicity of infection) of about 0.0001 to 10, preferably of
0.001 to 0.5.
[0052] Optionally, a macrolide polyene antibiotic or a derivative
can be present during the dilution step in the dilution solution
and/or during infection and/or cultivation. Specifically, the
antibiotic is amphotericin B or a derivative thereof. Specifically,
the macrolide polyene antibiotic can be added before infection, for
example about 60-30 minutes before, more preferably 30 minutes
before infection. Optimal concentration of antibiotic used for
virus incubation or cultivation is between 0.20 and 0.50 .mu.g/ml,
specifically 0.25 .mu.g/ml.
[0053] The temperature for incubating the virus for binding it to
the cells, especially to the cellular receptors, can be between
20.degree. C. and 38.degree. C. The pH for incubation is preferably
between 5.4 and 5.8. For determination of the time sufficient for
internalization of the virus into the cell, virus can be monitored
by standard procedures, like labelling with a dye or electron
microscopy. Specifically, the time period is between at least 5
minutes and 60 minutes, preferably between 20 and 60 minutes at
room temperature.
[0054] A protease which cleaves the precursor protein of
hemagglutinin can be added and the internalization of the viruses
to the cells can be carried out according to the invention shortly
before, simultaneously with or shortly after the infection of the
cells with influenza viruses. If the addition is carried out
simultaneously with the infection, the protease can either be added
directly to the cell culture to be infected or, for example, as a
concentrate together with the virus inoculate. The protease is
preferably a serine protease, and particularly preferably trypsin.
If trypsin is used, the final concentration added in the culture
medium is advantageously 1 to 200 .mu.g/ml, preferably 5 to 50
.mu.g/ml, more preferably 5 to 30 .mu.g/ml.
[0055] After infection, the infected cell culture is cultured
further to replicate the viruses, in particular until a maximum
cytopathic effect or a maximum amount of virus antigen can be
detected. The harvesting can alternatively be at any time point
during cultivation.
[0056] The pH for cultivation of the host cells, can be for example
between 6.5 and 7.5. The pH for cultivation depends on the pH
stability of the host cells used for cultivation. This can be
determined by testing of the host cells' viability under different
pH conditions.
[0057] The terms cultivation and propagation do have the same
meaning according to the invention.
[0058] For cultivation any medium useable for cultivation of cells
is appropriate. Specifically the medium can be SFM opti-pro.TM.
medium, a low protein medium for the culture of kidney epithelial
and related cells expressing virus. Cells can be cultivated at a
temperature between 20 and 40.degree. C., specifically between 30
and 40.degree. C.
[0059] The viruses can be passaged in the host cells for at least
one passage, yet usually several passages are needed, for example
at least three passages.
[0060] According to a specific embodiment of the method, the
harvesting and isolation of the replicated influenza viruses is
carried out 2 to 10 days, preferably 3 to 7 days, after infection.
The cells or cell residues can be separated and harvested from the
culture medium by means of methods known to the person skilled in
the art, for example by separators or filters. Following this
concentrating, the influenza viruses present in the cultivation
medium is carried out by methods known to the person skilled in the
art, such as, for example, gradient centrifugation, filtration,
precipitation and the like.
[0061] It was shown successfully by the inventors that diluting
enveloped viruses and infecting cells under low pH conditions lead
to enveloped viruses which show stability at low pH and increased
immunogenicity. These viruses can also show stability at increased
temperatures and/or high growth rates in cell culture and/or human
receptor specificity. This is surprising as Scholtissek (1985,
Archives Virol., 85, 1-11) showed at low pH values infectivity of
influenza viruses was irreversibly lost. It was also stated that
there is no correlation between the pH and heat stability.
[0062] As a further embodiment of the invention, an influenza virus
is also provided that is for example useful as seed virus or virus
useful for vaccination purposes. Said influenza virus retains
detectable hemagglutination activity at increased temperature,
retains infectivity stable at pH range of 5.4 and 5.8, is of high
growth rate in cell culture and has human receptor specificity.
[0063] "Seed virus" is defined as the virus used to inoculate a
cell culture.
