Note: Descriptions are shown in the official language in which they were submitted.
203829
The present invention relates to vaccines for horses
against infections with equine herpesviruses, based on
isolated glycoproteins and to process for the preparation
thereof.
Equine herpesviruses (EHV) are distributed enzootically
in all horse-breeding areas of the world and are of
predominant importance in the infectious diseases of
horses. Primary infection may be followed by life-long
persistence in the infected animal. To date, a total of
four herpesvirus serotypes have been identified in
horses.
EHV-1 (Equine abortion virus), .a pathogen belonging
to the alphaherpesviruses., previously called
EHV-1 subtype 1 (rhinopneLUmonitis virus),
EHV-2 (Equine cytomegalo-like v.irus), a betaherpes-
virus,
EHV-3 (Equine coital exanthema 'virus), belonging to
the alphaherpesviruses and
EHV-4 likewise an alphaherpesviros, previously called
EHV-1 subtype 2.
The equine herpesviruses of serotypes 1 to 4 persistently
cause economic damage in horsebreeding throughout the
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world.
This damage arises, in particular, from recurrent epi-
demics of abortion, of respiratory disorders and, in some
cases with a dramatic course, of encephalomyelitis. The
economic importance of the losses during rearing, the
missing of training and the losses of performance, which
are mainly caused by the respirator~r form of the infec-
tion with equine herpesviruses of types 1, 2 and 4,
should not be underestimated.
All herpesviruses have the property of being able to
persist in the host after the primary infection, and this
is also possessed by the equine herpesviruses. Horses
with latent infection thus play an important part as
virus reservoir. Stress factors such as chemotherapy,
parturition, castration or transport; may lead to reacti-
vation of latent EHV infections. Because such reactiva-
tion may, although associated with virus excretion, take
a clinically inapparent course, every horse once infected
must be regarded as a potential exc:retor and thus as a
possible focus of infection.
Herpesviruses display in general weak immunogenicity
which results in a quantitatively slight humoral immune
response. In particular, the respiratory form after EHV
4 infection frequently results in a serologically weak
immune response.
Investigations into the immunogenic structural components
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of herpesviruses initially showed that only the virus
coat has significance in the induction of immunity. In
recent years, information about the importance of the
individual viral glycoproteins of herpesviruses for the
immune response has been obtained. Thus, it has been
possible to transmit passive immunit:Y in each case using
monoclonal antibodies against the gA, gB, gC, gD, gE and
gF glycoproteins of herpes simplex viruses (HSV) to mice.
Although these antibodies showed no virus-neutralising
properties in vitro, they did protect the mice after
infection with a lethal dose of HSV (Balachandran et al.,
Infection and Immunity 37 (1982) pages 1132 to 1137).
In exgeriments with equine EHV-1 herpesviruses in the
hamster model, protection from homologous loading infec-
tions was achieved with live and inactivated, complete
virions and with purified completes coat proteins. By
contrast, administration of fractions of the coat pro-
teins resulted in only a partial protection by a high
molecular weight fraction (Papp-Vid and Derbyshire, Can.
J. Comp. Med. 42 (1978) pages 219 to~ 226).
The extracellular EHV virions are composed of 28 to 30
structural proteins which, apart from the 6 polypeptides
of the capsid, are all to be found in the virus coat. The
molecular weights of these coat proteins range from 16 to
270 kDa. The number of glycoproteins is stated to be from
12 to 14. Six of these glycoproteins have been identified
as major glycoproteins of EHV-1 and EHV-4.
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The molecular weights of the 6 major glycoproteins
identified for EHV-1 are 252 to 260 kDa for gp2, 127 to
138 kDa for gpl0, 92 to 97 kDa for gpl3, 81 to 87 kDa for
gpl4, 60 to 65 kDa for gpl8 and 42 t:o 4G kDa for gp22.
Shimizu et al . Arch. Virol 104 ( 1989 ) pages 169 to 174
describe the preparation of monoclonal antibodies against
gpl3 and gpl4 of EHV-1, which, on administration to
hamsters, confer passive immunity against experimentally
produced lethal EHV-1 infection. In addition, they
neutralised various EHV-1 strains in vitro.
Passive immunisation of hamsters with antibodies does not
allow any conclusions to be drawn about the immunogeni-
city of purified glycoproteins in horses. On the one
hand, the monoclonal antibodies were prepared in mice
and, on the other hand, preparation started from the
complete viruses.
