Note: Descriptions are shown in the official language in which they were submitted.
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VACCINATION AGAINST CANINE HERPESVIROSIS AND VACCINES THEREFOR
The present invention relates to vaccination
against canine herpesvirus. The invention relates in
particular to a vaccination method, to suitable
vaccines and to the use of vaccines in the context of
this vaccination method.
Canine herpesvirus (or CHV) was described for
the first time in 1965 (L.E. Carmichael, Am. J. Vet.
Res., 1965, 26, 803-814). Infection with canine
herpesvirus was then identified in many countries (USA,
France, United Kingdom, Switzerland, Italy, Japan,
Australia, New Zealand etc.). CHV is responsible for
reproductive problems in canine breeding and most
particularly for mortality in newborn puppies, which
may give rise to significant losses in certain breeding
kennels. It is also responsible for abortions and
still-births. Infection with CHV is moreover suspected
of reducing fertility in bitches. The transmission of
CHV takes place via the venereal route, the oronasal
route or the transplacental route.
CHV belongs to the family of Herpesviridae and
to the subfamily of Alphaherpesvirinae. To date, only
one type of CHV has been identified. This is an
enveloped virus containing a double-stranded DNA
molecule. The envelope contains several glycoproteins
of viral origin. The glycoproteins gE, gC and gD are
responsible for the induction of neutralizing
antibodies in mice.
CHV is antigenically similar to feline
herpesvirus (J.A. Lincumpao et al., Arch. Virol., 1990,
111, 165-176) but has no significant cross-
neutralization (X. Xuan et a/., Arch. Virol., 1992,
122, 359-365).
CHV is strongly dependent on its host and
multiplies only on cells of canine origin (Pierson et
a/., recueil de Medecine V6terinaire, March/April 1998,
174 No. 3/4, 87-94).
Newborn puppies are highly sensitive to
infection with CHV during the first 3 weeks of life.
The contamination occurs during whelping or the first
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few days of life. Consequently, it is not possible to
vaccinate the puppies. The only means of protection
available are hygiene measures such as heating the
puppies with an infrared lamp and serotherapy. However,
CHV is relatively non-immunogenic and it is difficult
to prepare a good serum (L.E. Carmichael, J. Am. Vet.
Med. Assoc., 1970, 156, 1714-1721).
Under natural conditions, puppies can be
protected with the CHV-neutralizing antibodies present
in the colostrum of seropositive bitches: in the
absence of these antibodies, the puppies are highly
sensitive to infection with CHV (D.L. Huxsoll and I.E.
Hemelt, J. Am. Vet. Med. Assoc., 1970, 156, 1706-1713).
The at-risk animals are puppies born from uninfected
bitches in contact with excreting animals, and puppies
born from latent but seronegative or weakly
seropositive infected bitches. The CHV-neutralizing
antibodies do not persist for a long time, and an
animal can be infected and seronegative.
In puppies less than 3 weeks old, CHV causes a
neonatal herpesvirosis which is often fatal. The
incubation is rapid and the puppies die within a few
days, usually with no symptoms other than sudden
hypothermia and devastating nervous disorders, in the
vast majority of cases before the 5-day-old stage. In
the less acute forms, the hypothermia is followed by
symptoms such as anorexia, depression, bradycardia,
hypoglycemia, subcutaneous edema, erythema and ventral
papules, soft yellowish-gray stools, abdominal pains,
vomiting, incessant whining, pedaling and death within
24 to 72 hours in opisthotonus. The necropsic
examination may reveal an enlarged spleen, serous
effusion in the pleural and peritoneal cavities and
visceral petechia (Pierson et a/., recueil de Medecine
Veterinaire, March/April 1998, 174 No. 3/4, 87-94).
This article recalls that no vaccine against canine
herpesvirosis is currently marketed and suggests that
it would be useful to be able to vaccinate latent
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infected breeders in order to attenuate the re-
excretion of the wild-type virus.
On the other hand, in her article (Rec. Med.
Vet., 1982, 158, 669-676), F. Delisle proposes
vaccinating bitches before gestation with an
inactivated vaccine or injecting the puppies with a
serum at whelping. She recalls, however, that no
commercial vaccine is available. See also L.E.
