Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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INACTIVATED VACCINE AGAINST FELINE CALICIVIRUS DISEASE
The present invention relates to. the use of
particular strains of feline caliciviruses for the
production of immunogenic preparations and of vaccines,
in particular inactivated or subunit vaccines, against
feline calicivirosis. These immunogenic preparations
and these vaccines may also be combined with
immunogenic preparations or vaccines prepared on the
basis' of other feline pathogens, for the production of
multivalent immunogenic preparations and vaccines.
Feline caliciviruses (FCV) were first described
in 1957 (Fastier L.B. Am. J. Vet. Res. 1957. 18, 382-
389). Feline caliciviruses are, with the feline
herpesviruses, the two principal sources of viral
diseases of the upper respiratory tract in cats. The
FCV viruses affect a large number of animals, with FCV
carrying rates of the order of 15 to 25%, and an anti-
FCV seroprevalence of 70 to 100% (Coutts et al. Vet.
Rec. 1994. 135. 555-556; Ellis T.M. Australian Vet. J.
1981. 57. 115-118; Harbour et al. Vet. Rec. 1991. 128.
77-80; Reubel et al. Feline Dendistry 1992. 22. 1347-
1360)_ After an initial phase of hyperthermia, these
respiratory diseases are generally accompanied by
buccal ulcerations (palate, tongue, lips, nose),
rhinitis, conjunctivitis, possibly anorexia and
asthenia. The FCV viruses can also cause pneumonia,
enteritis, and articular pain (lameness, syndrome).
The FCV virus is transmitted only horizontally,
there is no vertical transmission from the mother to
its kitten during gestation (Johnson R.P. Res. Vet.
Sci_ 1984. 31. 114-119). FCV is transmitted by contact
between infected animals and healthy animals or by the
airways during sneezing (Wardley RC_ Arch.' Virol. 1976.
52. 243-249).
Feline caliciviruses are naked viruses of the
Caliciviridae family, they possess a single-stranded
positive RNA of about 7.7 kilobase pairs (kbp) in size
(Radfor et al_ Proc. 1st Int. Symp. Caliciviruses ESVV
1997. 93-99).
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Like many RNA viruses, a large heterogeneity
exists within the viral population of FCV. The
antigenic variations, demonstrated since the beginning
of the 70s by cross-serum neutralization experiments,
make it possible to classify the FCVs into several
viral strains or quasispecies (Radford et al. Proc. 1st
Int. Symp. Caliciviruses ESVV 1997. 93-99).
Several FCV strains have been identified and
isolated, in particular strain F9 (deposited with the
American Type Culture Collection or ATCC under the
accession number VR-782), strain 2280 (ATCC VR-2057),
strain KCD (ATCC VR-651) and strain CFI (ATCC VR-654).
Vaccination against FCV was introduced since
the end of the 70s from attenuated FCV strains, mainly
strain F9 isolated in the USA in 1958 by Bittle (Bittle
et al. Am. J. Vet. Res. 1960. 21. 547-550) or strains
derived from F9 by passage in vitro or in vivo ("F9-
like") .
Inactivated vaccines are also available. They
mainly use strains 255 and 2280, which were isolated in
the USA respectively in 1970 in a cat with a pneumonia
(Kahn and Gillepsie. Cornell Vet. 1970. 60. 669-683;
Powvey et al. J. Am. Vet. Med. Assoc. 1980. 177. 347-
350) and in 1983 in a Cat suffering from lameness
(Pedersen et al. Fel. Prac. 1983. 13. 26-35; Pedersen
N.C_ and Hawkins K.F. Vet. Microbiol. 1995. 47. 141-
156).
Because of antigenic drift over time, antisera
produced against vaccine strains isolated in the 60-
70s, such as strains F9, 255 or 2280, neutralize only
few isolates of the 90s. For example, the anti-F9 serum
neutralizes 43% of the American isolates of the period
1990-1996, against 56% for the period 1980-89 and 86%
for the period 1958-79', and only 10% of the English
isolates of the period 1990-96 (Lauritzen et al. Vet.
Microbiol. 1997. 56. 55-63). Accordingly, attenuated
and inactivated vaccines from old FCV strains at
present no longer offer sufficient protection against
recent FCV strains.
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The objective of the present invention is the
detection of new FCV strains, which induce in cats
antibodies having a broad cross-neutralization
spectrum.
