U.S. patent application number 12/962375 was filed with the patent office on 2011-09-01 for method for producing the flu virus.
This patent application is currently assigned to SANOFI PASTEUR S.A.. Invention is credited to Michel Marie Joseph Bublot, Catherine Gerdil, Francois-Xavier Le Gros, Isabelle Legastelois, Catherine Moste.
Application Number | 20110212129 12/962375 |
Document ID | / |
Family ID | 39146849 |
Filed Date | 2011-09-01 |
United States Patent
Application |
20110212129 |
Kind Code |
A1 |
Gerdil; Catherine ; et
al. |
September 1, 2011 |
Method for Producing the Flu Virus
Abstract
The invention relates to a method for producing flu virus
according to which: a) immunizing a hen by administering a flu
vaccine to the hen, b) triggering embryogenesis in one or more eggs
of the immunized hen, c) infecting the one or more embryonated eggs
by inoculating a flu virus into the allantoic cavity of the eggs,
d) incubating the one or more infected embryonated eggs under
temperature and humidity conditions that allow replication of the
virus, and e) harvesting the allantoic fluid of the one or more
incubated eggs containing the virus.
Inventors: |
Gerdil; Catherine; (Tassin
la Demi Lune, FR) ; Moste; Catherine; (Charbonnieres
les Bains, FR) ; Legastelois; Isabelle; (Saint Andeol
le Chateau, FR) ; Bublot; Michel Marie Joseph;
(CHAPONOST, FR) ; Le Gros; Francois-Xavier; (SAINT
GENIS LAVAL, FR) |
Assignee: |
SANOFI PASTEUR S.A.
Lyon
GA
MERIAL LIMITED
Duluth
|
Family ID: |
39146849 |
Appl. No.: |
12/962375 |
Filed: |
December 7, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12238740 |
Sep 26, 2008 |
7871807 |
|
|
12962375 |
|
|
|
|
61063659 |
Feb 4, 2008 |
|
|
|
Current U.S.
Class: |
424/209.1 |
Current CPC
Class: |
C12N 2760/16134
20130101; A61K 2039/5256 20130101; A61K 39/12 20130101; C12N 7/00
20130101; A61K 2039/5252 20130101; A61P 31/16 20180101; A61K
2039/55 20130101; A61K 39/00 20130101; A61P 37/04 20180101; A61K
39/145 20130101; C12N 2710/24143 20130101; C12N 2760/16161
20130101 |
Class at
Publication: |
424/209.1 |
International
Class: |
A61K 39/145 20060101
A61K039/145; A61P 37/04 20060101 A61P037/04; A61P 31/16 20060101
A61P031/16 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 26, 2007 |
FR |
0757884 |
Claims
1. (canceled)
2. (canceled)
3. (canceled)
4. (canceled)
5. (canceled)
6. (canceled)
7. (canceled)
8. (canceled)
9. (canceled)
10. (canceled)
11. (canceled)
12. (canceled)
13. (canceled)
14. (canceled)
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. A flu vaccine composition which comprises a flu virus or a flu
virus-derived product obtained by a) immunizing a hen by
administering a flu vaccine to the hen, b) triggering embryogenesis
in one or more eggs of the immunized hen, c) infecting the one or
more embryonated eggs by inoculating a flu virus into the allantoic
cavity of the eggs, d) incubating the one or more infected
embryonated eggs under temperature and humidity conditions that
allow replication of the virus, e) harvesting the allantoic fluid
of the one or more incubated eggs containing the virus, f)
purifying the virus, and g) inactivating the virus.
21. A method for preventing infection against an epidemic or a
pandemic flu strain in a human subject, which method comprises
administering to the human subject an effective amount of a flu
vaccine composition as claimed in claim 20.
22. A method for preventing flu in an animal selected from the
group of equine family, porcine family, canine family, feline
family, mustelids and avian species, which method comprises
administering to the animal an effective amount of a flu vaccine
composition as claimed in claim 20.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
provisional application 61/063,659, filed Apr. 2, 2008.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to a method for producing the flu
virus using eggs originating from hens immunized against the flu
and also to the use of such a method for the manufacture of a flu
vaccine.
[0004] 2. Summary of the Related Art
[0005] Three types of flu virus (A, B and C) are currently known,
the type A viruses being responsible for animal and human
conditions while the type B and type C viruses are especially
pathogenic for humans. The type A viruses are subdivided into
subtypes according to the antigenic structure of hemagglutinin (HA)
and of neuraminidase (NA) which are the principal glycoproteins of
the viral envelope. Sixteen subtypes of HA (H1 to H16) and 9
subtypes of NA (N1 to N9) stand out. The subtype of a type A virus
is therefore defined by the HA subtype and the NA subtype which are
present in the viral envelope. Wild birds constitute the reservoir
of all influenza A subtypes. Certain subtypes of influenza virus
type A endemically or epidemically (annual epidemics) infect
domestic birds (various subtypes including H5N1 and H9N2), horses
(principally H3N8), pigs (principally H1N1, H3N2 and H1N2) and also
humans (principally H1N1 and H3N2). Dogs, cats and other wild
species can also occasionally be infected with certain subtypes
(H3N8 and H5N1 in dogs; H5N1 in cats).
[0006] In the veterinary field, poultry farms, and more
particularly chicks, chickens, hens and roosters, represent in
terms of number the largest population liable to be affected by the
flu virus. The avian flu strains of subtypes H5 and H7 may be of
two pathotypes: a low path (or LP) pathotype and a high path (or
HP) pathotype. The HP strains are responsible for avian flu and
derive from the LP strains H5 and H7 after mutations/insertions in
particular at the hemagglutinin cleavage site (presence of multiple
basic amino acids). Up until now, strict hygiene measures and
regular controls are strongly recommended in farms in order to
prevent avian flu, and in particular infection with the H5 and H7
subtypes.
[0007] In humans, immunization is recommended against the seasonal
circulating viral strains responsible for epidemics that are more
or less substantial according to the years. Most of the current
vaccines are produced using embryonated hen eggs, these eggs being
infected with three different flu virus strains (two strains of
type A flu virus having the H3N2 and H1N1 subtype and one strain of
type B virus). Eggs fro hens that have not been immunized against
the flu are used in order to prevent any phenomenon of interference
which could be harmful to the replication of the virus. It is in
fact known that the maternal antibodies are transferred to the
chicks after having spent time in the egg and protect them against
microbial infections during the first days of life, but, in return,
they are responsible for deficient immunity if chicks are
prematurely immunized against a microbial agent while protective
maternal antibodies still exist against this agent. H. Stone et al.
(1992, Avian Dis. 36: 1048-1051) have shown that newborn chicks can
be passively immunized against Newcastle disease by inoculating
yolk from eggs originating from hens immunized against the
Newcastle disease virus (NDV). However, if, following the
administration of the egg yolks, the chicks are immunized with the
NDV virus, a decrease in the immune response to the vaccine is
observed. It is also known, according to the studies by Hamal et
al. (2006, Poultry Science 65: 1364-1372), that the rates of
transfer of protective maternal antibodies to newborn chicks, in
particular the transfer of antibodies directed against the NDV
virus or the infectious bronchitis virus (IBV), is between 30% and
40% (percentage of the amount of antibodies in the hen's plasma
circulating in the blood of the three-day-old chick), which
indicates that a large amount of the maternal antibodies is
sequestered in the egg. This is confirmed by the studies of J. R.
Beck et al. (2003, Avian Dis. 47:1196-1199), which show that all
the eggs contain anti-HA antibodies, approximately 3 weeks after
having immunized hens with a strain of inactivated flu virus.
Finally, it is known, according to the studies by Fontaine et al.
(1963, Pathobiologie, 11/9: 611-613), that if embryonated eggs are
inoculated with anti-flu serum, the eggs are protected against
infection by the flu virus. All these reasons have led those
skilled in the art to consider that if eggs from hens immunized
against the flu were used, said eggs would, due to the transfer of
the maternal antibodies directed against the flu into the eggs,
become incapable of producing flu viruses.
[0008] Since the beginning of the 2000s, the economic consequence
of avian flu in domestic bird farms has not ceased to increase with
the appearance of highly contagious and pathogenic avian virus
strains which decimate entire poultry farms. The typing of the HAs
of highly pathogenic virus strains shows that almost all of them
have the H5 or H7 subtype. It is now feared that virus strains
having the H5 or H7 subtype will adapt to humans and may be
responsible for a real flu pandemic in humans; serious cases of
human flu, admittedly isolated, involving these subtypes have
already been reported.
[0009] Faced with the risk that the supply of eggs may no longer
always be ensured for manufacturing the flu vaccine, new methods of
producing vaccines against the flu are currently directed toward
the use of cell culture systems.
SUMMARY OF THE INVENTION
[0010] Despite new methods of producing vaccines, there still
exists a need to be able to produce, under any circumstances, in a
short period of time and in very large amount, flu virus for
manufacturing the flu vaccine. The present invention meets this
need by describing a method for producing flu virus based, against
all expectations, on the use of eggs originating from hens
immunized beforehand against the flu.
[0011] A subject of the invention is in fact:
[0012] A method for producing flu virus comprising: [0013] a)
immunizing a hen by administering a flu vaccine to the hen, [0014]
b) triggering embryogenesis in one or more eggs of the immunized
hen, [0015] c) infecting the one or more embryonated eggs by
inoculating a flu virus into the allantoic cavity of the eggs,
[0016] d) incubating the one or more infected embryonated eggs
under temperature and humidity conditions that allow replication of
the virus, and [0017] e) harvesting the allantoic fluid of the one
or more incubated eggs containing the virus.
[0018] Preferably, the vaccine protects the hens against avian
flu.
[0019] Typically, the flu vaccine comprises, in its composition,
the hemagglutinin of a flu virus in the form of protein and/or of a
gene encoding this protein.
[0020] According to one aspect, the composition of the flu vaccine
contains an inactivated whole flu virus.
[0021] According to another aspect, the composition of the flu
vaccine contains a product derived from a whole flu virus.
[0022] According to yet another aspect, the composition of the
vaccine also contains an adjuvant.
[0023] In another aspect, the composition of the flu vaccine
contains an attenuated flu virus.
[0024] According to another aspect, the flu vaccine comprises a
vector comprising a nucleic acid fragment encoding the
hemagglutinin of a flu virus.
[0025] According to another aspect, the composition of the vaccine
also contains an adjuvant.
[0026] Preferably, the vector is a poxvirus.
[0027] In a specific aspect, the vector also comprises a nucleic
acid fragment encoding the neuraminidase of a flu virus.
[0028] According to one embodiment of the method according to the
invention, the flu virus hemagglutinin in the form of protein
and/or of a gene encoding this protein contained in the composition
of the vaccine which is used to immunize the hens against the flu
and the hemagglutinin of the flu virus which is used to infect the
allantoic cavity of the embryonated eggs from the immunized hens
are of different subtypes.
[0029] According to another embodiment, the flu virus hemagglutinin
in the form of protein and/or of a gene encoding this protein
contained in the composition of the vaccine which is used to
immunize the hens against the flu and the hemagglutinin of the flu
virus which is used to infect the allantoic cavity of the
embryonated eggs from the immunized hens are of the same
subtype.
[0030] According to yet another embodiment, the flu virus
hemagglutinin in the form of protein and/or of a gene encoding this
protein contained in the composition of the vaccine which is used
to immunize the hens against the flu and the hemagglutinin of the
flu virus which is used to infect the allantoic cavity of the
embryonated eggs from the immunized hens are identical.
[0031] According to yet another embodiment, the flu virus contained
in the composition of the vaccine which is used to immunize the
hens against the flu is identical to the flu virus which is used to
infect the allantoic cavity of the embryonated eggs from the
immunized hens.
[0032] In a particularly preferred embodiment, the flu virus
hemagglutinin in the form of protein and/or of a gene encoding this
protein contained in the composition of the vaccine which is used
to immunize the hens against the flu and the hemagglutinin of the
flu virus which is used to infect the allantoic cavity of the
embryonated eggs from the immunized hens are independently selected
from those of the H5, H6, H7 or H9 subtype.
[0033] In another particularly preferred embodiment, the flu virus
hemagglutinin in the form of protein and/or of a gene encoding this
protein contained in the composition of the vaccine which is used
to immunize the hens against the flu and the hemagglutinin of the
flu virus which is used to infect the allantoic cavity of the
embryonated eggs from the immunized hens are independently of the
H5 or H7 subtype.
[0034] In a specific aspect, the method according to the invention
comprises an additional step of purification of the virus.
[0035] In another specific aspect, the method according to the
invention comprises an additional step of inactivation of the
virus.
[0036] The invention also comprises a flu vaccine obtained using a
method according to the invention.
[0037] A subject of the invention is also the use of a method
according to the invention for the manufacture of a vaccine for use
in preventing the flu.
[0038] In a specific aspect, the use of the method according to the
invention serves to manufacture a vaccine for use in preventing
pandemic human flu.
[0039] In another specific aspect, the use of a method according to
the invention serves to manufacture a vaccine for use in preventing
epidemic human flu.
[0040] In yet another aspect, the use of a method according to the
invention serves to manufacture a vaccine for use in preventing the
flu in members of the equine family, members of the porcine family,
members of the canine family, members of the feline family,
mustelids and avian species.
[0041] A subject of the invention is also the use of the eggs from
hens immunized against the flu for the production of a flu
virus.
[0042] Finally, a subject of the invention is the use of the eggs
from hens immunized against the flu for the manufacture of a flu
vaccine.