Carmichael, J. Am. Vet. Med. Assoc., 1970, 156, 1714-
1721. In his article ("Canine Herpesvirus" in Virus
Infections of Vertebrates, vol.1, M.C. Horzinek, Ed.
M.J. Appel, Elsevier Science Publisher, 1987, 5-15), A.
Appel proposes vaccinating bitches before or at the
very start of gestation with an inactivated vaccine.
H. Poulet and P. Dubourget (Point Vet., 1993,
25, 69-75) suggested in 1992, in general terms, the
vaccination of the mother with a booster vaccination at
the end of gestation. The authors also recall that
tests using viruses inactivated with formaldehyde or
attenuated variants were carried out (L.E. Carmichael
et a/., Infection and Immunity, 1978, 20(1), 108-114;
US-A-4,213,965; attenuated live vaccine, composed of a
heat-sensitive variant of the CHV virus, referred to as
being "of small range"), but these tests did not yield
any convincing results and did not lead to the
marketing of vaccines.
In his article (Vet. Med., April 1991, 4, 394-
403), J.O. Anvik points out that the production of
neutralizing antibodies in the mother and consequently
the protection of the litter is unpredictable on
account of the low immunogenicity of the CHV virus.
Faced with the difficulty of vaccination,
Pierson et al. (recueil de Medecine Veterinaire, March/
April 1998, 174 No.3/4, 87-94) even went as far as to
propose vaccinating the mother with an inactivated
vaccine against felid herpesvirus (FHV) or
administering anti-FHV antibodies to puppies at
whelping.
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To date, none of the vaccination hypotheses has been confirmed, no
vaccine is commercially available and no publication has presented convincing
vaccination results announcing the impending marketing of such a vaccine.
In addition, in gestating bitches, infection with CHV can lead to
embryonic resorption, fetal mummification, abortion or premature whelping,
still-birth
or neonatal mortality. Placental lesions may appear.
Although vaccines against various herpesviruses exist in other animal
species, including cats, it has never been possible to propose effective
vaccination
against canine herpesvirosis.
The object of the present invention is to develop a method for
vaccination against canine herpesvirosis which allows puppies to be protected.
Another object of the invention is to produce efficient and
pharmaceutically acceptable vaccines which can be used in the context of this
vaccination method.
In a particular embodiment, the invention relates to the use, for
protecting a puppy against Canine Herpes Virus (CHV), of a vaccine comprising
inactivated CHV or glycoprotein-extract from inactivated CHV, and an oil-in-
water
based emulsion comprising paraffin oil, wherein the vaccine is for
administration to
the mother of the puppy on two occasions, 10 days after insemination and 10
days
before the expected date of whelping.
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The Applicant has found, surprisingly, that it
is possible to vaccinate gestating bitches and newborn
puppies by administering a vaccine during the gestation
25 period and in particular as close as possible to
whelping, so as to have a high level of anti-CHV
antibodies in the gestating bitch at the, time of
whelping, this level providing protection to the
puppies at whelping by transmission of the maternal
30 antibodies during suckling. In addition, these
antibodies are substantially maintained in the puppies
during the first weeks of their life, this being the
period during which they are prone to disease (3 to 4
weeks). A titer of neutralizing antibodies in the bitch
35 of greater than or equal to 0.9 10g10 is preferred,
more particularly a titer greater than or equal to
1.2 10g10. The titers indicated are calculated
according to the method described in Example 7.
Vaccination of the bitch moreover leads to a reduction
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in the pressure of infection in the puppies. The
Applicant has moreover observed, in bitches vaccinated
and maintained in a contaminated environment, a higher
weight of puppies at whelping and a tendency toward a
higher level of gestation. These phenomena can be
explained by a reduction or absence of placental
lesions in the vaccinated bitches (A. Hashimoto et al.,
Am. J. Vet. Res., 1979, 40(9), 1236-1240; A. Hashimoto
et a/., Am. J. Vet. Res., 1982, 43(5), 844-850).