Currently employed in practice are nnonovalent EHV-1 live
vaccines or inactivated vaccines in combination with
other antigens. Despite the use of these vaccines,
clinical illnesses repeatedly occur because of infections
with EHV-1 and EHV-4. This is partly attributed to the
fact that the prescribed and very elaborate vaccination
schedule is not complied with, and, on the other hand,
the efficacy of the vaccines is not entirely satis-
factory, in particular there is no immunity to EHV-4.
Thus, overall, the practical situation with the vaccines
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employed at present is unsatisfactory.
It was desirable to find an EI3V vaccine which is
straightforward to use and has high efficacy.
The present invention relates to:
1. Vaccine for horses against infections with equine
herpesviruses based on isolated glycoproteins and/or
their complexes with lectins from EHV-infected cells
or extracellular EHV virions.
2. Process for the preparation of the vaccines accord-
ing to (1), characterised in that
a) equine herpesviruses (EHV) are grown,
b) the infected cells or the extracellular virus
is treated with detergents,
c ) the lysate obtained in this way is treated with
lectins
d) the lectin-ligand complex is separated from
remaining lysate,
e) where appropriate the lectin-ligand complex is
split up and the glycoprotein mixture is
isolated,
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f) subsequently the lectin-ligand complex and/or
the isolated glycoprotein mixture is formulated
in a customary manner.
3. Complexes of lectin with glycoproteins from equine
herpesviruses or from cells infected with equine
herpesviruses.
4. Glycoproteins from equine herpesviruses isolated and
purified with lectins.
The vaccines are based on EH virus types 1 to 4, EHV-1
and EHV-4 may be particularly mentioned, and EHV-1 is
preferred.
The vaccines can be prepared in principle from all EHV
virus strains.
Examples of EHV strains which may be mentioned are
1. The strain RAC-H (EHV-1), a virus which was isolated
from an aborted horse fetus. The strain is, for
example, also described in "Untersuchungen zur
Entwicklung eines Lebendimpfstoffes gegen die
Rhinopneumonitis (Stutenabort) der Pferde" [Investi-
gations into the development of a live vaccine
against equine rhinopneumon~tis (abortion in mares)]
(Mayer et.al., 1968; Zbl.Vet.Med. Vol. 5_, 406-408).
2. The strain DA8 (EHV-1) was deposited in compliance
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with the Budapest Treaty on 24.7.1990 under the
designation J-979 at the Institut Pasteur CNCM
Paris, France.
3. The strain Thein 400/3 (EHV-2) was deposited in
compliance with the Budapest '.treaty on 24.7.1990
under the designation J-981 at ithe Institut Pasteur
CNCM Paris, France.
4. The strain Thein 166 (EHV-3)
5. The strain Thein 252/1 (EHV-4) was deposited in
compliance with the Budapest 'treaty on 24.7.1990
under the designation J-982 at 'the Institut Pasteur
CNCM Paris, France.
6. The strain MAR 87 (EHV-1) was deposited in
compliance with the Budapest 'Treaty on 24.7.1990
under the designation J-980 at 'the Institut Pasteno
CNCM Paris, France.
The glycoproteins for preparing the vaccines according to
the invention can be obtained from ex.tracellular virus or
from infected cells. The virus is grown in a manner known
per se for this purpose.
The growing of the viruses is c3rri.ed out in the usual
way in tissue cultures of animal cells, for example in
horse cells, pig cells, monkey cells or rabbit cells,
preferably in equine dean cells such as the permanent
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205382'
equine dean cell ED (ATCC 57 or its derivatives) or
porcine kidney cells such as the permanent porcine kidney
cells PK15 (ATCC CCL 33 or its derivatives) or rabbit
kidney cells such as in the rabbit kidney cell RK-13
(ATCC CCL 37 or its derivatives) or in monkey kidney
cells.
The growing is carried out in a manner known per se in
stationary, roller or carrier cultures in the form of
united cell assemblages or in suspension cultures.
Employed as growing media are all cell culture media
known per se, for example described in the product
catalogue of Flow Laboratories Gn,~bH Post 1249, 5309
Meckenhei.m, such as, in particular, 'the minimal essential
medium (MEM) which contains as essential consistuents
amino acids, vitamins, salts and carbohydrates, completed
with buffer substances such as, for example, sodium
bicarbonate or (hydroxyetlzylpiperazine-N-2-
ethanesulphonic acid (herpes) and,. where appropriate,
animal sera such as, for example, sera of cattle, horses
or their foetuses. It is particularly preferable to
employ foetal calf serum in a concentration of 1-30% by
volume, preferably 2-10% by volume.