[0043] In one aspect, the eggs from immunized hens contain
antibodies against the hemagglutinin of flu virus.
[0044] In a further aspect, the eggs from immunized hens contain
antibodies against H5, H6, H7 or H9 subtype.
[0045] All patents, patent applications, and publications referred
to herein are hereby incorporated by reference.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING
[0046] FIG. 1. represents the complete nucleotide sequence of the
donor plasmid pJY1394.1.
[0047] FIG. 2. represents the nucleotide sequence of the insert in
the donor plasmid pJY1394.1 comprising the arms flanking the
insertion locus F8, and also the H6 vaccinia promoter followed by
the synthetic gene modified at the cleavage site encoding the HA of
the A/chicken/Indonesia/7/03 strain.
DETAILED DESCRIPTION OF THE INVENTION
[0048] The phrase "flu virus" denotes both flu virus originating
from a wild-type strain and flu virus originating from a
reassortant strain which results from the reassortment of the
genomic segments of one or more wild-type strains with a "master"
strain selected for its strong growth potential in eggs. The
reassortant strain acquires characteristics of the "master" strain
but keeps at least the characteristics of the HA and of the NA of
the wild-type strain, which means that the identity of the protein
structure of the HA and of the NA of the reassortant strain with
respect to the HA and to the NA of the wild-type strain, determined
by means of an overall alignment program, is at least 95%,
preferably at least 98%, more preferably at least 99%, and even
more preferably 100%. The reassortant strain can be obtained by
coinfection of a sensitive cell with the wild-type strain and the
"master" strain, followed by the appropriate means for selecting
the desired reassortant strain. It can also be obtained by reverse
genetics from the nucleic acids of the wild-type strain and of the
"master" strain and expression in multiplasmid expression systems
as described in WO 01/83794 and WO 03/091401 and in Proc. Nat.
Acad. Sci. USA 96:9345-9350 (1999). In the case of the high path H5
and H7 strains, a modification of the cleavage site comprising the
multiple basic amino acids may be carried out so as to render the
reassortant strain low path.
[0049] For ease of language, the term "vaccinal strain" or
"vaccinal virus" is used without distinction to denote the flu
virus used for the manufacture of the flu vaccine; similarly, the
term "infecting strain" or "infecting virus" is used to denote the
flu virus used to infect biological material (eggs, animals).
[0050] Moreover, the term "eggs from immunized hens" is used to
denote fertilized hen eggs originating from hens which have been
immunized beforehand and brought into contact with roosters.
[0051] In general, the flu vaccine used for the immunization of the
hens can be manufactured from any strain of flu virus. The vaccinal
strain may be a type A virus, but also a type B or C virus. When it
is a strain of type A virus, the virus may be any subtype of HA
and/or any subtype of NA. It may, for example, be viral strains
having the H1N1 or H3N2 subtype currently responsible for epidemic
human type A flu.
[0052] There is a great advantage in immunizing hens with a flu
virus provided that the immunization confers protection against
avian flu.
[0053] Avian flu can pass unnoticed or can be characterized by a
set of manifestations, often of respiratory and/or intestinal
nature, of more or less great intensity, which more or less impair
the general condition of the hens and which can result in the
animal's death when the virus strain is highly pathogenic. Avian
flu commonly results in a decrease or even a disappearance of the
egg-laying activity. The low path virus strains belonging to the
H6N2 or H9N2 or even H5 or H7 subtypes are commonly responsible for
mild forms of avian flu, generally resulting in a decrease or a
disappearance of egg production, but no great mortality. On the
other hand, the high path virus strains belonging to the H5 and H7
subtypes (in particular H5N1, H5N2, H5N9, H7N1, H7N4 or H7N7) are
highly virulent and cause a very high degree of mortality in hen
farms.
[0054] HA is an antigen that is essential in the development of
protective immunity against the flu. The vaccine used in the method
according to the invention comprises in its composition at least
the HA of a flu virus in the form of protein and/or of a gene
encoding this protein.
[0055] The term "gene" is intended to mean a nucleic acid
comprising nucleotide sequence corresponding to an open reading
frame (ORF) and encoding a protein. The gene under the control of
regulatory sequences for expression (promoter, enhancer,
polyadenylation signal, transcription stop, etc.) can be inserted
into the nucleic acid of a vector, in particular a plasmid or a
virus. The regulatory sequences can be of exogen or endogen origin
with respect to the ORF encoding the protein.
[0056] It may involve the HA of a human strain of flu virus but
which is not pathogenic for hens. Preferably, it contains an HA of
interest, i.e., an HA which has the same subtype as the HA of a
viral strain which is responsible for an avian flu and against
which it is sought to immunize and to protect the hens. Preferably,
the degree of identity between the protein sequence of the HA
present in the vaccine and that of the strain against which it is
desired to protect the hens is at least 80%, preferably at least
90%, and even more preferably at least 95%, determined by means of
an overall alignment program (such as the Blast program).
[0057] Conventionally, the vaccine comprising the HA is in the form
of a composition containing inactivated whole flu virus, or a
product derived from the inactivated whole flu virus.
[0058] The expression "product derived from an inactivated whole
virus" is intended to mean an inactivated (i.e. noninfectious)
vaccinal composition which is prepared from a strain of virus and
which comprises at least the HA of said strain of virus. The
product derived from a strain of whole virus may be fragmented (or
split) virus and, in this case, reference is made to a "split"
vaccine. Another product derived from a strain of whole virus is
the HA of this strain as such or associated with the NA which has
been obtained using extraction and purification methods and, in
this case, reference is made to a "subunit" vaccine (EP 0 776 362).
The HA may also be integrated secondarily into a virosome. The
vaccinal composition containing inactivated whole flu virus, or a
product derived from inactivated whole virus, may also contain one
or more adjuvants or adjuvant formulations. As an example of
nonlimiting adjuvant formulations, mention is made of water-in-oil
or oil-in-water emulsions, such as the MF59 emulsion (Vaccine
Design--The Subunit and Adjuvant Approach Edited by M. Powell and
M. Newman, Plenum Press, 1995 page 183), liposome-based
formulations, and formulations based on MPL (Vaccine Design--The
Subunit and Adjuvant Approach Edited by M. Powell and M. Newman,
Plenum Press, 1995 pages 1186-187), on avridine (Vaccine
Design--The Subunit and Adjuvant Approach Edited by M. Powell and
M. Newman, Plenum Press, 1995 page 148), on
dimethyldioctadecylammonium bromide (Vaccine Design--The Subunit
and Adjuvant Approach Edited by M. Powell and M. Newman, Plenum
Press, 1995 page 157), on Corynebacterium parvum, on saponin, on
lysolecithin, on pluronic derivatives (Hunter H. et al. 1991,
vaccine, 9: 250-256) (Vaccine Design--The Subunit and Adjuvant
Approach Edited by M. Powell and M. Newman, Plenum Press, 1995 page
200 and pages 297-311), on aluminum salts or on combinations
thereof (Vaccine Design--The Subunit and Adjuvant Approach Edited
by M. Powell and M. Newman, Plenum Press, 1995 pages 249-276).
Preferably, the water-in-oil emulsions are composed of liquid
paraffin, of a hydrophilic surfactant such as polysorbate 80 or
polysorbate 85 and of a lipophilic surfactant such as sorbitan
oleate or sorbitan trioleate.
[0059] Examples of emulsions used in the hen are described in Stone
et al. (1983, Avian Dis., 27: 688-697; 1993; Avian Dis., 37:
399-405; 1991, Avian Dis., 35: 8-16); in M. Brugh et al. (1983, Am.
J; Vet. Res., 44: 72-75); in Woodward L. et al. (1985, Vaccine, 3:
137-144); in (Vaccine Design--The Subunit and Adjuvant Approach
Edited by M. Powell and M. Newman, Plenum Press, 1995 page 219).
The vaccinal strain generally originates from a wild-type strain
which has been isolated in hens, turkeys, ducks, geese or in other
avian species, this strain most commonly being low path for hens.
As an example of isolates (wild-type strains) used for the
preparation of vaccines for protecting hens against avian flu
subtype H5 or H7, mention is made of the A/turkey/Wisconsin/68 or
A/chicken/Italy/22A/98 isolates which are viral strains having the
H5N9 subtype, the A/turkey/England/N-28/73,
A/chicken/Mexico/238/94/CPA, A/chicken/Mexico/232/94/CPA or
A/duck/Potsdam/1402/86 isolates which are viral strains having the
H5N2 subtype, the A/goose/Guandong/1/1996 isolate which is an HP
viral strain having the H5N1 subtype, the
A/chicken/Italy/AG-473/1999 or A/chicken/Italy/1067/1999 isolates
which are viral strains having the H7N1 subtype, the
A/chicken/Pakistan/95 isolate which is an HP strain having the H7N3
subtype, and the A/duck/Potsdam/15/80 isolate which is a viral
strain having the H7N7 subtype. As an example of H9N2 viral strains
used for protecting domestic birds against avian flu subtype H9,
mention is made of the A/chicken/Iran/AV12221/98 and
A/chicken/UAE/415/99 isolates. As an example of an H6N2 viral
strain used for protecting hens against avian flu subtype H6,
mention is made of the A/turkey/Italy/90 isolate. The vaccinal
strains used for the manufacture of vaccines may also be
reassortants of wild-type strains obtained in particular by reverse
genetics. Mention is in particular made of the Re-1 vaccinal strain
which is a reassortant strain obtained by reverse recombination of
the H5N1 wild-type strain A/goose/Guandong/1/96 with the "master"
strain A/PR/8/34, which reproduces very well in eggs (Tian et al.,
2005, Virology, 341: 153-162). Another example of a reassortant
strain obtained by reverse genetics is the H5N3 strain obtained by
genetic reassortment and containing the H5 hemagglutinin of the
A/chicken/Vietnam/C58/04 H5N1 strain, the neuraminidase of the
A/duck/Germany/1215/73 H2N3 strain and the internal genes of the
A/PR/8/34 "master" strain (Webster et al., 2006, Virology, 351:
301-311).
[0060] The vaccines against avian flu usually contain the virus of
a single inactivated flu virus strain (monovalent vaccines) but, in
certain cases, it may be advantageous to use multivalent vaccines
containing several inactivated flu virus strains. This is in
particular a vaccine based on H7N1 and H5N9 strains which is a
water-in-oil emulsion containing the inactivated vaccinal strains
A/chicken/Italy/22A/98 (H5N9) and A/chicken/Italy/1067/1999 (H7N1).
The inactivated vaccines may also contain different valences, for
example a divalent vaccine against H9N2 avian flu and Newcastle
disease. The inactivated vaccines are generally administered
parenterally (intramuscularly or subcutaneously). They can also be
administered in the form of a spray, as in the case of the Aerovac
AI vaccine sold by Investigacion Aplicada, which contains the
inactivated vaccinal strain A/chicken/Mexico/232/94/CPA (H5N2). The
immunization scheme usually comprises one or two administrations 2
to 4 weeks apart. The vaccinal dose administered varies according
to the age of the animals, but usually contains the equivalent of
10 to 200 .mu.l of allantoic fluid having a titer of 10.sup.8 to
10.sup.10 EID.sub.50/ml before inactivation. The vaccinal dose is
usually administered in a volume ranging from 0.05 to 1 ml. The
preparation of inactivated avian flu vaccines is described by H.
Stone (1987, Avian Dis. 31: 483-490). Insofar as the vaccinal
strain HA subtype is the same as that of the HA of the strain
responsible for pathogenic avian flu, and on the basis of a degree
of identity between the protein sequences of the two HAs of the
order of 80% to 90%, determined by means of an overall alignment
program, the protection rate obtained against the clinical symptoms
of avian flu (morbidity and mortality) is generally more than 80%,
and preferably more than 90%. This is confirmed by the studies of
M. Bublot et al., 2007, Avian Dis. 51: 332-337, which show that a
degree of identity of the order of 80% to 90% between the protein
sequence of the vaccinal strain HA and the protein sequence of the
pathogenic infecting strain HA is sufficient to obtain this
protection rate. Furthermore, it is not necessary for the NA
subtype of the vaccinal strain to be the same as that of the NA of
the pathogenic infecting strain in order to obtain this protection
rate. Moreover, the avian flu vaccines greatly reduce the excretion
of the virus in immunized animals challenged with infectious virus,
demonstrated by a very clear decrease in the viral load observed in
oral and cloacal samples (M. Bublot et al., 2007, Avian Dis. 51:
332-337). Another beneficial effect of avian flu vaccines is that
of reducing the diffusion of the virus in hen farms.
[0061] According to another embodiment of the method according to
the invention, the vaccine used to immunize the hens is in the form
of a composition comprising the virus originating from a live
attenuated flu virus strain. The vaccinal strain is generally in
the form of a reassortant which has been selected subsequent to
genetic reassortment between a wild-type strain expressing the HA
of interest and secondarily the NA of interest, and a "master"
strain which has been cold-adapted and/or which is
temperature-sensitive. The reassortant is a viral strain which
expresses at its surface the HA and secondarily the NA of interest
while at the same time retaining the phenotypic characteristics of
the "master" strain which relate to its ability to replicate only
within narrow temperature limits, below that of the internal
temperature of birds. As a result, the vaccinal strain, after
having been administered to hens, replicates in a limited manner
and locally. The methods for obtaining these reassortant strains
are well known to those skilled in the art and are described in
particular in WO 03/091401 and WO 2006/063053 and in 2002, Vaccine
20: 2082-2090. The administration of the vaccine is generally
carried out by nebulization. Another means of producing an
attenuated strain is to truncate the gene encoding the NS1 protein
(Richt J.A. et al., Vaccination of pigs against swine influenza
viruses by using an NS1-truncated modified live-virus vaccine,
2006, J. Virol., 80: 11009-18; Quinlivan M. et al., Attenuation of
equine influenza viruses through truncations of the NS1 protein,
2005, J. Virol., 79: 8431-9).
