Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR REPLICATING INFLUENZA VIRUS IN CULTURE
This is a divisional application of Canadian application number 2,671,869
filed on Decesaiber 14, 2007.
BACKGROUND OF THE INVENTION
Influenza epidemics and pandemics have been recognized for several centuries
and
have resulted in considerable loss of life. Influenza virus is a segmented RNA-
containing
virus belonging to the family Orthomyxoviridae. The epidemics and pandemics
are caused
by the appearance of viruses with new envelope components for which there is
little
immunity in the population. These new components are often the result of
mutation and/or
mixing of human and animal influenza viruses.
Whereas the capsid of the influenza virus is somewhat pleomorphic, the outer
surface is consistent for all viruses and consists of a lipid envelope from
which projects
prominent glycoprotein spikes of two types: hemagglutinin (HA or H) and
neuraminidase
(NA or N). There are three types of influenza viruses: A, B, and C. Only
influenza A viruses
are further classified by subtype on the basis of the two main surface
glycoproteins HA and
NA. Influenza A subtypes are further classified by strains. Influenza B
viruses infect
mammals only and cause disease in humans, but generally not as severe as A
types. Influenza
C viruses also infect mammals only, but cause only a very mild respiratory
disorder in
children. They are genetically and morphologically distinct from A and B
types.
Influenza A viruses infect a wide variety of animals, including mammals e.g.,
humans, horses, dogs, swine, ferrets and avians, e.g. ducks, chickens and
turkeys. There are
16 known HA serotypes and 9 known NA serotypes. Birds are particularly
important
reservoirs, generating pools of genetically/antigenically diverse viruses
which get transferred
back to the human population via close contact between humans and animals.
Pigs are
permissive to both human and bird influenza strains. Because of this unusual
feature, pigs
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are considered as "Mixing Vessels" allowing for genetic exchange between avian
and human
viruses when the same cell is infected with both types of virus.
The genome of the influenza virus consists of single strand (-)sense RNA in 8
segments (7 in Influenza Q. The structure of the genome is known in great
detail because of
the tremendous amount of genetic investigation (conventional and molecular)
which has been
done. Each segment encodes one or two viral proteins. Epidemics and pandemics
are
believed to be due to genetic change in the HA and NA proteins of the
influenza virus in two
different ways: antigenic drift and antigenic shift. Antigenic drift occurs
constantly, whereas
antigenic shift happens only occasionally. Influenza type A viruses undergo
both kinds of
changes; influenza type B viruses change only by the more gradual process of
antigenic drift.
Antigenic drift refers to small, gradual changes that occur through point
mutations
in the two genes that contain the genetic material to produce the main surface
proteins, HA
and NA. Antigenic shift refers to an abrupt, major change to produce a novel
influenza A
virus subtype in humans that was not currently circulating among people.
Antigenic shift can
occur either through direct animal (poultry)-to-human transmission or through
mixing of
human influenza A and animal influenza A virus genes to create a new human
influenza A
subtype virus through a process called genetic reassortment. Antigenic shift
results in a new
human influenza A subtype. Genetic reassortment occurs when two different
influenza
viruses infect the same cell and share or trade one or more RNA segments. If
the segment
that is transferred is the HA, for example, this can result in the appearance
of a new viral
strain that is antigenically new entering a population with little or no
immunity. The result
can lead to an epidemic and/or a pandemic.
The entry of influenza viruses into cells is facilitated by binding of the HA
spikes to
mucoproteins containing terminal N-acetyl neuraminic acid (NANA = sialic acid)
groups.
After binding, the particle is engulfed by endocytosis via coated pits into
endocytotic vesicle
and finally endosomes. These are acidified by the cell and at about pH 5.0,
the HA
monomers are cleaved by trypsin-like enzymes in the endosome to activate them
for
internalization. Once internalized the viral replication occurs and results in
the symptoms of
influenza.
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There is considerable concern about recent outbreaks of influenza. A severe
type of
respiratory disease has been identified in dogs, which is due to Canine
Influenza Virus (CIV).
This respiratory disease has proven to be highly contagious. Moreover, CIV can
cause 100%
infection with 80% morbidity, and up to 5-8% mortality in severe infections.
Since its first
detection in 2004 in greyhound racing dogs (Crawford et at., Science
310(5747):482-485
(2005)) CIV has rapidly spread across the United States with at least 25
states reporting CIV
outbreak, and twenty-seven states reporting CIV seroprevalence.
The serotype of the CIV causing the recent outbreak is H3N8. This CIV serotype
was originally discovered in horses, and is believed to have crossed the
species barrier into
canines. It is probable that the absence of an effective vaccine against
canine influenza virus
plays a major role in the rapid and widespread dissemination in dogs of this
virus.
Influenza A (H5NI, avian influenza) virus- also called "H5N1 virus"- is an
influenza A virus subtype that occurs mainly in birds, is highly contagious
among birds, and
can be deadly to them. HSN 1 virus does not usually infect people, but
infections with these
viruses have occurred in humans. To date, over 200 confirmed human cases that
resulted in
over 150 deaths have been reported in 10 countries, mainly in Asia.
Fortunately, as of yet,
the virus does not readily spread from birds to humans or from one human to
another.
However, this could happen with the result that an epidemic or pandemic could
occur. The
best strategy for prevention of morbidity and mortality associated with an
epidemic or
pandemic is vaccination.
The influenza vaccines presently administered to humans have a high benefit-to-
cost ratio in terms of preventing hospitalizations and deaths, however, the
world's annual
production capacity for seasonal vaccine is limited and does not realistically
cover the global
high-risk population. The present vaccines are made in eggs using virus
obtained from the
World Health Organization (WHO) or the Centers for Disease Control (CDC), who
provide
the virus seeds for vaccine manufacture every year. Changes in the HA of
circulating viruses
(antigenic drift) require periodic replacement of the vaccine strains during
interpandemic
periods. The WHO publishes semiannual recommendations for the strains to be
included for
the Northern and Southern Hemispheres. To allow sufficient time for
manufacture, the WHO
determines in February which vaccine strains should be included in the
following winter's
vaccine. In general, 1 dose for adults contains the equivalent of 45 g HA (I 5
g each for 3
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viruses). This dose is approximately the amount of purified virus obtained
from the allantoic
fluid of one infected embryonated egg. If 100 million doses of killed
influenza virus vaccine
are prepared, the manufacturer has to procure 1 00 million embryonated eggs.
This makes
vaccine production dependent on the timely availability of good quality
embryonated eggs
and the seed strains provided by the WHO/CDC. Most of the prototype seed
strains are not
easily grown to high titer even in embryonated eggs. To overcome this problem,
government
agencies first create high-yielding laboratory strains through classical
reassortment with high-
yielding laboratory strain A/PR/8/34 (in a 6:2 reassortment obtaining 6
segments from the
A/PR/8/34 strain). Unfortunately, this process can be difficult to do and may
effect the
antigenicity of the resulting vaccine. Therefore, there is a need to provide
alternative
methods of manufacturing vaccines that protect against clinical diseases
caused by influenza,
particularly highly pathogenic strains such as H5N 1. Furthermore, there
remains a need to
provide methods of manufacturing large quantities of life-saving influenza
vaccines in a time
period quick enough to effectively prevent possible epidemics and/or
pandemics. The
present invention addresses these and other needs.
The citation of any reference herein should not be construed as an admission
that
such reference is available as "prior art" to the instant application.
BRIEF SUMMARY OF THE INVENTION
The present invention relates to vaccines for the prevention of influenza A
and B
infections. The vaccines and the related methods of the invention provide a
number of
advantages over prior art vaccines and methods. For example, the vaccines of
the invention
are produced using tissue culture cells instead of embryonated eggs. The
inventive
production methods save critical time by bypassing the classical vaccine
manufacture
procedure. In addition, the vaccines of the invention are useful for those
that are allergic to
egg material. The present invention also provides new immunogenic compositions
that may
be used in the vaccines. These new immunogenic compositions can be used to
immunize
animals, including avians, against influenza virus. In particular embodiments
of the
invention, the recipient of the vaccine is a mammal. In one aspect, the
present invention
provides a vaccine that protects canines against the canine respiratory
disease due to Canine
Influenza Virus (CIV). In another aspect, the present invention provides a
vaccine that
CA 02766173 2012-01-23
protects humans against influenza virus strains naturally produced through
genetic
reassortment.
The invention provides influenza virus isolates that have been specifically
adapted
to grow in tissue culture cells. In a particular embodiment, the adapted
influenza virus
isolates are selected for their ability to grow on a chosen tissue culture
cell line using limit
dilution cloning. In one particular embodiment, a subpopulation of influenza
virus adapted
for growth on a cultured cell line is selected by serially diluting into a
multiplicity of aliquots,
a quantity of influenza virus that comprises a multiplicity of influenza
subpopulations. The
multiplicity of aliquots are then contacted with and/or grown in the cultured
cell line. A
subpopulation of influenza virus within the multiplicity of influenza
subpopulations is
identified as one that grows on the cultured cells at a low multiplicity of
infection (MOI) and
selected as the subpopulation of influenza virus that is adapted for growth on
the cultured cell
line. In related embodiments, the present invention provides methods that
include contacting
the tissue-culture adapted isolate with tissue culture cells, growing for a
time sufficient to
produce cytopathic effects (CPE). This method can also include harvesting the
influenza
virus. In some such embodiments, the limit dilution cloning process involves
serially diluting
an influenza virus isolate and contacting each dilution with cultured cells,
growing the cells
for a time sufficient to produce cytopathic effects (CPE), harvesting virus
from the highest
dilution that causes CPE, and repeating the process with the harvested virus.
In some
embodiments, the method also includes admixing the influenza virus isolate
with an effective
amount of trypsin before contacting the cultured cells. In some embodiments,
the mixture is
incubated for a time sufficient to allow the trypsin to cleave viral proteins,
without detaching
the cells from the substrate. The trypsin used for cleaving viral proteins can
be type IX
trypsin. In some cases, the step of contacting the tissue-culture adapted
isolate with the tissue
culture cells is carried out at a multiplicity of infection (MOI) of less than
about 0.01,
including less than about 0.001 and/or less than about 0.0001. In other cases,
the influenza
virus isolate is first tested on the tissue culture cells to determine the
optimal MOI. The
tissue culture cells can be mammalian embryonic kidney cells such as human
embryonic
kidney cells. The influenza virus can be an influenza A, B, or C virus. In one
particular
embodiment, the influenza A virus is an H5N1 strain. The influenza virus
isolate can be
obtained from any number of sources including from a nasal swab, a lung
tissue, and/or can
be provided by a third party, e.g., the WHO. In some embodiments, the
influenza virus
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isolate is initially grown in embryonated eggs to obtain a large inoculum for
adaptation to
tissue culture. Some methods include purifying the harvested virus. In one
such method, the
step of purifying is carried out using size exclusion chromatography. The
methods of the
invention can also involve admixing a second isolate of influenza virus with
the first isolate
prior to, during or subsequent to purification, such that the second isolate
is a different strain
from the first isolate. In some methods dose titration studies are performed
prior to admixing
the two viral isolates to determine a mixture that allows equal immunogenicity
of the viral
proteins. In some methods the influenza is inactivated. In some embodiments of
this type,
the influenza virus is treated with an amount of binary ethyleneimine
effective to inactivate it.
In some methods, the harvesting is performed when the hemagglutinin protein
content is
maximal.
Further embodiments include a vaccine prepared by harvesting the viral isolate
prepared by the method of selecting an influenza virus for growth on tissue
culture cells by
titrating an influenza virus isolate using limit dilution cloning such that an
influenza virus
isolate that is adapted to the tissue culture cells is selected. In some
embodiments the virus is
prepared by inoculation of embryonated specific pathogen free chicken eggs by
chorioallantoic (also called the allantoic cavity) or amniotic membrane
inoculation routes
prior to limited dilution cloning. In one embodiment the influenza virus is
replicated first by
inoculation via the amniotic membrane of embryonated eggs to obtain an
inoculum for
adaptation to tissue culture cells.
In addition, the invention provides methods of selecting an influenza virus
for
growth on human embryonic kidney cells. In one such method an influenza virus
isolate is
titrated using limit dilution cloning such that an influenza virus isolate
that is adapted to the
HEK cells is selected. This method can include contacting the HEK-adapted
isolate with
HEK cells and growing the cells for a time sufficient to produce cytopathic
effects (CPE). In
a particular embodiment of this type, the resulting influenza virus is
harvested. The invention
also provides a vaccine that includes the influenza virus isolate obtained by
these methods.
The method can also include admixing the influenza virus isolate with an
effective amount of
type IX trypsin before contacting the cultured cells for a time sufficient to
allow the trypsin to
cleave viral proteins, without detaching the cells from the substrate. In one
embodiment, the
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step of contacting the HEK-adapted isolate with the HEK cells is carried out
at an MOI of
less than about 0.001. .
In some embodiments, the invention further provides vaccines comprising human
influenza virus formulated at less than 4 .tg of human influenza HA per dose.
In a related
embodiment the invention provides vaccines comprising human influenza virus
formulated at
less than 3 pg of human influenza HA per dose. In another embodiment the
invention
provides vaccines comprising human influenza virus formulated at less than 2
g. of human
influenza HA per dose. In still another embodiment, the invention provides
vaccines
comprising human influenza virus formulated at 1.5-3.5 g. of human influenza
HA per dose.
In particular vaccine embodiments the adjuvant is an ISCOM. In other vaccine
embodiments
at least 70% of the viruses comprise HA that have the same amino acid
sequence. In still
other vaccine embodiments at least 80% of the viruses comprise HA that have
the same
amino acid sequence. In yet other vaccine embodiments at least 90% of the
viruses comprise
HA that have the same amino acid sequence. In still other vaccine embodiments
greater than
95% fo the viruses comprise HA that have the same amino acid sequence.
The present invention further provides combination vaccines for eliciting
protective
immunity against influenza virus, e.g., canine influenza virus (CIV) and other
diseases, e.g.,
other canine infectious diseases. The present invention further provides for a
method of
immunizing a mammal, for example, a dog, cat, or horse against influenza.
Methods of
making and using the vaccines to the infectious diseases, e.g., canine
infectious diseases are
also provided.
In a particular embodiment an immunogenic composition of the present invention
comprises an immunogenic composition comprising an inactivated CIV H3N8 and an
adjuvant. Typically, the adjuvant comprises an oil in water emulsion. In one
such
embodiment, the adjuvant further comprises aluminum hydroxide. In a particular
embodiment of this type, the adjuvant is Emunade . In another embodiment the
immunogenic composition is a vaccine.
The vaccine composition may include from about 100 hemagglutination units
(HAU) to about 1500 HAU per dose. This can vary widely depending on the size
and other
health considerations of the individual receiving treatment. The composition
is typically
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between 250 and 750 HAU per dose. In one embodiment, the vaccine composition
includes
about 500 HAU per dose.