Another. objective of the invention is the
production of immunogenic preparations and of vaccines
against feline calicivirosis from these FCV strains.
Yet another objective of the invention is the
production of multivalent immunogenic preparations and
of multivalent vaccines against feline calicivirosis
and against at least one other feline pathogen.
The Applicant has selected four FCV strains
obtained by pharyngeal swabs taken in France, the
United Kingdom and the USA on cats exhibiting signs of
infection by feline calicivirus. They are respectively
strain G1 (deposited at the Collection Nationale de
Cultures de Microorganismes (or CNCM) of the Institut
Pasteur, Paris, France, under the accession number I-
2167) and strain 431 (deposited at the CNCM under the
accession number 1-2166), both deposited on 12 March
1999. The latter two strains are American and
designated RMI6 and RMI9. The FCV G1 strain isolated
in France does not correspond to the FCV strain
isolated in the United Kingdom in 1978 by Tohya Y.
(Tohya Y. et al. Jpn. J. Sci., 1990, 52, 955-961) and
also called Gi.
The selection of the FCV 431, G1, RMI6 and RMI9
strains was carried out by cross-serum neutralization
tests with respect to the FCV isolates of a reference
panel. This reference panel is composed of 18 current
isolates of FCV -taken from cats exhibiting signs of
infection with feline calicivirus and coming from three
distinct geographical regions.- 7 isolates are American,
these isolates are identified RMI1, RMI2, RMI3, RMI5,
RMI6, RMI7 and RMI9. 7 isolates are French, they are
designated A2, F1, G1, G3, F3031, H3-2 and H1-4. The
last 4 isolates are English, they are designated 431,
388b, 337 and J5.
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The panel strains and the RMI6 and RMI9 strains
are accessible from the Applicant simply on request.
They have also been published in a review
article "Archives of Virology" (Poulet et al. Arch.
Virol. February 2000. 145(2). 243-261), available
online on Internet on the date of filing with the
editor.
During cross-serum neutralization tests between
the 18 FCV isolates of the reference panel, it was
found, surprisingly, that the antiserum for isolate 431
neutralizes 14 of the 17 heterologous isolates of the
reference panel (the homologous serum neutralization
titer is not taken into account). By comparison, the
antisera for the "historical" vaccine strains 255 and
F9 neutralize only 2 of the 18 panel isolates each.
Unexpectedly, the Applicant has therefore found
with the FCV 431 strain a dominant strain which can be
used for the protection of the Felidae and in
particular of cats against most FCV strains. By virtue
of the panel of FCV strains disclosed here, it is
possible for persons skilled in the art to select other
dominant FCV strains. By way of equivalence, the
invention also covers through the FCV 431 strain the
FCV strains which are equivalent thereto, which have
antibodies with broad cross-neutralization spectrum.
Equivalence exists when the antiserum for an
FCV strain seroneutralizes at least 13 of the 18
heterologous isolates of the reference panel (that is
to say including FCV 431), preferably when it
seroneutralizes at least 14 of the 18 heterologous
isolates of the reference panel, still more preferably
when it seroneutralizes at least 15 of the 18
heterologous isolates of the reference panel.
It is generally considered that an FCV strain
seroneutralizes another FCV strain when the
heterologous serum neutralization titer is greater than
or equal to 1.2 log10 VNsp (Povey C. and Ingersoll J.,
Infection and Immunity, 1975, 11, 877-885). The
Applicant took this value as the positivity threshold.
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However, the cross-serum neutralization results
obtained with an FCV isolate having a homologous serum
neutralization titer of less than or equal to 2 log10
VNso cannot be interpreted.
A second method for establishing the
equivalence of an FCV strain with respect to the FCV
431 strain is to use monoclonal antibodies specific for
the FCV 431 strain and to test the candidate FCV strain
by indirect immunofluorescence (IIF). The Applicant has
thus succeeded in producing several monoclonal
antibodies which have proved specific for the 431
strain. One of them was called 44. There is equivalence
if there is reactivity in immunofluorescence with
monoclonal antibodies specific for 431, for example
with the monoclonal antibody 44. This monoclonal
antibody and the corresponding hybridoma are available
from the Applicant upon simple request and are also
disclosed in the article by Poulet et al. Arch. Virol.