[0062] The vaccinal strains can be produced by any means, using
culture techniques on cells such as Vero cells, MDCK cells, PER.C6
cells or chicken embryo cells (CEK, PCJ) and/or using conventional
methods of production on embryonated eggs. The methods for
harvesting, purifying and, as appropriate, inactivating the virus
are also well known to those skilled in the art.
[0063] According to another embodiment of the method according to
the invention, the vaccine is in the form of proteins produced in
an in vitro expression system. For example, the HA can be produced
in an expression system using a recombined baculovirus in insect
cells (Crawford J. et al., Baculovirus-derived hemagglutinin
vaccines protect against lethal influenza infections by avian H5
and H7 subtypes, 1999, Vaccine, 17: 2265-74). The hemagglutinin can
also be expressed in vitro in the form of "virus-like particles"
(VLPs) (Prel A. et al., Assessment of the protection afforded by
triple baculovirus recombinant coexpressing H5, N3, M1 proteins
against a homologous H5N3 low-pathogenicity avian influenza virus
challenge in Muscovy ducks, 2007, Avian Dis., 51: 484-9; Pushko P.
et al., Influenza virus-like particles comprised of the HA, NA, and
M1 proteins of H9N2 influenza virus induce protective immune
responses in BALB/c mice, 2005, Vaccine, 23: 5751-9) or of a
retrovirus-based pseudotype (Szecsi J. et al., 2006, Virol. J., 3:
70). The proteins produced in vitro or the viral particles produced
are more or less purified and then adjuvanted with various
adjuvants, such as those used in inactivated vaccines.
[0064] According to another embodiment of the method according to
the invention, the flu vaccine comprises a vector comprising a
nucleic acid fragment encoding the flu virus HA.
[0065] The term "vector" refers to nucleic acid structures which
can be propagated in and/or transferred into organisms, cells or
cell components. This includes in particular plasmids, viruses,
bacteriophages, proviruses, phagemids and artificial chromosomes
which are capable of replicating autonomously or which can
integrate into the chromosome of a host cell.
[0066] The expression "vector comprising a gene encoding the flu
virus HA and/or NA" is intended to mean a vector containing the
nucleic acid encoding the HA and/or NA of interest and which, after
introduction into an avian cell, expresses the HA and/or NA in this
cell. It may be a plasmid expressing the HA of interest, but it is
generally a viral vector containing, in its genome, the nucleic
acid encoding the HA of interest and expressing the HA of interest
in the infected cells. The integration of the nucleic acid encoding
the HA into the genome of the viral vector is generally carried out
by molecular biology techniques, in particular genetic
recombination, cloning, reverse genetics, etc. The HA may or may
not be expressed at the surface of the viral vector. Preferably,
the viral vector has been conventionally attenuated by multiple
passages in vitro or by deletion of certain genes so that the
replication of the vector virus in avian cells is sufficiently
limited and has no effect on the general state of the hens and is
thus considered to be nonpathogenic. As an example of viral
vectors, mention is made of avian paramyxoviruses (Ge J., et al.,
Newcastle disease virus-based live attenuated vaccine completely
protects chickens and mice from lethal challenge of homologous and
heterologous H5N1 avian influenza viruses, 2007, J Virol., 81:
150-8); the turkey herpesvirus (HVT) or Marek's disease herpesvirus
(Sondermeijer et al., 1993, Vaccine, 11, 349-358); the infectious
laryngotracheitis virus (ILTV) (Veits J., et al., Deletion of the
non-essential ULO gene of infectious laryngotracheitis (ILT) virus
leads to attenuation in chickens, and ULO mutants expressing
influenza virus haemagglutinin (H7) protect against ILT and fowl
plague, 2003, J Gen. Virol., 84: 3343-52; Luschow D. et al.,
Protection of chickens from lethal avian influenza A virus
infection by live-virus vaccination with infectious
laryngotracheitis virus recombinants expressing the hemagglutinin
(H5) gene, 2001, Vaccine, 19: 4249-59); adenoviruses (Francois A.
et al., Avian adenovirus CELO recombinants expressing VP2 of
infectious bursal disease virus induce protection against bursal
disease in chickens, 2004, Vaccine, 22: 2351-60; Gao W. et al.,
Protection of mice and poultry from lethal H5N1 avian influenza
virus through adenovirus-based immunization, 2006, J Virol., 80:
1959-64; Toro H. et al., Protective avian influenza in ovo
vaccination with non-replicating human adenovirus vector, 2007,
Vaccine, 25: 2886-91); coronaviruses (Cavanagh, 2007, Vet Res. 38:
281-97; Eriksson, 2006, Clin. Dev. Immunol. 13: 353-60), but use is
preferably made, for immunizing hens, of poxviruses, in particular
the vaccinia virus, NYVAC (deleted vaccinia virus), the vaccinia
virus MVA strain, particularly avipoxes, especially canary pox,
ALVAC (attenuated canary pox), pigeon pox, quail pox, turkey pox,
sparrow pox and most particularly fowl pox, TROVAC (attenuated
fowlpox), which are described in particular in AU 701599B and AU
701781B and in U.S. Pat. No. 5,756,103. As appropriate, the
attenuated viral vectors express only the HA of interest: the
vaccine involved is in particular the vaccine containing a TROVAC
fowlpox vector expressing the HA of the H5N8 flu virus strain
(A/turkey/Ireland/1378/83). In other cases, the attenuated viral
vector expresses both the HA of interest in combination with an NA
of interest, such as the recombinant poxvirus described in 2003,
Avian Pathology, 32: 25-32, which expresses both the HA and the NA
originating from an H5N1 flu virus strain. An NA of interest is an
NA which has the same subtype as the NA of the viral strain against
which it is sought to immunize and protect the hens. In yet other
cases, the attenuated vector expresses several HAs of interest
belonging to different subtypes, as in the case of the recombinant
poxvirus described in 2006, Vaccine, 24: 4304-4311, which expresses
both the H5 and H7 subtypes. The immunogenic capacity of these
vectors can be further reinforced by introducing therein the genes
encoding cytokines and/or chemokines which exert an
immunostimulatory capacity, such as IL-1, IFN, CSF, GM-CSF, IL-2,
IL-12, IL-18 or TNF 5 (2006, Vaccine, 24: 4304-4311). The vaccines
based on vectors encoding the flu virus HA can also be adjuvanted
in order to increase their immunogenicity.
[0067] The vaccinal compositions containing viral vectors can be
administered by various routes, which depend in particular on the
vector: for example, transfixion of the alar membrane (poxvirus
vector), via the intramuscular, subcutaneous or transdermal route
with or without needle (any vector), via the in ovo route (in the
17- to 19-day embryonated egg; for example, HVT/Marek and
adenovirus vector), via the ocular or oronasal route, by spray, or
in the drinking water (paramyxovirus, coronavirus, adenovirus
vector), in one or two injections at least 15 days apart. The
vaccinal dose(s) administered is (are) of the order of 1 to 7
log.sub.10 50% infectious unit with a preference for a dose of 2 to
4 log.sub.10 for fowlpox vectors. The advantage of an immunization
based on a viral vector compared with a conventional immunization
using inactivated or attenuated whole flu virus lies in the fact
that the immunized animals can be distinguished from the infected
animals. Furthermore, immunization with a viral vector promotes the
development of a cellular immunity that can reinforce the
protection of the animals. As is illustrated in 2007, Avian Dis.,
51: 325-331 and 2007, Avian Dis., 51: 498-500, the degree of
protection obtained in the hens and the decrease in diffusion of
the virus in poultry farms are of the same order as that which is
observed with a conventional vaccine containing inactivated flu
virus.
[0068] When the immunization of the hens comprises several
injections, the vaccine used in the first administration may be
different than that used in the second injection or the subsequent
injections. Two different attenuated viral vectors may be used, for
instance using a recombinant ALVAC vector expressing the HA of
interest in the first immunization and a recombinant TROVAC or
NYVAC vector expressing the same HA of interest in subsequent
immunizations such that the antibody response directed against the
ALVAC vector does not prevent the infection of the hen cells by the
recombinant TROVAC or NYVAC vector and, consequently, the
expression of the HA in the infected cells. It is also possible to
use the "prime boost" method, which consists in using, in the first
injection, an attenuated viral vector expressing an HA and in
using, in the boost injection(s), a vaccine containing for example
one or more inactivated vaccinal strains belonging to the same
subtype as the HA used in the first immunization, or alternatively
carrying out the process in reverse order. Finally, it is possible
to carry out a DNA immunization in the first injection, using a
plasmid expressing the HA of interest, followed by boost injections
using a vaccine containing an inactivated vaccinal strain and/or an
attenuated viral vector which express an HA belonging to the same
subtype as the HA used in the first immunization or which is
identical to the HA used in the first immunization.
[0069] Whatever the type of vaccine administered or the
immunization scheme adopted, the protection of the hens against
avian flu is provided quite rapidly, generally within a period of 7
to 18 days after the administration of a vaccinal dose. However, in
order to ensure protection of the hens against avian flu throughout
their egg-laying activity, which lasts approximately one year, one
or two booster immunizations, which are carried out within a period
of 3 to 16 weeks after the first vaccinal administration, are
recommended. Several immunization schemes with inactivated vaccines
can be used in egg-laying hens: for example, 2 injections, the
first at 3 to 6 weeks old and the second at 16 to 19 weeks old,
just before beginning egg laying, or 3 injections, the first around
2 to 4 weeks, the second 3 to 4 weeks later and the third at 16 to
19 weeks old just before beginning egg laying. A booster can also
be administered during egg laying. In the "prime-boost" scheme
using 2 different vaccines, the chicks can be immunized at 1 day
old with a fowlpox vector-based vaccine; they subsequently receive
one (at 16 to 19 weeks old just before beginning egg laying) or two
(at 3 to 6 weeks old and at 16 to 19 weeks old just before
beginning egg laying) immunizations with a vaccine containing
inactivated flu virus.
[0070] In the implementation of the method according to the
invention, the eggs are preferably taken from the hens once the
protection of the hens against avian flu is ensured, which usually
occurs within a period of 7 to 18 days after the administration of
the vaccine (Bublot M. et al. (2006, Annals of the New York Academy
of Sciences 1081: 193-201); Van der Goot et al. (2005, Proc. Natl.
Acad. Sc., 102: 18141-6); Ellis et al. (Avian Pathol. 2004, 33,
405-412)).
[0071] Despite the presence of flu antibodies in the eggs of
immunized hens, especially of antibodies directed against HA, and
in particular of antibodies inhibiting hemagglutination (IHA) which
block the penetration of the flu virus into sensitive cells, the
method used for producing flu virus from embryonated eggs
originating from hens immunized against the flu is conventional. 9-
to 14-day embryonated eggs, originating from immunized hens which
have been reared preferably in a controlled environment, are used.
The embryogenesis process is controlled in the following way: eggs
which have been conserved at a temperature between 10 and
20.degree. C., preferably between 16 and 18.degree. C., for a
period which does not in general exceed one week after laying, are
used. The embryogenesis process is triggered by incubating the eggs
at a temperature of 37.5.degree. C..+-.1.degree. C. in a humid
chamber having a relative humidity of 70.+-.10% for a period of
between 9 and 14 days. The embryonated eggs are selected by
candling, and their allantoic cavities are infected with a dose of
virus generally between 2 and 7 log.sub.10 TCID.sub.50 in a small
volume (approximately 0.1 to 0.2 ml). The virus is allowed to
multiply for a period generally ranging from 1 to 4 days depending
on the virulence of the viral strain, and at a temperature which
can also vary according to the phenotype of the virus strain and
its degree of cold- or hot-adaptation. The temperature for
multiplication of the flu virus is generally in a range of from 28
to 39.degree. C. and normally in a temperature range of from 33 to
39.degree. C. The infectious allantoic fluids are then harvested
and processed according to the uses intended to be made
thereof.
[0072] This method can be used to produce a flu virus whose HA
subtype is different than the subtype of the HA contained in the
vaccine which was used to immunize the hens. This is, for example,
the case where the hens are immunized with an inactivated H5N9
virus in order to protect the hens against H5 avian flu, while the
eggs from these hens are infected with an H1N1 or H3N2 flu virus,
or even a type B flu virus in order to prepare a vaccine against
the epidemic forms of current human flu. According to a variant of
the method according to the invention, the HA subtype of the flu
virus which is produced using the embryonated eggs from immunized
hens has a subtype that is different than the HA which was used to
immunize the hens, but, on the other hand, the NA of the flu virus
produced has the same subtype as the NA of the virus used in the
vaccine. This is, for example, the case where the hens are
immunized with an inactivated H5N1 virus strain or a recombinant
poxvirus expressing H5 and N1 in order to protect the hens against
avian flu, while the embryonated eggs from the immunized hens are
infected with an H1N1 virus strain.