The objects of the invention already mentioned,
as well as others, are obtained with the aid of a
method for vaccinating gestating bitches which
comprises the administration of a vaccine to a
gestating bitch as close as possible to whelping, in
particular during the final third of gestation (the
average duration of gestation in dogs is about 63
days), notably 1 to 20 days, in particular 1 to 15
days, more particularly 5 to 15 days, preferably 5 to
10 days before the presumed date of whelping, typically
about 10 days before, so as to have a high level of
anti-CHV antibodies in the gestating bitch at whelping,
which induces protection of the puppies by transfer of
the maternal antibodies during suckling. These
antibodies are substantially maintained during the
first weeks of life of the puppies, this being the
period during which they are prone to disease (3 to 4
weeks).
By definition, an antigen, in an effective
amount in the vaccine according to the invention, makes
it possible to induce a high and protective level of
antibodies under the conditions of the protocol in the
gestating bitch and the newborn puppy. This may be an
- antigen comprising the inactivated CHV virus, the
attenuated CHV virus, one or more subunits of the CHV
virus or one or more antigens expressed by
recombinants.
Preferably, the vaccine is intended to produce
= in the gestating bitch, at the time of whelping, a
level of anti-CHV antibodies (determined according to
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example 7) of greater than or equal to about 0.9 10g10,
preferably greater than or equal to about 1.2 log10.
Especially in the case of an animal which has
never been vaccinated against CHV, another' (or several
other) administration(s) of anti-CHV vaccine is
preferably carried out before the administration
described above in order to induce the immune response
in the animal. The interval between these two
administrations must be sufficient to induce a booster
effect, which generally requires at least 2 to 3 weeks.
The first administration of vaccine is notably done
within a period which goes from oestrus and at least 2
or 3 weeks before the above described administration,
in particular within the period which goes from oestrus
to the first third of the gestation, including the
first third of gestation, which period includes
administration before insemination. Preferably this
administration is done about 7 to 10 days after the
insemination. According to a preferred embodiment, this
vaccination scheme is applied for each whelping
occurrence.
If the animal has already been vaccinated against
CHV, a single administration close to whelping may
suffice.
The administration, or at least one of the
additional administrations, can also be carried out
with a vaccine whose composition is different from that
used as close as possible to whelping, the difference
possibly lying in the antigen and/or the formulation of
the vaccine.
The vaccine can be administered especially via
the parenteral route, preferably via the subcutaneous
=or intramuscular route.
The dose volumes can be, in particular, between
0.2 and 2 ml, preferably about 1 ml.
A subject of the invention is also the use of
CHV antigen to prepare a vaccine against canine
herpesvirosis, which is intended to be administered
according to the protocol described above.
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In one aspect, the present invention relates to use, for protecting a
puppy against Canine Herpes Virus (CHV) of a vaccine comprising inactivated
CHV
or glycoprotein-extract from inactivated CHV, and an adjuvant, wherein the
vaccine
is for administration to the mother of the puppy on two occasions, 1 to 20
days prior
to whelping.
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A person skilled in the art has the competence
required to accurately define the doses of each vaccine
as a function of the type of vaccine.
When several administrations are performed on
the animal, the vaccines used can be of different
types, chosen in particular from those mentioned above.
A subject of the invention is also inactivated
vaccines or subunit vaccines, which can be used in
particular in the context of this method of
vaccination.
The use of CHV subunits has only been described
in mice (J.A. Limcumpao et a/., J. Vet. Med. Sc.,
1991, 53(3), 423-432). However, the data obtained in
mice cannot be extrapolated to the canine species, as
stated in that document. Although the antibody titers
obtained with the purified glycoproteins gp145/112,
gp80 and gp47 induce a detectable antibody response, it
is not possible, however, to determine whether or not
this antibody titer may be reflected by a protection
since, as mice are not sensitive to the CHV virus, it
is not possible to develop a virulent challenge model
in this species. Furthermore, no data are available
regarding the cellular immunity induced by CHV.
Moreover, the relationship between the antibody titer
obtained in mice and that obtained in dogs is unknown
and these titers may prove to be different. Thus, it
has been published that rabbits immunized with type 1
bovine herpes glycoprotein gI have a higher
neutralizing titer than rabbits immunized with gill,
whereas in cattle, the glycoprotein gIV induces the
highest neutralizing titer and the glycoprotein gI is
the least immunogenic. These data show the difficulty
of transposing results from laboratory animals to the
target species.
In accordance with the invention, the use of an
inactivated vaccine or of subunits allows the
vaccination of bitches during the gestation phase
without any risk to the bitch or its offspring.