2000. 145. 1-19. .The corresponding hybridoma was also
deposited on 11 August 1999 at the CNCM under the
accession number 1-2282. It goes without saying,
however, that persons skilled in the art are perfectly
capable of producing monoclonal antibodies by
conventional techniques and of selecting, relative to
the panel, those which are specific for the 431 strain.
...The first subject of the present invention is
therefore immunogenic preparations and vaccines
prepared from feline calicivirus strain 431, which
includes its equivalents as defined above, preferably
in inactivated or subunit form, in a veterinarily
acceptable vehicle or excipient, and preferably in the
presence of an adjuvant. The notion of immunogenic
preparation covers here any preparation capable, once
administered to cats, of inducing an immune response
directed against the feline pathogen considered.
Vaccine is understood to mean a preparation capable of
inducing effective protection.
The other FCV G1, RMI6 and RMI9 strains were
chosen for their complementarity to the FCV 431 strain,
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namely that the combination of the antisera for 431 and
for one of these three FCVs seroneutralize 100% of the
isolates of the reference panel, that is to say that
these three FCV strains have a homologous serum
neutralization titer greater than or equal to 2 loglo
VN50 and heterologous serum neutralization titers
greater than or equal to 1..2 log,() VNSO with respect to
the FCV isolates of the reference panel against which
the 431 antiserum does not seroneutralize ' or
seroneutralizes weakly (value less than 1.2 loglo VN50) .
The invention also covers the equivalent FCV strains
having the same complementarity with respect to the FCV
431 strain. It is also possible to produce and select
monoclonal antibodies specific for these strains, in
particular for G1, which makes it possible to determine
equivalents on this other basis.
. The second subject of the invention is
therefore immunogenic preparations and vaccines
-comprising, in addition to the antigens of the FCV 431
strain or one* of its equivalents' according to the
invention, antigens of at least one other FCV strain,
especially a complementary strain, in particular chosen
from the group comprising G1, RMI6, RMI9, which
includes their equivalents, in a veterinarily
acceptable vehicle or excipient, and optionally an
adjuvant. Preferably, the antigens obtained from the
other FCV strain(s) comprise inactivated virus or
subunits.
The subject of the invention is in particular
the combination of the two FCV 431 and G1 strains for
the production of. immunogenic preparations or of
inactivated or subunit vaccines.
Surprisingly, the combination of the two FCV Gi
and 431 strains causes advantageously a synergistic
effect. During studies on the complementarity of the
FCV G1 and 431 strains, the immune responses induced by
G1 alone, 431 alone or the combination of both (G1 +
431) were compared- The group of animals which were
immunized with the combination of the two FCV G1 and
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431 strains had the benefit of a better clinical
protection.
The culture and propagation of the FCV viruses
is preferably carried out on feline cells, more
particularly on Crandell-Reese Feline Kidney or CRFK
cells (accessible from the American Type Culture
Collection under the number CCL-94) with a multiplicity
of infection (moi) of 2 to 0.01 cell culture infectious
doses 50% (CCIDso) per cell, preferably 0.5 CCID50/cell.
After harvesting and clarifying, the FCV
viruses intended to produce an inactivated immunogenic
preparation or an inactivated vaccine are inactivated
by a chemical treatment (e.g. formalin or formaldehyde,
0-propiolactone, ethylenimine, binary ethylenimine
(BEI)) and/or a heat treatment. Preferably, the viruses
according to the invention are inactivated by the
action of ethylenimine formed immediately before use
from bromoethylamine (BEA) . The viral particles may be
concentrated by conventional concentration techniques,
in particular by ultrafiltration and then optionally
purified by conventional. purification means, in
particular gel filtration techniques or selective
precipitation techniques in particular in the presence
of polyethylene glycol (PEG) . A purification without
previous concentration can also be done.
For the production of an immunogenic
preparation or of an inactivated or subunit vaccine,
the viral particles are taken up in a veterinarily
acceptable vehicle or excipient, and optionally
supplemented with an adjuvant. The quantity of antigen
is in particular equal to a preinactivation titer of
about 105 to about 1010 CCID50 per dose, preferably of
about 108 to about 109 CCID50 per dose.
To supplement the immunogenic preparations and
vaccines- according to the invention with adjuvants, it
is possible to use as adjuvant (1) aluminum hydroxide,
(2) a polymer of acrylic or methacrylic acid, a polymer
of maleic anhydride and of alkenyl derivative, or (3)
to formulate the immunogenic preparation or vaccine in
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the form of an oil-in-water emulsion, in particular the
emulsion SPT described p 147 "Vaccine Design, The
Subunit and Adjuvant Approach". edited by M. Powell,
M. Newman, Plenum Press 1995, and the emulsion MF59
described p 183 in the same book.