[0073] According to another mode of the method according to the
invention, the HA of the flu virus produced on embryonated eggs
from immunized hens has the same subtype as the HA contained in the
vaccine which was used to immunize the hens. This is, for example,
the case where the hens are immunized with inactivated H5N1 virus
or a recombinant poxvirus expressing H5, while the eggs from the
immunized hens are infected with an H5N1 or H5N9 flu virus. Despite
the presence of "subtype"-specific crossreactive anti-HA antibodies
in the egg (this is the case when the HA contained in the vaccinal
composition has the same subtype as the HA of the flu virus strain
infecting the embryonated eggs from immunized hens), this has no
negative effect on the replication of the virus.
[0074] Furthermore, even more surprisingly, this replication is
also not affected in the case where there is complete identity
between the flu virus which was used to infect the embryonated eggs
from immunized hens and the flu virus which was used to immunize
these hens. In this case, this is reflected by the presence in the
eggs of a panel of anti-HA antibodies which is even broader since
both subtype-specific crossreactive anti-HA antibodies and anti-HA
antibodies that are highly specific for the strain (also called
strain-specific antibodies) are found (see example 3). Contrary to
widely established opinion, the presence of flu antibodies in the
egg and, in particular, the presence of anti-HA antibodies does not
therefore affect the replication of the flu virus. The amounts of
virus and/or of hemagglutinating antigen harvested in the infected
allantoic fluids originating from eggs from hens immunized against
the flu are of the same order as those harvested in infected
allantoic fluids originating from eggs from nonimmunized hens (see
examples 1 and 2).
[0075] The method according to the invention can also be taken
advantage of for manufacturing a reassortant virus strain. In this
case, in a first step, embryonated eggs from immunized hens are
coinfected with a wild-type virus strain and a master virus strain
which replicates well in embryonated eggs, for instance the
A/PR/8/34 strain. In a second step, the infected allantoic fluids
containing essentially a mixture of reassortants and the master
strain, while the wild-type strain which is not as capable of
replicating is generally in very small amount, are collected. The
reassortant strain expressing at the same time the phenotypic
characteristics of the A/PR/8/34 strain (i.e., its good ability to
replicate in embryonated eggs) and the HA and also the NA of the
wild-type strain is then selected with successive cloning steps by
mixing at each cloning step the harvested infectious allantoic
fluid with anti-HA and anti-NA antibodies specific for A/PR/8/34
according to methods well known to those skilled in the art. It is
also possible to manufacture a cold-adapted and heat-sensitive
reassortant strain with a view to a live attenuated virus vaccine.
In this case, in a first step, embryonated eggs from immunized hens
are coinfected with a wild-type virus strain and a master strain
which has the phenotypic characteristic of being cold-adapted and
heat-sensitive. In this case, the temperature for incubation of the
infected eggs is a temperature that is lower than normal (the
temperature is commonly below 35.degree. C., or even below
30.degree. C.). In a second step, the infected allantoic fluids
containing essentially a mixture of reassortants and the master
strain, since the cold-sensitive wild-type strain has not
replicated, are collected. The reassortant expressing at the same
time the phenotypic characteristics of the master strain (in
particular the cold-adaptation and/or its thermosensitivity) and
the HA and also the NA of the wild-type strain is then selected
with successive cloning steps by mixing at each cloning step the
harvested infectious allantoic fluid with anti-HA and anti-NA
antibodies specific for the master strain according to methods well
known to those skilled in the art.
[0076] The method according to the invention can be carried out as
first line for preparing vaccines intended to protect breeding
colonies of hens and more generally breeding colonies of domestic
birds (ducks, turkeys, geese, etc.) against avian flu. The viral
strains implicated in the asymptomatic or mild forms of avian flu
may be of any subtype, and in particular the H9N2, H6N2, H7N2, H7N3
or H7N1 subtypes. They do not cause any substantial mortality in
the breeding colonies but may be the cause of a transient decrease
in egg production. The high path viral strains implicated in the
serious forms of avian flu which cause substantial mortality in
breeding colonies belong generally to the H5 and H7 subtypes, and
in particular H5N1, H5N2, H5N8, H5N9, H7N1, H7N3, H7N4 or H7N7.
[0077] Generally, the flu virus hemagglutinin contained in the
vaccinal composition which is used to immunize the hens against
avian flu of the method according to the invention and the
hemagglutinin of the virus which is used to infect the embryonated
eggs of the method have the same subtype and are selected in
particular from the H5, H6, H7 and H9 subtypes, since it is those
which are found principally in the strains of virus responsible for
avian flu.
[0078] In a specific aspect, the flu virus hemagglutinin contained
in the vaccinal composition which is used to immunize the hens
against avian flu of the method according to the invention and the
hemagglutinin of the virus which is used to infect the embryonated
eggs of the method have the same subtype and are selected from the
H5 and H7 subtypes, since it is those which are found in the
strains of virus responsible for the serious forms of avian flu and
of human flu. The strains of virus responsible for the serious
forms of avian flu and/or of human flu which have been
characterized generally belong to the H5N1, H5N2, H7N1, H7N3 or
H7N7 subtypes.
[0079] Thus, very specifically, a subject of the invention is:
[0080] A method for producing the flu virus comprising: [0081] a)
immunizing a hen with an inactivated flu virus, wherein the
hemagglutinin of the virus has the H5 or H7 subtype, [0082] b)
triggering embryogenesis in one or more eggs of the immunized hen,
[0083] c) infecting the one or more embryonated eggs by introducing
into the allantoic cavity of the embryonated eggs a flu virus
identical to that that used for the immunization, [0084] d)
incubating the one or more infected embryonated eggs under
temperature and humidity conditions that allow replication of the
virus, and [0085] e) harvesting the allantoic fluid containing the
virus of the one or more incubated eggs.
[0086] When the immunization scheme of the hens comprises two
injections, the first injection may be made with a vaccine
composition wherein the inactivated flu virus is replaced by a
vector, preferably a poxvirus comprising a gene encoding the H5 or
H7 subtype of hemagglutinin.
[0087] When the infected allantoic fluids are intended for the
production of a flu vaccine, the method according to the invention
generally comprises an additional step of purifying the virus
strain and is optionally followed or preceded by a viral
inactivation step using methods well known to those skilled in the
art such as those described in FR 2201079 or in FR 1538322.
[0088] The purification may be brief and may be limited to a step
of concentrating the virus by centrifugation after having generally
clarified the infected allantoic fluids. The purification may be
supplemented with a zonal centrifugation step carried out for
example by means of sucrose density gradients (EP 0 7760362).
Chromatographic methods may also be carried out in order to purify
the virus. A suspension of purified whole viruses which go to make
up the composition of the inactivated whole vaccines or of
attenuated vaccines is thus obtained.
[0089] The inactivation of the viral suspension can be carried out
by conventional means, using .beta.-propiolactone (E. Budowsky et
al. 1991, Vaccine, 9: 319-325; 1991, Vaccine, 9: 398-402; 1993,
Vaccine, 11: 343-348), ethyleneimine or derivatives (D. King 1991,
Avian Dis. 35: 505-514) or formol (EP 0 776 0362).
[0090] The vaccinal composition based on inactivated whole viruses
can be formulated with one or more adjuvants. Although
conventionally these vaccines may be formulated with aluminum salts
or in a water-in-oil or oil-in-water emulsion (in the case of avian
flu vaccines), a water-in-oil emulsion composed of liquid paraffin,
of a hydrophilic surfactant such as polysorbate 80, polysorbate 83
or polysorbate 85 and of a lipophilic surfactant such as sorbitan
oleate, sorbitan sesquioleate or sorbitan trioleate is normally
used. Any adjuvant capable of increasing the humoral and/or
cellular response against the flu may be used. As an example of
nonlimiting adjuvant formulations, mention is made of the MF59.RTM.
emulsion, the liposome-based formulations, and formulations based
on MPL, on Corynebacterium parvum, on saponin, on lysolecithin, on
pluronic derivatives, or on combinations thereof.
[0091] The purified virus suspension may also undergo subsequent
treatments and "flu virus-derived products" produced. For example
the viral suspension may be fragmented using detergents or lipid
solvents according using methods well known to those skilled in the
art in order to manufacture, for example, vaccines based on
fragmented or split viruses, virosomes, or subunit vaccines
containing the flu virus hemagglutinin. Fragmented or split
viruses, virosomes that contain the hemagglutinin of the flu virus,
or subunit vaccines containing the flu virus hemagglutinin derived
from the purified flu virus suspension are considered as "flu
virus-derived products." These flu virus-derived products may
similarly be formulated with one or more adjuvants.
[0092] The vaccines obtained by means of the method according to
the invention are for use in protecting humans and animals against
the flu.
[0093] In the veterinary field, the vaccine can be mainly used in
the avian flu prevention field, but it may also be used for
preventing or reducing flu symptoms and/or viral secretion in
members of the equine family, in particular horses, members of the
canine family, in particular dogs, members of the feline family, in
particular cats, members of the porcine family, in particular pigs,
mustelids, in particular minks and ferrets, and avian species, in
particular hen, duck, turkey, quail, guinea-fowl, goose and
ostrich. When the vaccinal composition contains an inactivated
whole virus strain or a derived product, it is generally
administered subcutaneously or intramuscularly, or optionally in
the form of nebulized material in poultry breeding colonies. When
the vaccine is in the form of a live attenuated virus, it is
generally administered oronasally, by spray, in the drinking water
or as a drop in the eye. The immunization scheme generally provides
for an injection or an injection followed by a booster. The
vaccinal dose administered depends on the size and the age of the
animal. It usually contains between 20 and 200 .mu.l of allantoic
fluid having a titer of 10.sup.8 to 10.sup.10 EID.sub.50/ml before
inactivation, injected in a volume of between 0.05 and 1 ml.
[0094] In humans, the vaccine can be used in the field of epidemic
flu and pandemic flu prevention. While epidemic flu affects a human
population already sensitized by contact (by infection) or by
immunization with one (or more) strain(s) of flu virus for which
there exists an antigenic relationship with the HA of the virus
strain responsible for the epidemic and in which there exists a
certain immunity, even if it is only partially effective, pandemic
flu affects a human population not sensitized to a new strain of
virus because the HA of this new strain has no or too little an
antigenic relationship with the prior circulating virus
strains.
[0095] The epidemic flu vaccine is intended to protect the human
population against seasonal flu forms brought about by circulating
seasonal flu virus strains that have an antigenic relationship with
prior virus strains that have already circulated. Currently, the
flu virus strains responsible for epidemic flu, also called
epidemic flu strains are of type A and belong to the H1N1 or H3N2
subtypes or are of type B.
[0096] The pandemic flu vaccine is intended to prevent the
infection of the human population against a pandemic flu strain,
which is a flu virus strain that has no antigenic relationship in
terms of the HA with prior circulating flu virus strains.
[0097] The epidemic or pandemic flu vaccine may be in the form of a
live attenuated vaccine or an inactivated vaccine, although an
inactivated vaccine is preferred for the prevention of pandemic
flu. The vaccine may be in the form of a monovalent vaccine
(vaccine prepared from a single flu virus strain) or of a
multivalent vaccine (vaccine prepared from several flu virus
strains). The composition of the epidemic flu vaccine is currently
in the form of a trivalent vaccine prepared from the H3N2 and H1N1
strains and from a type B virus strain. The inactivated vaccine is
generally in the form of whole virus, of fragmented virus (split
virus) or of virosomes, or in a subunit form containing HA, and
optionally contains one or more adjuvants such as those mentioned
above. While the live attenuated vaccine is generally administered
orally or nasally in order to promote the development of mucosal
immunity, the inactivated vaccine can be administered parenterally
(intramuscularly or subcutaneously), intradermally or even
mucosally (intranasally), or even by combining two different routes
of administration as described in WO 01/22992. The immunization
scheme generally provides for an injection or an injection followed
by a booster. The vaccinal dose administered depends on the age of
the individual and on the presence or absence of an adjuvant.
Conventionally, the vaccinal dose contains the equivalent of 15
.mu.g of HA of each vaccinal strain contained in the vaccine. This
dose may be reduced to approximately 1 to 2 .mu.g of HA when the
vaccine is adjuvanted, or increased to 30 .mu.g of HA or even more
in elderly individuals or individuals suffering from an immune
deficiency.
[0098] Finally, a subject of the invention also comprises:
[0099] The use of eggs from hens immunized against the flu, for the
production of flu viruses or for the manufacture of a flu
vaccine.
[0100] The following examples illustrate in a nonlimiting manner
various embodiments of the invention.
[0101] FIG. 1 represents the complete nucleotide sequence of the
donor plasmid pJY1394.1.
[0102] FIG. 2 represents the nucleotide sequence of the insert in
the donor plasmid pJY1394.1 comprising the arms flanking the
insertion locus F8, and also the H6 vaccinia promoter followed by
the synthetic gene modified at the cleavage site encoding the HA of
the A/chicken/Indonesia/7/03 strain.
[0103] The various origins of these sequences are the following:
[0104] From 1 to 53: partial sequence of the cloning plasmid
comprising the sequence of the M13F primer (underlined). [0105]
From 54 to 1483: sequence of the "left arm" flanking the F8
insertion locus in the genome of the TROVAC fowlpox vector. [0106]
From 1484 to 1568: linker sequence between the left arm and the H6
promoter. [0107] From 1569 to 1692: sequence of the vaccinia virus
H6 promoter. [0108] From 1693 to 3387: sequence of the modified
synthetic HA gene of the A/chicken/Indonesia/7/03 strain; the amino
acid sequence is indicated above the nucleotide sequence using the
1-letter code per amino acid. [0109] From 3388 to 3414: linker
sequence between the HA gene and the right arm comprising a TTTTTAT
transcription stop sequence of the fowlpox early genes. [0110] From
3415 to 4790: sequence of the "right arm" flanking the F8 insertion
locus in the genome of the TROVAC fowlpox vector. [0111] From 4791
to 4885: partial sequence of the cloning plasmid comprising the
sequence of the M13R primer (underlined).