Optionally, the vaccines of the present invention can also include a
pharmaceutically acceptable immune stimulant, e.g., cytokines, growth factors,
chemokines,
supernatants from cell cultures of lymphocytes, monocytes, or cells from
lymphoid organs,
cell preparations and/or extracts from plants, bacteria or parasites, or
mitogens.
The vaccines of the present invention may be administered by a route such as:
parenteral administration, intramuscular injection, subcutaneous injection,
peritoneal
injection, intradermal injection, oral administration, intranasal
administration, scarification
and combinations thereof. In a preferred embodiment of the invention, the
vaccine is
administered by intramuscular injection.
The invention also provides serum obtained from a vaccinated animal that
contains
antibodies that bind to CIV H3N8 and the purified antibodies themselves. In a
particular
embodiment of the invention, the purified antibody that binds to CIV H3N8 is a
chimeric
antibody.
The present invention further provides combination vaccines that include one
or
more strains of inactivated CIV, e.g., CIV H3N8, in combination with one or
more other
canine pathogens and/or immunogens, including, e.g., immunogens for eliciting
immunity to
canine distemper virus; canine adenovirus; canine adenovirus type 2; canine
parvovirus;
canine parainfluenza virus; canine coronavirus; canine influenza virus; and/or
Leptospira
serovars, e.g., Leptospira kirschneri serovar grippotyphosa, Leptospira
interrogans serovar
canicola, Leptospira interrogans icterohaemorrhagiae, and/or Leptospira
interrogans serovar
pomona. Additional canine pathogens that can be added to a combination vaccine
of the
present invention include Bordetella bronchiseptica; Leishmania organisms such
as
Leishmania major and Leishmania infantum; Borrelia species (spp.) spirochetes,
including B.
burgdorferi sensu stricto (ss), B. burgdorferi ss, B. garinii, and B. afielii;
a Mycoplasma
species (e.g., Mycoplasma cynos); rabies virus; and Ehrlichia canis.
The present invention provides for methods of growing CIV H3N8 in cultured
cells.
In some embodiments, the cultured cells are non-canine mammalian kidney cells.
In one
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embodiment, the cells are Madin-Darby bovine kidney (MDBK) cells. In another
embodiment, the cells are Vero cells.
In some embodiments, the invention further provides vaccines comprising CIV
H3N8 formulated at least than 500 HAU per dose. In these embodiments, the
adjuvant is
usually aluminum hydroxide, and at least 70%, typically at least 90%, of the
HA has the same
amino acid sequence. In other vaccine embodiments at least 80% of the viruses
comprise HA
that have the same amino acid sequence. In still other vaccine embodiments
greater than
95% of the viruses comprise HA that have the same amino acid sequence.
These and other aspects of the present invention will be better appreciated by
reference to the following Figures and the Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the average of clinical scores following CIV challenge in dogs.
Non-vaccinated control and vaccinated dogs were challenged with CIV and
monitored daily
from day -2 through 10 days post-challenge for clinical signs such as ocular
and nasal
discharge, sneezing, coughing, depression and dyspnea. The clinical signs were
scored as per
the guidelines described in Example I and average clinical scores for each
treatment group
were plotted against days.
Figure 2 demonstrates post-challenge nasal CIV shedding in dogs. Non-
vaccinated
control and vaccinated dogs were challenged with CIV. Nasal swabs were
collected on the
day before challenge (day -1) to confirm that the dogs were CIV free. Nasal
virus shedding
was monitored in challenged dogs by collecting nasal swabs daily for 10 days
(day I through
post-challenge) and performing titration on MDCK monolayers. The average virus
titers
for each treatment group, expressed as Loglo TCIDSd mL, were calculated and
plotted
against days post-challenge.
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DETAILED DESCRIPTION OF THE INVENTION
Traditional methods of producing influenza vaccines involve growth of an
isolate
strain in embryonated hens' eggs at least partly because the use of eggs is
cheap and efficient
and because there is no readily apparent alternative choice for growth of
influenza in large
amounts. In the case of human influenza this is particularly true because many
cell lines that
can be used to propagate influenza virus have not been approved by the FDA for
human
vaccine manufacture and only very low titers have been obtained in tissue
culture.
When the vaccine is made in eggs, it is generally made as follows. Initially,
the
virus is recovered from a throat swab or similar source and isolated in eggs.
The initial
isolation in egg is difficult, but the virus adapts to its egg host and
subsequent propagation in
eggs takes place relatively easily. After growth in the egg, the virus is
purified and formalin
or beta-propiolactone inactivated. Growing evidence suggests that the egg is
not optimal for
virus propagation. For example, the conventional laying flocks used in egg-
based
manufacture are at high risk for contaminating the virus preparation with
endogenous viruses
routinely found in these settings. Also, the separate inoculation and harvest
of millions of
eggs as well as the complicated downstream processing results in vast
opportunities for
environmental contaminants to be introduced into the virus preparation and is
likely the
reason there was a recall of vaccine in 2004. As mentioned previously, it is
difficult to
completely remove egg material and this can result in sensitivity to the
vaccine. In addition,
the process completely lacks flexibility if demand suddenly increases because
of the logistical
problems due to the non-availability of large quantities of suitable eggs.
There is also
evidence that growth in eggs can reduce the antigenicity of the virus.
Consistently, growing
influenza A or B viruses in eggs results in a heterogenous viral product that
has a spectrum of
HA mutations. In direct contrast, the corresponding growth in mammalian host
cells results
in influenza viruses that are structurally identical to those originally
isolated (Rocha, et al. J.
Gen. Virol 1993;74:2513-2518). Moreover, influenza viruses grown in mammalian
cells
elicit neutralizing and HA inhibition antibodies in human sera more readily
and with a higher
titer than do their egg-grown counterparts (Oxford, et al. Bull WHO
1987;65:181-187).
Unfortunately, whereas all influenza viral strains seem to grow in eggs,
heretofore,
many do not grow well in tissue culture cells, and of those that do grow in
tissue culture cells
often do not grow in the quantities necessary to produce an effective vaccine.
The present
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inventors now disclose that when influenza virus is grown in tissue culture
using limiting
dilution cloning, isolates can be produced that are tissue culture-adapted.
Surprisingly, the
inventor's discovered that replication of a virus resulting from inoculation
through the
amniotic membrane of embryonated eggs resulted in a virus population that
could replicate
and produce high levels of HA when propagated on tissue culture cells (e.g.-
Vero cells). The
resulting viral isolate produces an HA titer that is almost equal to that
obtained using
embryonated eggs and HA titer is one important measure of vaccine potential.
Therefore,
one important aspect of the present invention is directed to methods of
producing viral
isolates of influenza virus that are tissue-culture adapted and suitable for
use in the
production of influenza vaccines, particularly mammalian vaccines. The methods
involve the
use of limit dilution cloning to isolate and identify tissue-culture adapted
viruses. The
resulting isolate can be treated to produce a vaccine. Thus, the present
invention is also
directed to methods of producing improved Influenza virus vaccines.
To this end, methods are provided for selecting tissue-culture adapted
influenza
virus by titrating the virus using limit dilution cloning and repeating the
process 2 or more
times. In some methods, the tissue culture cell used is a HEK cell. Trypsin or
an equivalent
protease can be used to increase the efficiency of viral entry into the cells.
Further methods
involve titrating the trypsin to identify the best concentration for the
trypsin lot used and for
the cells used. Identification of the best multiplicity of infection (MOI) for
each influenza
virus used and for the specific cells also contributes to successful tissue
culture propagation.
The isolated tissue-culture adapted virus can be used to produce a vaccine
according to
standard methods. In some embodiments, the vaccines include the use of the
adjuvant
ISCOM. In some embodiments, the vaccines include the use of the adjuvant
aluminium
hydroxide. When more than one viral strain or isolate is included in the
vaccine, methods can
involve mixing the two in an immunologically equal amount. Methods and
compositions are
provided herein for the tissue-culture adapted viral isolates and for vaccines
made therefrom.
1. Methods of Selecting Tissue Culture Adapted virus
The source of virus used in the methods of the invention is not critical to
the
invention. For example, the virus can be obtained by isolation from an
infected animal or
patient, as a seed virus stock from WHO, by purchase from an appropriate
agency (e.g.
ATCC), or from research laboratories. In particular, CIV is known to cause
severe
CA 02766173 2012-01-23
12
respiratory disease including pneumonia. Thus, dogs showing these symptoms are
useful
sources. Appropriate specimens for isolating virus include: nasal wash
/aspirate,
nasopharyngeal swab, throat swab, broncheoalveolar lavage, tracheal aspirate,
pleural fluid
tap, sputum, cloacal smears, and autopsy specimens. Specimens from living
animals
optimally should be collected early, and in some cases, within 4 days after
illness onset.
Specimens can be collected in an appropriate transport media to be stored
until use or used
immediately. If stored, the virus can be kept at a reduced temperature, such
as at 4 C to
ensure viability. To isolate the influenza virus from the specimen, large
contaminants can be
removed (for example, by centrifugation) and the supernatant inoculated onto a
variety of
cells at a variety of dilutions. Alternatively the virus from the animal can
initially be grown
in hens eggs. Methods can be used to confirm that the virus isolate is indeed
influenza at any
point in the process.
To confirm the presence of the desired influenza virus at any time in the
process of
preparing a tissue-culture adapted isolate, a variety of screening methods can
be used. The
virus can be screened by assaying using any known and/or suitable assay for
influenza virus.
Such assays include (alone or in combination), viral replication, quantitative
and/or
qualitative measurement of inactivation (e.g., by antisera), transcription,
replication,
translation, virion incorporation, virulence, HA or NA activity, viral yield,
and/or
morphogenesis, using such methods as reverse genetics, reassortment,
complementation,
and/or infection.
A. Tissue culture cells
Any mammalian host cells that allow the growth of influenza virus can be used
for
the methods herein. Typically, the host cells suitably exclude adventitious
agents and are of a
passage number that can be certified according to the WHO requirement for
vaccine
production. A number of cell lines can be used to isolate and propagate
influenza viruses.
Some cell lines that have been used include: Vero cells (monkey kidney cells),
MDCK cells
(Malin-Darby canine kidney cells), BHK-21 cells (baby hamster kidney) and BSC
(monkey
kidney cell) and HEK cells (human embryonic kidney cells). Thus, any tissue
culture cell
that allows the growth of influenza virus can be used. Suitable cells include,
but are not
limited to: Vero, MDBK, BK21, CV-1, and any mammalian embryonic kidney cell
(e.g.
CA 02766173 2012-01-23
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HEK). In some embodiments, Vero cells or mammalian embryonic kidney cells are
used. In
some embodiments, human embryonic kidney cells (HEK cells) are used.
The appropriate tissue culture medium for propagation of the aforementioned
cell
lines will be known to those of skill in the art. Such medium may contain an
appropriate
serum (e.g., fetal bovine serum) at a concentration up to 20% v/v. It will be
appreciated by
those of skill, that a medium containing less than 20% v/v serum (2-5% v/v)
can be used to
propagate the aforementioned cell lines.
B. Optimizing the trypsin for the cells
In order to grow a high titer stock of the virus in cell cultures, an
appropriate
amount of protease can be used to activate the hemagglutinin for
internalization into cells.
The protease containing solution can be added to the isolate directly or the
isolate can be
diluted into the protease for growth of an isolate for production of a
vaccine. The protease
can be used to dilute the virus to the appropriate MOI for growth.
The protease can be any protease that is capable of activating the HA for
internalization without damaging viral proteins such that they cannot grow
and/or infect cells.
Such proteases include, but are not limited to, prokaryotic protease, pronase,
trypsin, and
subtilisin (A), for example, Trypsin IX.
The amount of protease to be used should be enough to activate the virus with
very
little toxic effect to the cells. Toxic effect can be analyzed by identifying
characteristics of
damage to the cell such as detachment from the plate or substrate, presence of
cellular debris,
appearance of dead cells and lack of viable cells. Thus, an "effective amount
of trypsin" is
one which, when used for a time sufficient, allows the trypsin to cleave viral
proteins, without
detaching the cells from the substrate or causing other toxic effects.
Titrating the protease
can also be used to increase production of active virus in tissue culture.
Titration involves
identifying the maximum amount of trypsin that is minimally damaging to the
cells. This
amount can vary with the tissue culture cells used and with the protease lot.
Therefore, new
protease lots can be titrated to establish the optimal level prior to using
and each protease can
be titrated for each tissue culture cell. Titration involves inoculating the
tissue culture cells
with step-wise dilutions of the protease and incubating them for an
appropriate time. For
example, half-step dilutions (10-0'5) of protease may be used. Incubation time
will vary with
CA 02766173 2012-01-23
14
the cell line, but, typically will be between about 2 days and 7 days. The
protease level can
be determined using the typical incubation time for the cells being used. For
example, if a 4
day incubation is best for the influenza virus, the protease can be tested by
incubating for
about 4 days in the presence of protease alone. The lowest dilution of
protease that has no
toxicity or very little toxicity for the cells can then be used. The range of
trypsin
concentrations that can be used in mammalian tissue culture cells, for example
Vero cells, is
from about 0.5 g/mL to about I Op.g/mL, but more commonly about 2.5gg/mL of
medium.
Once the optimal level of protease is identified, the virus can be diluted in
the
infection medium with an appropriate amount of protease, (e.g., trypsin) such
that the optimal
level is reached when the virus is added to the cellular media. The optimal
amount of trypsin
can be used for the limit dilution cloning to produce a tissue-culture adapted
isolate as well as
growth and harvesting of the isolate.
C. Limiting dilution cloning
A typical virus culture is heterogeneous. Thus, for example the individual
viral
particles in the well of a microtiter plate can vary with respect to
infectivity, replication, and
the like. Serial dilution is used to select subpopulations of viruses in a
culture,
subpopulations that are best adapted to cells, for example. Serial dilution
involves diluting a
virus culture serially up to extinction, for example, to determine the best
MOI. Typically this
involves a series of 10-fold dilutions, but can vary depending upon the titer
of virus.
Typically limit dilution is used to identify the highest dilution which
produces viral
effect on the cell. The viral effect can be a cytopathic effect (CPE).
Cytopathic effects are
any effects on the cell caused by influenza virus infection. These include,
but are not limited
to: cell rounding, degeneration, sloughing, apoptosis, induction of reactive
oxygen species
(ROS), cells becoming granular and then fragmented, and detachment of cells
from a support
(such as a tissue culture dish). The well of the highest dilution is
harvested. This harvested
virus is then diluted to extinction and the process is repeated. Typically,
serial 10-fold
dilutions are made in an appropriate medium (with or without trypsin), and 0.2
mL of each
dilution is added to a plate or wells of a microtiter plate containing the
tissue culture cells and
incubated for a time sufficient to identify CPE of the cells. Because serum
inactivates
trypsin, the medium typically does not contain serum. The well or plate
containing the
CA 02766173 2012-01-23
highest dilution that causes CPE is harvested, then diluted and the process is
repeated. The
process is typically repeated at least two times, but can be repeated up to 5
times. In some
cases, the process is repeated 3 times.