The oil-in-water emulsion may in particular be
based on light liquid paraffin oil (European
Pharmacopeia type); isoprenoid oil such as squalane,
squalene; oil resulting from the oligomerization of
alkenes, in particular of isobutene or of decene;
esters of acids or alcohols containing a linear alkyl
group, more particularly vegetable oils, ethyl oleate,
propylene glycol di(caprylate/caprate), glyceryl
tri(caprylate/caprate), propylene glycol dioleate;
esters of branched fatty alcohols or acids, in
particular esters of isostearic acid. The oil is used
in combination with emulsifiers to form the emulsion.
The emulsifiers are preferably nonionic surfactants, in
particular the esters of sorbitan, mannide, glycerol,
polyglycerol, propylene glycol and of oleic,
isostearic, ricinoleic or hydroxystearic acid, which
are optionally ethoxylated, the polyoxypropylene-
polyoxyethylene block copolymers, in particular the
Pluronic copolymers, especially L121-
The polymers of acrylic or methacrylic acid are
crosslinked, in particular with polyalkenyl ethers of
sugars or polyalcohols. These compounds are known under
the term carbomer (Pharmeuropa vol. 8, No. 2, June
1996). Persons skilled in the art can also refer to US-
A-2,909,462 describing such acrylic polymers crosslinked
with a polyhydroxylated compound having at least 3 hydroxyl
groups, preferably not more than 8, the hydrogen atoms
of at least three hydroxyls being replaced with
unsaturated aliphatic radicals having at least 2 carbon
atoms. The preferred radicals are those containing 2 to
4 carbon atoms, e.g. vinyls, allyls and other
ethylenically unsaturated groups. The unsaturated
radicals may themselves contain other substituents,
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such as methyl. The products sold undei the name
Carbopol (BF Goodrich, Ohio, USA) are particularly
appropriate. They are crosslinked with an allyl sucrose
or with allylpentaerythritol. Among them, there may be
mentioned Carbopol 974P, 934P and 971P.
Among the copolymers of maleic anhydride and of
alkenyl derivative, the EMA copolymers (Monsanto)
which are copolymers of maleic anhydride and of
ethylene, which are linear or crosslinked, for example
crosslinked with divinyl ether, are preferred.
Reference may be made to J.. Fields et al., Nature, 186:
778-780, 4 June 1960. From the point of view of their
structure, the polymers of acrylic or methacrylic acid and the
EMA copolymers are preferably formed of basic units of
the following formula:
R, R2
I I
-----C (CH2)x - Ci (CH2)y .---
COOH COOH
in which:
- R1 and R2, which are identical or different,
represent H or CH3
- x = 0 or 1, preferably x = 1
- y= 1 or 2, with x+ y 2
For the EMA copolymers, x = 0 and y = 2. For
the carbomers, x = y = 1.
The concentration of polymer in the final
vaccine composition will be from 0.01% to. 1.5% W/V,
more particularly from 0.05 to 1% W/V, preferably from
0.1 to 0.4% W/V.
It is of course possible to also combine
-30 inactivated virus and subunits of the same FCV strain
in accordance with the invention and/or of different
FCV strains in accordance with the invention.
The subject of the invention is also a
multivalent vaccine comprising one inactivated feline
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calicivirus valency, comprising. at least the FCV 431
strain, which includes its equivalents, and optionally
at least one other FCV strain, in particular a strain
which is complementary within the meaning of the
invention, in particular chosen from G1, RMI6 and RM19,
and at least one valency for another feline pathogen,
in a veterinarily acceptable vehicle or excipient and
'preferably with an adjuvant, in particular one of those
described above. It is likewise possible to produce
subunit-based multivalent vaccines.
Said feline pathogens are in particular chosen
from the group comprising the feline rhinotrachitis
virus or the feline herpesvirus (FHV), the feline
leukemia virus (FeLV), feline panleukopenia virus or
feline parvovirus (FPV), the feline infectious
peritonitis virus (FIPV), the feline immunodeficiency
virus (FIV), the rabies virus, Chlamydia.