Vaccination with booster at the end of gestation is the
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means for assuring an optimum titer of neutralizing
antibodies at whelping. This titer conditions the
protection obtained by transfer of neutralizing
antibodies from the bitch to its puppies at the start
of suckling.
The CHV virus is preferably cultured and
propagated on primary canine cells or on a cell line,
more particularly on canine kidney cells of a line such
as MDCK (Madin-Darby Canine Kidney, available from the
American Type Culture Collection (ATCC) under the
number CCL-34) in particular with a multiplicity of
infection (moi) of from 0.1 to 0.001 tissue culture
infective doses 50% (TCID50 per cell, preferably 0.01
TCIDso/cell. The CHV virus is harvested 3 to 4 days
later. A viral titer of about 106 to 108 TCID50/m1 and
in general from 106'6 to 107'5 TCID50/m1 is obtained.
In order to obtain an inactivated vaccine, the
CHV virus is inactivated, preferably after harvesting
and clarification, by means of a chemical treatment
(e.g. formalin or formaldehyde, p-propiolactone,
ethyleneimine, binary ethyleneimine (BEI)) and/or a
heat treatment. Preferably, the virus according to the
invention is inactivated by the action of
ethyleneimine. The viral particles can be concentrated
by conventional concentration techniques, in particular
by ultrafiltration and/or purified by conventional
purification means, in particular techniques of gel-
filtration, ultracentrifugation on a sucrose gradient
or selective precipitations, in particular in the
presence of polyethylene glycol (PEG).
The subunit vaccine is preferably an extraction
subunit vaccine, namely is prepared from the whole
virus or from a main fraction of this virus (for
instance after elimination of capsid). In particular,
this vaccine may comprise the various envelope
glycoproteins or a mixture of envelope glycoproteins.
The subunit vaccines are preferably produced from an
inactivated viral suspension, in particular inactivated
as described above. Preferably, in order to obtain a
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subunit vaccine, mainly containing the envelope
glycoproteins, the suspension of viral particles,
preferably inactivated, obtained above, is subjected to
the action of a suitable detergent, in particular a
nonionic detergent, preferably an ethoxylated alcohol
advantageously with an HLB of between 12 and 15. The
viral capsids are then removed, in particular by
ultracentrifugation. Other equivalent methods for
removing the capsids and collecting the envelope
glycoproteins are available to those skilled in the
art.
In order to develop an inactivated vaccine or a
subunit vaccine, the antigen is taken up in a
veterinarily acceptable vehicle or excipient and a
veterinarily acceptable adjuvant is preferably added
thereto. The amount of antigen is equivalent in
particular to a titer before inactivation of about 105
to about 109*5 TCID50, particularly from about 105 to
about 109 TCID50, per dose. More particularly, for an
inactivated vaccine, the titer before inactivation is
of about 105 to about 109 TCID50, particularly from
about 106 to about 108 TCID50, per dose. More
particularly for a subunit vaccine, the titer before
inactivation is of about 106 to about 109'5,
particularly from about 107 to about 109, preferably
from about 107'5 to about 108'5, per dose. Preferably,
for a subunit vaccine, the vaccine has a glycoprotein
gB titre as measured in ELISA of about 0.01 lig to about
g, in particular from about 0.1 pg to about 10 g,
30 preferably from about 0.3 lig to about 3 g per dose.
The vaccines according to the invention are
conserved and stored either in formulated form at 5 C,
or in lyophilized form, preferably with a stabilizer,
in particular SPGA (sucrose, phosphate, glutamate,
albumin, EP-B1-0 008 255. These vaccines may be taken
up subsequently in solvent (vehicle or excipient and/or
adjuvant).
In order to adjuvate the vaccines according to
the invention, it is possible to use (1) aluminum
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hydroxide, (2) an acrylic acid or methacrylic acid
polymer, a polymer of maleic anhydride and of alkenyl
derivative, (3) an immunostimulating sequence (ISS), in
particular an oligodeoxyribonucleotidic sequence
bearing one or more non-methylated CpG groups (Klinman
D. M. et al., Proc. Natl. Acad. Sci. USA, 1996, 93,
2879-2883 ; WO-A-9816247), or (4) to formulate the
immunogenic or vaccine preparation in the form of an
oil-in-water emulsion, in particular the SPT emulsion
described on page 147 of "Vaccine Design, The Subunit
and Adjuvant Approach" edited by M. Powell and M.