Virus cultures produced by the methods of the invention are characterized by
homogeneity of the sequence of the HA proteins in the viruses. A number of
methods can be
used to measure the degree of sequence homogeneity. For example, sequence the
HA
proteins themselves or by sequencing the RNA encoding such proteins.
Typically, the
viruses preparations produced by the methods of the invention will contain
viruses in which
at least 70% of the HA proteins have the same amino acid sequence. In some
embodiments
at 80%, or at least 90% of the HA proteins have the same amino acid sequence.
D. Methods of testing Influenza virus
Tests can be performed to confirm activity and the presence of influenza virus
at
any time in the process for the methods herein. For example, hemagglutination
can be
identified as follows. If the virus has a surface HA protein it can attach to
RBCs and
agglutinate them. If the concentration of virus in a sample is high, when the
sample is mixed
with RBCs, a lattice of viruses and RBCs will be formed. This phenomenon is
called
hemagglutination. It is a simple way to detect the presence and titer of
viruses that
hemagglutinate such as influenza viruses. If there is not enough virus in the
sample to
hemagglutinate the RBCs, they form a pellet at the bottom of the well. The
highest dilution
showing complete hemagglutination is taken as the end point. The viral titer
is expressed in
HA units (HAU), which are the inverse of the dilution per milliliter. For
example if there is
complete hemagglutination in the well, at a 1/32 dilution in 50 Ls, but not
in the well with
the next highest dilution, the titer of the virus is 32 HAU per 50 Ls or 640
HAU per mL..
Other assays that can be used to identify and quantify influenza virus include
identification of CPE (as discussed herein), Western blot, ELISA, PCR, and
other methods
for identification of influenza virus using antibodies and/or probes that are
specific for some
part of the virus, particularly the HA antigen.
II. Methods of growing and harvesting
After production of a virus isolate, the isolate can be harvested. Standard
methods
can be used. The harvested isolate can be stored for future use or used to
produce a vaccine
CA 02766173 2012-01-23
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using standard methods. The virus can be harvested when a maximum amount of
virus is
produced; when a maximal amount of hemagglutinin is produced, as measured by
HA assay;
and/or when the cells are lysed.
After obtaining a tissue-culture adapted isolate, the isolate can be grown and
harvested. The method of growing and harvesting the tissue-culture adapted
virus is not
critical to the invention and standard methods can be used. However, in some
embodiments,
the virus is grown in the tissue culture cell it is best adapted to. The
harvested viral isolate
can be stored for future use or used to produce a vaccine using standard
methods. The virus
can be grown on the appropriate tissue culture cells by adding the virus at an
appropriate
MOI in the appropriate amount of protease (e.g., trypsin) to cells for a time
sufficient to
produce a high titer of virus and/or until the cells are lysed. The virus can
be harvested when
a maximum amount of virus is produced, when a maximal amount of hemagglutin is
produced and/or when the cells are lysed. It has been found herein that an
optimal pre-
growth of the virus in eggs (in the allantoic cavity or inoculation through
the amniotic
membrane) can increase the adaptation of virus in the tissue culture cells.
A. Pre-growth in eggs
Passage in egg cultures has been shown to facilitate the adaptation of virus
in the
tissue culture cell. Thus, it may be desirable to pre-grow the viral isolate
in embryonated
hens' eggs according to standard technologies. For example, the virus is
injected into the
allantoic cavity or via the amniotic membrane of 9-12 day old embryonated eggs
and allowed
to multiply for about three days. Then the allantoic or amniotic fluid is
collected and the
collected material can be grown in tissue culture at the appropriate MOI for
use in production
of a vaccine. Alternatively, the collected material can be directly used in
limit dilution
cloning. It has been found that inoculation of the eggs through the amniotic
membrane
enriches for virus that can replicate and can produce high levels of HA
protein when grown in
Vero cells.
B. MOI
It was identified herein that a low MOI results in a better and/or higher
titer viral
isolate. Without limitation to a specific theory, it is believed that a low
MOI reduces the
amount of defective virus particles and results in a more efficient infection
process. In some
CA 02766173 2012-01-23
17
embodiments, the MOI used is less than about 0.01 (one virus per 100 cells).
In other
embodiments, the MOI is less than about 0.003. In some embodiments, the MOI is
less than
about 0.001. The MOI can be chosen as being the lowest MOI that results in a
high titer of
virus and/or that lyses the cells in about 3 to 4 days.
III. Vaccine Production
Once a desired isolate is obtained from the tissue-culture adapted virus, the
virus
can be used to produce a vaccine. Many types of viral vaccines are known,
including but not
limited to attenuated, inactivated, subunit, and split vaccines.
A. Methods of producing attenuated virus
Attenuated vaccines are live viral vaccines that have been attenuated or
changed to
no longer cause disease. These can be produced in many ways, for example,
growth in tissue
culture for repeated generations and genetic manipulation to mutate or remove
genes
involved in pathogenicity. The tissue-culture adapted isolate can be used to
produce an
attenuated virus using standard methods. For example, once viral genes and/or
proteins are
identified that are involved in pathogenicity or involved in the disease
manifestation, these
can be mutated or changed such that the virus is still able to infect and
replicate within a cell,
but it cannot cause disease. An example of this is to mutagenize the HA1/HA2
cleavage site.
The tissue-culture adapted virus can be attenuated using any standard methods,
for example
cold adapting the virus.
After production of the attenuated virus, the vaccine can be prepared using
standard
methods (for example, the methods herein). The virus can be purified using
standard
methods, for example using size exclusion chromatography. A vaccine can then
be prepared
using standard adjuvants and vaccine preparations, for example, ISCOM, nano-
beads,
mineral oil, vegetable oil, aluminum hydroxide, saponin, non-ionic detergents,
squalene, and
block co-polymers can be used alone or in combination as adjuvents. Current
commercial
vaccines in the United States and Europe do not contain any adjuvant (both
live and killed
vaccines) and this is partly why such a high concentration of HA (15 g of HA
per virus
strain, 45 g of HA for trivalent formulation) is needed in the vaccine.
CA 02766173 2012-01-23
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B. Methods of producing inactivated, subunit, and split virus vaccines
Once a desired virus is obtained, it can be used to produce an immunogenic
composition, for example, a vaccine. Examples of "killed" vaccines are
inactivated, split and
subunit vaccines. These can be prepared to treat influenza using standard
methods.
For example, subunit vaccines generally involve isolating only the part of the
virus
that activates the immune system. In the case of Influenza, subunit vaccines
have been
prepared using purified HA and NA, but any mixture of viral proteins can be
used to produce
a subunit vaccine. Generally, the viral protein, such as HA is extracted from
recombinant
virus forms and the subunit vaccine is formulated to contain a mixture of
these viral proteins
from strains recommended by WHO. For example, the 1995-1996 vaccine contained
the HA
and NA from two A strains and one B strain (A/Singapore/6/86 (H IN 1);
A/Johannesburg/33/94 (H3N2); and BBeijing/84/93). For H3N8 CIV, the H3 and/or
N8
antigens can be used.
Generally, the viral protein(s) are extracted from the virus and the subunit
vaccine is-
formulated to contain a mixture of these viral proteins. Proteins can be
isolated from a tissue
culture adapted viral isolate for a subunit vaccine using standard methods.
Alternatively, the
proteins may be produced using recombinant techniques. Techniques for
producing a
particular protein are known in the art.
Split vaccines generally involve treating enveloped viruses with detergent to
solubilize the proteins therein. In the case of influenza virus, HA and NA
become
solubilized. For example, nonionic detergents such as Triton X-100 can be used
for
producing split vaccines.
Inactivated viral vaccines are prepared by inactivating the harvested virus
and
formulating it using known methods for use as a vaccine to induce an immune
response in a
mammal. The inactivation step, purification of subunits, and/or splitting can
be performed
before or after purification of the virus by size exclusion. For example,
production of an
inactivated vaccine, may involve removal of cellular material, inactivation of
virus,
purification and solubilization of the viral envelope., Other embodiments may
involve
purification of virus and then inactivation, for example using formaldehyde.
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CA 02766173 2012-01-23
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Once prepared, any of the vaccines (for example, attenuated, split, subunit or
inactivated) can be tested to identify that the virus and/or vaccine has
maintained similar
antigenicity, produces a serological response in a mammal, and/or provides
protection from
disease in a mammal.
C. Further processing of harvested virus
1. Clarification of harvested virus
After harvesting and/or after inactivation of the harvested virus, the
cellular material
and other interfering materials can be removed, for example, by sedimenting to
remove the
microcarriers and concentrating the supernatant by ultrafiltration. Influenza
grown in tissue
culture cells will contain host cellular proteins. Some further clarification
of the supernatant
may be needed. Cellular DNA can be removed by enzymatic treatment (e.g.
Benzonase).
After the initial removal of interfering material, virus can be inactivated
using standard
methods. Alternatively, inactivation can be performed after further
purification by, for
example, size exclusion chromatography.
2. Inactivation of virus
The influenza virus may be inactivated in any number of ways and by any number
of agents. The method of inactivation is not critical to the invention.
Inactivation can occur
after contaminating or interfering material is removed. Inactivation can
include the use of
known inactivating agents. Such inactivating agents include, but are not
limited to: UV
irradiation, formaldehyde, glutaraldehyde, binary ethyleneimine (BEI), and
beta-
propiolactone. In some embodiments BEI is used because it is known to destroy
the viral
nucleic acid without damaging the viral proteins. In addition, BEI is not
affected by protein
content and temperature. Inactivating agents are used at a concentration high
enough to
inactivate every viral particle in the solution. For example, BEI can be used
at a final
concentration of between about 0.5 and 10mM, including but not limited to:
1.5, 3, 4, 5, and 6
mM and including ranges of about I to 6, and I to 3 mM. In one embodiment, the
BEI is
used at a concentration of about 6mM. Typically, the BEI is used at a
concentration of about
1.5 mM and incubated at 37 C for 48 hours. In the preparation of vaccines to
CIV, BEI can
be used at a final concentration of between about 0.5 and 10mM usually between
4 to 8, and
often between 5 to 7mM. In one embodiment, the BE[ is used at a concentration
of about
CA 02766173 2012-01-23
6mM. In some embodiments inactivation occurs at the appropriate pH and
temperature for
the inactivating agent. The pH and temperature can be chosen to ensure the
resulting
inactivated virus is still immunogenic. Inactivation can proceed with an
appropriate amount
of agitation to ensure that the agent contacts all virus particles in the
solution.
After inactivation, the inactivating agents can be removed using methods
including,
but not limited to, inactivation of the inactivating agent, precipitation of
the inactivating
agent, filtration of the inactivating agent, and chromatography, or a mixture
of these methods.
For example, BEI can be inactivated by the addition of sodium thiosulfate.
Residual BEI can
also be separated from virus/viral proteins using size exclusion methods. Once
innocuity
(absence of live virus) is confirmed, the viral solution can be further
processed to produce a
vaccine.
3. Further processing
The viral solution can be further processed, for example, to remove
contaminants,
further concentrate the virus and provide for a stronger immune response. Some
examples of
further processing include initial removal of cellular material, removal of
cellular DNA,
concentration, and formulation in adjuvant using standard methods. Influenza
grown in
tissue culture cells will contain host cellular proteins. For example,
influenza propagated on
human embryonic kidney (HEK) cells will possess HEK proteins or bovine or
simian
proteins if grown in MDBK or VERO cells respectively. These proteins can be
detected by
methods known to those of skill in the art. Many methods are known for removal
of DNA,
including addition of a variety of DNase enzymes known to degrade cellular
DNA, for
example, Benzonase. An initial concentration step can be performed to provide
the viral
solution at a concentration best suited for additional purification by
chromatography. This
can be done using any standard methods, including but not limited to ultra
filtration using a
membrane having a molecular weight cut off of about l 00K (e.g. polysulfone
membrane with
a MWCO of I OOK). The viral solution can be concentrated up to about 100 fold,
including
but not limited to 90 fold, 80 fold, 70 fold, 60 fold, 50 fold, 40 fold, 30
fold, 20 fold, 10 fold,
and 5 fold. In some embodiments, the viral solution is concentrated up to
about 50 fold, but
more typically including 20 fold and 30 fold.
CA 02766173 2012-01-23
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4. Purification
The virus can be purified using standard methods such as density
centrifugation. In
some embodiments, the virus is purified by size exclusion gel chromatography.
One
advantage of using size exclusion is that the yields are better than when
using density
centrifugation. Any size exclusion gel can be used that results in
purification of virus. Any
standard gel can be used, such as Sepharose gel (e.g. Sepharose CL-2B). In
some
embodiments, the column is about 70 to 120 cm in length to achieve the
required separation,
including but not limited to about 80, 90, 100, and 110. In other embodiments,
the column is
from about 80 to 100 cm in length, for example the column is about 90 cm in
length. In some
embodiments, the length is achieved by multiple columns in series, for example
two 45 cm
columns or three 30 cm columns (e.g., a column 30-32 cm in length by 30 cm in
diameter).
The concentrated virus can be applied using standard methods, for example, the
virus is
applied at 5-10% of the column volume (CV), typically 5-7% CV. The viral peak
from the
column can then be collected and further concentrated using standard methods,
for example
ultra filtration. In some embodiments, 2 to 3 columns in series having a total
of 90 cm in
length are used and the final peak is pooled and concentrated by
ultrafiltration.
5. Solubilization of the envelop proteins
The concentration virus peak material can be solubilized using standard
methods,
such as, with a non-ionic detergent. The solubilization can be performed to
prepare the
material for formulation of ISCOMSf(see below). Examples of non-ionic
detergents,
includes but are not limited to, Nonanoyl-N-Methylfucamide (Mega 9), Triton X-
100,
Octylglucoside, Digitoniri C12E8, Lubrol, Nonidet'`P-40, and Tween (for
example Tween 20,
80 or 120). After solubilization, the virus can be used to produce a vaccine,
and/or an
adjuvant can be added. For example, for production of an ISCOM adjuvant, a
lipid mixture
can be added to assist ISCOM formation. The lipid mixture can include a
phosphatidyl
choline and synthetic cholesterol. In some embodiments, the virus is disrupted
with Mega 9
at room temperature with stirring and then the lipid mixture (phosphatidyl
choline and
cholesterol) can be added and stirring continued.
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CA 02766173 2012-01-23
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6. Formation of adjuvants
Appropriate adjuvants can be added to the vaccine and/or pharmaceutical
composition. Examples of adjuvants include those containing an oil and water
emulsion, as
well as those that further comprise aluminum hydroxide. In the latter case,
aluminum
hydroxide from a commercial source can be used, for example, Alhydrogel;
(Superfos
Biosector, Frederikssund, Denmark) and Rehydrogel (Reheis Inc.). The oil and
water
emulsion typically comprises mineral oil or metabolizable oils (e.g.,
vegetable oil, fish oil).