The FCV vaccines according to the invention may
be mixed immediately before use with the other feline
valency (valencies) which may be in the form of
attenuated live, inactivated, subunit, recombinant or
polynucleotide vaccines.
The subject of the invention is therefore also
a multivalent vaccination kit or box comprising,
packaged separately, an FCV valency according to the
invention in a veterinarily acceptable vehicle or
excipient, and preferably with an adjuvant, and at
least one valency of another feline pathogen. The FCV
valency can serve as solvent for another feline
valency, in particular attenuated, recombinant or
polynucleotide valency provided in freeze-dried. form.
In accordance with a feature of the invention,
it is- possible to produce immunogenic preparations or
subunit vaccines, by extraction of the capsid from the
virus, with optionally inactivation before or after the
extraction. These preparations and vaccines therefore
comprise, as sole active ingredient or otherwise, such
a product of extraction containing predominantly capsid
protein and optionally subfragments, optionally
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inactivated, produced from the strains according to the
invention, in particular strain 431, which includes its
equivalents, optionally also from another FCV strain,
in particular Glor equivalents. These subunit vaccines
and preparations are advantageously supplemented with
adjuvant, for example as described supra. It is also
possible to mix whole inactivated vaccine or
preparation and subunit vaccine or preparation.
The subject of the invention is also an
immunogenic preparation or a vaccine based on the G1
strain, in particular which. is inactivated or a subunit
of extraction.
The subject of the invention is also a method
of immunizing cats against diseases caused by the
feline caliciviruses.
This method of immunization comprises the
administration of a combined multivalent, subunit or
inactivated FCV vaccine according to the invention to
cats. The administration of said. vaccine may be carried
out in particular by the parenteral route, preferably
by the subcutaneous or intramuscular route.
Persons skilled in the art have the competence
necessary to define precisely the number of injections
and the doses of each vaccine to be used for each
vaccination protocol.
The dose volumes may be in particular between
0.2 and 2 ml, preferably of the order of 1 ml.
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According to one aspect of the invention, there is provided an
immunogenic preparation or vaccine against feline calicivirus (FCV) comprising
an
isolated FCV that binds to monoclonal antibody 44 secreted by the hybridoma
deposited at the CNCM under the access number 1-2282, or a capsid protein of
an
isolated FCV that binds to monoclonal antibody 44 secreted by the hybridoma
deposited at the CNCM under the access number 1-2282, and a veterinarily
acceptable vehicle or excipient.
According to another aspect of the present invention, there is
provided an immunogenic preparation or vaccine against feline calicivirus
(FCV)
comprising an isolated FCV strain 431 deposited at the CNCM under the
accession number 1-2166, and a veterinarily acceptable vehicle or excipient.
According to still another aspect of the present invention, there is
provided a hybridoma as deposited at the CNCM under the accession number
1-2282.
The invention will now be described in greater detail with the aid of
the embodiments taken by way of nonlimiting examples giving a table of the
cross-serum neutralization titers.
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Examples:
Example 1 : Viral isolates and hybridomas
The feline caliciviruses (FCV) were obtained by
pharyngeal swabs taken on cats exhibiting signs of
infection with feline caliciviruses. These FCVs have
different geographical origins.
The FCV 431, 337, J5, 388b, 220 and 393 strains
were isolated in Great Britain and provided by
Professor O. Jarrett of the University of Glasgow, UK.
The FCV A2, G1, G3, F3031, Fl, H3-2 and H1-4
strains were isolated in France by the Applicant.
The FCV RMI1, RMI2, RMI3, RMI5, RM16, RMI7 and
FMI9 strains were isolated in the USA by the Applicant.
The pharyngeal samples were collected in 2 ml
of Dulbecco's modified Eagle's minimum medium (DMEM,
Gibco BRL), supplemented with 5% fetal calf serum
(Bayer Diagnostic), with antibiotics, more particularly
with 50 mg/l 'of gentamycin. Each isolate is frozen at
-70 C while waiting to be tested. The monoclonal
antibody 44, obtained from the hybridoma identified
431 2 0 17 E9 T, is specific for the FCV 431 strain.
Example 2 : Amplification of the viral isolates
Cells of the cat kidney line (Crandell-Reese
Feline Kidney or CRFK No. ATCC CCL-94, Crandell et al.