EXAMPLE 1
Method for Producing Two Vaccinal Strains A/New Caledonia/20/99
(H1N1) and A/New York/55/04 (H3N2) Used to Prepare Vaccines Against
Human Epidemic Flu Using Embryonated Eggs from Hens which have been
Immunized, Either with Two Injections of an Inactivated Vaccine
Containing an Avian Flu Virus Strain A/Chicken/Italy/22A/98 (H5N9),
or by an Injection of a Recombinant Avipoxvirus Encoding the HA of
an H5N1 Strain Followed by a Second Injection of an Inactivated
Vaccine Containing an Avian Flu Virus Strain A/Chicken/Italy/22A/98
(H5N9)
[0112] 1.1) Operating Protocol
[0113] 1.1.1) Construction of the Recombinant Vector vFP2211
[0114] vFP2211 is a recombinant fowlpox virus, into the genome of
which has been inserted a synthetic gene encoding the hemagglutinin
(HA) of the A/chicken/Indonesia/7/03 H5N1 strain. The HA gene was
synthesized so as to obtain an open reading frame which encodes an
amino acid sequence identical to the native sequence of the
A/chicken/Indonesia/7/03 strain described in the GenBank nucleotide
sequence database under the reference EF473080 (or the GenPept
protein sequence database under the reference ABO30346), with the
exception of the cleavage site. The RERRRKKRG amino acid sequence
located between positions 339 and 347 (corresponding to the
cleavage site of a high path strain) was replaced with the RE - - -
TRG sequence. This cleavage site thus modified corresponds to the
cleavage site of the low path strains of subtype H5.
[0115] In a first step, a donor plasmid pJY1394.1 comprising the
modified synthetic HA gene under the control of the H6 vaccinia
promoter (Taylor J. et al., Vaccine, 1988, 6: 504-508; Guo P. et
al., J. Virol., 1989, 63: 4189-4198; Perkus M. et al., J. Virol.,
1989, 63: 3829-3836) and bordered by the flanking arms of the F8
insertion locus so as to allow insertion of the HA gene into the F8
insertion locus of the fowlpox vector genome, was constructed. The
F8 insertion locus corresponds to the fowlpox gene encoding
photolyase described by Srinivasan and Tripathy (2005, Veterinary
Microbiology 108: 215-223); this gene is also described under the
name FPV158 in the complete sequence of the fowlpox genome
(GenBank, reference AF198100). The insertion of the HA gene and of
the H6 promoter into the F8 locus results in the deletion of the
FPV158 gene from the recombinant fowlpox virus genome. The complete
nucleotide sequence of the plasmid pJY1394.1 and also the series of
nucleotide sequences bordering the HA gene (which also appear in
the complete sequence of the plasmid) are described respectively in
FIGS. 1 and 2.
[0116] The recombinant virus vFP2211 was then obtained by double
recombination between the flanking arms of the plasmid pJY1394.1
and the fowlpox genome. For this, primary chicken embryo cells were
transfected with the plasmid pJY1394.1 linearized with NotI and
infected (multiplicity of infection of 10) with the parental
fowlpox strain (TROVAC vector). The TROVAC vector derives from the
vaccinal strain of the Diftosec vaccine produced by Merial against
fowlpox in chickens. The recombinant viruses were selected by
specific hybridization on lysis plaques with a probe for detecting
the inserted HA gene. The vFP2211 thus isolated was then produced
in rolling bottles on chicken embryo cells.
[0117] 1.1.2) Preparation of the Inactivated H5N9 Vaccine
[0118] The inactivated H5N9 vaccine consisting of the low path
A/chicken/Italy/22A/98 H5N9 strain (provided by the laboratory of
Ilaria Capua (Istituto Zooprofilattico Sperimentale delle Venezie,
Laboratorio Virologia, Padua, Italy)), which was produced on
embryonated eggs and inactivated with beta-propiolactone (BPL), and
of a water-in-oil emulsion composed of liquid paraffin, of sorbitan
oleate and of polysorbate 80, was prepared according to a method
equivalent to that described by H. Stone (1987, Avian Dis. 31:
483-490). The HLB (lipophilic hydrophilic balance) of the mixture
of surfactants of the emulsion has a value of 5.3. A vaccinal dose
is equivalent to 60 .mu.l of allantoic fluid having a titer of
10.sup.8.9 EID.sub.50/ml before inactivation.
[0119] 1.1.3) Immunization of Egg-Laying Hens
[0120] Two groups (G1 and G2) of 1-day (D0) Leghorn chicks, having
the "specific pathogen free" (SPF) status, were formed. The
following immunization scheme was applied to group G1: the 1-day
(D0) chicks received subcutaneously, at the base of the neck, using
an insulin syringe, 0.2 ml of a viral suspension of vFP2211 having
a titer of 3.0 log.sub.o CCID.sub.50/0.2 ml. At the age of 8 weeks
(W8) the chickens were sexed. Approximately 25 pullets and 16
roosters were conserved and put together. At the age of 17 weeks
(W17) they received a booster injection of a vaccinal dose in a
volume of 0.5 ml of the monovalent inactivated H5N9 vaccine.
[0121] The following immunization scheme was applied to group G2:
the 3-week-old SPF chicks received, by IM injection, in the
wishbone, using an insulin syringe, a vaccinal dose in a volume of
0.3 ml of the monovalent inactivated H5N9 vaccine.
[0122] At the age of 8 weeks (W8), the chickens were sexed.
Approximately 25 pullets and 16 roosters were conserved and put
together. At the age of 17 weeks (W17) they received a booster
injection of a vaccinal dose in a volume of 0.5 ml of the
monovalent inactivated H5N9 vaccine.
[0123] 1.1.4) Protocol for Infection of Eggs Originating from the
Hens of Groups G1 and G2 and from Nonimmunized Hens, with the A/New
Caledonia/20/99 IVR-116 (H1N1) or A/New York/55/04 X-157 (H3N2)
Virus Strains
[0124] The ability of the reassortant vaccinal strains A/New
Caledonia/20/99 (xPR8) called A/New Caledonia/20/99 IVR-116 (H1N1)
and A/New York/55/04 (xPR8) called A/New York/55/04 X-157 (H3N2) to
replicate was evaluated in the eggs from the hens of group G1 which
were laid at 40-41 (test 2) and 49-50 (test 3) weeks of age and in
the eggs from the hens of group G2 which were laid at 26-27 (test
1), 40-41 (test 2) and 49-50 (test 3) weeks of age. In parallel,
the ability of the virus strains A/New Caledonia/20/99 (xPR8)
(H1N1) and A/New York/55/04 (xPR8) (H3N2) to replicate was
evaluated in the same manner in the control eggs derived from
nonimmunized SPF hens (control group).
[0125] All the eggs laid during the week were conserved in a
temperature-controlled chamber (12 to 15.degree. C.). The eggs of
the same test and originating from the same group of animals were
grouped together and then the embryogenesis process was triggered
by incubating the eggs for 10 days at 37-38.degree. C. in a chamber
in which the relative humidity was approximately 70-80%. After
incubation for 10 days, the vitality of the embryo was verified in
each egg by means of a candling device, and the allantoic cavity
was pinpointed with a cross. The embryonated eggs of the same test
and originating from the same group of animals were grouped
together in pools of 8 eggs. The embryonated eggs of the same pool
were infected with the same infectious dose of flu virus injected
in a volume of 200 .mu.l at the level of the cross after having
disinfected and then pierced the shell of the egg. Infectious doses
of 10.sup.2, 10.sup.3 and 10.sup.4 EID.sub.50 (egg infectious dose
50%) of A/New Caledonia/20/99 (xPR8) (H1N1) and A/New York/55/04
(xPR8) (H3N2) virus were tested. Infectious doses of 10.sup.3 and
10.sup.4 EID.sub.50 per egg were tested in the eggs of tests 1 and
2. Infectious doses of 10.sup.2 and 10.sup.3 EID.sub.50 per egg
were tested in the eggs of test 3. The infected embryonated eggs
were then incubated at 34.degree. C. for 48 hours in a chamber in
which the relative humidity was 80%, and then placed at +4.degree.
C. overnight. The infected allantoic fluid was then taken from each
egg. The infecting titer was evaluated on each allantoic fluid
taken by measuring the HAU (hemagglutinating unit) titer. Turkey
red blood cells were used to measure the HAU titers of the
allantoic fluids infected with the A/New York/55/04 (xPR8) (H3N2)
strain. Hen red blood cells were used to measure the HAU titers of
the allantoic fluids infected with A/New Caledonia/20/99 (xPR8)
(H1N1). The HAU titer was expressed by the inverse of the last
dilution of the infected allantoic fluid which showed visible
hemagglutination in the presence of a suspension of hen or turkey
red blood cells at 0.5% in phosphate buffer.
[0126] 1.2) Results
[0127] The means of the HAU titers obtained in the eggs originating
from the various groups of immunized and nonimmunized hens as a
function of the infectious dose of virus injected into the eggs and
as a function of the time at which the eggs were sampled are given
in tables I and II.
TABLE-US-00001 TABLE I Hemagglutinating titers obtained in the
allantoic fluids as a function of the infecting dose of A/New
Caledonia/20/99 (xPR8) (H1N1) used, of the origin of the eggs (G1,
G2, nonimmunized control) and of the moment at which the eggs were
laid (test 1, test 2, test 3). Infecting dose (EID.sub.50/egg) 100
1000 10 000 Group test 3 test 1 test 2 test 3 test 1 test 2
Nonimmunized 640* 1050 1660 1280 861 1522 control G1 (vFP2211/ 1140
NA 1974 830 NA 1974 Vac. Inact. H5N9) G2 (Vac. Inact. 806 905 1660
1159 905 1974 H5N9/Vac. Inact. H5N9) *Hemagglutinating titer (HAU)
expressed in the form of the geometric mean of 8 HAU titers/50
.mu.l obtained on the allantoic fluids of the 8 eggs included in
each condition. NA: Not applicable
TABLE-US-00002 TABLE II Hemagglutinating titers obtained in the
allantoic fluids as a function of the infecting dose of A/New
York/55/04 (xPR8) (H3N2) used, of the origin of the eggs (G1, G2,
nonimmunized control) and of the time at which the eggs were laid
(test 1, test 2, test 3) Infecting dose (EID.sub.50/egg) 100 1000
10 000 Group test 3 test 1 test 3 test 1 Nonimmunized control 211*
861 349 830 G1 (vFP2211/Vac. Inact. H5N9) 88 NA 254 NA G2 (Vac.
Inact. H5N9/Vac. 254 987 557 844 Inact. H5N9) *Hemagglutinating
titer (HAU) expressed in the form of the geometric mean of 8 HAU
titers/50 .mu.l obtained on the allantoic fluids of the 8 eggs
included in each condition. NA: Not applicable
[0128] The results obtained in tables I and II show that the means
of the HAU titers of the allantoic fluids of the eggs originating
from immunized hens (G1 and G2) are equivalent to the means of the
HAU titers of the allantoic fluids of the eggs originating from
nonimmunized hens.
[0129] A statistical analysis was carried out in order to study the
immunization and dose factors. The individual values of the HAUs
were converted to log.sub.2 and then analyzed using a model of
variance. The variance heterogeneity was tested using the test for
reduced size, performed with the residues of the model (SAS v8.2
software).
[0130] The statistical analysis carried out on the individual
values of the HAU titers showed that there was no immunization
effect. Neither was there any effect related to the dose of virus
which was used to infect the eggs. The HAU titers did not
substantially fluctuate during the egg-laying period studied
(ranging from 26 weeks (test 1) to 50 weeks (test 3)).
EXAMPLE 2
Method for Producing Two Pandemic Vaccinal Strains
A/Chicken/Italy/22A/98 (H5N9) and A/Vietnam/1194/04 NIBRG14 (H5N1)
Using Embryonated Eggs from Hens which have been Immunized, Either
with Two Injections of an Inactivated Vaccine Containing an Avian
Flu Virus Strain A/Chicken/Italy/22A/98 (H5N9), or with an
Injection of a Recombinant Avipoxvirus Encoding the HA of an H5N1
Strain Followed by a Second Injection of an Inactivated H5N9
Vaccine Containing an Avian Flu Virus Strain
[0131] 2.1.1) Immunization of Hens
[0132] The protocol for immunizing the hens was identical to that
which was used in example 1.
[0133] 2.1.2) Protocol for Infection of Eggs Originating from the
Hens of Groups G1 and G2 and from Nonimmunized Hens, with the
A/Chicken/Italy/22A/98 (H5N9) and A/Vietnam/1194/04 NIBRG14 (H5N1)
Virus Strains
[0134] The infection protocol was the same as that which was used
in example 1, except for the following modifications:
[0135] The low path A/chicken/Italy/22A/98 (H5N9) avian strain
originating from the Laboratoire Istituto Zooprofilattico
Sperimentale delle Venezie, Laboratorio Virologia, Padua, Italy and
an attenuated pandemic reassortant vaccinal strain
A/Vietnam/1194/04 NIBRG14 (H5N1) were used to infect the eggs. The
latter strain was provided by the National Institute for Biological
Standards and Control (NIBSC) laboratory, South Mimms, Potters Bar,
Herts EN6 3QG, UK and was obtained by reverse genetics, as is
described by C. Nicolson et al., Generation of influenza vaccine
viruses on Vero cells by reverse genetics: an H5N1 candidate
vaccine strain produced under a quality system, 2005, Vaccine, 23:
2943-2952.