The cells and cell lawns used for growing the viruses are
grown in a customary manner almost to confluence or to
optimum cell density. Before their infection with
viruses, preferably the cell-growing medium is removed,
and the cells are washed preferably with virus-growing
medium. Employed as virus growing media are, all cell
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culture media known per se, such as,. in particular, the
abovementioned MEM. Infection with a virus suspension is
then carried out. The virus is present in the virus
suspension diluted in the virus growing medium in such a
way that infection is with a MOI (= multiplicity of
infection corresponds to infectious virus particles for
each cell present) of 0.001 - 50, preferably 0.10 - 10.
The viruses are grown with or without addition of aminal
sera. In the case whose serum is used, it is added to the
growing medium in a concentration oi: 1 - 30~ by volume,
preferably 2-10~ by volume.
Infection and virus growing is carried out at tempera-
tures between room temperature arid 40°C, preferably
between 32 and 39°C, particularly preferably at 37°C.
In the case where the intention is to work subsequently
with extracellular virus, the growing is carried out
until the cellular substrate is completely destroyed. In
the case where the intention is to work with virus
proteins from infected cells, growing is carried out
until the maximum content of virus-;specific antigens is
reached. The maximum content of virus-specific antigens
is reached, for example in the case of monolayer
cultures, at 30 to 100$ cytopathogenic alterations (cpE).
The region around 70 to 90$ cpE is preferred.
In the case where the intention is to work with extra-
cellular virus, the virus is worked up by removing the
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cell detritus and isolating and concentrating the virus
particles from the virus-growing medium. This is carried
out in a manner known per se by filtration such as, for
example, ultrafiltration with membrane or depth-type
filters, or by centrifugation. Cell detritus is removed,
for example, by centrifugation at between 500 and
15,000 x g, preferably between 5,000 and 15,000 x g.
Virus isolation is achieved by zonal centrifugation or
isopycnic centrifugation in, for example, sucrose density
gradients. For this purpose, for example, the virus-
containing growing medium is, after removal of the cell
detritus, subjected to a zonal centrifugation of
100,000 x g until the virus particles sediment. A higher
degree of purity is achieved when the zonal
centrifugation for pelleting the virus particles is
carried out through an aqueous solution with higher
density than the virus-containing medium. It is possible
to use as aqueous solution, for example, a 10-40% w/w,
preferably 25-40% w/w, buffered solution of sucrose.
An even more efficient purification is achieved by
centrifugation in a density gradient. For this purpose,
the virus material which has lbeen, for example,
prepurified by a low-form centrifugation at
5,000-15,000 x g and sedimented by a subsequent
centrifugation at 100,000 x g, is isolated by a zonal or
isopycnic gradient centrifugation. An isopycnic
centrifugation through a sucrose density gradient of, for
example, 30 to 50% w/w in buffered aqueous solution at a
centrifugal acceleration of, for example, 100,000 to
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150, 000 x g is preferred. In the case: where the intention
is to work with virus proteins from infected cells, the
infected cells used to grow the viruses are, if
necessary, suspended, and the suspended cells are
concentrated. Concentration is carried out by filtration
or centrifugation. Centrifugation i;s preferably carried
out at 300 to 2,000 g. The centrifugation is carried out
at a volume of about 1 1 for about 1.5-45 minutes.
After the concentration, the cells are preferably washed,
for example with physiological saline.
The virus or cell concentrates obtained in this way are
treated with detergents.
Suitable detergents are:
anionic surfactants such as Na lauryl sulphate, fatty
alcohol ether sulphates, mono/dialkyl polyglycol ether
orthophosphoric ester monoethanolamine salt, calcium
alkylarylsulphonate, sodium deo~ycholate, cationic
surfactants such as cetyltrimethylammonium chloride,
ampholytic surfactants such as di-Nay N-lauryl-:-iminodi-
propiont or lecithin, non-ionic surfactants, for example
polyoxyethylated castor oil, polyo:xyethylated sorbitan
monooleate, sorbitan monostearate, g:Lycerol monostearate,
polyoxyethylene stearate, alkylphenol polyglycol ether.