[0064] Detectable hemagglutination activity according to the
embodiment of the invention is defined as a not more than fourfold
decrease of hemagglutination activity compared to the source virus
as used. The source virus can be for example virus directly
isolated from a nasal swab.
[0065] According to a further embodiment virus can be provided that
is useful as vaccine virus. Said virus particles are stable at low
pH and show immunogenicity which is similar or increased compared
to virus obtained by known cell culture procedures, for example
from Vero cells, MDCK or MDBK cells. Specifically the virus shows
increased growth rate in cell culture compared to virus that has
not been exposed to the method according to the present invention.
Further, the virus is temperature stable and has human receptor
specificity.
[0066] Temperature stability according to the present invention
means that hemagglutination activity is not significantly decreased
at a temperature of up to 60.degree. for a time period of up to 15
minutes. pH stability is defined as stability of the virus at a pH
of 5.6, preferably between 5.4 and 5.8, preferably between 5.2 to
5.9. High growth rate means a growth rate of up to 6 log TCID
50/ml, preferably above 7 log TCID 50/ml.
[0067] Said virus can be obtained by the method as described in the
present application. The viruses are particularly useful for
vaccine formulations or therapeutic formulations. Influenza virus
contained in these formulation can be either attenuated virus or
inactivated virus. Inactivation can be performed by any method as
known in the art, like treatment with formalin or other agents used
in the manufacturing of killed virus vaccines or treatment with
non-ionic detergents or exposure to UV light. Influenza virus
comprising preparations can be administered by any route like for
example subcutaneously, intranasally or intramuscularly.
[0068] Alternatively, the preparations containing the influenza
virus can further comprise pharmaceutically acceptable carriers or
adjuvants known to enhance immunogenicity of the administered
preparation.
[0069] Preferably, the preparations are administered via the
mucosa, specifically by intranasal application as these viruses are
of high immunogenicity being a result of the above listed
characteristics, i.e. pH stability, temperature stability, high
growth rate and human receptor specificity.
[0070] An influenza virus comprising these characteristics has
never been described or indicated before.
[0071] The foregoing description will be more fully understood with
reference to the following examples. Such examples are, however,
merely representative of methods of practicing one or more
embodiments of the present invention and should not be read as
limiting the scope of invention.
EXAMPLES
Example 1
[0072] Two influenza strains bearing surface glycoproteins from
epidemic strain A/Wisconsin/67/05 (H3N2) and all other genes from
WHO vaccine strain IVR-116 (reassortant of A/New Caledonia/20/99
and A/Puerto Rico/8/34) in combination with NS gene lacking the NS1
open reading frame (.DELTA.NS1) were constructed by reverse
genetics. Obtained viruses differed by one amino acid substitution
in the sequence of HA molecule, due to different passaging
conditions on Vero cells. First virus, named Wisc..DELTA.NS1 was
always passaged on Vero cells with pre treatment of virus inoculum
with low pH buffer, namely:
[0073] the virus was diluted in a MES infection buffer (0.1 M MES,
150 mM NaCl, 0.9 mM CaCl.sub.2, 0.5 mM MgCl.sub.2; pH=5.6)
supplemented with 0.25 .mu.g/ml amphotericin B to appropriate moi.
Vero cells were washed with infection buffer and the virus inoculum
was applied to the cells and incubated for 30 min. Then the
inoculum was removed and cells were incubated in serum free
Opti-pro medium supplemented with 0.25 .mu.g/ml amphotericin B and
5 .mu.g/ml trypsin.
[0074] This method resulted in the preservation of the original
virus HA sequence found in the clinical swab. Second virus was
propagated by standard way at neutral conditions and acquired one
substitution in HA2 subunit, namely G75R (Table.1). Table 1 shows
the sequence comparison of HA molecule comparing to virus present
in the swab.
[0075] The HA nucleotide sequences of two viruses cultivated at
different conditions were compared. Virus Wisc..DELTA.NS did not
acquire any mutations in HA molecule, while one mutation G75R
located in HA2 subunit was identified in the virus
Wisc..DELTA.NS_HA2_G75R.