Non-ionic detergents or surfactants may be used as emulsifiers. Examples of
emulsifiers
include Tween 80/ Span 80, Arlecel 80/ Tween 80, and Montanides (Seppic,
Paris, France).
In the case of adjuvant emulsions, generally 5-25% of the volume is oil and 75-
95% of the
volume is aqueous. In some embodiments, the adjuvant emulsion is 20% oil and
80%
aqueous by volume. The amount of aluminum hydroxide is usually between about
5% and
15% of the aqueous phase. In some embodiments, Emunade is the adjuvant.
For some embodiments, ISCOM is used as an adjuvant. ISCOM is an acronym for
Immune Stimulating Complex and the technology was described by Morein et al.
(Nature
308:457-460 (1984)). ISCOM's are a novel vaccine delivery system and are
unlike the
conventional adjuvant techniques. An ISCOM can conveniently formed in one of
two ways.
In some embodiments, the antigen is physically incorporated in the structure
during its
formulation. In other embodiments, an ISCOM-matrix (as supplied by, for
example,
Isconova) does not contain antigen but is mixed with the antigen of choice by
the end-user
prior to immunization. After mixing, the antigens are present in solution with
the ISCOM-
matrix but are not physically incorporated into the structure.
Generally, in an ISCOM, purified antigens are presented in a multimeric form
based
on the ability of Quil A to spontaneously form micelles at a critical
concentration and by a
hydrophobic/hydophilic link, entrap the purified immunogens. These micellar
structures are
in the order of 35 nm in size and are easily recognized by the immune system.
Unlike
conventional depot adjuvants, ISCOMS are rapidly cleared from the injection
site and illicit
local, humoral and cell-mediated immune responses. In particular embodiments,
ISCOMs
are formed as follows. The virus is solubilized using standard methods, such
as with a non-
ionic detergent (e.g., Mega-9, TritoiTX-100, Octylglucoside, Digitonin Nonidet
P-40, C12Eg,
Lubrol, Tween 80). A lipid mixture is added to assist ISCOM formation. The
lipid mixture
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CA 02766173 2012-01-23
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can include a phosphatidyl choline and a synthetic cholesterol. In some
embodiments, the
mixture is first treated with non-ionic detergent at room temperature with
stirring, then the
lipid mixture (equal parts phosphatidyl choline and cholesterol, for example)
is added and
stirring continued. Quil*A (a purified glycoside of saponin) is added to the
virus lipid
mixture and stirring is continued. The Quil A can be added to give a final
concentration of
Quil A of about 0.01 to 0.1 %, including but not limited to 0.02, 0.03, 0.04,
0.05, 0.06, 0.07,
0.08, and 0.09. In some embodiments, the final concentration is about 0.05%.
The non-ionic
detergent is removed (for example, by diafiltration with ammonium acetate).
The matrix of
the ISCOM is formed by Quil A. The morphology of an ISCOM particle, as viewed
by
electron microscopy, shows a typical cage like structure of approximately 35
nm in size. The
ISCOM formation stage can be refined by the use of tangential flow
diafiltration. ISCOMs
present purified antigens in a.multimeric form based on the ability of Quil A
to spontaneously
form micelles at a critical concentration and by a hydrophobic/hydophilic link
that entrap the
purified antigens. Formation of ISCOMs can be verified by electron microscopy
to verify
that the typical cage-like structures have been formed. The immune response
from an
ISCOM presentation was shown to be at least ten times better than from a
similar antigen
payload presented as micelles of aggregated membrane protein alone. ISCOMs
were also
found to elicit a cell mediated response, not seen with conventional whole
virus vaccines. In
some embodiments, the final concentration is about 0.05%.
Immune stimulants may also be added to the vaccine and/ or pharmaceutical
composition. Immune stimulants include: cytokines, growth factors, chemokines,
supernatants from cell cultures of lymphocytes, monocytes, or cells from
lymphoid organs,
cell preparations and/or extracts from plants, cell preparation and/or
extracts from bacteria,
parasites, or mitogens, and novel nucleic acids derived from other viruses
and/or other
sources (e.g., double stranded RNA, CpG), block co-polymers, nano-beads, or
other
compounds known in the art, used alone or in combination.
Particular examples of adjuvants and other immune stimulants include, but are
not
limited to: lysolecithin; glycosides (e.g., saponin and saponin derivatives
such as Quil A or
GPI-0100); cationic surfactants (e.g. DDA); quaternary hydrocarbon ammonium
halogenides;
pluronic polyols; polyanions and polyatomic ions; polyacrylic acids, non-ionic
block
polymers (e.g., Pluronic F-127); and MDP (e.g., N-acetyl-muramyl-L-threonyl-D-
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CA 02766173 2012-01-23
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isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine, N-
acetylmuramyl-
L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'-dipalmitoyl-sn -glycero-3-
hydroxyphosphoryloxy)-ethylamine).
D. Efficacy of vaccines
Methods are well-known in the art for determining whether a subunit,
attenuated,
split, and/or inactivated virus vaccine has maintained similar antigenicity to
that of the
clinical isolate or tissue culture adapted isolate derived there from. Such
known methods
include the use of antisera or antibodies, HA and NA activity and inhibition
and DNA
screening (such as probe hybridization or PCR) to confirm that donor genes
encoding the
antigenic determinants are present in the inactivated virus. Methods of
identifying whether
the vaccine induces a serological response are also well known in the art and
include
immunization of a test animal with the vaccine, followed by inoculation with a
disease-
causing virus and identification of the presence or absence of symptoms of the
disease. Thus,
efficacy of the influenza vaccine can be tested in animals, usually ferrets,
mice, and guinea
pigs are used. Antibody titer can be tested using Hemagglutinatin inhibition
(HI) or
Neuraminidase inhibition (NI) method, or testing for virus neutralizing
antibodies (micro
neutralization test) in tissue culture, and such methods that are generally
known. Challenge
studies can provide important information to evaluate the vaccine.
Pharmaceutical compositions and/or vaccines suitable for treatment include
virus or
viral subunit in admixture with sterile aqueous or non-aqueous solutions. The
process of
producing a pharmaceutical composition and/or vaccine can involve isolating a
tissue culture
adapted isolate, growing and purifying the viral isolate, inactivating and or
attenuating the
virus and mixing an appropriate titer with a physiologically acceptable
diluent and an
immune stimulating agent. Alternatively, viral proteins can be purified for a
subunit vaccine
and an appropriate amount mixed with a physiologically acceptable diluent and
an immune
stimulating agent. The virus can be purified enough that there will be no
contaminating
material or substance that could interfere with the inactivation step and/or
the
immunogenicity of the virus.
An appropriate titer of virus or concentration of viral proteins can be
admixed with
the diluent and the immune stimulating agent. Measurement of TCID5o is one way
to
CA 02766173 2012-01-23
measure virus titer (50% tissue culture infective dose). For example a titer
of from about 105
to 1012 TCID50 (based on pre-inactivation titers) can be used, including but
not limited to 106,
107, 108, 109, 1010 and 1011. Optionally the titer can be analyzed by the HA
titer and can
contain from about I to 30 g of HA per virus included in the vaccine,
including but not
limited to I to 10 g for adjuvanted formulations and I to 30 g for non-
adjuvanted vaccines.
In some embodiments, the titer is about 15 g. Thus, for example, when 3
viruses are
included in an unadjuvanted vaccine, I dose for adults contains the equivalent
of 45 g HA
(15 g for each of the 3 virus strains). In other embodiments, the amount from
each strain
may differ (for example depending on the antigencity), but the final
concentration is from
about Ito about 60 g HA, including 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45,
50 and 55 pg. In
another embodiment, adjuvanted vaccines are expected to contain quantities of
HA from
about I to 30 g, including 2, 5, 10, and 20 g. The vaccine is typically of a
volume between
about 50 l and 5000 1., including 100, 500, 1000, 2000, and 5000 1s.
Standard physiologically acceptable diluents can be used, including, for
example,
EMEM, Hank's Balanced Salt Solution, and Phosphate Buffered Saline (PBS) and
Normal
Saline.
Appropriate immune-stimulating adjuvants can be added to the vaccine and/or
pharmaceutical composition. Examples of immune-stimulating adjuvants, include,
but are
not limited to: mineral oil, vegetable oil, aluminum hydroxide, saponin, non-
ionic detergents,
squalene, block co-polymers, nano-beads, ISCOM, ISCOM matrix or other
compounds
known in the art, used alone or in combination.
In addition to adjuvant, any appropriate antiviral agents that are useful
against
influenza can also be included in a pharmaceutical composition. Such antiviral
agents
include, for example, rimantadine, amantadine, neuraminidase inhibitors (such
as zanamivir
and oseltamivir), gamma interferon, guanidine, hydroxybenzimidazole,
interferon alpha,
interferon beta, thiosemicarbarzones, methisazone, rifampin, ribavirin,
pyrimidine or purine
analogs, and foscarnet.
Vaccines having more than one virus or strain of viral proteins can be
produced
using the methods herein. The mixtures can be immunogenically titrated to
provide
approximately equivalent imrnunogenicity. Immunogenically titrated means that
the final
CA 02766173 2012-01-23
26
product is produced to even out differences in immunogenicity. For example, if
a mixture of
strain A and strain B is prepared and strain A is 5 times more immunogenic,
the strains are
mixed at a ratio of strain A:strain B of 1:5.
IV. Administration of vaccine
The administration of the vaccine composition and/or the pharmaceutical
composition can be for prophylactic purposes. When provided prophylactically,
the
compositions are provided before any symptoms of influenza viral infection is
evident. The
prophylactic administration of the composition serves to prevent or attenuate
any subsequent
infection. The pharmaceutical composition and/or vaccine can be administered
in any way
known in the art, including via inhalation, intranasally (for example with
attenuated
vaccines), orally, and parenterally. Examples of parental routes of
administration include
intradermal, intramuscular, intravenous, intraperitoneal and subcutaneous. In
some
embodiments, the vaccine is administered by intramuscular or deep subcutaneous
injection in
the upper arm. A second dose can be given after an interval of 2 to 4 weeks to
some children
who were not previously vaccinated for or exposed to influenza (unprimed). One
or more
booster vaccinations may be administered at an appropriate time after the
initial
immunization.
An effective amount of the vaccine and/or pharmaceutical composition is
administered. An effective amount is an amount sufficient to achieve a desired
biological
effect such as to induce enough humoral or cellular immunity. This may be
dependent upon
the type of vaccine, the age, sex, health, and weight of the recipient.
Examples of desired
biological effects include, but are not limited to, production of no symptoms,
reduction in
symptoms, reduction in virus titer in tissues or nasal secretions, complete
protection against
infection by influenza virus, and partial protection against infection by
influenza virus.
In some embodiments, an immunologically effective amount of a CIV vaccine is
from about 100 HAU to about 1500 HAU per dose. The composition typically is
between
250 and 750 HAU per dose. In one embodiment, the vaccine composition includes
about 500
HAU per dose.
When administered as a solution, the vaccine can be prepared in the form of an
aqueous solution, a syrup, an elixir, or a tincture. Such formulations are
known in the art, and
CA 02766173 2012-01-23
27
are prepared by dissolution of the antigen and other appropriate additives in
the appropriate
solvent systems. Such solvents include water, saline, ethanol, ethylene
glycol, glycerol, and
Al fluid, for example. Suitable additives include certified dyes, flavors,
sweeteners, and
antimicrobial preservatives, such as thimerosal (sodium
ethylmercuithiosalicylate). Such
solutions may be stabilized using standard methods, for example, by addition
of partially
hydrolyzed gelatin, sorbitol, or cell culture medium and may be buffered using
standard
methods, using, for example reagents such as sodium hydrogen phosphate, sodium
dihydrogen, phosphate, potassium hydrogen phosphate and/or potassium
dihydrogen
phosphate. Liquid formulations may also include suspensions and emulsions. The
preparation of suspensions, for example using a colloid mill, and emulsions
for example
using a homogenizer.
V. Viral strains and WHO
In order to give time for adequate vaccine stocks to be produced, a decision
must be
made well before flu season, as to which influenza A and B strains to use for
this years
vaccine (for the winter season). There is an elaborate and sophisticated
epidemiological
monitoring systems worldwide, which helps these decisions. In addition, the
WHO usually
prepares seed virus for use in producing vaccines.
There are 16 known HA subtypes and 9 known NA subtypes. Many different
combinations of HA and NA proteins are possible. Only some influenza A
subtypes (i.e.,
H I N 1, H I N2, and H3N2) are currently in general circulation among people.
Other subtypes
are found most commonly in other animal species. For example, H7N7 and H3N8
viruses
cause illness in horses, and H3N8 also has recently been shown to cause
illness in dogs.
Three prominent subtypes of the avian influenza A viruses that are known to
infect
both birds and people are Influenza A H5- H5 infections, such as HPAI H5NI
viruses,
Influenza A H7, and Influenza A H9. However, the next strains that infect
humans and cause
epidemics or pandemics could come from any subtypes.
Virus can be obtained using standard methods, for example, from patient
samples,
the American Type Culture Collection (or other collection) or from specific
laboratories
working on the virus. In some embodiments, the virus is obtained from the WHO
or CDC,
including seasonal viruses and possible pandemic strains.
CA 02766173 2012-01-23
28
VI. Detection of serological response
Methods of identifying whether the vaccine induces a serological response are
also
well known in the art. For example, one can inject a test animal with the
immunogenic
compound/vaccine and identify antiviral antibodies in the blood serum. Methods
of
identifying whether the vaccine is protective are well known in the art and
include
immunization of a test animal with the vaccine, followed by inoculation with a
disease-
causing virus and identification of the presence or absence of symptoms of the
disease.
The Hemagglutination inhibition test can be performed to identify the presence
of a
serological response to hemagglutinin. The hemagglutination inhibition (HAI)
assay may be
performed with turkey red blood cells (RBCs) on all test serum samples, for
example by
using a known influenza subtype such as CIV(H3N8). Briefly, a serial two-fold
dilution of
test serum is performed in PBS in V-bottomed 96-well microtiter plates. An
equal volume of
virus suspension containing 4-8 HAU/50 t of CIV is added to each well
containing test
serum, and the plates incubated at room temperature for 30 minutes. Then, an
equal volume
of 0.5 % turkey RBC suspension was added. The plates are then incubated at
room
temperature for 30 min and HAI results are read. The reciprocal of the highest
dilution of the
serum showing HA inhibition is considered as the HAI titer of the test sample.
Other methods of determining the presence of antibodies to influenza virus
include
the Neuraminidase inhibition test, Western blot, ELISA, PCR, and other methods
for the
identification of influenza virus antibodies. These assays are known in the
art.