Non-ionic detergents may be particu:Larly mentioned:
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non-ionic, water-soluble emulsifiers with an HLB (hydro-
philic/lipophilic/balance value) gi:eater than 10, for
example Emulgator NP 40° (Bayer AG), alkylaryl polyglycol
ether; Renex 678° (Atlas Chemical Industries), polyoxy-
ethylene alkylaryl ether; Tween 20° (Atlas), polyoxy-
ethylene sorbitan monopalmitate; Myri 53° (Atlas), poly-
oxyethylene stearate; Atlas G 3707°, polyoxyethylene
lauryl ether; Atlas G 3920°, polyoxyEahylene oleyl ether;
Atlas G 9046 T°, polyoxyethylene mannitan monolaurate;
Emulgator 1371 H~ (Bayer AG), alkyl polyglycol ether;
Emulgator 1736° (Bayer AG), alkyl po7lyglycol ether (oleyl
polyglycol ether); Emulgator OX° (Bayer AG), alkyl poly-
glycol ether (dodecyl polyglycol ether); Ninox BM-2°
(Stepan Chemical Co.), ethoxylated nonylphenol; Triton X-
100° (Rohm an Haas Co. ), isooctylphenol polyethoxyethanol;
Cremophor EL°, Nonidet P 40° (She.ll), glycopyranoside
(Sugena), N-decantoyl-N-methyl-glycoside.
The detergents are used in the fo~zn of dilute aqueous
solutions. Solutions which may be mentioned are those
containing 0.1 to 10 percent by volume, preferably 0.5 to
5 percent by volume, particularly preferably about 1
percent by volume of detergent.
The detergent solution is added in the ratio of about
1 : 1 to about 10 : 1 by volume t:o the cell or virus
concentrate. The preferred ratio of detergent solution to
cell or virus concentrate is about 3 : 1.
To improve the detergent treatment, the solution can
additionally be subjected to an ultrasound treatment.
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The detergent treatment is carried out at temperatures
between 0 and about 24°C, preferably between 2 and 8°C.
The detergent treatment lasts 15 minutes to 2 days,
preferably 12 to 24 hours.
The particles not dissolved by 'this treatment are
removed, preferably by centrifugation or filtration. When
centrifugation is used, the lysate obtained above is
exposed to a centrifugal acceleration of, for example,
100,000 x g for 1 hour, for example.
The centrifugation supernatant obtained in this way or
the filtrate can be stored at low temperatures (0 to
70°C) before it is processed further.
To isolate the glycoproteins contained in the solution,
it is treated with lectins . For this, it is previously
mixed with sufficient sodium chloride and with the known
lectin-stabilising salts to result in a concentration of
sodium chloride of 0.5 to 2, preferably 0.7 to 1.2 molar.
The required concentration of lectin--stabilising salts is
known from the state of the art and specific for the
lectins to be employed. The concentration is preferably
adjusted by dialysis of the lysates.
Lectins are proteins or glycoproteins from plants,
specifically their seeds, microorgan:lsms, vertebrates and
invertebrates which specifically bind sugars and their
conjugates. Used according to the invention are lectins
which recognise and bind glycoprotei.ns from~EHV-infected
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CA 02053829 2000-10-OS
23189-7283
cells or from EHV virions and which have mitogenic effects on
lymphocytes.
It is possible in principle to use all lectins
according to the invention. The lectins which are preferably
used are those which recognise mannose and/or glucose and their
conjugates.
Specific mention may be made of the lectins from
Canavalia ensifornis, Lens culinaris, Lathyros odoratus, Pisum
sativum, Vicia faba, Sambucus nigra, Glycine max, Ulex
europaens, Helix pomatia, Phytolacca americana, Lycopersicon
esculentum, Datura stramonium, Bandeiraea simplicifolia.
The lectins can be used for the process according to
the invention in a form, which is soluble in water or insoluble
in water. In the form insoluble in water, they are preferably
employed immobilised by coupling to inert matrices such as, for
example, dextrans, agaroses, celluloses as gels.
Specific mention may be made of concanavalin
A-agarose, concanavalin A-Sepharose*, lentil lactin-Sepharose*,
pokeweed mitogen-agarose.
The lectins are employed in the form of a detergent-
and salt-containing solution, suspension or of a gel.
To prepare this solution, suspension or this gel, the
*Trade-mark
14
~.... .
203829
lectins are mixed with the detergent employed for the
treatment of the cell lysates or virus lysates in a salt
solution. This salt solution contains about 0.5 to 2,
preferably 0.7 to 1.2 mole, of sodium chloride in addi-
tion to the known salts which are customary and necessary
for the stability and reactivity of the lectins. The salt
and detergent solution of the lecti.ns is thus identical
to the solution, the solution used for adjusting the salt
and detergent concentration in the cell and virus
lysates.
About 1 to 150 mg, preferably 1 to 50 mg, particularly
preferably 5 to 20 mg, of pure lectin are used per ml of
solution, suspension or gel which contains or the
detergent in the concentration employed in the treatment
of the cell lysates or virions.