TABLE-US-00001 TABLE 1 Substitutions in HA subunits Viruses HA1 HA2
Wisc..DELTA.NS none None Wisc..DELTA.NS_HA2_G75R none G75R
[0076] Next both viruses were compared in their stability toward
low pH by the next method. The virus was diluted in the infection
MES buffer having the pH range 5.6-7.5 in order to get a defined
moi and applied to the Vero cells with subsequent incubation for 30
min to allow the virus to infect cells. Afterwards the inoculum was
removed, cells were incubated at 37.degree. C. for 4-9 h (depending
on the strain) then fixed and influenza NP protein was detected by
immunofluorescence.
[0077] This test revealed that virus Wisc..DELTA.NS1 appeared to be
stable at pH 5.6 infecting the cells with the same efficiency as at
neutral conditions, while mutant virus Wisc..DELTA.NS1_HA2_G75R
completely lost the ability to infect cells at pH 5.6 infecting
cells only at pH 5.8 with the same efficiency as at neutral
conditions (results are not shown).
[0078] The immunogenicity of Wisc..DELTA.NS1 and
Wisc..DELTA.NS1_HA2_G75R viruses was compared in ferrets after
single intranasal immunization with the dose of 6.0 log
TCID50/animal. The obtained results demonstrated that virus with
intact sequence of HA Wisc..DELTA.NS1 induced significantly higher
serum antibody titers, measured by HA1 test (GMT 202.9), than the
virus Wisc..DELTA.NS1_HA2_G75R (GMT 27.9) (FIG. 1).
Example 2
[0079] Two H1N1 influenza strains bearing surface glycoproteins
from epidemic strain A/Brisbane/59/07 (H1N1) and all other genes
from WHO strain IVR-116 in combination with NS gene lacking the NS1
open reading frame (.DELTA.NS1) were constructed by reverse
genetics. Obtained viruses differed by one amino acid substitution
in the sequence of HA molecule, appeared due to different passaging
conditions on Vero cells. First virus, named Brisbane.DELTA.NS1 was
passaged at low pH conditions in the presence of amphotericin B.
This procedure resulted in the preservation of the HA sequence,
which appeared to be similar to that of virus isolated in MDCK
cells from clinical sample (passage 1).
[0080] Second virus, named Brisbane.DELTA.NS1_HA2_N16I was passaged
by standard method and acquired one substitution in HA2 subunit,
namely N16I (Table 2). Tab. 2 shows the sequence comparison of HA
molecule comparing to initial isolate.
TABLE-US-00002 TABLE 2 Substitutions in HA subunits Viruses HA1 HA2
Brisbane.DELTA.NS none none Brisbane.DELTA.NS_HA2_N16I none
N16I
[0081] The HA nucleotide sequences of two viruses cultivated at
different conditions was compared. Virus Brisbane.DELTA.NS did not
acquire any mutations in HA molecule, while one mutation N16I
located in HA2 subunit was identified in the virus
Brisbane.DELTA.NS_HA2_N16I.
[0082] Comparison of virus stability towards low pH revealed that
virus Brisbane.DELTA.NS1 appeared to be stable. In immune
fluorescent assay the same amount of stained Vero cells at pH as
low as 5.6 and pH 7.5 was observed. Mutant virus
Brisbane.DELTA.NS1_HA2_N16I was less stable infecting cells only at
pH 5.8 and did not infect any at pH 5.6. No immune fluorescent
signal was visible on the cells infected with the virus combined
with buffer pH 5.6 (results are not shown).
[0083] Immunogenicity of both viruses was compared after single
intranasal immunization of mice with virus dose 5.6 log
TCID50/animal. The obtained results revealed that virus
Brisbane.DELTA.NS1 was more immunogenic than
Brisbane.DELTA.NS1_HA2_N16I inducing higher levels of serum
antibodies (measured by HA1 test) and better protection of animals
indicated by reduced replication of the challenge virus in the
lungs and nasal turbinates of mice (FIGS. 2A, B). FIG. 2B
specifically discloses the reproduction of challenge virus in the
lungs and nasal turbinates of immunized mice.