Newman, Plenum Press, 1995, and the emulsion MF59
described on page 183 of this same book.
The oil-in-water emulsion can be based in
particular on light liquid paraffin oil (European
Pharmacopea type); isoprenoid oil such as squalane or
squalene; oil resulting from the oligomerization of
alkenes, in particular of isobutene or decene; esters
of acids or of alcohols containing a linear alkyl
group, more particularly plant oils, ethyl oleate,
propylene glycol di(caprylate/caprate), glyceryl
tri(caprylate/caprate) or propylene glycol dioleate;
esters of branched fatty acids or alcohols, in
particular isostearic acid esters. The oil is used in
combination with emulsifiers to form the emulsion. The
emulsifiers are preferably nonionic surfactants, in
particular esters of sorbitan, of mannide (e.g.
anhydromannitol oleate), of glycerol, of polyglycerol,
of propylene glycol and of oleic, isostearic,
ricinoleic or hydroxystearic acid, which are optionally
ethoxylated, and
polyoxypropylene-polyoxyethylene
copolymer blocks, in particular the Pluronic products,
especially L121.
The acrylic acid or methacrylic acid polymers
are crosslinked in particular with polyalkenyl ethers
of sugars or of polyalcohols. These compounds are known
under the term "carbomer" (Pharmeuropa vol. 8, No. 2,
June 1996). A person skilled in the art may also refer
to US-A-2 909 462
* Squalane, Pluronic L121, Tween' 80
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describing such acrylic polymers crosslinked with a
polyhydroxylated compound containing at least 3
hydroxyl groups, preferably not more than 8, the
hydrogen atoms of at least 3 hydroxyls being replaced
with unsaturated aliphatic radicals containing at least
2 carbon atoms. The preferred radicals are those
containing from 2 to 4 carbon atoms, e.g. vinyls,
allyls and other ethylenically unsaturated groups. The
unsaturated radicals can themselves contain other
substituents, such as methyl. The products sold under
the name Carbopol (BF Goodrich, Ohio, USA) are
particularly suitable. They are crosslinked with an
allyl sucrose or with allylpentaerythritol. Among
these, mention may be made of the products Carbopol
974P, 934P and 971P.
Among the copolymers of maleic anhydride and of
an alkenyl derivative, those which are preferred are
the EMA products (Monsanto) which are copolymers of
maleic anhydride and of ethylene, which may be linear
or crosslinked, for example crosslinked with divinyl
ether. Reference may be made to J. Fields et al.,
Nature, 186, 778-780, 4 June 1960. As regards their
structure, the acrylic acid or methacrylic acid polymers
and the EMA products are preferably formed from units
based on the following formula:
R2
___________________________________ (CH2) ____ C(CH 2) y
COON COOH
in which:
- R1 and R2, which may be identical or different,
represent H or CH3
- x = 0 or 1, preferably x = 1
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- y = 1 or 2, with x + y = 2.
For the EMA products, x = 0 and y = 2. For the
carbomers, x = y = 1.
The polymer concentration 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.
One form of vaccine which is particularly
preferred comprises an inactivated vaccine or a subunit
vaccine formulated in the form of an oil-in-water
emulsion comprising anhydromannitol oleate, ethoxylated
oleic acid and light liquid paraffin oil. In
particular, a method for immunizing gestating bitches
according to the invention comprises the administration
of such a vaccine. Two injections are given, preferably
subcutaneously, in particular in accordance with the
above-described scheme, for example with the first
injection during the first third of the gestation,
preferably about 10 days after the date of insemination
and in particular the second injection during the final
third, preferably about 10 days before the presumed
date of whelping.
A subject of the invention is also the process
for preparing the inactivated vaccine or the subunit
vaccine described.
A subject of the invention is also multivalent
vaccines comprising the inactivated canine herpesvirus
valency or the canine herpesvirus subunit valency, and
at least one valency for at least one other canine
pathogen, in a veterinarily acceptable vehicle or
excipient and preferably with an adjuvant, especially
one of those described above.