VII. Examples
The following examples provide procedures for isolation, adaptation and
purification of influenza virus to create a homologous virus population for
the production of a
master seed. The examples use Vero cells (American Type Culture Collection,
CCL 81 ), but
any cell type could be used that is permissive for influenza virus.
EXAMPLE 1: Chemicals and Biologicals
The infection medium used contained 1 Liter DMEM (Cambrex, Catalog No. 04-
096) or equivalent; stored at 2-7 C, 20 mL L-Glutamine (Cellgro, Catalog No.
25-005-CV) or
CA 02766173 2012-01-23
29
equivalent; stored frozen at -10 C or colder. Once thawed, it was stored at 2-
7 C for up to 4
weeks, and Type IX Trypsin (Sigma Product No. T0303, CAS No. 9002-07-7) or
equivalent;
was aliquoted and stored frozen at -5 to -30 C. The infection medium was
freshly made
before infecting the cells.
The cell culture medium preparation was as follows: 1 Liter DMEM, 20 mL L-
Glutamine, 50 mL Fetal Bovine Serum (Gibco Catalog No. 04-4000DK) or
equivalent (Note:
Sourced from a BSE free country). The complete medium was stored at 2-7 C for
no more
than 30 days after preparation.
EDTA-Trypsin (Cellgro Catalog No. 98-102-CV or equivalent) used for passaging
the cells was stored at -5 to -30 C, expiration date was assigned by the
manufacturer.
EXAMPLE 2: Cell Culture preparation
Because the dilution of protease to be used for the virus infection step can
vary by
lot, new trypsin lots were titrated to establish the optimal level prior to
use. An example of
the titration is to serial dilute type IX trypsin in DMEM containing L-
glutamine, using half
log dilutions (10-x, 10-15, 10-1, 10"2.5, etc.). Using a 96 well plate
containing a freshly
confluent monolayer of Vero cells, wash each well of the plate 2 times using
280 L of PBS.
Immediately after washing, add 200 L of each dilution of type IX trypsin to a
row of the
plate. The plate is then incubated at 37 C plus or minus 2 C with 5% CO2 and
the cells are
observed after 4 days. The lowest dilution of trypsin that shows no or little
effect on the
health of the cells is selected as an appropriate concentration of trypsin to
use for both
isolation and optimization of infection with influenza. It is desirable and
common to see little
or no variation between wells inoculated within each concentration.
For limit dilution cloning of virus in the Vero cells, a confluent monolayer
was
used, typically 3-4 days of age; grown in 96 well Falcon microtest plates. The
cells were
derived from ATCC CCL 81 and used between passages 132 and 156.
Preparation of Vero cells from liquid nitrogen (LN2) was as follows: one
ampule of
Vero cells was removed from LN2 and thawed in a 36 C plus or minus 2 C water
bath. The
entire contents of the vial was pipetted into a 25 cm2 tissue culture flask
containing 10mL of
cell culture media that is supplemented with 10% fetal bovine serum. The flask
was
incubated at 36 C plus or minus 2 C in 4-6% CO2. After about 1 hour, the
supernatant and
CA 02766173 2012-01-23
unattached cells were gently removed and 10 mL of fresh tissue culture media
added. Cells
were incubated at 36 C plus or minus 2 C in 4-6% CO2 until 90-100% confluency
was
reached.
Passage of the Vero cells was as follows: Monolayers were washed using 10-20
mL
of PBS for approximately 3 minutes. PBS was decanted and replaced with 3 mL of
EDTA-
trypsin (Cellgro, Catalog No. 98-102-CV), the monolayer was incubated for
approximately 3
minutes or until the cells were detached from the flask. The suspension was
diluted by
adding 17 mL of prepared growth media (containing FBS) to dilute and
neutralize the trypsin.
Cells were then counted using a hemocytometer to determine the cell count per
mL of
suspension. The number of flasks that could be prepared using this suspension
was
calculated by: Cells per mL (suspension) X mLs of desired suspension = mLs of
plate for
each vessel. Cells per mL (desired) Total the mLs of suspension, the total mLs
of suspension
must be less the volume available. If not the plating cell density can be
adjusted to
accommodate this, however it should be noted that the length of time until the
cells are
confluent will be longer with a lower cell density at plating. Vessels were
usually plated
using cells suspensions with a cell density of 1 x l 04 to 1 x 105 cells per
mL. Cells were
incubated for 3-4 days, or until confluent.
These techniques for maintaining and propagating Vero cells were similarly
applied
to other cell lines used such as the Madin Darby Canine Kidney (MDCK) and HEK
293.
HEK 293 cells particularly suited for propagation of viral isolates was
cloned. This
HEK 293 subclone, designated GT-D22 (or D22), was isolated from an initial
preparation of
HEK293 cells (ATCC No. CRL-1573; ATCC Batch No. F-11285 at Passage 33). HEK
293
cells were subcloned and selected on the basis of improved productivity of
recombinant
adenovirus Type 5 carrying the gene for expression of p53. Clones having
normal
morphologies and adequate growth rates were trypsinized and seeded at 1-2 x
106 cells per
well for further analysis. Top producing clones were subjected to further
subcloning and
selection. The D22 subclone was finally selected. The ability of the D22
subclone to
propagate Influenza has been demonstrated using Swine Influenza Virus (SIV).
Biosafety precautions were taken when working with live influenza virus.
Influenza
is a Class 2 etiologic agent and recommendations found in CDC-NIH, HHS
Publication No.
CA 02766173 2012-01-23
31
(CDC) 88-8395 (Biosafety in Microbiological and Biomedical Laboratories) for
handling the
virus in the laboratory were followed.
EXAMPLE 3: Limit Dilution Cloning
Preparation of dilution tubes and sample dilution for limit dilution cloning
was as
follows. Test tubes (12 x 75 mm) were set up in racks and labeled. I sample
was run per
plate and the dilution series for each sample was 10-1 to 10-10. Dilution
medium was
dispensed in 1.8 mL amounts into each test tube using a serological pipette.
The first tube
was labeled with the virus identification. Several additional tubes were
prepared for use as
diluent controls and for replacement if any errors are made during dilution
performance.
Samples were vortexed for approximately 5 seconds, then the initial dilution
was made by
pipetting 200 pL of sample into the 10.1 dilution tube. Serial dilutions were
continued to
1010. For each dilution, the sample was vortexed and the pipette tip was
changed between
dilutions.
Dilutions were transferred to cell plates as follows. Immediately prior to
use, the
medium was aseptically poured from the cell plates. Each well was rinsed using
a 12-channel
pipetter 2-3 times with 280 L of sterile PBS. The plates were aseptically
emptied, but they
were not allowed to dry out. Plates were labeled with the virus
identification, date, and
dilution scheme. Each dilution was vortexed briefly prior to addition to the
plate. Using a
motorized Finnpipette or other appropriate pipetter with 1000 L tips, 200 pL
per well of
sample was inoculated into a single row of the plate. The samples were loaded
according to
virus concentration. First, 2 rows of diluent controls were added to the plate
followed by the
highest dilution of virus (10"10) and continuing with the remainder of the
samples. Samples
were loaded in sequence from 10.10 through 10-1.
EXAMPLE 4: Virus Preparation
Harvesting of the virus and the CPE was evaluated as follows. Following the 4
day
incubation period, cytopathic effect (CPE) was read by microscopic
examination. The
presence of cellular debris, appearance of dead cells and lack of viable cells
were used to
characterize CPE because they are typical for influenza virus. Occasionally
nonspecific
interference was observed in the initial virus dilutions, therefore it was
important to examine
several dilutions for CPE. When evaluating CPE it was important to determine
the extent of
CA 02766173 2012-01-23
32
CPE in the wells of the highest dilution exhibiting CPE. The highest levels of
success was
achieved by selecting the wells of the highest sample dilution that exhibited
CPE, but of those
wells, preference was given to the well or wells that showed the smallest
extent of CPE.
Once selected, the contents of the well were harvested by using a single
channel 1000 L
pipette to aspirate the fluids. Usually the fluids were pipetted repeatedly to
remove loosely
attached cells from the well bottom and to help break up any clumps of cells
or virus in the
suspension. The harvested fluids were then used to perform an additional
round, or rounds,
of limiting dilution cloning or were sometimes used to inoculate a fresh
monolayer for the
production of a post cloning seed. It was typical to perform 2 to 3 rounds of
limiting dilution
cloning in immediate succession to produce a uniform population of virus. HA
titer was
analyzed at each step in the process and the results are shown in Table 1.
A/lndonesia/05/2005 (INDOH5NI), A/Vietnam/1203/04 (VNH5NI), A/New
Caledonia/20/99 (A/NC/20/99(R)), and A/Wisconsin/67/05 (A/Wis/67/058) are
reassortment
influenza viruses provided by the CDC. For the reassortment viruses the genes
encoding
surface glycoproteins HA and NA are from the influenza strain (INDOH5N1,
VNH5NI,
A/New Caledonia/20/99 and A/Wis/67/05) while the remaining internal genes are
from
A/PR/8/34. Limiting dilution cloning was also applied to wildtype (without PR8
reassortment) influenza strains A/New Caledonia/20/99 (A/NC/20/99),
A/Wisconsin/67/05
(A/Wis/67/05) and B/Malaysis/2506/04. The viruses were generated by using
reverse genetic
techniques and passaged in embryonated eggs. The egg materials were directly
used for limit
dilution cloning in Vero cells (A procedure for HA titration is shown in
Example 5).
CA 02766173 2012-01-23
33
TABLE 1
HA Titer* before and after Limit Dilution Cloning (LDC)
influenza strain Original** Before*** After After Roller Bioreactor
materials LDC 2nd 3rd bottle
LDC LDC
IndoH5N1 20,480 160 1,280 2,560 5,120 7,680
VNH5N 1 2560 <40 640 1,280 5,120 7,680
A/NC/20/99 5,600 640 1,280 ------- ------- -------
A/NC/20/99 (R) 10,240 1,280 1,280 ------- 2,560 10,240
A/Wis/67/05 (H3) 2,560 640 1,280 ------- ------- --------
A/Wis/67/05(H3R) 7,680 640 1,280 ------- 2,560 5,120
B/Malaysia/2506/ 2,560 <40 640 ------- -------- 5,120
04
*HA titer is expressed as Unit/mL. **HA titer in egg embryonic material
provided by CDC. ***HA titer from Vero cells infected directly with egg
embryonic
material provided by CDC.
As seen in the above Table 1, a cell culture system can produce a virus titer
as high
as that from eggs. Additionally, virus strains which showed no detectable
growth on Vero
cells at the outset, VNH5NI and B/ Malaysia/2506/04, could be adapted to grow
on Vero
cells by limiting dilution cloning. Propagation of VNH5NI and B/
Malaysia/2506/04 on the
amniotic membrane of eggs prior to limiting dilution cloning enhanced the
adaptation of
these viruses to growth on Vero cells.
Using SRID (Single Radial Immunodiffusion) to quantitate hemagglutinin, up to
132 gg/mL hemagglutinin protein was obtained for B/Malaysia/2506/04 in the
concentrated
Vero cell culture medium. The yield in g of HA/mL of culture was I Ox better
than known
published methods using Vero cells. Currently, a dose of human vaccine is
equivalent to
about 15 g/strain of virus. Therefore about 8-9 doses can be obtained from
one ml. of
concentrated Vero cell culture medium.
CA 02766173 2012-01-23
34
EXAMPLE 5: Passage of Influenza through Amniotic Membrane Enhances for Virus
Able to Grow on Tissue Culture Cells
Influenza virus VNH5NI-PR8/CDC-RG was received from the CDC. This is a
reassortant virus composed of the H5 and Ni genes from a Vietnam strain of
avian H5N 1
influenza virus on the PR8 virus backbone. This virus was expanded in II day
old
embryonated eggs inoculated by the allantoic cavity. Allantoic fluids were
harvested 2-3
days after inoculation and had a virus yield of 2560 hamagglutinin units
(HAU)/ml.
Allantoic fluids (-200 l) were diluted in 5 mL DMEM containing 1.25 jig/ml
Type
IX Trypsin and allowed to absorb to a confluent monolayer of Vero Cells (ATCC
CCL No.
81 at passage 147) for 60 minutes at 36 2 C. Cultures were fed with 25 mL
DMEM
containing 1.25 g/ml Type IX Trypsin and incubated for 3 days and culture
supernatants
harvested. There was no detectable hemagglutinin activity in the harvested
fluids.
Virus-containing allantoic fluids were diluted 1:10,000 and = I O0 1
inoculated into
both the allantoic cavity and onto amnionic membranes of 11 day-old
embryonated eggs.
Eggs were incubated at approximately 39 C for three days and allantoic and
amniotic fluids
harvested separately.
Vero cells (passage 132 to 152) were planted into 96-well plates at I x 104 to
I x
105 cells/m1, 200 gl/well in DMEM containing 5% fetal bovine serum and
incubated for 3-4
days (until confluent) at 36 2 C, 3-5% CO2. VNH5N 1 virus in allantoic or
amnionic fluids
was serially diluted 10"' to 10-10 in DMEM containing 1.25 pg/ml type IX
trypsin. Media
was removed from Vero cells, wells rinsed with 280 pl of phosphate buffered
saline and then
200 pl of each dilution of virus was inoculated into 8 replicate wells. Plates
were incubated
for 4 days at 36 2 C, 3-5% CO2. Amniotic fluids resulted in virus that grew
to higher titers
on Vero cells as evidenced by cytopathic effects up to the 10-9 dilution for
the amnion source
virus compared to 10'' dilution for the allantoic source virus (see Table 2).
Virus from one
well at the highest dilution showing cytopathic effects was harvested and
cloned by limiting
dilution a second time. Again, the amnionic fluid sourced virus showed
cytopathic effects at
higher dilutions than seen for allantoic source virus (approximately 10 times
more virus).