Example 3
[0084] Cultivation of influenza B strains on Vero cells at standard
conditions also leads to the appearance of destabilizing mutations
either at the interface HA1-HA2 or HA2-HA2 regions in HA molecule
related to the decreased stability and in turn decreased
immunogenicity of mutated virus in animal models (data not
shown).
Example 4
[0085] Previously it was found that H5N1 avian highly pathogenic
viruses circulated during last decade do not stand treatment with
human nasal washings, having a pH of 5.6. They also did not stand
treatment with acidic buffer with the same pH 5.6 during
inoculation of Vero cells. It was found that the reason of this
instability is high pH at which HA molecule changes the
conformation in order to perform fusion with the cell membrane,
which for H5N1 virus has the value pH 5.6 while for human viruses
it is in the range of 5.2-5.4.
[0086] Steinhauer et al., have demonstrated that one substitution
in HA2, namely K58I of H7N7 virus could decrease significantly the
pH of fusion by 0.7 units. Introduction of this mutation in H3N2
virus had a similar effect.
[0087] This change was introduced by site-directed mutagenesis to
the HA protein of the A/VN1203/04 .DELTA.NS1 (H5N1) virus
(reassortant, inheriting the HA, NA, and M genes from A/VN/1203/04
and the remaining genes from the IVR-116 vaccine strain in
combination with ANSI gene) and named the rescued virus VN1203 HA
K58I (FIG. 3A). FIG. 3A shows the sequence comparison of HA
molecule of original and mutant viruses)
[0088] HA of both viruses was modified in a trypsin dependent
manner. The pH of fusion for mutated virus VN1203 HA K58I was
reduced on 0.3 units in hemolisis experiment with human
erythrocytes (data not shown).
[0089] Moreover, virus VN1203 HA K58I showed reduced loss of
infectivity at pH 5.6. In immune fluorescent assay it was observed
almost similar amount of stained cells after infection made at pH
5.6 and 7.5 with virus VN1203 HA K58I, while no stained cells were
visible when infection was done with VN1203 virus at pH 5.6 (data
not shown).
[0090] The ability of both viruses was compared to induce the
immune response after intranasal immunization of mice. After 4
weeks post immunization mouse sera and nasal washings were obtained
and HA1 and IgA antibodies were measured. As presented on FIG. 3B,
VN1203 HA K58I virus induced 4 times higher titers of IgA
antibodies than virus VN1203 with original HA sequence.
[0091] FIG. 3B shows the IgA antibody titers in mouse nasal washes
after immunization with VN1203 and VN1203 K58I viruses.
[0092] FIG. 3C shows HA1 antibody titers in mouse sera after
immunization with VN1203 and VN1203 K58I viruses.
[0093] In order to prove that increased HA stability towards low pH
leads to better virus infectivity for mammals, two analogous
reassortants VN1203R and VN1203R HA K58I comprising competent NS
gene were constructed. Presence of competent NS gene was necessary
for the efficient viral growth in respiratory tract of
immuno-competent organisms. The viral growth of both viruses in the
upper respiratory tract after intranasal inoculation with each of
these viruses taken in different doses was compared. It was found
that virus having mutated HA VN1203R HA K58I was 100 times more
infectious for mice growing in the upper respiratory tract at
MID.sub.50 (mouse infectious dose--dose infecting 50% of mice)
value 2.5 log in comparison to 4.5 log MID.sub.50 of non-modified
VN1203R virus (FIG. 3E).