The amount of this lectin solution, suspension or gel
which is added to the detergent-containing cell lysates
or virus lysates is sufficient to employ 0.01 to 50 mg,
preferably 0.1 to 20 mg, particularly preferably 0.5 to
5 mg, of lectin per mg of total protein. In each case it
is necessary for the amount of lectin to be chosen so
that all glycoproteins in the cell or virus lysates can
be bound.
The lectin treatment is carried out at -5 to +30°C,
preferably at 2 to 8°C for about 10 min to 3 days,
preferably at 1 hour to 2 days.
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The reaction of the lectins with the glycoproteins can
also be carried out via column chromatography. For this,
the immobilised lectins, preferably bound to a gel-like
matrix, are brought into contact with the cell lysates or
virus lysates in a chromatography column. About 0.1 to
100 mg, preferably 1 to 50 mg, particularly preferably 5
to 20 mg, of lectin are employed pe.r ml of packed gel.
The glycoprotein-lectin complex i.s removed from the
complete solution or suspension in a customary manner.
This can be carried out by centrifugation, filtration or,
in the case of chromatography, by washing.
The suspensions or gels which contain the lectin-glyco-
protein complexes and which are obtained in this process
step are Washed to reduce the salt and detergent concen-
tration. For this, the suspension or the gel is mixed
with a solution which contains 0.1 to 0.01 times the
detergent concentration and sodium clhloride concentration
which was employed in the treatment of the cell or virus
lysate, so that there is reduction to 0.1 to 0.01 times
the concentration of detergent and sodium chloride in the
suspension or gel containing the lectin-glycoprotein
complexes.
The suspensions or gels of the lectin-glycoprotein
complexes obtained in this way can be employed directly
or, where appropriate, after addition of adjuvant for
immunisation of horses. Depending on the content of
lectin-bound glycoprotein, the suspensions or gels
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employed for the immunisation can be further concentrated
or diluted. The content of bound glycoprotein is adjusted
so that each vaccine dose contains :10 to 250 mg, prefer
ably 10 to 100 mg, particularly preferably 25 to 50 mg,
of glycoprotein.
In the resulting suspensions or gels of the lectin-
glycoprotein complexes, the concentration of the deter-
gent can be altered, as far as eliminating it, by filtra-
tion, centrifugation, dialysis or other washing pro-
cesses . The same methods can also be used to alter the
salt concentration in the physiologically tolerated
range.
The suspensions or gels of the lectin-glycoprotein
complexes can be stored at temperatures below 8°C. They
can also be freeze-dried.
The glycoproteins can be isolated from the resulting
suspensions or gels of the glycoprot:ein-lectin complexes
to prepare the vaccine. For this, the suspensions or gels
are treated with a sugar-containing, aqueous salt solu
tion.
The nature of the sugar to be employed depends on the
specificity of the lectins used. The concentration of the
sugar is 0.1 to 1 molar, preferably 0.1 to 0.5 molar,
particularly preferably 0.3 to 0.5 molar. The concentra-
tion and composition of the salt content and detergent
content of the sugar solution corresponds to that of the
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gels or suspensions containing they glycoprotein-lectin
complexes.
The treatment with the sugar solution is carried out at
-5 to +30°C, preferably between 2 and +g°C. The treatment
amounts to about 15 min to 4 days, preferably 1 hour to
2 days, particularly preferably 10 to 24 hours.
The glycoproteins eluted in this way are isolated from
the lectins by centrifugation, filtration or by other
customary separation processes (for example chromato
graphy).
The isolated glycoproteins obtained in this way in
detergent- salt- or sugar-containing solution can be
employed directly or after addition of adjuvant for the
immunisation of horses depending on the glycoprotein
content.
Depending on the content of glycoproteins, the solutions
employed for the immunisation can be concentrated or
diluted. The content of glycoproteins is adjusted so that
each vaccine dose per horse contains 10 to 250 mg,
preferably 10 to 100 mg, particularly preferably 25 to 50
mg, of glycoprotein.
In the case of preparations from virus lysates, the
content of glycoprotein can be adjusted so that it is 0.1
to 0.01 times lower per vaccine dose..
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The concentrations of detergent and salts in the prepara-
tions can be altered as describs~d hereinbefore. The
preparations are stored in the form of their solutions at
low temperatures (below 0°C) or in lyophilised form.
The lectin-glycoprotein complexes and isolated glycopro-
teins can be mixed with customary adjuvants before they
are administered to enhance the immune response.
Adjuvants which may be mentioned are: aqueous and oily
adjuvants such as, for example, altuninium hydroxides or
~Havlogen.
Administration is carried out in a customary parenteral
manner such as intramuscularly, subcutaneously, intra-
nasally.