CA 02766173 2012-01-23
TABLE 2
Limiting Dilution Passage 1
Allantoic Fluid Virus Amnionic Fluid Virus
ldilution C to ethic Effect + resent ldilution C to ethic Effect + resent
10'10 ---10
- - -
+ -
t 0"6 10+ +
t- - - + + 1-+ + + + + + + +
10-, + + + + 10-6 + + + + + + + +
10'5 + + + + + + + + 10"5 + + + + + + + +
104 + + + + + + + + 104 + + + + + + + +
to-, + + + + + + + + 1o + + + + + + + +
102 + + + + + + + + 10'2 + + + + + + + +
10'1 + + + + + + + + 10'1 + + + + + + + +
A B C D E F G H A B IC D E F G H
Virus from well H7 was harvested Virus from well G9 was harvested
Limiting Dilution Passage 2
Allantoic H7 clone Amnion G9 clone
ldilution C o ethic Effect + resent ldilution C o ethic Effect + resent
1010 - - - - - - 10.10 - - - - - -
10"9 - - - - - - - 10 9 - - - - - - - -
104 to-, - - - - - -
to'' - - - - - - t0' - - - - - - -
10~ - - - - - 10. - - - - - --- -
10.5 to 5 - - - - -
104 --f. IV + + + + + + + +
to-, + + + to-, + + + + + + + +
t 0"2 + + 10"2 + + + + + + + +
10.1 + + + + + + + + + +
J__F_ G 111 A B C D E F G H
Virus from well H3 was harvested Virus from well G4 was harvested
CA 02766173 2012-01-23
36
Limiting Dilution Passage 3
Allantoic H7H3 clone Amnion G9G4 clone
1-dilution C o athic Effect + resent ldilution C to athic Effect + resent)
lo'ro - - - - - - - 100 - - -
I o"9 - - - - - - to-, - - - - - -
to-B --10.*
to'
Ion - - - - - - -
10-6 - - - - - - 10 - - - - -
t o"3 --IO
10.1 - - - - - -
to-' 10-4 + + + + + + + +
I0 to+ + + + + + + +
t0.
2 + + + + + + + +
to-' + + + + + + + + to-' + + + + + + + +
A B C D E F G H A B C D E F G H
Virus from wells B4 and F4 was harvested Virus from wells C5 & G5 was
harvested
Influenza clones, in Table 2, were identified based upon a combination of the
column designation A-H and row 1-10 (dilution series 10") through 10-10,
respectively). For
example the clone name B4 represents virus harvested from the well found at
column B and
row 4 (104 dilution).
Virus harvested from the third limiting dilution (-100 l) were diluted in 5
ml of
DMEM containing 1.25 g/ml type IX trypsin and inoculated onto confluent Vero
cells
(passage 132-152) and incubated for 60 minutes at 36 2 C. After absorption,
45 ml of
DMEM containing 1.25 g/ml type IX Trypsin was added and cultures were
incubated for
four days at 36 2 C. Harvested culture supernatants were tested for
hemagglutinin and both
clones derived from amnion expanded virus resulted in four to eight times
higher yields of
HA (see Table 3).
TABLE 3
Source virus Clone Designation Yield (HAU/ml)
Allantoic fluid H7H3F4 320
H7H3B4 160
Amniotic fluid G9G4C5 1280
G9G4G5 1280
CA 02766173 2012-01-23
37
The G9G4C5 virus was subsequently expanded by growth in Vero cells in roller
bottles (resulted in 5120 HAU/ml) and 5 liter bioreactors (resulted in 7680
HAU/ml).
Similar results were obtained with the Type B Influenza virus,
B/Malaysia/2506/04.
This virus also failed to yield any measurable hemagglutinin when virus
obtained from
allantoic fluid was propagated in Vero cells. However, virus derived from
amnion expansion
resulted in 640 HAU/ml after two limited dilution clonings and 5120 HAU/ml
after
expansion in a 5 liter bioreactor.
EXAMPLE 6: Quantification of hemagglutinin
The purpose of this procedure was to quantitate influenza virus hemagglutinin
activity in viral fluids in the final product.
The materials used included: PBS, Cambrex, 517-16Q or equivalent, Alsevers
Solution, E8085 or equivalent, fresh Rooster erythrocytes in Alsevers Solution
(1:1 ratio).
Allow to sit overnight in Alsevers to stabilize the receptors. Wash 2 times
and store as a 10%
suspension in PBS, or as a 50% suspension in Alsevers. Use within 4 days of
collection,
Microtiter plates, Falcon U-bottom plates, Catalog No. 3911 or equivalent, 8
channel
micropipette, 5-50 L, or equivalent, Centrifuge Beckman TJ-6, or equivalent,
20-200 L
micropipette or equivalent, Disposable 200 L pipette tips, Positive Control
Virus with
known titer, inactivated antigen. Stored at 2-7 C and used as is on the day of
the test.
A. A standardized 0.5% Rooster Red Blood Cell (rRBC) suspension in PBS was
prepared by first allowing the rRBC solution to equilibrate to room
temperature (15-30 C).
Rooster RBC's in Alseveres has a 4 day expiration period from date of
collection. A
sufficient volume of rRBC's in Alsevers was transferred to a 50 mL conical
centrifuge tube.
The rRBC's were washed by filling the tube to the 45 mL mark with PBS or
Alsevers and
mixed by inverting tube several times, then centrifuged at 400 x g, at 4 C for
10 minutes.
The supernatant was removed with a pipette. If there was any hemolysis in the
supernatant,
the wash steps were repeated up to three times. After the final wash, 0.25 mL
of packed
rooster RBCs were added to 49.75 mL PBS and inverted to mix. The cell
suspension was
labeled with the date of preparation, PBS lot number used, and 0.5% Rooster
Red Blood
Cells in PBS, store at 2-7 C (maximum storage time is 4 days).
*Trade-mark
CA 02766173 2012-01-23
38
B. The number of microtiter plates necessary to test the sample material was
determined. All test samples were tested using two rows each of a 1:2 and 1:3
dilution
scheme. Two rows of the positive control virus at a 1:2 dilution scheme and
two rows as a
PBS control were also used. Other samples were tested as requested. Using a
black
permanent marker the row designation on the microtiter plate was indicated. An
example is
shown below as Table 2.
Table 2: microtiter late
1 2 3 4 5 6 7 8 9 10 11 12
Serial No. A 0 0 0 0 0 0 0 0 0 0 0 0
1:2 B 0 0 0 0 0 0 0 0 0 0 0 0
Dilution
Serial No. C 0 0 0 0 0 0 0 0 0 0 0 0
1:3 D 0 0 0 0 0 0 0 0 0 0 0 0
Dilution
Positive E 0 0 0 0 0 0 0 0 0 0 0 0
Control F 0 0 0 0 0 0 0 0 0 0 0 0
Virus
1:2
Dilution
PBS G 0 0 0 0 0 0 0 0 0 0 0 0
PBS H 0 0 0 0 0 0 0 0 0 0 0 0
50gL of PBS was added to each well of the microtiter plate. An additional 50 L
of
PBS was added to well number one of those rows using a 1:3 dilution scheme and
the PBS
control rows. Each microtiter plate from the point of sample addition up to
the addition of
rRBC's was completed before proceeding to the next microtiter plate. 50 L of
sample and
positive control virus was added to well one of the designated rows. Serial
two-fold dilutions
of the samples were prepared using the multi-channel pipettor, transferring
50gL aliquots.
The appropriate tips were aseptically pushed onto the multi-channel pipettor,
assuring that the
pipettor was set at 50 L. The well contents of one column were mixed by
drawing up and
expelling the material a minimum of seven times. The tips used for mixing were
discarded.
More tips were pushed on and 50 L of material in each well was transferred to
the next
column of wells. These steps were repeated until all rows were sequentially
diluted.
Removal of 50 L from row twelve of the microtiter plates was assured. 50 1 of
0.5% rRBC
suspension was delivered to each well using the multi-channel pipettor. The
rRBC
suspension was added from the wells of the highest dilution to the lowest
dilution. Each plate
CA 02766173 2012-01-23
39
was agitated gently to mix the contents. A lid cover was placed over the top
microtiter plate
and the plates were stacked and incubated at room temperature (approxiamately
20-25 C) for
45-60 minutes.
After the incubation period, the plates were set on a microtiter plate viewer
to read
and to determine whether the PBS control wells are acceptable (the PBS wells
should not
exhibit any hemagglutination). The characteristics were also determined. The
rRBC's must
have settled out as a complete button without any shield. A shield reaction is
a dispersion of
the rRBC's. If the PBS control wells were acceptable, the other wells were
scored for
hemagglutination. If the PBS control wells were not acceptable the test was
invalid and was
repeated. The hemagglutination results were recorded as positive (+) for
hemagglutination,
partial (+/-), and negative (0). A positive reaction showed a complete shield
of the rRBC's or
total dispersion of the cells. A negative reaction showed a total button
formed by the rRBC's
Wells that exhibited a "+/-" were considered to be negative for purposes of
endpoint
calculation. The highest dilution at which agglutination (no button) occurred
was identified
for each of the replicates of each dilution (i.e. the 1:2 and 1:3) and the
titers were computed
as the inverse of the last dilution demonstrating complete agglutination. The
titer level of the
samples and positive control virus tested were identified. The arithmetic mean
of the
endpoint titer of each set of duplicate dilutions was determined. The HA units
per 0.5 mL
(50 L) of the Sample, PBS and Positive Control Virus were also determined. The
HA units
per I mL was calculated by multiplying the 0.05 mL value by 20.
The calculation was performed as follows: For each of the 1:2 and 1:3 test
sample
dilution, the arithmetic mean of the duplicates was recorded. The highest
titer was recorded.
HA/0.05mL x 20 = HA/1 mL was multiplied. The Off Test Date and the Results as
HA units
per I mL (virus fluids) or HA units per dose (final product) were recorded. A
valid test for
bulks or final products contains complete agglutination (no button) at the
lowest dilution and
no agglutination (button) at the highest dilution. The positive Control lot
should be within
the established range. A titer range outside the listed parameters constitutes
a "no test" or an
invalid test and should be repeated without bias.
CA 02766173 2012-01-23
EXAMPLE 7: Production of a vaccine
The virus strain is a pandemic strain or seasonal strains designated by the
WHO,
CDC, or other governmental organizations. For the purposes of validation of
the human
vaccine manufacturing process, the influenza virus reassortant VNH5NI-PR8/CDC-
RG
reference strain provided by CDC is used. Phosphate buffered saline is used as
a diluent for
the vaccine preparation and ISCOM as the adjuvant. A lipid mixture of equal
parts of
cholesterol and phosphatidyl choline is used to assist the
hydrophilic/hydrophobic
complexing process in the ISCOM formation. The non-ionic detergent which is
used to
disrupt the virus is removed by diafiltration. Formation of ISCOMS is verified
by electron
microscopy. Binary ethylenimine (BEI) is used to inactivate the virus and then
the BET is
neutralized with sodium thiosulfate. The process is set out in more detail in
steps 1-19 below.
The influenza strain is produced in Vero (African Green Monkey Kidney) cells,
inactivated with the aziridine compound binary ethyleneimine (BEI),
concentrated, purified
and purified by filtration and gel chromatography. The virus is formulated
with an adjuvant
with Quil A and a lipid mixture to make the drug product.
Stage I A - Vero cells are revived from the Working Cell Bank (WCB). Passage
number is limited to 20 passages from Master Cell Bank (MCB). One ampule is
thawed from
WCB in liquid nitrogen and seeded at 4-5 x 104 cells/cm2 in Dulbecco's
Modified Minimum
Essential medium (DMEM) containing 20% v/v irradiated fetal bovine serum of
New
Zealand or Australian origin, 4mM L-glutamine in (typically) a 25cm2 Nunc
flask. The flask
is incubated at 36 C plus or minus 2 C, the supernatant and unattached cells
are removed
after about 1 hour, and the flask is refed with fresh media and incubated as
before.
Stage 2A - Cell expansion is continued by harvesting of confluent monolayers
using
a Trypsin/EDTA solution, and re-planting cells into more static flasks or
roller bottles.
Further expansion may be performed in bioreactors by the use of microcarriers
at 20-30 g/L.
Stage 3A - When the desired production volume of culture substrate is achieved
in
either the bioreactor or in roller bottles, cells are washed twice with
Dulbecco's Modified
Minimum Essential medium (DMEM) to remove residual serum, that will inactivate
trypsin
in the infection medium.
CA 02766173 2012-01-23
41
Stage I B - Working Virus Seed (WVS) is prepared separately and frozen in
advance
of large-scale manufacturing. Master Seed Virus or Working Seed Virus (MSV+1)
stored at
-70 C is thawed and diluted in virus infection medium containing Type IX
porcine trypsin to
achieve the desired MOI. A predetermined volume is inoculated onto confluent
Vero cell
monolayer in the roller bottles or bioreactor and incubated at 36 C plus or
minus 2.0 C,
typically for 40-72 hours, when up to 100% cytopathetic effect (CPE) is
identified. Virus is
harvested and frozen at -50 C or colder.
Stage 4 - Working seed virus (not higher than passage MSV+2) is thawed and
diluted in Virus Infection Medium containing 0.5-5.0 g/mL of Type IX porcine
trypsin to
achieve the desired multiplicity of infection. The use of trypsin in the
medium will assist the
attachment and penetration of the virus into the cell.
Stage 4A-production of influenza virus using a bioreactor. A 5- liter
bioreactor was
prepared with SoloHill Plastic Plus microcarriers at a density of 30 g/L. The
bioreactor was
planted at 2 x 105 Vero cells/mL in Dulbecco's Modified Minimum Essential
medium with
5% v/v irradiated fetal bovine serum of New Zealand or Australian origin, 4mM
L-glutamine
and incubated at 36 C plus or minus 2 C. After the cell confluency reached 80-
100%, the
microcarriers containing the Vero cells was settled and washed twice with 2
liters per wash of
serum-free DMEM. Infection medium containing 2.5 .tg/mL of trypsin IX was
added to the
bioreactor. Virus seed, for example VNH5N 1, also was added to the bioreactor
at a MOI of
0.0001-0.0003. The preparation of virus was continued for 5 days. The
bioreactor was
sampled daily an for CPE observation and HA titration. The virus was harvested
after 80-
100% CPE was reached.
Stage 5- Binary ethyleneimine (BEI) is added to the harvested virus to give a
final
concentration of 1.5mM, and held at 36 C plus or minus 2 C for 1 hour (with
agitation) at a
pH of 7.3 plus or minus 0.3.
Stage 6 - After stage 5 is complete, the harvest is transferred to a second
vessel and
the inactivation process continued at 36 C 2 C for 48 hours (with
agitation). After this
time sodium thiosulfate is added to a final concentration of 3mM to neutralize
any residual
BEI.
CA 02766173 2012-01-23
42
Stage 7 - The culture is clarified through 7 and I p filters and stored at 2
C - 8 C
pending innocuity clearance. The innocuity test takes ten days to perform.
Stage 8 - Antigen is concentrated using a tangential flow ultra filtration
system,
using a polysulfone membrane with a molecular weight cut off(MWCO) of 100 K.
Up to
approximately a 50 fold concentrate is achieved.
Stage 9 -The resulting concentrated culture fluids are equilibrated in an
appropriate
buffer and treated with DNase (Benzonase) to degrade cellular DNA.
Stage 10 - The concentrate is puri fied using size exclusion gel
chromatography. The
gel currently used is a cross linked Sepharose (CL-2B, Pharmacia). The column
is 90 cm in
length to achieve the required separation. CL-2B is a `soft' gel which depends
on the wall of
the column for support. Typically the 90 cm length is achieved by 2 x 45 cm or
3 x 30 cm
columns (e.g., 30-32 cm in height by 30 cm in diameter) in series. The
concentrated virus is
applied at approximately 5-7% of the column volume.