In Vitro 1973. 9. 176-185) are cultured in a 96-well
plate or in a 25-cm2 Falcon (Falcon) with DMEM medium
supplemented with 5% fetal calf serum, containing about
100,000 cells per ml. The cells are cultured at 37 C in
an atmosphere containing 5% CO2. After 3 days, the cell
layer arrives at confluence. The culture medium is then
replaced with serum-free DMEM medium supplemented with
50 mg/l of gentamycin and the thawed aliquote of the
FCV viral isolates (Example 1) are added at the rate of
a volume of 100 l of four-fold serial dilutions per
well for the limiting dilution cloning of the FCV
viruses or of 1 ml per Falcon.
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When the cytopathic effect (ECP) is complete
(24-48 hours after the start of the culture), the viral
suspensions are harvested and frozen at -70 C. 3 to 4
successive passages are generally necessary for the
production of a viral batch. The viral batch is stored
at -70 C.
Example 3 : Production of serum
For each FCV virus, an antiserum was produced
by inoculating kittens by the oronasal route with 106.0
CCID50 of the relevant FCV virus. The specific pathogen-
free (SPF) kittens were 10 to 14 weeks old. The serum
of each animal was collected one month after the
infection. The sera were heat-inactivated (30 minutes
at 56 C), distributed, aliquoted and stored at -20 C.
Example 4 : Cross-serum-neutralization in vitro
Cross-serum neutralization tests were carried
out between 18 field isolates obtained by pharyngeal
swabs performed on cats exhibiting signs of feline
calicivirosis. 7 of them have as geographical origin
France, they are the isolates identified A2, F3031, G1,
G3, Fl, H3-2 and H1-4. 4 have as geographical origin
Great Britain, they are the isolates identified J5,
337, 388b and 431. Finally, 7 have as geographical
origin the USA, they are the isolates identified RMI1,
RMI2, RMI3, RMI5, RMI6, RMI7 and RMI9_
The serum obtained for each isolate (Example 3)
was tested for its ability to neutralize the 18
isolates. The sera were three-fold serially diluted
with DMEM medium in 96-well cell culture plates.
0.05 ml of culture medium containing approximately
100*CCID50 of the selected viral strain was added to
0.05 ml of the dilute serum produced as in Example 2.
This mixture was incubated for 2 hours at 37 C in an
incubator under an atmosphere containing 5% CO2.
0.15 ml of a suspension of CRFK cells
containing about 100,000 cells per ml was then added to
each mixture. The cytopathic effect was observed by
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phase contrast microscopy after 4 days of culture at
37 C in an atmosphere containing 5% CO2. The
neutralizing titers of each serum were calculated
according to the Karber method. The titers are given in
the form of the highest dilution inhibiting the
cytopathic effect for 50% of the wells. The titers are
expressed in logio. The minimum titer thus found was
0.7 log10 VN50. Each serum was titrated at least twice,
preferably three times.
Example 5 : Indirect immunofluorescence (IIF) tests
The IIF tests are carried out on 96-well plates
containing the CRFK cells cultured in monolayers
infected with the FCV viruses to be tested.
200 gl per well of a suspension of CRFK cells
containing 90,00'0 cells/ml in F15 medium (Gibco BRL,
Cat # 045-1075) containing 5% fetal calf serum are
cultured in a 96-well plate. At confluence, 320 CCID50
of FCV are inoculated in 100 l of F15 medium. When the
first CPE foci appear, the cells are then rinsed with
cold PBS with no calcium or magnesium (PBS, Sigma), and
then fixed at -20 C for 30 minutes with cold acetone
containing 5% v/v of water. After drying, the infected
and fixed cells are brought into contact for 30 minutes
at 37 C with 100 l per well of ascitic fluid
corresponding to the anti-FCV 431 monoclonal antibody
44 (hybridoma 431 2 0 17 E9 T, diluted 1/5000
approximately in 50 mM TRIS-HCl buffer, pH 7.6.
After two rinses in PBS, the attachment of the
antibodies is visualized by incubation under' the same
conditions of a goat anti-mouse IgG antibody conjugated
with fluorescein isothianate (Biosys, FITC conjugated
at 2.mg/ml) and diluted 1/150 in 50 mM TRIS-HC1 buffer,
pH 7.6. The reading is made under an optical microscope
under UV light.
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This monoclonal antibody was tested with
respect to each of the isolates of the panel. It is
attached exclusively to the CRFK cells infected with
FCV 431.