Examples
1. Preparation of the cell cultures
1.1 Media and solutions
Culture medium: E-MEM 0.85 g (Earle minimal
essential medium (cf. Virol.
Arbeitsmethoden [Virol. Working
Methods ] Vol . 1, Gustav Fischer-
Verlag, ~~tuttgart 1974, A.
Mayer, P.A. Bachmann, B. Bibrack
+ G. Wittmann) + 0.85 g NaHC03/1
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Trypsin EDTA: 0.2 g of EDTA-ethylenediamino-
tetraaceti.c acid sodium salt;
1 g of glucose; 0.4 g of KC1; 8
g of NaCl; 0.58 g of NaHC03; 0.5
g of tryp;sin to 1 1 distilled
water.
Fetal calf serum (FCS): supplied by "Gibco"
1.2 Cell culturing
The virus cultures and the virus titrations were
carried out with ED (equine dermal) and PK15 cells
(porcine kidney). The medium used to culture the
cells was E-MEM 0.85 g with the addition of 10$
fetal calf serum. At confluence, the cells were
detached from the monolayer assemblage by trypsin-
EDTA and passaged in a density corresponding to the
culture vessel.
2. Preparation of the virus cultures
2.1 Media and solutions
Maintenance medium: E-MEM (2.0 g (Earle minimal
essential medium) (Virol.
Arbeitsmethoden [Virol. Working
Methods ] Vol . 1, Gustav Fischer-
Verlag, Stuttgart 1974, A. Mayer,
P.A. Bachmann) + 2.0 g NaHC03/1)
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PBS ~ 8 g of Na~Cl, 0 . 2 g of KHZP04,
1.44 g of Na2HP04, 0.2 KC1 to 1 1
distilled water.
2.2 Virus culturing
At confluence, the culture medium of the cells was
decanted off, and the cells were washed with main-
tenance medium. The subsequent infection was carried
out by adding the virus stock diluted in maintenance
medium. This entailed EHV-1 being diluted 1:100,
corresponding to an m.o.i. (mu.ltiplicity of infec-
tion) of 0.1 and EHV-4 being diluted 1:50 (in an
m.o.i. of 0.001). The inoculated cell cultures were
incubated at 37°C. The cells inoculated with EHV-1
reached a 100 cpE (cytopathic effect) after about
24 hours after infection, while the EHV-4-infected
cells required at least 48 hours.
3. Preparation of the cell lysate~
3.1 Chemicals and solutions
Extraction buffer: 25 mM tris~ HC1 (tris(hydroxy-
methyl)-aminomethane HC1), 1$
NP40; pH 8.6
NP40: Nonident P 40 (ethylphenyl
polyethylene glycol)
Storage buffer: 0.1 M NaCH3C00, 1 mM CaCl2, 1 mM
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MgCl2, 1 mM MnClz, 1 M NaCl, pH
6
Elution buffer: 0.1 M NaCH3C00, 1 mM CaCl2, 1 mM
MgCl2, 1 mM MnCl2, 0.1 M NaCl,
0.1% NP40, 0.3 M methyl manno-
pyranoside, pH 6
3.2 Detergent treatment
The ED monolayer cell cultures cultured in roller
bottles (850 cm2 Falcon) or dish stacks (6000 cm2
, Nunc) were inoculated at 90 to 100% confluence with
EHV-1 or EHV-4. According to t;he results of kinetic
investigations (harvesting at .a cpE of 10 to 100%),
the infected cells were harve;ated at a cpE of 80%
for the preparative experiment:.. For this, the cells
from roller cultures were scraped off the vessel
walls, using a sterile rubber scraper (cell-scraper,
supplied by Costar) into the medium, or were suspen-
ded in the culture medium by vigorously shaking the
dish stacks. The suspension was subsequently centri-
fuged at 3,000 g for 20 min, and the cell pellet
obtained in this way was washed 2 x in PBS before it
was finally taken up 1:2 in 1?eS v/v and stored at
-70C. Extraction was carried out by adding 3 ml of
extraction buffer per 1 ml of cell suspension and
subsequently treating with ultrasound (3 bursts of
5 sec each at 7 microns). This cell lysate was
stirred at 4C for 24 hours before insoluble
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particles were removed by sedimentation by centrifu-
gation in an SW41 rotor ( 38, OCIO rpm, 1 hour ) . The
supernatant material was stored at -70°C. The
protein concentration in the membrane lysates was
determined after ultrafiltration of an aliquot of
the supernatant.