= Said canine pathogens are chosen in particular
from the group comprising canine distemper virus,
canine parvovirus and canine adenovirus, but other
valencies may also be combined.
The inactivated CHV vaccines or subunit
vaccines according to the invention can be administered
simultaneously in the same preparation or successively
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in different preparations with the other canine valency
(valencies) which may be in the form of attenuated live
vaccines, inactivated vaccines, subunit vaccines,
recombinant vaccines or polynucleotide vaccines.
A subject of the invention is thus also a
multivalent vaccine kit or pack comprising, separately
packaged, a CHV valency according to the invention in a
veterinarily acceptable vehicle or excipient, and
preferably with an adjuvant, in particular those
described above, and at least one valency of at least
one other canine pathogen. The CHV valency according to
= the invention can serve as a solvent for the other
canine valency, in particular for an attenuated,
recombinant or polynucleotide valency in lyophilized
form.
The invention will now be described in greater
detail with the aid of embodiments given as non-
limiting examples.
Examples :
Example 1 : Viral strain
The strain of canine herpesvirus used is the
strain F205 (L.E. Carmichael, Am. J. Vet. Res., 1965,
26, 803-814), available from the American Type Culture
Collection (ATCC) under the number VR-647.
Example 2 : Amplification of the canine herpesvirus
Cells of a canine line (Madin-Darby Canine
Kidney or MDCK, available from the ATCC under the
number CCL-34) are cultured in 2-liter rolling flasks
(850 cm2) with Dulbecco modified Eagle's minimum
essential medium (DMEM - Gibco BRL) supplemented with
5% fetal calf serum (Bayer Diagnostic) and gentamycin
at a concentration of 50 mg/1 in a proportion of
200,000 cells per ml under a volume of 350 ml. The
cells are cultured at +37 C in an atmosphere containing
5% CO2.
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After 3 or 4 days, the cell layer reaches
confluence. The culture medium is then replaced with
serum-free DMEM medium still supplemented with 50 mg/1
of gentamycin, and the cells are inoculated with canine
herpesvirus (Example 1) with a multiplicity of
infection (moi) of 0.01 TCID50/cell. The viral culture
is maintained at 37 C.
When the cytopathic effect (CPE) is complete
(about 96 hours after the start of culturing), the
viral suspension is harvested and frozen at -70 C.
Several cell amplification passages can be carried out
prior to the viral inoculation depending on the amount
of virus desired. The cell passages and the viral
culturing can also be carried out by culturing the
cells on microcarriers in a biogenerator.
The virus is conserved at 5 C until the
following steps.
The titer of the CHV virus at harvest is
107.2 TCID50 per ml.
Example 3 : Titration of the CHV virus
Titration of the CHV virus is carried out in
96-well microplates in F15 medium supplemented with 5%
fetal calf serum and 50 mg/1 of gentamycin. Dilutions
with a rate of about 3 of the viral suspension are
mixed with a suspension of MDCK cells in the microplate
in a proportion of 0.10 ml of virus dilution per
100,000 cells in 0.15 ml per well. After incubation for
4 to 5 days at 37 C with 5% CO2, the wells with a
generalized CPE are counted and the titer is calculated
as tissue culture infectious doses (50%) (TCID50) by the
Karber method.
Example 4 : Inactivation of the virus
The CHV virus amplified as described in Example
2 is inactivated with ethyleneimine at a concentration
of about 12 mM, at 37 C for 6 hours.
The ethyleneimine is prepared at the time of
use by dissolving 28.0 g of sodium hydroxide pellets in
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200 ml of distilled water and adding 68.1 g of
bromoethylamine (BEA) corresponding to a 1.2 M solution
of ethyleneimine (H. Bahnemann, Arch. Virol., 1975, 47,
47-56). The inactivated viral suspension is
concentrated 100-fold on an ultrafiltration cassette of
the Ultrasette type with a cutoff threshold of 300 kDa
(Filtron). The concentrated inactivated virus can be
frozen and stored at -40 C.