This test may be used to determine the
equivalents of the FCV 431 strain. These equivalents
are those to which the monoclonal antibody 44.attaches.
Example 6 : Synergy
32 non-vaccinated SPF kittens about 9 weeks old
are divided by randomization into 4 groups (identified
from A to D) of 8 kittens each, each group is housed in
an isolated box.
After thawing the viral suspensions (Example 2) and
diluting in PBS so as to obtain the desired titer, the
cats are vaccinated by subcutaneous injection of 1 ml
of FCV G1 inoculum at 103.3 CCID50/ml for group B, of
1 ml of FCV 431 inoculum at 103'5 CCID50/ml for group C,
of 0.5 ml of FCV Gl inoculum at 1033 CCID50/ml and
0.5 ml of FCV 431 inoculum at 103'5 CCID50/ml (at a
different injection site) for group D. Group A serves
as control group.
Half of each group A to D is randomly
distributed into two groups 1 and 2 and housed in
separate boxes. The animals are challenged on the 31st
day after vaccination (d31).
The animals in group 1 are challenged by
administration of 1 ml of challenge viral strain FCV
220 having a titer of 107.2 CCID50/ml by the oronasal
route (0.5 ml by the oral route and 0.25 ml into each
nostril).
The animals in group 2 are challenged by
administration of 1 ml of challenge viral strain FCV
393 having a titer of 106 8 CCID50/ml by the oronasal
route (0.5 ml by the oral route and 0.25 ml into each
nostril).
The virulent strains FCV 220 and 393 were
chosen because they are distant in cross-serum
neutralization from the viral strains FCV G1 and 431.
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Any cross-contamination between the two boxes
is carefully avoided. Clinical monitoring of the
animals in both groups is done by taking the rectal
temperature and clinical examinations of the animals
(general state, presence of ulcers of the tongue and of
the palate, presence of gingivitis, presence of
rhinitis, presence of conjunctivitis, presence of
lameness, death of the animal).
The total clinical score for each animal was
calculated by adding the scores obtained for each group
of clinical signs according to the following scale:
rectal temperature:
0 - less than 39 C
1 - greater than or equal to 39 C and less than
39.5 C
2 - greater than or equal to 39.5 C and less than
40 C
3 - greater than'or equal to 40 C
- general state:
0 - normal behavior
1 - exhaustion
- ulcers of the tongue and of the palate (some of the
diameters of all the ulcers, if there are several):
0 - absence of ulcer
1 - diameter of 1 to 5 mm
2 - diameter of 6 to 10 mm
3 - diameter greater than 10 mm
gingivitis:
0 - absence of gingivitis
1 - gingivitis
rhinitis:
0 - absence of rhinitis
1 - rhinitis with serous nasal discharge
2 - rhinitis with mucous to mucopurulent nasal
discharge
- conjunctivitis:
0 - absence of conjunctivitis
1 --conjunctivitis with serous discharge
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2 - conjunctivitis with mucopurulent discharge
- lameness:
0 - absence of lameness
1 - lameness
- death:
0 - survival
5 - death.
The mean clinical scores obtained are the
following:
Group/challenge FCV 220 FCV 393
Control (group A) 31 30
FCV G1 (group B) 5 23
FCV 431 (group C) 6 18
FCV G1 + FCV 431 (group D) 2 9
The results thus obtained show synergy between
the FCV G1 and FCV 431 strains by a significant
difference between the mean value obtained for the best
strains and that obtained for the combination of the
two strains (Kruskal-Wallis test).
Example 7 : Production of inactivated vaccine
The CRFK cells are cultured at 37 C in 2-liter
roller flasks (850 cm2) in modified Eagle's medium
(MEM, Gibco BRL) supplemented with 2.5% of lactalbumin
hydrolysate (Gibco BRL) and 5% fetal calf serum (Gibco
BRL): 300 ml of a cellular suspension in MEM medium
contaiiing about 100,000 cells/ml are added per roller
flask. After 3 days, the cell layer becomes confluent.
The cell culture medium is then replaced with serum-
free MEM medium and the FCV virus added at a
multiplicity of infection (moi) of 0.5 CCID50/cell. The
viral culture is maintained. at 37 C for 24 to 48 hours
until a cytopathic effect is obtained for the whole
cellular lawn. The viral suspension is harvested and
then clarified on a bag filter having a porosity of
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1.5 pm. The FCV virus titer at harvest is -8.5 +/- 0.3
loglO CCID50/ml.