[0136] Infectious doses of 10.sup.2, 10.sup.3, 10.sup.4 and
10.sup.5 EID.sub.50 (egg infectious dose 50%) were tested for these
two strains. Infectious doses of 10.sup.3, 10.sup.4 and 10.sup.5
EID.sub.50 per egg were tested in the eggs of test 1. Infectious
doses of 10.sup.4 and 10.sup.5 EID.sub.50 per egg were tested in
the eggs of test 2. Infectious doses of 10.sup.2 and 10.sup.3
EID.sub.50 per egg were tested in the eggs of test 3.
[0137] Hen red blood cells were used to measure the HAU titers of
the allantoic fluids infected with A/chicken/Italy/22A/98 (H5N9) or
A/Vietnam/1194/04 NIBRG14 (H5N1).
[0138] 2.2) Results
[0139] The HAU titers obtained in the eggs originating from the
various groups of immunized and nonimmunized hens, as a function of
the infectious dose of virus injected into the eggs and as a
function of the time at which the eggs were taken, are given in
tables III and IV.
TABLE-US-00003 TABLE III Hemagglutinating titers obtained in the
allantoic fluids as a function of the infecting dose of
A/chicken/Italy/22A/98 (H5N9) used, of the origin of the eggs (G1,
G2, nonimmunized control) and of the time at which the eggs were
laid (test 1, test 2, test 3) Infecting dose (EID.sub.50/egg) 100
1000 10 000 100 000 Group test 3 test 1 test 3 test 1 test 2 test 1
test 2 Nonimmunized 35* 53 32 53 64 54 53 control G1 (vFP2211/ 64
NA 54 NA 59 NA 86 Vac. Inact. H5N9) G2 (Vac. Inact. 102 64 57 59 70
102 59 H5N9/Vac. Inact. H5N9) *Hemagglutinating titer (HAU)
expressed in the form of the geometric mean of 8 HAU titers/50
.mu.l obtained on the allantoic fluids of the 8 eggs included in
each condition. NA: Not applicable
TABLE-US-00004 TABLE IV Hemagglutinating titers obtained in the
allantoic fluids as a function of the infecting dose of
A/Vietnam/1194/04 (H5N1) RG14 used, of the origin of the eggs (G1,
G2, nonimmunized control) and of the time at which the eggs were
laid (test 1, test 2, test 3) Infecting dose (EID.sub.50/egg) 1000
10 000 100 000 Group test 1 test 1 test 2 test 1 test 2
Nonimmunized control 194* 232 197 172 181 G1 (vFP2211/Vac. Inact.
NA NA 197 NA 166 H5N9) G2 (Vac. Inact. 152 279 215 181 235
H5N9/Vac. Inact. H5N9) *Hemagglutinating titer (HAU) expressed in
the form of the geometric mean of 8 HAU titers/50 .mu.l obtained on
the allantoic fluids of the 8 eggs included in each condition. NA:
Not applicable
[0140] The results given in tables III and IV and also the
statistical analysis show that the HAU titers of the allantoic
fluids originating from the eggs from the immunized hens (G1 and
G2) are similar to the HAU titers of the allantoic fluids of the
eggs from the nonimmunized hens, even in the situation where the
hens were immunized with a strain identical to that which was used
to infect the embryonated eggs originating from these hens (in the
case of groups G1 and G2 immunized with the A/chicken/Italy/22A/98
(H5N9) strain). There is no effect, either, related to the dose of
virus which is used to infect the eggs. The HAU titers did not
substantially fluctuate during the egg-laying period studied
(ranging from 26 weeks (test 1) to 50 weeks (test 3)).
[0141] The statistical analysis was carried out as in example
1.
EXAMPLE 3
Analysis of the Anti-H5 Maternal Antibody Response in the Eggs from
Hens Which Have Been Immunized with the A/Chicken/Italy/22A/98
(H5N9) Vaccinal Strain
[0142] 3.1) Operating Protocol
[0143] The presence of anti-H5 antibodies in the eggs from the hens
of groups G1 and G2 which were immunized with the
A/chicken/Italy/22A/98 (H5N9) strain was measured according to the
protocol described in paragraph 1.1.1). In parallel, the anti-H5
response was also analyzed in the yolks of eggs from nonimmunized
hens (control group).
[0144] The anti-H5 response was analyzed in the yolks of eggs (or
vitelline fluids) which were taken at the time of tests 1 (W26-27),
2 (W40-41) and 3 (W49-50). The analysis of the anti-H5 response in
the yolks of eggs of test 1 was carried out before embryogenesis
(D0) and after embryogenesis (D10) (i.e. after the phase of
incubation for 10 days at 37.degree. C.). The analysis of the
anti-H5 responses in the yolks of eggs of tests 2 and 3 was carried
out only after embryogenesis (D10). The same analysis was also
carried out with eggs originating from nonimmunized hens (see
paragraph 3.2.2)). The anti-H5 response was also studied in the
allantoic fluids of the eggs of test 2 after embryogenesis (D10).
The egg yolks were removed by suction using a pipette with a
disposable tip. With the exception of test 1, where the egg yolks
were kept frozen without having been prediluted, the egg yolks of
tests 2 and 3 and the allantoic fluids were prediluted to 1/5th in
phosphate buffer before freezing. The diluted egg yolks and the
allantoic fluids were conserved frozen until the time of the
anti-H5 antibody assay which was carried out using the method of
assaying by inhibition of hemagglutination (IHA) of hen red blood
cells which takes place in the presence of the
A/chicken/Italy/22A/98 (H5N9) strain. The assay was based on the
ability of the neutralizing antibodies directed specifically
against the HA of the virus to inhibit the "hemagglutinating"
activity of the virus. In this test, anti-A/Vietnam/1194/04 sheep
serum provided by the NISBC was used as positive control and naive
mouse serum was used as negative control. Successive two-fold
dilutions of the samples (diluted egg yolks or allantoic fluids)
were carried out in a conical-bottomed microplate in order to
obtain 50 .mu.l of each of the dilutions per well. 50 .mu.l of a
viral suspension having a titer of 4 hemagglutinating units (4HAU)
and originating from a clarified allantoic fluid which was infected
with the A/chicken/Italy/22A/98 (H5N9) strain provided by the
laboratory of Ilaria Capua (Istituto Zooprofilattico Sperimentale
delle Venezie, Laboratorio Virologia, Padua, Italy), were added to
each well. This was left to incubate for 1 hour at laboratory
temperature before adding 50 .mu.l of a solution of hen red blood
cells at 0.5% in phosphate buffer. After leaving to stand for 1
hour at +4.degree. C., the test was read. The presence of
inhibition of hemagglutination was reflected by the presence of a
red spot at the bottom of the microwell, while the presence of
hemagglutination was reflected by the presence of a pinkish halo in
the microwell. The IHA antibody titer was represented by the
inverse of the last dilution where no hemagglutination is observed
in the microwell.
[0145] 3.2) Results
[0146] 3.2.1) Analysis of the Anti-H5 Response in the Egg Yolks of
Test 1
[0147] At D0, 8 of the 8 egg yolks analyzed of the nonimmunized
group were negative in the IHA test, whereas 6 of the 8 egg yolks
originating from group 2 were positive in the IHA test, which
indicated the presence of anti-H5 antibodies.
[0148] At D10, 4 of the 4 egg yolks analyzed of the nonimmunized
group were negative in the IHA test, whereas 4 of the 4 egg yolks
originating from group 2 were positive in the IHA test.
[0149] 3.2.2) Analysis of the Anti-H5 Response in the Egg Yolks and
the Allantoic Fluids of Test 2
[0150] At D10, 5 of the 5 egg yolks analyzed of the nonimmunized
group were negative in the IHA test, whereas 4 of the 5 egg yolks
originating from group 1, and 4 of the 5 egg yolks originating from
group 2, were positive in the IHA test, which indicated the
existence of anti-H5 antibodies in these groups. The individual
values of the IHA titers obtained are given in table V.
TABLE-US-00005 TABLE V IHA titers for the yolks of eggs taken after
embryogenesis (D 10) Egg yolks D 10* Group 1 160 (vFR2211/Vac.
Inact. H5N9) 80 320 80 <5 Group 2 80 (Vac. Inact. H5N9/Vac. 160
Inact. H5N9) 40 40 <5 Nonimmunized group <5 <5 <5 <5
<5 *The titers at D 10 originate from different egg yolks
[0151] On the other hand, all the allantoic fluids originating from
groups 1 and 2 were negative in the IHA test, which means that
there were no antibodies inhibiting the hemagglutination of the
H5N9 flu virus that were detectable in the allantoic fluids.
[0152] 3.2.3) Analysis of the Anti-H5 Response in the Egg Yolks of
Test 3
[0153] At D10, 5 of the 5 egg yolks analyzed of the nonimmunized
group were negative in the IHA test, whereas 5 of the 5 egg yolks
originating from group 1 were positive in the IHA test.
[0154] The individual values of the IHA titers are given in table
VI.
TABLE-US-00006 TABLE VI IHA titers of the yolks of eggs taken after
embryogenesis (D 10) as a function of the origin of the eggs Egg
yolks D 10 Group 1 80 (vFP2211/Vac. Inact. H5N9) 80 160 5 320
Nonimmunized group <5 <5 <5 <5 <5
[0155] In conclusion, on the basis of the results of examples 2 and
3, the presence of maternal anti-H5N9 antibodies, which was
revealed in the egg yolks of the 3 tests, has no effect on the
viral productivity of the allantoic fluids infected with an H5N1 or
H5N9 strain.
EXAMPLE 4
Analysis of the Serological Response of Hens Immunized with the
A/Chicken/Italy/22A/98 (H5N9) Vaccinal Strain and of the Maternal
Anti-H5 Antibodies Present in the Eggs from These Immunized
Hens
[0156] Blood samples were taken, at 28 and 36 weeks of age, from
the immunized hens of groups 1 and 2. The eggs laid by these hens
were collected at weeks 27 and 37 in order to evaluate the
importance of the transfer of the maternal antibodies into the egg
yolks. The anti-H5 antibody response was evaluated by means of the
hemagglutination inhibition test using as antigen the
A/chicken/Italy/22A/98 H5N9 strain homologous to the inactivated
vaccine strain. The results are expressed as log.sub.10 in table
VII.
TABLE-US-00007 TABLE VII Hemagglutination-inhibiting antibody
titers (A/chicken/Italy/22A/98 H5N9 antigen) in the serum of
immunized egg-laying hens and in the vitellus of the eggs laid by
these hens Serum Vitellus Serum Vitellus Weeks of age 28 27 36 37
G1 2.10 .+-. 0.61 1.96 .+-. 0.94 1.92 .+-. 0. 91 1.63 .+-. 0.89
(15)* (21)** (15) (10) G2 2.20 .+-. 0.62 1.81 .+-. 0.88 2.26 .+-.
0.65 1.43 .+-. 0.89 (15) (13) (15) (14) *geometric mean of the
hemagglutination-inhibiting antibody titers, expressed as
log.sub.10 .+-. standard deviation (number of samples tested per
group) **the lowest dilution tested on the vitelli is 1/10 (1
log.sub.10); for the calculation of the means and standard
deviation, the values of the eggs that were negative at the 1/10
dilution were placed at 0.7 log.sub.10.
TABLE-US-00008 TABLE VIII Hemagglutination-inhibiting antibody
titers (A/turkey/Wisconsin/68 H5N9 antigen) in the vitellus of the
eggs laid by the immunized hens of groups 1 and 2 Vitellus Weeks of
age 27 G1 1.67 .+-. 0.79 (21)* G2 1.39 .+-. 0.80 (13) *geometric
mean of the hemagglutination-inhibiting antibody titers, expressed
as log.sub.10 .+-. standard deviation (number of samples tested per
group); the lowest dilution tested is 1/10 (1 log.sub.10); for the
calculation of the means and standard deviation, the values of the
eggs that were negative at the 1/10 dilution were placed at 0.7
log.sub.10.
[0157] These results confirm the transmission of the maternal
antibodies into the egg yolks of the eggs laid by the immunized
hens of groups 1 and 2.
[0158] The homologous anti-H5N9 antibody titers (table VII) in the
vitellus are greater than the heterologous anti-H5N9 titers (table
VIII), although this difference is not statistically
significant.
EXAMPLE 5
Production of the A/Turkey/Wisconsin/68 H5N9 Strain on Eggs Laid by
Hens Immunized with an Inactivated H5N9 Vaccine
(A/Chicken/Italy/22A/98 Strain) of Group 2
[0159] 5.1. Operating Protocol
[0160] The eggs (approximately 220-230) from the hens of group 2
immunized twice at 3 and 17 weeks of age with the inactivated H5N9
vaccine (A/chicken/Italy/22A/98 strain) were taken during weeks 28
and 29. These eggs, after storage at controlled temperature
(between 12 and 15.degree. C.), were incubated at 37.degree. C. for
11 days. After candling in order to verify that the embryos had
good viability, the eggs were inoculated in the allantoic cavity
with 0.2 ml of a dilution of a stock viral solution of the
A/turkey/Wisconsin/68 H5N9 strain having a titer of 9.68 log.sub.10
EID.sub.50/ml (inoculum). Seven groups of approximately 30 eggs
were formed. Four groups (G1 to G4) were inoculated with the
following dilutions of the inoculum: 10.sup.-5, 10.sup.-4,
10.sup.-3 and 10.sup.-2. Three other groups (G5 to G7) were
inoculated with the 10.sup.-3 dilution of the inoculum. After
inoculation, the eggs were incubated at 37.degree.