4. Isolation of g~lycoproteins
The purification of glycoproteins on con A-Sepharose
by affinity chromatography was described by TODD et
al., Arch. Virol 96 (1987), pages 215 - 224, for the
preparation of glycoproteins i:rom Aujeszky virus.
The cell lysates were initially dialysed at 4°C for
24 hours against the storage buffer of the con A-
Sepharose 4B completed with the detergent NP40 (1%
final concentration). The concanavalin A-Sepharose
4B (Sigma C9017) was introduced bubble-free into a
column (Pharmacia 1.5 cm diameter) and equilibrated
with 20 times the bed volume of the storage buffer
described above (+1% NP40) at 4"C. The glycoproteins
were subsequently adsorbed onto the lectin by
applying the cell lysates to t;he column ( 15 mg of
protein/ml of packed gel) . For 1=his, the cell lysate
(average protein content 2.1 mg~/ml) was introduced,
by means of a peristaltic pump with a pumping rate
of 1 ml/hour, into the gel and incubated stationary
for 12 hours. Following this, the column was washed
with equilibration buffer in order to wash out
unbound protein. This, and all following chromato-
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graphic operations were carried out at about 4°C. To
reduce the detergent and salt concentration, the gel
adsorbed with glycoproteins was reequilibrated with
20 times the column volume of a modified equilibra-
tion buffer with reduced NaCl (0.1 M) and NP40
content (0.1~). The glycoproteins were eluted with
this reduced buffer after addition of 0.1 to 0.5 M
methyl mannopyranoside. The elution buffer was
introduced into the gel with a pumping rate of 2 to
S ml/hour, and the column was subsequently incubated
stationary with the elution buffer overnight. The
eluate was subsequently obtained with a pumping rate
of 2 ml/hour, monitoring by photometry (275 nm) in
a flow photometer. Since even with high methyl man-
nopyranoside concentrations the adsorbed glycopro-
teins could not be quantitatively detached from the
lectin in one elution run, another elution was
carried out by stationary incubation of the gel bed
once again in the elution buffer. This second elu-
tion run yielded amounts of glycoprotein comparable
in quality and quantity. These glycoprotein eluate
fractions were stored at -20°C. On the basis of the
results in the optimisation tests, the elution
buffer containing 0.1 M NaCl, 0.1~ NP40 and 0.3 m
methyl mannopyranoside described above was routinely
used.
5. Protein determination
Since the NP40 content of 1$ in. the cell lysate and
of 0.1$ in the eluates impeded photometric
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23189-7283
determination of proteins, it was necessary to dilute the NP40
in the sample by preceding ultra-filtration. For this, an
aliquot of 50 ~l (in the case of lysates containing 1% NP40) or
500 ~l (in the case of eluates containing 0.1% NP40) of the
sample to be determined was made up to 2 ml with distilled
water in Centricon* 10 tubes (supplied by Amicon) and
centrifuged at 8,000 rpm (A8.24 rotor) for 30 min. The
retentate was washed twice with 2 ml of water each time and
then made up to exactly 1 ml with distilled water, and the
protein content was determined by photometry at 595 nm after
addition of Bio-Rad protein assay. The protein content in the
initial sample could be calculated from this by simple
computation.
6. Preparation of lectin-bound glycoproteins
Packed concanavalin A-Sepharose* 4B (Sigma C9017) was
washed five times with twice the volume each time of storage
buffer (completed with 1% NP40) in Falcon tubes by pelleting
(3,000 rpm) and resuspending before the cell lysate, which had
been dialysed against the same buffer, was added. The
adsorption of the glycoproteins to the lectin took place while
agitating gently on a tumbler at 4°C for 16 hours. Unbound
protein was subsequently removed by repeated washing in twice
the volume of storage buffer (+1% NP40). To reduce the salt
content and to eliminate the detergent, the lectin was
repeatedly washed,
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first with the modified storage buffer (containing
O.1M NaCl and 0.1~ NP40) and subsequently in the
same buffer without NP40. This suspension of the
lectin adsorbed with glycoproteins was stored at
4°C. The proteins bound to con-A were quantified as
follows : an aliquot of 200 ~1 of the lectin bound
with glycoproteins was incubated twice with 500 ~1
each time of elution buffer {storage buffer contain-
ing O.1M NaCl, 0.5M methyl mannopyranoside) for 16
hours each time. The amount of protein in the
eluates collected from this was determined by
photometry and extrapolated by simple computation to
the amount of protein bound to 1 ml of packed con-A.