Example 5 : Extraction of the viral glycoproteins
The inactivated virus (Example 4) is clarified
by centrifugation at 2500 x g for 30 min. The
supernatant liquid is then concentrated 50-fold on an
ultrafiltration cassette of the Ultrasette type with a
cutoff threshold of 300 kDa (Filtron). The concentrate
is supplemented with polyethylene glycol 8000 (PEG
8000) at a concentration of 8% W/V and 4% W/V sodium
chloride. After contact for 16 hours at 4 C, the
precipitated virus is sedimented by centrifugation at
2500 x g for 60 min and then taken up in phosphate
buffer (PBS: 8 g/1 NaCl; 0.2 g/1 KC1; 0.2 g/1 K1-12PO4;
1.44 g/1 Na2HPO4 = 2H20). It is then taken up in
isotonic phosphate buffer in a proportion of one
hundredth of the initial volume. The viral concentrate
then undergoes a zonal ultracentrifugation on a cushion
of sucrose (35% and 60% W/W PBS, for 60 min at 200,000
x g) with a zonal rotor or a Ti45 fixed-angle rotor.
The virus is harvested at the interface and is diluted
approximately four-fold with PBS. Next, 10 EO cetyl
alcohol (ethoxylated alcohol of HLB 13) at a
concentration of 1.5% W/V is added, and the mixture is
incubated for one hour at 37 C. The capsids are then
= . sedimented by ultracentrifugation for one hour at
230,000 x g with a Ti45 rotor. The supernatant
containing the glycoproteins is collected. The
glycoprotein solution can optionally be concentrated by
ultrafiltration.
The solution of purified glycoproteins is
stored at -40 C until formulation of the vaccine.
*Trade-mark
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Example 6 : Formulation of the vaccines
The concentrated inactivated antigen (Example
4) or the solution of subunits (Example 5), after
thawing, is diluted in PBS buffer as a function of the
required amount of antigens per dose.
The adjuvant-containing vaccine is prepared in
the following way: 167 ml of aqueous phase consisting
of 8 ml of subunit or inactivated viral antigen and
159 ml of PBS are emulsified in 83 ml of oily phase
containing 7% w/v of anhydromannitol oleate, 8% w/v of
oleic acid ethoxylated with 11 EO (ethylene oxide) and
85% v/v of light liquid paraffin oil (European
Pharmacopea type) with the aid of a Silverson*
emulsifying turbomixer at 32 C for 2 minutes. The
vaccine formulated in the form of an oil-in-water
emulsion is then stored at 5 C.
An alternative method for preparing the vaccine
consists in emulsifying, by three passages through a
model Y110* high-pressure homogenizer (Microfluidics
CoLp.) at a pressure of 600 bar and at a temperature of
between 30 and 40 C, a mixture of 5% w/v squalane, 2.5%
w/v Pluronic L121, 0.2% w/v of an ester of oleic acid
and of hydrosorbitol ethoxylated with 20 EO, 92.3% v/v
of the viral antigen diluted 30-fold in PBS buffer
after thawing. The vaccine formulated in the form of an
oil-in-water emulsion 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 (9 g/1 NaCl) . The pH is
adjusted to 7.3-7.4 with sodium hydroxide. This
Carbopol solution is then mixed in equal parts with the
viral antigen diluted 16-fold after thawing. The
vaccine formulated in the form of a suspension is then
stored at 5 C.
0.5 ml of a solution of subunits (Example 5),
after thawing, diluted 15-fold in PBS buffer, is added
to 0.5 ml of SPGA lyophilization substrate (sucrose,
phosphate, glutamate, albumin, EP-B1-0 008 255). This
*Trade-mark
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mixture is divided among flasks and lyophilized and
then stored at 5 C. The diluent consists of the
adjuvants described above incorporating only PBS buffer
in the aqueous phase.
Example 7 : Titration of the anti-CHV neutralizing
antibodies
The sera to be titrated are tested for their
ability to neutralize the CHV virus. The sera taken
from the animals are diluted in cascade three-fold with
DMEM medium supplemented with 5% fetal calf serum in
96-well cell culture plates. 0.05 ml of culture medium
containing approximately 200 TCID50/m1 of CHV is added
to 0.05 ml of diluted serum. This mixture is incubated
for 2 hours at 37 C in an oven under an atmosphere
containing 5% CO2.