C..+-.1.5.degree. C. in 70%.+-.10% humidity. After incubation for
20 h, the eggs were candled in order to eliminate the dead eggs
derived from the inoculation and they were then reincubated under
the same conditions. Forty-two hours after the inoculation, the
eggs were placed in the cold before harvesting the allantoic fluid.
The allantoic fluids of the eggs of groups 1 to 4 were pooled (one
pool of fluid per group). The allantoic fluids of the 4 groups of
eggs were stored at -70.degree. C. before being titered in terms of
hemagglutinating units and of egg infectious dose 50%
(EID.sub.50).
[0161] 5.2. Results
[0162] The results are given in table IX.
TABLE-US-00009 TABLE IX Hemagglutinating and infectious titers
obtained after production of the A/turkey/Wisconsin/68 H5N9 strain
on eggs originating from hens immunized with an inactivated vaccine
containing the A/chicken/Italy/22A/98 H5N9 strain Inoculum Titer in
hemagglutinating Infectious titer Group dilution units (log.sub.10)
EID.sub.50 (log.sub.10/ml) 1 10.sup.-5 2.25 9.50 2 10.sup.-4 2.40
9.33 3 10.sup.-3 2.25 9.67 4 10.sup.-2 2.40 9.88
[0163] These results show very similar titers between the groups
and there is therefore no effect of the dilution of the inoculum.
Furthermore, the titers obtained are very high and are comparable
to those obtained on eggs having no maternal anti-H5N9 antibodies
(historical laboratory data). These results therefore confirm that,
under the conditions tested, the presence of maternal antibodies in
the eggs does not interfere with the production of a flu virus of
the same subtype.
Sequence CWU 1
1
51564PRTAvian influenza virusMISC_FEATURE(1)..(564)Amino acid
sequence encoded by the modified synthetic HA gene at the level of
the cleavage site; sequence of the HA protein of
theA/chicken/Indonesia/7/03 avian flu virus 1Met Glu Lys Ile Val
Leu Leu Leu Ala Ile Val Ser Leu Val Lys Ser1 5 10 15Asp Gln Ile Cys
Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln Val 20 25 30Asp Thr Ile
Met Glu Lys Asn Val Thr Val Thr His Ala Gln Asp Ile 35 40 45Leu Glu
Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp Gly Val Lys 50 55 60Pro
Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp Leu Leu Gly Asn65 70 75
80Pro Met Cys Asp Glu Phe Ile Asn Val Pro Glu Trp Ser Tyr Ile Val
85 90 95Glu Lys Ala Asn Pro Ala Asn Asp Leu Cys Tyr Pro Gly Asn Leu
Asn 100 105 110Asp Tyr Glu Glu Leu Lys His Leu Leu Ser Arg Ile Asn
His Phe Glu 115 120 125Lys Ile Gln Ile Ile Pro Lys Ser Ser Trp Ser
Asp His Glu Ala Ser 130 135 140Ser Gly Val Ser Ser Ala Cys Pro Tyr
Gln Gly Lys Ser Ser Phe Phe145 150 155 160Arg Asn Val Val Trp Leu
Ile Lys Lys Asn Ser Ala Tyr Pro Thr Ile 165 170 175Lys Arg Ser Tyr
Asn Asn Thr Asn Gln Glu Asp Leu Leu Val Leu Trp 180 185 190Gly Ile
His His Pro Asn Asp Ala Ala Glu Gln Thr Arg Leu Tyr Gln 195 200
205Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr Ser Thr Leu Asn Gln Arg
210 215 220Leu Val Pro Lys Ile Ala Ile Arg Ser Lys Val Asn Gly Gln
Ser Gly225 230 235 240Arg Met Glu Phe Phe Trp Thr Ile Leu Lys Pro
Asn Asp Ala Ile Asn 245 250 255Phe Glu Ser Asn Gly Asn Phe Ile Ala
Pro Glu Tyr Ala Tyr Lys Ile 260 265 270Val Lys Lys Gly Asp Ser Ala
Ile Met Lys Ser Glu Leu Glu Tyr Gly 275 280 285Asn Cys Asn Thr Lys
Cys Gln Thr Pro Met Gly Ala Ile Asn Ser Ser 290 295 300Met Pro Phe
His Asn Ile His Pro Leu Thr Ile Gly Glu Cys Pro Lys305 310 315
320Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr Gly Leu Arg Asn Ser
325 330 335Pro Gln Arg Glu Thr Arg Gly Leu Phe Gly Ala Ile Ala Gly
Phe Ile 340 345 350Glu Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr
Gly Tyr His His 355 360 365Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala
Asp Lys Glu Ser Thr Gln 370 375 380Lys Ala Ile Asp Gly Val Thr Asn
Lys Val Asn Ser Ile Ile Asp Lys385 390 395 400Met Asn Thr Gln Phe
Glu Ala Val Gly Arg Glu Phe Asn Asn Leu Glu 405 410 415Arg Arg Ile
Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp 420 425 430Val
Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg 435 440
445Thr Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val
450 455 460Arg Leu Gln Leu Arg Asp Asn Ala Lys Glu Leu Gly Asn Gly
Cys Phe465 470 475 480Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met
Glu Ser Ile Arg Asn 485 490 495Gly Thr Tyr Asn Tyr Pro Gln Tyr Ser
Glu Glu Ala Arg Leu Lys Arg 500 505 510Glu Glu Ile Ser Gly Val Lys
Leu Glu Ser Ile Gly Thr Tyr Gln Ile 515 520 525Leu Ser Ile Tyr Ser
Thr Val Ala Ser Ser Leu Ala Leu Ala Ile Met 530 535 540Met Ala Gly
Leu Ser Leu Trp Met Cys Ser Asn Gly Ser Leu Gln Cys545 550 555
560Arg Ile Cys Ile27618DNAArtificialdonor plasmid pYJ1394.1
2ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
60attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
120gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
acgtggactc 180caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc
ccactacgtg aaccatcacc 240ctaatcaagt tttttggggt cgaggtgccg
taaagcacta aatcggaacc ctaaagggag 300cccccgattt agagcttgac
ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 360agcgaaagga
gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
420cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat
tcaggctgcg 480caactgttgg gaagggcgat cggtgcgggc ctcttcgcta
ttacgccagc tggcgaaagg 540gggatgtgct gcaaggcgat taagttgggt
aacgccaggg ttttcccagt cacgacgttg 600taaaacgacg gccagtgaat
tgtaatacga ctcactatag ggcgaattgg gtgacccttt 660acaagaataa
aagaagaaac aactgtgaaa tagtttataa atgtaattcg tatgcagaaa
720acgataatat attttggtat gagaaatcta aaggagacat agtttgtata
gacatgcgct 780cttccgatga gatattcgat gcttttctaa tgtatcatat
agctacaaga tatgcctatc 840atgatgatga tatatatcta caaatagtgt
tatattattc taataatcaa aatgttatat 900cttatattac gaaaaataaa
tacgttaagt atataagaaa taaaactaga gacgatattc 960ataaagtaaa
aatattagct ctagaagact ttacaacgga agaaatatat tgttggatta
1020gtaatatata acagcgtagc tgcacggttt tgatcatttt ccaacaatat
aaaccaatga 1080aggaggacga ctcatcaaac ataaataaca ttcacggaaa
atattcagta tcagatttat 1140cacaagatga ttatgttatt gaatgtatag
acggatcttt tgattcgatc aagtatagag 1200atataaaggt tataataatg
aagaataacg gttacgttaa ttgtagtaaa ttatgtaaaa 1260tgcggaataa
atacttttct agatggttgc gtctttctac ttctaaagca ttattagaca
1320tttacaataa taagtcagta gataatgcta ttgttaaagt ctatggtaaa
ggtaagaaac 1380ttattataac aggattttat ctcaaacaaa atatgatacg
ttatgttatt gagtggatag 1440gggatgattt tacaaacgat atatacaaaa
tgattaattt ctataatgcg ttattcggta 1500acgatgaatt aaaaatagta
tcctgtgaaa acactctatg cccgtttata gaacttggta 1560gatgctatta
tggtaaaaaa tgtaagtata tacacggaga tcaatgtgat atctgtggtc
1620tatatatact acaccctacc gatattaacc aacgagtttc tcacaagaaa
acttgtttag 1680tagatagaga ttctttgatt gtgtttaaaa gaagtaccag
taaaaagtgt ggcatatgca 1740tagaagaaat aaacaaaaaa catatttccg
aacagtattt tggaattctc ccaagttgta 1800aacatatttt ttgcctatca
tgtataagac gttgggcaga tactaccaga aatacagata 1860ctgaaaatac
gtgtcctgaa tgtagaatag tttttccttt cataataccc agtaggtatt
1920ggatagataa taaatatgat aaaaaaatat tatataatag atataagaaa
atgattttta 1980caaaaatacc tataagaaca ataaaaatat aattacattt
acggaaaata gctggtttta 2040gtttaccaac ttagagtaat tatcatattg
aatctatatt gctaattagc taataaaaac 2100ccgggttaat taattagtca
tcaggcaggg cgagaacgag actatctgct cgttaattaa 2160ttagagcttc
tttattctat acttaaaaag tgaaaataaa tacaaaggtt cttgagggtt
2220gtgttaaatt gaaagcgaga aataatcata aattatttca ttatcgcgat
atccgttaag 2280tttgtatcgt aatggagaaa atcgtgctgc tgctggccat
cgtgagcctg gtgaaaagcg 2340atcagatctg catcggctac cacgccaaca
acagcacaga gcaagtggac acaatcatgg 2400aaaagaacgt gaccgtgaca
cacgcccagg acatcctgga aaagacacac aacgggaagc 2460tgtgcgatct
ggatggagtg aagcctctga tcctgagaga ttgcagcgtg gccggatggc
2520tgctggggaa cccaatgtgc gacgaattca tcaacgtgcc cgaatggagc
tacatcgtgg 2580agaaggccaa cccagccaac gacctgtgct acccagggaa
cctgaacgac tacgaagaac 2640tgaaacacct gctgagcaga atcaaccact
ttgagaaaat ccagatcatc cccaaaagca 2700gctggtccga tcacgaagcc
agcagcggag tgagcagcgc ctgcccatac cagggaaagt 2760ccagcttttt
tagaaacgtg gtgtggctga tcaaaaagaa cagcgcctac ccaacaatca
2820agagaagcta caacaacacc aaccaggaag atctgctggt gctgtggggg
atccaccacc 2880ctaacgatgc cgccgagcag acaaggctgt accagaaccc
aaccacctac atctccgtgg 2940ggacaagcac actgaaccag agactggtgc
caaaaatcgc catcagatcc aaagtgaacg 3000ggcagagcgg aagaatggag
ttcttctgga caatcctgaa acccaacgat gccatcaact 3060tcgagagcaa
cggaaacttc atcgccccag aatacgccta caaaatcgtg aagaaagggg
3120acagcgccat catgaaaagc gaactggaat acggcaactg caacaccaag
tgccagaccc 3180caatgggggc catcaacagc agcatgccat tccacaacat
ccaccctctg accatcgggg 3240aatgccccaa atacgtgaaa agcaacagac
tggtgctggc caccgggctg agaaacagcc 3300ctcagagaga gaccagagga
ctgtttggag ccatcgccgg ctttatcgag ggaggatggc 3360agggaatggt
ggatggctgg tacggatacc accacagcaa cgagcagggg agcggatacg
3420ccgccgacaa agaatccacc cagaaggcca tcgacggcgt gaccaacaaa
gtgaacagca 3480tcatcgacaa aatgaacacc cagtttgagg ccgtgggaag
ggagtttaac aacctggaaa 3540ggagaatcga gaacctgaac aagaagatgg
aggacggatt cctggatgtg tggacctaca 3600acgccgaact gctggtgctg
atggaaaacg agagaaccct ggactttcac gacagcaacg 3660tgaagaacct
gtacgacaaa gtgaggctgc agctgaggga taacgccaag gagctgggca
3720acggctgctt cgagttctac cacaaatgcg ataacgaatg catggaaagc
atcagaaacg 3780gaacctacaa ctacccccag tacagcgaag aagccagact
gaaaagagaa gaaatctccg 3840gagtgaaact ggaatccatc ggaacctacc
agatcctgag catctacagc acagtggcct 3900cctccctggc cctggccatc
atgatggccg gactgagcct gtggatgtgc tccaacggaa 3960gcctgcagtg
cagaatctgc atctgactcg agtttttatt gactagttaa tcataagata
4020aataatatac agcattgtaa ccatcgtcat ccgttatacg gggaataata
ttaccataca 4080gtattattaa attttcttac gaagaatata gatcggtatt
tatcgttagt ttattttaca 4140tttattaatt aaacatgtct actattacct
gttatggaaa tgacaaattt agttatataa 4200tttatgataa aattaagata
ataataatga aatcaaataa ttatgtaaat gctactagat 4260tatgtgaatt
acgaggaaga aagtttacga actggaaaaa attaagtgaa tctaaaatat
4320tagtcgataa tgtaaaaaaa ataaatgata aaactaacca gttaaaaacg