Stage 11 - Virus peak material is reconcentrated using a bench scale
tangential flow
ultra filtration system with a 100 K MWCO polysulfone membrane.
Stage 12 - The re-concentrated virus peak material is solubilised by adding 5
ml. of
a 10% (w/v) detergent (Nonanoyl-N-Methylglucamide) Mega 9 solution to 200 mis.
of an
antigen solution. The solution is mixed slowly with a magnetic stirring bar
for 1 hour at 20 -
25 C in a glass container.
Stage 13 - A lipid mixture is added at 50 l per 20 mLs of re-concentrated
virus
peak. The lipid mixture contains 10 mg/mL each of egg derived
phosphatidylcholine and
cholesterol. Stirring is continued at 20-25 C to ensure even distribution of
the lipids in the
re-concentrated virus. Quil A (from 10% w/v stock solution) is added to give a
final
concentration of 0.05%. The solution is stirred for approximately 30 minutes
at 20-25 C.
Stage 14 - The Mega 9 detergent is removed from this mixture (to allow ISCOM
formation) by diafiltration with 50 mM ammonium acetate. This is performed
using the
bench scale tangential flow ultrafiltration system with a 100 K MWCO
polysulfone
membrane. The volume of ammonium acetate used is a minimum of about t OX the
volume of
re-concentrated virus peak mixture. Diafiltration is effected by maintaining a
constant volume
CA 02766173 2012-01-23
43
throughout, by balancing feed and permeate flow. Detergent interferes with the
formation of
ISCOM. Electron microscopy verifies that the typical cage-like structures have
been formed.
Stage 15 - The ISCOM is re-concentrated as the final step of diafiltration.
Stage 16 - After satisfactory QC release, batches of ISCOMs are formulated to
make the vaccine at I to 20 g human influenza HA per I mL dose. Phosphate
buffered
saline (PBS) is used as the diluent.
Stage 17 - The blended vaccine is filled into single dose final containers
under Class
A conditions. Samples are taken for sterility, safety in lab animal species,
extractable
volume and visual appearance. Vaccine is labeled and packed and held in
quarantine at 2 to
7 C.
Stage 18 - After final QA clearance, product is released to finished goods'
cold store
(2 to 7 C) pending dispatch.
EXAMPLE 8: Efficacy of Vero Cell Derived Vaccine in Ferrets
The ability of Vero derived influenza vaccine to seroconvert ferrets was
evaluated.
A trivalent human influenza vaccine based on the 2006-2007 seasonal influenza
strains
(A/NC/20/99, A/Wis/67/05 both PR8 reassortments and B/Malaysia, all obtained
from the
CDC) was produced in Vero cells after limit dilution cloning. The resultant
vaccine,
"SPflu0607," with or without ISCOM adjuvant, was injected into the left hind
leg of 4-6
month old female ferrets. As control comparisons, commercially available human
influenza
vaccines Fluzone (manufactured by Sanofi-Pasteur) and Fluvirin (manufactured
by
Chiron) were tested along side SPflu0607. Single Radial Immune Diffusion
(SRID) was used
to measure the quantity of Hemagglutinin (HA) protein in the vaccine.
Seroconversion of the
ferrets was measured by hemagglutinin inhibition (HI). HI titers of greater or
equal to 1:40
are considered positive. See Table 3
CA 02766173 2012-01-23
44
Table 3
Days after
vaccination-4 14 days 28 eta s 60 days 90 days
Vaccine GMT Positive GMT Positive GMT Positive GMT Positive
kWIT607, ~010
1 6 1,18. `8.Q a 4_ x _6 91 x 6.
7 _ 403
K, r
Uffm
54 1'Q 1' on 4
~=
'SPfluO6O7
15 mL 118 67 43 67 29 56 20 33
7.5 gglmL 137 89 52 78 23 44 16 44
M > 48 .... 8 ~ 63_ r. 67,..x:. 3: ,_ X29,. ;56. ,
Sanofi
mL 101 89 101 89 25 44 15 22
7.5 g/mL 135 88 80 88 28 50 12 25
PBOni : Rst7 r . 6 - t .t. .OAS = `6' OT ,=5
GMT = Geometric mean antibody titer
% Positive - Percentage of animals with Hi antibody levels > 1:40
The above results indicate that Vero cell-derived vaccine is comparable to
commercially
available egg derived vaccines.
Example 9: Method of Canine Influenza Virus Propagation for Preparing a CIV
Vaccine
Canine influenza was isolated from nasal secretions of a sick dog. Nasal swabs
were taken and placed in 2 mL of tissue culture media containing gentamicin
and
amphotericin. 0.8 mL of the resultant swab material was inoculated onto
confluent Madin
Darby Canine Kidney (MDCK) cells in 10 mL of DMEM tissue culture media
containing 1.3
tg/mL type IX Trypsin and incubated for 2 days at 36 2 C. The flask was
harvested by
decanting the media and the virus was identified as H3N8 by the National
Veterinary
Services laboratory using standard anti-sera. The MDCK passaged virus
contained 160
hemagglutinin units per ml. The virus was cloned by inoculating 10-fold serial
dilutions onto
confluent MDCK cells in 96-well plates and harvesting a single well at the
highest dilution
CA 02766173 2012-01-23
showing cytopathic effects (tissue culture media was DMEM containing I.3gg/mL
type IX
trypsin). This procedure was repeated a second time. The clone was then
expanded on
MDCK cells in a 75 em2 flask. The resultant virus (passage 4) yielded 640
hemagglutinin
units per ml. The virus was then passaged onto Madin Darby Bovine Kidney cells
by
inoculating 0.23 MOI onto a confluent monolayer in a 1050 cm2 roller bottle
using 300 mL
DMEM containing 0.8 gg/mL type IX trypsin. The roller bottle was incubated for
3 days at
36 2 C. The harvested virus yielded 2560 HAU/ml. Due to the increased yield
of HA on
MDBK cells, this cell line was chosen for scale-up of virus for vaccine
preparation. The
virus was propagated in a bioreactor. A 5L bioreactor was seeded with MDBK
cells at 3.0 x
105 cells/mL attached to Cytodex III microcarriers at 5 grams per liter. Cells
were grown for
4 days in DMEM containing 5% fetal bovine serum with no antibiotics at 36 2
C. After
settling the microcarriers, 90% of the media was removed and replaced with
serum free
DMEM. Type LX trypsin was added to a concentration of I O g/mL in the final
5,000 mL.
The cells were infected with virus at an MOI of 0.01. The virus was incubated
with the
MDBK cells on microcarriers for 2 days at 36 2 C and then the supernatant
was harvested.
The resulting virus yielded 10,240 HAU/ml.
EXAMPLE 10: Limiting Dilution Cloning of Clinical Isolate Without Passaging in
Eggs
Creates a Uniform Population and Improves TCID50/mL and HA titer
Canine Influenza Virus H3N8 (wild type) was received from a diagnostic
laboratory
and isolated from a nasal swab. Upon receipt the nasal swab was processed and
used to
inoculate a 25 cm2 flask containing a confluent monolayer of MDCK cells.
Infection media
was comprised of: DMEM, 4 mM L-glutamine/ml, 1.3 gg/ml Type IX, and
gentamicin. The
flask was incubated at 36 2 C with 3-5% CO2 and harvested at onset of CPE.
An HA assay
was preformed on the harvest fluids with a result of 160 HAU/ml and a
TCID50/ml titer of
7.94.
MDCK cells were planted into 96-well plates at 1 x 104 to 1 x 105 cells/ml,
200
l/well in DMEM containing 5% fetal bovine serum and incubated for 3-4 days
(until
confluent) at 36 2 C, 3-5% CO2. CIV H3N8 virus was diluted as specified in
the section:
Limiting Dilution Round 1. Dilutions were performed in DMEM containing 1.3
g/ml type
IX trypsin, 4 mM L-glutamine, and gentamicin. Media was removed from MDCK
cells,
wells rinsed with 280 l of phosphate buffered saline and then 200 l of each
dilution of
CA 02766173 2012-01-23
46
virus was inoculated into 8 replicate wells. Plates were incubated for 4 days
at 36 2 C, 3-
5% CO2. Two rounds of limiting dilution cloning were preformed, limiting
dilution round 2
immediately following limiting dilution round 1. The process yielded a virus
isolate that
produced both higher TCID50/ml and hemagglutination titers. Virus from a well
at the higher
dilutions demonstrating the lowest extent of cytopathic effect was harvested
and cloned by
limiting dilution a second time.
Limiting Dilution Round 1
Pass I material was titrated with a result of 7.5 TCID50/ml. Using this value
the virus was
diluted to yield 5 samples.
A. 104
B. 10'5
C 10 virus particles/ well (10-6-5)
D. 3 virus particles / well (10-7.02)
E. i virus particle/ well (10'75)
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D 2 0 b 10.6.5 10-7.02 0.7.5
E c
F
G
H
Well A 11 was harvested in preparation for round 2 of limiting dilution
cloning.
Limiting Dilution Round 2
1 2 3 4 5 6 7 8 9 10 11 12
A
B o
C
c c
r c~
U o 0 o d o 0 0 0 o V
E C r e- r r r r r r C
F
H
Virus from well A5 was harvested
CA 02766173 2012-01-23
47
Virus harvested from the second round of limiting dilution cloning (-200 1)
was
used to inoculate a 75 cm2 flask containing a confluent monolayer of MDCK
cells and
DMEM supplemented with; 1.3 g/ml type IX trypsin, 4 mM L-glutamine/ml, and 25
Vg/ml
gentamicin. Harvested culture supernatants were titrated to determine the
TCID5o/ml and
hemagglutination titer (see Table 4).
TABLE 4
Virus Passage TCID%/ml HAU/ml
Pass I from field isolate 7.94 160 HAU/ml
Pass 4, Pre-master seed 7.69 640 HAU/mL
In laboratory experiments performed for immunogenicity trials the virus was
grown on
MDBK cells in a 5L bioreactor with a resulting hemagglutination titer of
10,240 HAU/ml.
EXAMPLE 11: Efficacy of an Inactivated Canine Influenza Vaccine in Dols
Canine influenza virus (CIV) serotype H3N8 causes severe respiratory disease
in
dogs. However, there is no effective vaccine against CIV currently available.
The objective
of this study is to evaluate the efficacy of an inactivated CIV vaccine,
prepared by limiting
dilution and propagation of CIV in tissue culture cells in preventing clinical
disease and lung
lesions induced by a virulent CIV challenge. The vaccine consists of binary
ethyleneimine
(BEI)-inactivated CIV antigen adjuvanted with Emunade adjuvant with an
antigen input
level of 500 hemagglutination units (HAU) per dose. A group of eight 7-week-
old CIV
seronegative dogs was vaccinated intramuscularly with the vaccine and booster
dose was
administered 21 days after the primary vaccine. Two weeks following booster
vaccination,
the vaccinated dogs exhibited significantly higher levels of HA inhibiting
antibody titer
compared to their non-vaccinated counterparts demonstrating stimulation of
immune
response by the vaccine in dogs. The non-vaccinated control and the vaccinated
dogs were
challenged with a heterologous virulent CIV isolate 16 days post-booster
vaccination, and
monitored daily for 10 days post-challenge for clinical signs, rectal
temperature and nasal
CIV shedding. All control dogs (100%) developed clinical signs including
ocular and nasal
discharge, sneezing and coughing indicating virulence of challenge virus. The
vaccinated
group exhibited significantly lower clinical signs (median score=4.3) compared
to control
CA 02766173 2012-01-23
48
group (median score=6.8; p=0.0051). Only one of the dogs in the vaccinated
group (12.5%)
showed nasal CIV shedding and it only was for one day, whereas, all the dogs
in the control
group (100%) had significantly higher virus shedding compared to the
vaccinates (p=0.0003).
The virus shedding lasted for 7 days following challenge in the control group.
All dogs were
euthanized 10 days after challenge, and necropsy was performed for evaluation
of lung
lesions. All dogs in the control group (100%) showed varying degrees of lung
consolidation,
whereas, only one in the vaccinated group (12.5%) exhibited a mild lung
consolidation. The
lung scores were significantly higher in control dogs (median score=4.9)-when
compared to
vaccinated dogs (median score=0; p=0.0005). These results unequivocally
demonstrate that
the vaccine formulation tested in this study protects dogs against CIV
infection by
significantly decreasing clinical signs, reducing virus shedding, and
preventing CIV-induced
lung consolidation.
Study Overview
The objective of the study was to test the efficacy of Emunade -adjuvated CIV
vaccine formulation to protect against CIV challenge in dogs.
Dogs were initially acclimatized for eight days. For the test group, the first
vaccination occurred on day 0 and a booster was given on day 21. The control
group was not
vaccinated. Challenge with CIV was presented to both groups on day 37. The
dogs were
monitored and observed throughout the study, as described below. The dogs were
euthanized
days after challenge and a necropsy was performed.
Test animals
Test animals were an average of 48.25 days old on day 0. There were 8 dogs in
the
control group and 8 dogs in the test group. Average weight was 1.8 kg. The
dogs used in the
study did not have a history of respiratory infection or CIV vaccination. To
confirm that the
dogs were negative for CIV antibodies (HA titer <10), blood samples were
collected on day -
1, and tested by a hemagglutination inhibition assay. Nasal swabs were also
taken on day -1
to ensure that the dogs were free of CIV at the time of vaccination.
Pre-vaccination monitoring
Blood samples were collected from all dogs in evacuated serum separation tubes
on
the day before administration of the first vaccine in order to confirm that
the dogs were
CA 02766173 2012-01-23
49
negative for CIV antibodies. Nasal swabs were taken on the same day to confirm
that the
dogs were free of CIV.
The general health of the dogs was assessed by physical examination of the
dogs
two days prior to first vaccination. Clinical assessments and rectal
temperature were
performed from two days before the initial vaccination and booster vaccination
through the
day of vaccination.
Vaccination
Canine influenza virus vaccine CIV H3N8-Emunade was used in this study.
Canine Influenza Virus (H3N8) was initially isolated from a dog with severe
respiratory
disease. Madin-Darby Bovine Kidney (MDBK)-KC cells at MCS+19 passage level,
i.e.,
following 19 passages of the Master Cell Stock, were used to propagate the CIV
H3N8. The
virus then was inactivated with 6 mM BEI for 60 hours at 36 C. The BEI was
neutralized
with 60mM Sodium thiosulfate.
The vaccine for this study was prepared in an 800mL stock for aliquoting into
800
one mL doses. The 800 mL solution was prepared as shown in Table 5.