0.15 ml of an MDCK cell suspension containing
about 100,000 cells per ml is then added to each
mixture. The cytopathic effect (CPE) is observed by
phase-contrast microscopy after culturing for 5 days at
37 C under an atmosphere containing 5% CO2. The
neutralizing titers of each serum are calculated
according to the Karber method. The titers are given in
the form of the highest dilution which inhibits the
cytopathic effect for 50% of the wells. The titers are
expressed as 10g10 VN50. Each serum is titrated at
least twice and preferably four times.
Example 8 : Efficacy
**
12 non-vaccinated EOPS Beagle bitches were
included in the study and divided randomly into 2
groups of six animals. The bitches of the first group
are vaccinated with the CHV subunit vaccine adjuvanted
with the oil-in-water emulsion based on paraffin oil
(Example 6), and the bitches of the other group are
vaccinated with a placebo vaccine.
The bitches were vaccinated twice
subcutaneously about 10 days after insemination, and
then about 10 days before the presumed date of
* SO% virus neutralization
** "Exempts d'Organismes Pathogenes Specifique"
(free of specific pathogenic organisms)
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whelping. The lyophilizate taken up in 1 ml of oily
solvent is administered to the bitches. It corresponds
to a titer equivalent of 107'7 TCID50 before
inactivation.
After whelping, the newborn puppies are tested
at the 3-day-old stage with 50 to 100 TCID50 of a
virulent strain of CHV, for example F205 isolated by
L.E. Carmichael (Am. J. Vet. Res., 1965, 26, 803-814)
or CHV 270 (H. Hill, Am. J. Vet. Res., 1974, 35, 669-
673) or CHV C4012SE, via the oronasal route (0.5 ml
orally and 0.25 ml in each nostril). The puppies are
then observed for 3 weeks. The clinical signs and
mortality are recorded. After their death, all the
puppies are autopsied. The macroscopic lesions
characteristic of canine herpesvirosis are searched for
in the kidneys, spleen, liver, lungs, digestive tract,
heart and mesenteric ganglia, and samples are taken for
isolation of CHV on MDCK cells. The diagnosis of canine
herpesvirosis is based on the death of the puppy and
the presence of characteristic macroscopic lesions and
is confirmed by a viral isolation.
All the vaccinated bitches underwent
seroconversion and had a high titer of neutralizing
antibodies at the time of whelping. These titers are
measured by the method described in Example 6 and are
expressed as 10g10 VN50.
Average Day of the Day of the Day of the 3 weeks
anti-CHV SN first booster test after test
titers in vaccination vaccination
the bitches
control <0.24 + <0.24 + <0.29 + <1.36 +
group = 0.00 0.00 0.13 0.65
vaccinated <0.35 <1.04 + 1.76 + 0.20 2.11 0.51
group 0.17 0.34
Among the puppies born from vaccinated mothers,
none of them died from canine herpesvirosis (0/27). No
CHV virus was re-isolated from these puppies.
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Among the puppies born from non-vaccinated
mothers (placebo group), a mortality rate due to CHV of
62% (18/29) was observed. Viral isolation is also
observed in 55% of these puppies (16/29) : samples from
two puppies were contaminated with bacteria and could
not be tested for the viral isolation of CHV.
The difference in the mortality rate between
the 2 groups is highly significant (Fisher exact test;
p<0.0001).
Yorkshire terrier bitches divided randomly into
2 groups are kept in an infected environment. The 14
bitches of the first group are vaccinated with the CHV
subunit vaccine adjuvated with an oil-in-water emulsion
based on paraffin oil (Example 6) and the 6 bitches of
the other group are vaccinated with a placebo vaccine.
The vaccination protocol is identical to that described
above.
The weight of the puppies is measured at
whelping.
The weight of the 28 puppies born from a
vaccinated mother is substantially higher (average
weight of 141.8 g) than that of the 14 puppies born
from a non-vaccinated mother (average weight of
85.0 g), i.e. a weight gain of 166.8%. The difference
is significant (Kruskal-Wallis, p<0.01).
The results show, in a contaminated
environment, a tendency to increase the level of
gestation in the vaccinated bitches.
The level of gestation is 82% in the vaccinated
bitches (50/61) and 68% in the non-vaccinated bitches
(placebo group) (19/28) (Fisher exact test, p=0.11).
It should be clearly understood that the invention
is not limited to the specific embodiments indicated in the
description above, but encompasses the variants which are
within the scope of the present invention, as defined in the
appended claims.