[0094] FIG. 3E shows the reproduction of viruses in the upper
respiratory tract of mice after intranasal infection with different
doses
Example 5
Thermostability
[0095] Propagation of viruses with acidic inoculation preserved
also virus stability to thermo inactivation. Thermo stability was
checked by titration of virus hemagglutination titer after
incubation of viruses at elevated temperatures for 15 min. It was
found that viruses which were cultivated with low pH inoculation
(Brisbane.DELTA.NS1 H1N1, Wisconsin.DELTA.NS1 H3N2,
Brisbane.DELTA.NS1 H3N2) retained hemagglutination activity even
after exposure to 60.degree. C. Viruses cultivated at standard
conditions and acquired destabilizing mutations in HA (New
Caledonia .DELTA.NS1_HA2.sub.--113,
Wisconsin.DELTA.NS1_HA.sub.--218.sub.--225.sub.--75.sub.--81 H3N2)
were not able to stand treatment at 60.degree. C. and completely
lost hemagglutination activity after 15 min (Table 1).
[0096] As regards to avian influenza viruses, it was found that
strains containing HA with the only modification of polybasic
cleavage site (in a trypsin dependent manner) did not stand even
treatment at 55.degree. C. (HongKong156.DELTA.NS1 H5N1, VN1203(6:2)
(H5N1), VN1203 H5N1) loosing completely the ability to agglutinate
erythrocytes in 15 min of treatment. The threshold for these
viruses was 50.degree. C. Introduction of mutation K58I in HA2
subunit of HA ectodomain increased virus stability until 55.degree.
C.
TABLE-US-00003 TABLE 3 stability Temperature of virus treatment
Viruses at pH 5.6 35.degree. C. 40.degree. C. 45.degree. C.
50.degree. C. 55.degree. C. 60.degree. C. 65.degree. C.
Brisbane.DELTA.NS1 H1N1 stable 64 64 64 64 64 64 0
Wisconsin.DELTA.NS1 H3N2 stable 16 16 16 16 8 4 0
Brisbane.DELTA.NS1 H3N2 stable 16 16 16 16 8 4 0 New Caledonia
.DELTA.NS1_HA2_113 n/stable 64 64 64 64 32 0 0
Wisconsin.DELTA.NS1_HA1_218_225_75_81 H3N2 n/stable 64 64 64 64 8 0
0 HongKong156.DELTA.NS1 H5N1 n/stable 32 32 32 16 0 0 0 VN1203
(6:2) (H5N1) n/stable 64 64 64 64 0 0 0 VN1203 H5N1 n/stable 64 64
64 32 0 0 0 VN1203/04 HA K58I (H5N1) stable 64 32 32 32 16 0 0
[0097] Human influenza viruses Brisbane.DELTA.NS1 H1N1,
Wisconsin.DELTA.NS1 H3N2, Brisbane0NS1 H3N2, New Caledonia
.DELTA.NS1_HA2.sub.--113,
Wisconsin.DELTA.NS1_HA1.sub.--218.sub.--225.sub.--75.sub.--.sub.81
H3N2 were obtained as 6:2 reassortants bearing surface antigens HA
and NA from the corresponding epidemic viruses and all other genes
from IVR-116 strain. IVR-116--strain recommended by WHO for
production of inactivated vaccine comprising surface glycoproteins
from A/New Caledonia/20/99 (H1N1) virus. NS gene of all viruses was
lacking NS1 open reading frame.
[0098] Avian viruses were obtained as 5:3 (VN1203 H5N1 and
VN1203/04 HA K581 H5N1) or 6:2 (VN1203(6:2) H5N1) reassortants
inheriting HA, NA and M (in case of 5:3) genes of avian viruses
A/Vietnam/1203/04 or A/Hong Kong/156/97 and all other genes from
IVR-116 strain. HA cleavage site of highly pathogenic avian strains
was substituted to that of low pathogenic avian viruses. All avian
viruses in this study contained NS gene lacking the NS1 open
reading frame.
Example 6
[0099] Two influenza 6:2 reassortants containing HA and NA genes
from epidemic virus A/Vienna/28/06 (H3N2) and all other genes from
WHO strain IVR-116 in combination with NS gene lacking the NS1 open
reading frame (.DELTA.NS1) were constructed by reverse genetics.
Obtained viruses differed by one amino acid substitution in the
sequence of HA2 subunit of HA molecule appeared due to different
passaging conditions on Vero cells. First virus, named Vienna/28
was cultivated in the presence of medium having pH 6.5, while
second virus (Vienna/28_HA2_G75R) was cultivated at standard
conditions. Virus Vienna/28.sub.--.sub.HA2_G75R was different in
the sequence of HA as compared to Vienna/28 at position G75R in the
HA2 subunit which is not present in the original wild type virus.