Table 5
Component Volume Used
Aqueous Phase:
Inactivated CIV 156 mL
(2560 HAU/mL)
Saline 342 mL
Aluminum hydroxide (2.1 % Aluminum 77 mL
oxide)
Ethyl Alcohol 16 mL
Glycerin 40 mL
HEPES 1M 8 mL
I% Red Dye Solution 0.8 mL
Oil Phase:
Mineral Oil 107 mL
Tween 80 34.5 mL
San 80 18.4mL
Preservative:
Gentamicin 0.373 mL
Total Volume 800.0 mL
CA 02766173 2012-01-23
The inactivated CIV H3N8 virus was diluted in normal saline and then
adjuvanted
with aluminum hydroxide. The remaining components in aqueous phase were added
to the
adjuvanted antigen. The oil phase was prepared separately and then added to
the aqueous
phase over a period of 10 minutes with continuous mixing for one hour. The
serial was
homogenized using Silverson homogenizer for 30 minutes.
The CIV vaccine vials that were stored at 2-7 C were equilibrated to room
temperature for a minimum of 30 minutes. The vaccine was loaded into 3 mL
syringes (1 mL
per syringe) and used for immunization. The first dose of vaccine was
administered
intramuscularly into right hind leg on day 0 and the second dose was
administered
intramuscularly into left hind leg on day 21.
Post vaccination procedures
Complete clinical assessments including rectal temperatures and injection site
observation were performed on all dogs within 3 - 6 hours after each
vaccination to measure
any immediate reactions. The clinical assessments were continued daily for 7
days following
each vaccination and scored according to the Clinical Assessment Guide below.
Dogs were
bled on study days 20 and 36, and the serum samples were used to measure CIV
antibodies
by hemagglutination inhibition.
Pre challenge procedures
Clinical assessments were performed and rectal temperatures were recorded for
all
dogs for two days prior to challenge (days 35 and 36) and on the day of
challenge (day 37)
before challenge administration. The clinical signs were scored according to
the Clinical
Assessment Guide.
Challenge
Challenge material: The C1V 14-06A virus isolated from MDCK cells and used to
challenge vaccinated dogs, was originally isolated from field sample collected
from dog
suffering with canine respiratory disease. The average titer of the challenge
virus was 7.7
Logi0TCID50/mL. On the day of challenge, the challenge material was diluted to
1:4 in
sterile, cold Dulbecco's Minimum Essential Medium (DMEM) to target a challenge
dose of
7.4 Logi0TCID50 per dog.
CA 02766173 2012-01-23
51
Challenge administration: All dogs were challenge-administered on day 37. Four
dogs were placed in a Plexiglas chamber and 8 mL of challenge virus (2 mL/dog)
was used to
generate aerosol over a period of approximately 20 minutes. The dogs were
exposed to
aerosol for a total of 40 minutes.
Post-challenge monitoring
Rectal temperature was recorded and clinical assessments were performed for
each
dog daily for 10 days post-challenge. Nasal swabs were collected daily from
each dog for 10
days post-challenge. Nasal swabs were processed and titrated daily after each
collection as
described herein. Blood samples were collected in evacuated serum separation
tubes on day
post-challenge just before euthanasia.
Necropsy
All challenged dogs were euthanized on day 10 post-challenge (Day 47) using an
AVMA approved method (Ketamine cocktails and Beuthanasia-D), and necropsy was
performed. Immediately following euthanasia, the lungs were evaluated. Areas
of visible
consolidation were assessed and scored as a percent consolidation of each lung
lobe. The
percentages were converted to weighted scores, and total score for each dog
was. calculated.
During necropsy, the lung tissue was collected for virus isolation and
titration, and for
histopathology.
Virus titration
Hemagglutination (HA) assay was performed for virus titer. The virus was
serially
two-fold diluted in a V-bottomed microtiter plate and an equal volume of 0.5 %
turkey red
blood cell (RBC) suspension was added to the virus suspension. The plates were
incubated at
room temperature for 30 min and HA results read. The highest dilution of the
virus showing
HA activity was considered as I HA unit. All assays were performed in
duplicate and the
endpoint HA titer was measured
To confirm the potency of challenge material and to measure virus shedding in
challenge-administered dogs, the virus titer in challenge material, nasal swab
and lung tissue
was determined by titration in MDCK cells. MDCK cells were seeded in 96-well
tissue
culture plates for two days, and were inoculated with ten-fold serially
diluted virus
suspension or samples prepared from lung tissue and nasal swabs. The plates
were incubated
CA 02766173 2012-01-23
52
at 36 2 C temperature and 5 % CO2. Seven days post-infection, the plates were
observed for
cytopathic effect (CPE), and the 50 % end-point for infectivity was calculated
using
Spearman-Karber method. The virus titers were expressed as LogioTCID50/mL.
Detection of serological response
Antibodies to CIV in dog serum samples were measured by hemagglutination
inhibition (HAI) assay. Briefly, a serial two-fold dilution of test serum was
performed in
PBS in V-bottomed 96-well microtiter plates. An equal volume of virus
suspension
containing 4-8 HAU of CIV25-06B was added to each well containing test serum,
and the
plates were incubated at room temperature for 30 min for antigen-antibody
reaction to occur.
Then, an equal volume of 0.5 % turkey RBC suspension was added. The plates
were
incubated at room temperature for 30 min and HAI results were read. The
reciprocal of the
highest dilution of the serum showing HA inhibition was considered as the HAI
titer of the
test sample. All assays were performed in duplicate and the endpoint HAI titer
was
determined.
Results: Clinical scores
All dogs were monitored daily, from two days prior to challenge through 10
days
post-challenge, for clinical signs including ocular discharge, nasal
discharge, sneezing,
coughing, dyspnea and depression. Daily clinical scores for ocular discharge,
nasal discharge,
sneezing, coughing, depression and dyspnea for 10 days post-challenge were
summed to
obtain a summed clinical score for each dog. Summed clinical scores for
vaccinated and
control groups were compared using Wilcoxon Exact Rank Sum tests and two sided
p-values
were calculated.
Dogs in both control and vaccinated groups exhibited a range of clinical signs
starting from two days post-challenge (Figure 1). All eight dogs (100%) in the
control group
exhibited varying degrees of coughing that lasted up to five days within 10-
day post-
challenge observation period. On the other hand, only two dogs (25%) in the
vaccinated
group exhibited a mild cough that was observed only one day during the entire
10-day post-
challenge period. Coughing was the predominant sign exhibited by dogs in
control group.
On the contrary, only mild ocular discharge was the predominant clinical sign
exhibited by
vaccinated dogs. The clinical scores were significantly higher (p--0.005 1) in
control group
CA 02766173 2012-01-23
53
(median score=6.8) compared to vaccinated dogs (median score=4.3). These data
suggest
that the CIV vaccine protects dogs against CIV-induced clinical signs.
Results: Nasal virus shedding
Nasal virus shedding was monitored in all dogs by collecting and processing
nasal
swabs on the day before challenge (day-1), and then, daily from day I through
day 10 post-
challenge. The virus titers (Logio TC[Dso/mL) of the nasal swabs were
determined and
plotted against time. Area under the curve was compared between control and
vaccinated
groups using Wilcoxon Exact Rank Sum tests. The average virus titer for each
group,
expressed as LogioTCID50/mL, was plotted against days post-challenge (Figure
2).
The control group started shedding virus in nasal secretions from day I post-
challenge. The virus shedding reached its peak on day 5 post-challenge (1.25
Log,0TCID50/mL) followed by a precipitous drop on day 7 (Figure 2). All dogs
in control
group (100%) were positive for virus shedding at one or more time points
during 10-day
post-challenge observation. On the other hand, only one dog (12.5%) in
vaccinated group
(ID No CXTAMM) shed virus in nasal secretions only for one day (day 3). Non-
vaccinated
control dogs exhibited significantly higher nasal virus shedding compared to
vaccinated dogs
(p=0.0003). These results clearly indicate that the CIV vaccine significantly
inhibits nasal
virus shedding by vaccinated dogs.
Results: Serological response
Geometric mean antibody titers (GMT) from HAI assays were calculated following
primary and booster vaccinations. The fold increase in titers between
immunizations was
reported. The antibody titers were compared between control and vaccinated
groups using
Wilcoxon Exact Rank Sum tests. All 16 dogs enrolled in the study were healthy
and
seronegative (i.e., negative for CIV antibodies) (HAI titer of <10) at the
time of primary
vaccination. Nasal swabs collected on the day before vaccination (Day -1)
confirmed that the
dogs were free of nasal CIV shedding. Control dogs remained seronegative at
the time of
challenge.
The HAI antibody titers were tabulated and compared between control and
vaccinated groups. All vaccinated dogs generated measurable levels of antibody
titers
following first vaccination. The HAI antibody titers ranged between 10 and 40
with a GMT
CA 02766173 2012-01-23
54
of 22, and the titers were significant when compared to control dogs
(p=0.0070). Second
vaccination boosted the antibody titers by six fold (GMT=135) which were
significantly
higher than control (p=0.0002). The antibody titers ranged between 80 and 160,
with most of
the dogs exhibiting an HAI titer of 160 (75%). All the control dogs remained
free of CIV
antibodies until the time of challenge (HAI titer <10). Following challenge,
antibody titers in
vaccinated dogs reached very high levels (GMT=546) demonstrating efficacy of
the vaccine
in priming the immune system against virulent CIV. The HAI titers in these
dogs ranged
between 120 and 1920. The non-vaccinated controls also made antibodies
following CIV
challenge with GMT of 149.
Results: Lung consolidation, virus isolation, and histopathology
Lung consolidation/pneumonia is the major pathological lesion in all influenza
infection. In the previous study on development of challenge model, we
observed severe
lung consolidation in dogs on 6 and 14 days post-challenge. Therefore, in
order to assess
whether the CIV vaccine protects against CIV-induced lung consolidation, all
dogs in control
and vaccinated groups were euthanized on day 10 post-challenge, and necropsy
was
performed. Lung lesions were evaluated and scored as percent consolidation of
each lung
lobe. The percent consolidation of each lung lobe scored during necropsy was
converted into
weighted scores based on the lung scoring system for dogs (similar to Swine
influenza virus
lung scoring system in Diseases of the Swine (1999) 8th Ed., Ch. 61, p. 913-
940). Median
lung scores for vaccinated and control groups were compared using Wilcoxon
Exact Rank
Sum tests and two sided p-values were reported. Mitigated fraction estimate of
vaccine
efficiency relative to controls and the 95% confidence interval for the
estimate was also
reported.
All dogs in control group (100%) exhibited varying degree of lung
consolidation
whereas only one dog in vaccinated group showed a mild lung consolidation
(12.5%). Lung
lesions in non-vaccinated control dogs were characterized by hemorrhages and
reddish
consolidation and hepatization. The lung scores in control group ranged
between 0.10 and
14.70 with median lung score of 4.9. The lung scores in control group were
significantly
higher than that in vaccinated group (p=0.0005; mitigating fraction estimate
is 93.5%). The
lung scores unequivocally demonstrate that the CIV vaccine formulation used in
this study
protects dogs against CIV-induced lung consolidation.
CA 02766173 2012-01-23
In addition to scoring lesion during necropsy, lung tissues were also
collected
aseptically for virus isolation and in formalin for histopathology. There was
no detectable
CIV in lung tissue samples from both vaccinated and control dogs, which
correlated with the
absence of nasal virus shedding. This was expected since influenza viruses
cause acute
infection with peak virus shedding and clinical signs within the first seven
days. The virus
was cleared completely from lung tissue by 10 days post-infection. These
results are in
agreement with the results of the previous study. Histopathological
examination revealed
varying degrees of histopathological changes suggestive of inflammation in
lung tissues from
control as well as vaccinated dogs. This finding was not unexpected because
since immune
response to any pathogen is likely to induce a certain degree of inflammatory
response even
in the presence of pathogen-specific immunity. Furthermore, the severity of
histopathologic
lung lesions in control and vaccinated dogs could not be compared since the
tissues were not
collected selectively from areas with lung lesions. Therefore, histopathology
could not be
used as a criterion to evaluate efficacy of the vaccine in this study.
Conclusions
The vaccination induced a significantly higher antibody response, as
determined by
HAI assay, following first (GMT=22) and second vaccinations (GMT=135)
indicating
stimulation of immune response by the vaccine.
The vaccine significantly decreased CIV-induced clinical signs, especially
coughing, at a dose of 500 HAU per dog demonstrating effectiveness of the
vaccine in
controlling CIV-induced clinical disease.
The vaccine significantly reduced nasal virus shedding in vaccinated dogs,
which
demonstrates the efficacy of the vaccine in reducing infection.
The vaccine successfully protected dogs against CIV-induced lung consolidation
proving the efficacy of the vaccine against the most severe clinical outcome,
pneumonia.
The vaccine caused no major adverse reactions in dogs demonstrating the safety
of
the vaccine.
CA 02766173 2012-01-23
56
Clinical Assessment Guide
Nasal Discharge
0 = Absent
0.5 = Serous discharge: Water fluid dribbles from the nostril. Fluids running
out of the nose
are recorded here.
1 = Mucopurulent discharge, mild to moderate: Cloudy fluid mixed with mucous
runs at least
halfway down from the nose to the mouth.
2= Mucopurulent discharge, severe: Mucous run past the mouth.
Ocular Discharge
0 = Absent: Small amount of dried crusted material in the corner of the eye is
not considered
an ocular discharge.
0.5 = Serous discharge: Clear fluid discharge running outside of the eye.
1 = Mucopurulent discharge, mild to moderate: Cloudy fluid mixed with mucous
that runs at
least halfway down from the eye to the mouth.
2 = Mucopurulent discharge, severe: Fluid or mucous runs halfway down the nose
or rim the
eye and soak the hair at inner or outer corner of the eye.
Cough
0 = Absent
0.5 = Mild: Only one brief cough is observed.
1.0 = Moderate: Cough is persistent, occurring repeatedly in the observation
period.
2.0 = Severe: Cough is accompanied by choking or retching sounds.
Sneezing
0 = Absent
2 = Present
Dyspnea
0 = Absent (Normal breathing)
2 = Present (Panting)
Depression
0= Absent (Normal activity)
2 = Present: Dog is less active or playful, compared to normal. Dogs are
recorded if
lethargic or lying down and reluctant to stand while observation was
conducted.
Although the foregoing invention has been described in detail for purposes of
clarity
of understanding, it will be obvious that certain modification may be
practiced within the
scope of the appended claims.
From the foregoing it will be apparent that the invention provides for a
number of
uses. For example, the invention provides for the use of any of newly
identified pathogenic
CA 02766173 2012-01-23
57
influenza virus strains for preparation of cell culture-adapted isolates
and/or for vaccines as
well as known pathogenic strains. The invention provides for the use of any
newly identified
cell culture cells that are or can be made permissible to influenza growth.
The invention
provides for the preparation of the tissue-culture adapted strains into
vaccines having at least
attenuated, subunit, split or killed virus, but also having adjuvants,
carriers, excipients, anti-
influenza pharmaceuticals, and other agents that increase the immune response
to the virus.
The vaccines can be used before contact with influenza or after contact.