The virus is inactivated with ethylenimine at
the concentration of about 8 mM at 22 C for 18 hours.
The ethylenimine is prepared immediately before
use by dissolving 28 g of sodium hydroxide pellets in
200 ml of distilled water and adding 68.1 g of
bromoethylamine (BEA) corresponding to a 1.2 M solution
approximately (H. Bahnemann, Arch. Virol., 1975, 47,
47-56). The =inactivatec: viral suspension is
concentrated 100-fold on an Ultrasette-type
ultrafiltration cartridge with a cut-off of 100 kDa
(Filtron) and then frozen at -70 C.
The inactivated viral suspension after thawing
is diluted 1/33 in PBS buffer (NaCl 8 g/l; KC1 0.2 g/l;
KH2PO4 0.2 g/l; Na2HPO4, 2 H2O 1.44 g/1) . The vaccine is
prepared in the same manner: 167 ml of aqueous phase
consisting of the dilution of the inactivated virus are
emulsified in 83 ml of an oily phase containing 7% w/v-
of anhydromannitol oleate, 8% w/v of ethoxylated oleic
acid containing 11 molecules of ethylene oxide (EO) on
average and 85% v/v of light liquid paraffin oil
(European Pharmacopeia type) with the aid of a
Silversor*turbine emulsifier at 32 C for 2 minutes. The
vaccine is then stored at 5 C.
An alternative method for preparing the vaccine
consists in forming into an emulsion by three passes
through a model Y110 high-pressure homogenizer
(Microfluidics Corp.) at a pressure of 600 bar and a
temperature of between 30 and 40 C the mixture 5% w/v
squalane, 2.5% w/v Pluronic L121, 0.2% w/v Tween*80,
92.3% v/v of inactivated viral suspension diluted 1/46
in PBS buffer after thawing. The vaccine is then stored
at 5 C.
Another alternative method consists in
preparing a solution containing 0.4% w/v of Carbopol
974P in physiological saline (NaCl 9 g/l). The pH is
adjusted to 7.3-7.4 with sodium hydroxide. This
solution of Carbopol is then mixed in equal parts with
*Trade-mark
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the suspension of inactivated FCV virus diluted 1/25
after thawing. The vaccine is then stored at 5 C.
The aqueous phase of the emulsions or the
aqueous phase mixed with Carbopol consists of a
dilution in PBS of the concentrated inactivated viral
suspension corresponding either to the FCV 431 strain
or to the FCV G1 strain or a mixture in equal parts of
the FCV 431 and Gi strains.
Example 8 Control of the immunogenicity of
inactivated FCV 431
19 nonvaccinated SPF kittens about 9 weeks old
are divided by randomization into 2 groups (identified
from A and B), the first with 12 kittens and the second
with 7 kittens, each group is housed in an isolated
box.
The vaccine is prepared with the adjuvant
composed of anhydromannitol oleate, ethoxylated oleic
acid and light liquid paraffin oil as described in
Example 7. -
The cats are vaccinated twice (DO and D28) by
subcutaneous injection of 1 ml of FCV 431 inoculum at
10' CCID50/ml for group A.. Group B serves as control
group.
The animals are challenged on the 42d day after
the first vaccination (D42) by administration of 1 ml
of challenge viral strain FCV 431 having a titer of 106
CCID50/ml by the oronasal route (0.5 ml by the oral
route and 0.25 ml-into each nostril).
The level of anti-FCV 431 neutralizing
antibodies and the clinical score were monitored. The
total clinical score for each animal was calculated by
adding. the scores obtained for each group of clinical
signs according to the scale given in Example 6.
The results obtained are the following:
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Anti-FCV 431 neutralizing antibody titers expressed as
login VN50/ml:
Group Antibody Antibody on Antibody on
on DO D28 D42
FCV 431 vaccine 0.24 1.61 2.87
(group A)
Controls 0.24 0.24 0.24
(group B)
Mean clinical scores over the period D42 to D56:
Group Clinical score
FCV 431 vaccine 0.7
(group A)
Controls 33.7
(group B)
These results show an excellent clinical
protection against the homologous challenge and good
seroconversion.
It should be clearly understood that the
invention defined by the appended claims is not limited
to the specific embodiments indicated in the
description above, but encompasses the variants which
do not depart from the scope or the spirit of the
present invention.