gatatgatta 4380tatacgttaa ggatattgat cataaaggaa gagatacttg
cggttactat gtacaccaag 4440atctggtatc ttctatatca aattggatat
ctccgttatt cgccgttaag gtaaataaaa 4500ttattaacta ttatatatgt
aatgaatatg atatacgact tagcgaaatg gaatctgata 4560tgacagaagt
aatagatgta gttgataaat tagtaggagg atacaatgat gaaatagcag
4620aaataatata tttgtttaat aaatttatag aaaaatatat tgctaacata
tcgttatcaa 4680ctgaattatc tagtatatta aataatttta taaattttaa
taaaaaatac aataacgaca 4740taaaagatat taaatcttta attcttgatc
tgaaaaacac atctataaaa ctagataaaa 4800agttattcga taaagataat
aatgaatcga acgatgaaaa attggaaaca gaagttgata 4860agctaatttt
tttcatctaa atagtattat tttattgaag tacgaagttt tacgttagat
4920aaataataaa ggtcgatttt tattttgtta aatatcaaat atgtcattat
ctgataaaga 4980tacaaaaaca cacggtgatt atcaaccatc taacgaacag
atattacaaa aaatacgtcg 5040gactatggaa aacgaagctg atagcctcaa
tagaagaagc attaaagaaa ttgttgtaga 5100tgttatgaag aattgggatc
atcctctcaa cgaagaaata gataaagttc taaactggaa 5160aaatgataca
ttaaacgatt tagatcatct aaatacagat gataatatta aggaaatcat
5220acaatgtctg attagagaat ttgcgtttaa aaagatcaat tctattatgt
atagttatgc 5280tatggtaaaa ctcaattcag ataacgaaac attgaaagat
aaaattaagg attattttat 5340agaaactatt cttaaagaca aacgtggtta
taaacaaaag ccattaccct agagcggccg 5400ccaccgcggt ggagctccag
cttttgttcc ctttagtgag ggttaatttc gagcttggcg 5460taatcatggt
catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
5520atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
ctaactcaca 5580ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
acctgtcgtg ccagctgcat 5640taatgaatcg gccaacgcgc ggggagaggc
ggtttgcgta ttgggcgctc ttccgcttcc 5700tcgctcactg actcgctgcg
ctcggtcgtt cggctgcggc gagcggtatc agctcactca 5760aaggcggtaa
tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
5820aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
tttccatagg 5880ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
gtcagaggtg gcgaaacccg 5940acaggactat aaagatacca ggcgtttccc
cctggaagct ccctcgtgcg ctctcctgtt 6000ccgaccctgc cgcttaccgg
atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 6060tctcatagct
cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
6120tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
ctatcgtctt 6180gagtccaacc cggtaagaca cgacttatcg ccactggcag
cagccactgg taacaggatt 6240agcagagcga ggtatgtagg cggtgctaca
gagttcttga agtggtggcc taactacggc 6300tacactagaa ggacagtatt
tggtatctgc gctctgctga agccagttac cttcggaaaa 6360agagttggta
gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
6420tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
gatcttttct 6480acggggtctg acgctcagtg gaacgaaaac tcacgttaag
ggattttggt catgagatta 6540tcaaaaagga tcttcaccta gatcctttta
aattaaaaat gaagttttaa atcaatctaa 6600agtatatatg agtaaacttg
gtctgacagt taccaatgct taatcagtga ggcacctatc 6660tcagcgatct
gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
6720acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
agacccacgc 6780tcaccggctc cagatttatc agcaataaac cagccagccg
gaagggccga gcgcagaagt 6840ggtcctgcaa ctttatccgc ctccatccag
tctattaatt gttgccggga agctagagta 6900agtagttcgc cagttaatag
tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 6960tcacgctcgt
cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
7020acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
gatcgttgtc 7080agaagtaagt tggccgcagt gttatcactc atggttatgg
cagcactgca taattctctt 7140actgtcatgc catccgtaag atgcttttct
gtgactggtg agtactcaac caagtcattc 7200tgagaatagt gtatgcggcg
accgagttgc tcttgcccgg cgtcaatacg ggataatacc 7260gcgccacata
gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
7320ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
tgcacccaac 7380tgatcttcag catcttttac tttcaccagc gtttctgggt
gagcaaaaac aggaaggcaa 7440aatgccgcaa aaaagggaat aagggcgaca
cggaaatgtt gaatactcat actcttcctt 7500tttcaatatt attgaagcat
ttatcagggt tattgtctca tgagcggata catatttgaa 7560tgtatttaga
aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccac
761834885DNAArtificialnucleotide sequence of the insert of the
plasmid pYJ1394.1 3gtaaaacgac ggccagtgaa ttgtaatacg actcactata
gggcgaattg ggtgaccctt 60tacaagaata aaagaagaaa caactgtgaa atagtttata
aatgtaattc gtatgcagaa 120aacgataata tattttggta tgagaaatct
aaaggagaca tagtttgtat agacatgcgc 180tcttccgatg agatattcga
tgcttttcta atgtatcata tagctacaag atatgcctat 240catgatgatg
atatatatct acaaatagtg ttatattatt ctaataatca aaatgttata
300tcttatatta cgaaaaataa atacgttaag tatataagaa ataaaactag
agacgatatt 360cataaagtaa aaatattagc tctagaagac tttacaacgg
aagaaatata ttgttggatt 420agtaatatat aacagcgtag ctgcacggtt
ttgatcattt tccaacaata taaaccaatg 480aaggaggacg actcatcaaa
cataaataac attcacggaa aatattcagt atcagattta 540tcacaagatg
attatgttat tgaatgtata gacggatctt ttgattcgat caagtataga
600gatataaagg ttataataat gaagaataac ggttacgtta attgtagtaa
attatgtaaa 660atgcggaata aatacttttc tagatggttg cgtctttcta
cttctaaagc attattagac 720atttacaata ataagtcagt agataatgct
attgttaaag tctatggtaa aggtaagaaa 780cttattataa caggatttta
tctcaaacaa aatatgatac gttatgttat tgagtggata 840ggggatgatt
ttacaaacga tatatacaaa atgattaatt tctataatgc gttattcggt
900aacgatgaat taaaaatagt atcctgtgaa aacactctat gcccgtttat
agaacttggt 960agatgctatt atggtaaaaa atgtaagtat atacacggag
atcaatgtga tatctgtggt 1020ctatatatac tacaccctac cgatattaac
caacgagttt ctcacaagaa aacttgttta 1080gtagatagag attctttgat
tgtgtttaaa agaagtacca gtaaaaagtg tggcatatgc 1140atagaagaaa
taaacaaaaa acatatttcc gaacagtatt ttggaattct cccaagttgt
1200aaacatattt tttgcctatc atgtataaga cgttgggcag atactaccag
aaatacagat 1260actgaaaata cgtgtcctga atgtagaata gtttttcctt
tcataatacc cagtaggtat 1320tggatagata ataaatatga taaaaaaata
ttatataata gatataagaa aatgattttt 1380acaaaaatac ctataagaac
aataaaaata taattacatt tacggaaaat agctggtttt 1440agtttaccaa
cttagagtaa ttatcatatt gaatctatat tgctaattag ctaataaaaa
1500cccgggttaa ttaattagtc atcaggcagg gcgagaacga gactatctgc
tcgttaatta 1560attagagctt ctttattcta tacttaaaaa gtgaaaataa
atacaaaggt tcttgagggt 1620tgtgttaaat tgaaagcgag aaataatcat
aaattatttc attatcgcga tatccgttaa 1680gtttgtatcg taatggagaa
aatcgtgctg ctgctggcca tcgtgagcct ggtgaaaagc 1740gatcagatct
gcatcggcta ccacgccaac aacagcacag agcaagtgga cacaatcatg
1800gaaaagaacg tgaccgtgac acacgcccag gacatcctgg aaaagacaca
caacgggaag 1860ctgtgcgatc tggatggagt gaagcctctg atcctgagag
attgcagcgt ggccggatgg 1920ctgctgggga acccaatgtg cgacgaattc
atcaacgtgc ccgaatggag ctacatcgtg 1980gagaaggcca acccagccaa
cgacctgtgc tacccaggga acctgaacga ctacgaagaa 2040ctgaaacacc
tgctgagcag aatcaaccac tttgagaaaa tccagatcat ccccaaaagc
2100agctggtccg atcacgaagc cagcagcgga gtgagcagcg cctgcccata
ccagggaaag 2160tccagctttt ttagaaacgt ggtgtggctg atcaaaaaga
acagcgccta cccaacaatc 2220aagagaagct acaacaacac caaccaggaa
gatctgctgg tgctgtgggg gatccaccac 2280cctaacgatg ccgccgagca
gacaaggctg taccagaacc caaccaccta catctccgtg 2340gggacaagca
cactgaacca gagactggtg ccaaaaatcg ccatcagatc caaagtgaac
2400gggcagagcg gaagaatgga gttcttctgg acaatcctga aacccaacga
tgccatcaac 2460ttcgagagca acggaaactt catcgcccca gaatacgcct
acaaaatcgt gaagaaaggg 2520gacagcgcca tcatgaaaag cgaactggaa
tacggcaact gcaacaccaa gtgccagacc 2580ccaatggggg ccatcaacag
cagcatgcca ttccacaaca tccaccctct gaccatcggg 2640gaatgcccca
aatacgtgaa aagcaacaga ctggtgctgg ccaccgggct gagaaacagc
2700cctcagagag agaccagagg actgtttgga gccatcgccg gctttatcga
gggaggatgg 2760cagggaatgg tggatggctg gtacggatac caccacagca
acgagcaggg gagcggatac 2820gccgccgaca aagaatccac ccagaaggcc
atcgacggcg tgaccaacaa agtgaacagc 2880atcatcgaca aaatgaacac
ccagtttgag gccgtgggaa gggagtttaa caacctggaa 2940aggagaatcg
agaacctgaa caagaagatg gaggacggat tcctggatgt gtggacctac
3000aacgccgaac tgctggtgct gatggaaaac gagagaaccc tggactttca
cgacagcaac 3060gtgaagaacc tgtacgacaa agtgaggctg cagctgaggg
ataacgccaa ggagctgggc 3120aacggctgct tcgagttcta ccacaaatgc
gataacgaat gcatggaaag catcagaaac 3180ggaacctaca actaccccca
gtacagcgaa gaagccagac tgaaaagaga agaaatctcc 3240ggagtgaaac
tggaatccat cggaacctac cagatcctga gcatctacag cacagtggcc
3300tcctccctgg ccctggccat catgatggcc ggactgagcc tgtggatgtg
ctccaacgga 3360agcctgcagt gcagaatctg catctgactc gagtttttat
tgactagtta atcataagat 3420aaataatata cagcattgta accatcgtca
tccgttatac ggggaataat attaccatac 3480agtattatta aattttctta
cgaagaatat agatcggtat ttatcgttag tttattttac 3540atttattaat
taaacatgtc tactattacc tgttatggaa atgacaaatt tagttatata
3600atttatgata aaattaagat aataataatg aaatcaaata attatgtaaa
tgctactaga 3660ttatgtgaat tacgaggaag aaagtttacg aactggaaaa
aattaagtga atctaaaata 3720ttagtcgata atgtaaaaaa aataaatgat
aaaactaacc agttaaaaac ggatatgatt 3780atatacgtta aggatattga
tcataaagga agagatactt gcggttacta tgtacaccaa 3840gatctggtat
cttctatatc aaattggata tctccgttat tcgccgttaa ggtaaataaa
3900attattaact attatatatg taatgaatat gatatacgac ttagcgaaat
ggaatctgat 3960atgacagaag taatagatgt agttgataaa ttagtaggag
gatacaatga tgaaatagca 4020gaaataatat atttgtttaa taaatttata
gaaaaatata ttgctaacat atcgttatca 4080actgaattat ctagtatatt
aaataatttt ataaatttta ataaaaaata caataacgac 4140ataaaagata
ttaaatcttt aattcttgat ctgaaaaaca catctataaa actagataaa
4200aagttattcg ataaagataa taatgaatcg aacgatgaaa aattggaaac
agaagttgat 4260aagctaattt ttttcatcta aatagtatta ttttattgaa
gtacgaagtt ttacgttaga 4320taaataataa aggtcgattt ttattttgtt
aaatatcaaa tatgtcatta tctgataaag 4380atacaaaaac acacggtgat
tatcaaccat ctaacgaaca gatattacaa aaaatacgtc 4440ggactatgga
aaacgaagct gatagcctca atagaagaag cattaaagaa attgttgtag
4500atgttatgaa gaattgggat catcctctca acgaagaaat agataaagtt
ctaaactgga 4560aaaatgatac attaaacgat ttagatcatc taaatacaga
tgataatatt aaggaaatca 4620tacaatgtct gattagagaa tttgcgttta
aaaagatcaa ttctattatg tatagttatg 4680ctatggtaaa actcaattca
gataacgaaa cattgaaaga taaaattaag gattatttta 4740tagaaactat
tcttaaagac aaacgtggtt ataaacaaaa gccattaccc tagagcggcc
4800gccaccgcgg tggagctcca gcttttgttc cctttagtga gggttaattt
cgagcttggc 4860gtaatcatgg tcatagctgt ttcct 488549PRTArtificial
SequenceSynthetic 4Arg Glu Arg Arg Arg Lys Lys Arg Gly1
555PRTArtificial SequenceSynthetic 5Arg Glu Thr Arg Gly1 5
* * * * *