This substitution resulted in reduced infectivity of
Vienna/28_HA2_G75R virus at low pH in comparison to Vienna/28 virus
measured by preincubation of viruses in acidic buffers (for 30 min)
with subsequent titration of infectious titer (FIG. 4).
Example 7
[0100] Virus A/Brisbane/10/2007 (egg derived, obtained from NIBSC,
UK) was passaged five times on MDCK and Vero cells in parallel.
After 5 passages both resulting variants were compared with the
original virus for their infectivity at different values of pH in
immune fluorescent assay. The obtained data clearly demonstrate
that original virus A/Brisbane/10/2007 infected cells with the same
efficiency at pH 5.6 and at neutral pH 7.5. But after 5 passages on
corresponding cell lines both viruses lost the ability to infect
cells at pH 5.6. Positive staining of the cells was observed only
at pH 5.8 but no stained cells were visible when buffer with pH 5.6
was used (results are not shown). Sequencing of HA genes revealed
that both variants acquired the same mutation at position 160
D.fwdarw.E in HA molecule.
Example 8
[0101] Virus A/Solomon Island/3/06 (egg derived, obtained from
NIBSC, UK) was adapted to Vero cells by consecutive passages made
by two different ways: at standard (neutral) conditions or at
acidic (infection at pH 5.6). Resultant virus passaged at neutral
pH improved the growth capacity on Vero cells from 4.7 log
TCID50/ml to 6.7 log TCID50/ml, but lost the ability to infect
cells at pH 5.6 in immune fluorescent assay. No stained cells were
observed after infection of cells with the virus combined with
buffer pH 5.6, while at neutral infection the whole monolayer was
stained. Virus A/Solomon Island/3/06 adapted to growth on Vero
cells using infection at acidic conditions reached the titer 7.6
log TCID50/ml and in the same time preserved the infectivity at low
pH conditions. In immune fluorescent assay it was observed similar
distribution of stained cells after the infection at pH 5.6 and
7.5.
Example 9
[0102] Virus A/California/7/09 of new H1N1 subtype (egg derived,
obtained from CDC) was adapted to Vero cells by several consecutive
passages made at acidic conditions (infection of the cells at pH
5.6). Resultant virus was named A/California/7/09-acid. Original
and adapted viruses were used for the small scale (10 L) production
in bioreactor with subsequent purification. The yield of
A/California/7/09-acid virus measured by hemagglutination titer
(HA) was higher than that of A/California/7/09 virus after each
production step in 2-8 times. Results are presented in the table
4.
TABLE-US-00004 TABLE 4 Yield of A/California/7/09 and
A/California/7/09-acid viruses HA titer A/California/ Production
step A/California/7/09 7/09-acid 1. Roller bottle, passage 1 8 64
2. Roller bottle, passage 2 16 64 3. Bioreactor 10 L (before
harvest) 64 128 4. Bioreactor 10 L (after harvest) 8 64
[0103] Both viruses were harvested, purified according to standard
procedure used for the purification of inactivated vaccines and
compared. The obtained results revealed that virus
A/California/7/09-acid was better than A/California/7/09 in all
measured parameters (Table 5).
TABLE-US-00005 TABLE 5 Comparison of purified preparations of
A/California/7/09 A/ A/ California/ California/ Parameters 7/09
7/09-acid 1. HA.sub.50 .mu.l before/after clarification 64/8 128/64
2. Titer TCID.sub.50/ml before/after clarification 6.82/6.13
8.08/7.71 3. Yield (mg HA/L) 1.8 5.4 4. HA/protein ratio 0.58 0.66
5. Host cell protein impurities (Vero protein/HA) 0.09 0.04
Sequence CWU 1
1
2115DNAInfluenza A virus 1attgacaaaa tgaac 15215DNAInfluenza A
virus 2attgacatta tgaac 15
* * * * *