7. Antigenicity analyses
Transfer 50 mM tris{hydroxymethyl)aminomethane
buffer: 40 mM glycine
1.3 mM Na dodecyl sulphate
20$ methanol
pH 9.3
Sample 15 mM tris(hydroxymethyl)aminomethane
buffer: 2$ w/v Na dodecyl sulphate
10~ w/v p-mercaptoethanol
6 M urea
10$ glycerol
0.02$ w/v bromophenol blue
pH 6.8
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7.1 Protein fractionation by electrophoresis
The SDS Page (sodium dodecyl sulphate polyacrylamide
gel electrophoresis) was carried out by the method
described by Lammli, Nature 227 (1970), pages 680 -
685. 7.5~ to 15$ polyacrylamide gradient gels were
used to achieve adequate separation of all the
proteins present in the solutions.
The samples to be investigated were concentrated in
vacuo and resolubilised in 25 ~1 of a denaturing
sample buffer in each case. After ultrasound treat-
ment for 5 min, they were boiled for 4 min and
loaded into the sample recesses.
The marker proteins used were Bio-Rad "Prestained
SDS-Page low range standards" with protein weights
of 110, 84, 47, 33, 24 and 16 kDa.
After the electrophoresis, the SDS polyacrylamide
gels were stained for 16 hours in a Coomassie
staining solution, shaking gently at room tempera-
ture. Non-protein-specific stains were then
eliminated by incubation in a destaining solution
for 2 to 3 hours. The gels were then dried on filter
paper, heating in vacuo.
7.2 Transfer of the fractionated proteins to
nitrocellulose
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After evaluation of the stained gel, proteins
fractionated by SDS Page were transferred to nitrocellulose
membranes in accordance with the principle described by
Burnette A., Anal. Biochem. 112 (1981), pages 195 - 203.
The gel was first equilibrated in the transfer
buffer. Then a sandwich was prepared in the sequence filter
paper, gel, nitrocellulose, filter paper, and placed bubble-
free on the cathode of the Sartoblot* II S (Sartorius) and,
after the anode plate had been placed on, the transfer was
started. It was stopped after 1 hour at room temperature and
constant 1 mA/cm2 of gel.
7.3 Immunostaining
The nitrocellulose strips to be stained were
initially agitated for 1 hour in the blocking buffer (3s bovine
serum albumin (BSA), 0.05% Tween 20 in PBS) to block free
protein-binding sites. The nitrocellulose was subsequently
incubated with the antisera in their specific dilutions (1:150
to 1:10,000) in the same buffer at room temperature for 2
hours. After thorough washing with PBS, the peroxidase-
labelled anti-species IgG conjugate (supplied by Sigma)
corresponding to the antisera used was placed in a dilution of
1:150 in PBS on the nitrocellulose, and the latter was
incubated for a further 2 hours. After washing three times in
PBS, the substrate
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solution was added. The latter was made up fresh
before use with 10 mg of 3-amino-9-ethylcarbazole,
3 ml of dimethyl sulphoxide (DMSO) in 50 ml of 20 mM
Na acetate. The staining reaction was stopped by
dilution in distilled water. After the distance
migrated by the marker proteins in the separating
gel had been determined, these values were plotted
semilogarithmically against the molecular weights
belonging thereto, and this calibration plot, which
is specific for each gel, was used to determine the
molecular weights of the i.mmunostained proteins.
The horse sera used for the immunological investiga-
tions were sera from the following animals:
"Maienschein" a warm-blood horse immunised
with experimentally prepared,
inactivated, bivalent vaccine (EHV-1
and EHV-4), and with a high EHV-1 SN
titre (1:112) and low EHV-4 SN titre
(1:6).
"Arfe" a horse from the test described
hereinafter ( 8 . y with a high EHV-1 SN
titre (1:80) and low, cross-reacting
EHV-4 SN titre (1:6).
"Ellis" a mare which was experimentally
infected in the: USA with EHV-1 and
aborted during this infection, with
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a high EHV-1 SN titre (1:144) and a
low EHV-4 SN tit:re (<1:8).
"Feldserum" a saddle horse with clinical signs of
respiratory disorder and with moder-
ately high EHV-7. SN titre (1:64) and
EHV-4 SN titre (1:14).
Monospecific goat sera were obtained from animals
hyperimmunised against various EHV-1 strains or
EHV-4.
Monospecific hyperimmune sera against EHV-1 were
prepared with band-purified virus material and with
rabbits of the "large white New Zealand" breed. For
this, 100 ~g of dissolved virus protein/dose were
homogeneously formulated in each case with Freund's
incomplete adjuvant 3 x at 1.4-day intervals and
administered to the rabbit subcutaneously. Serum was
obtained likewise at 2-week intervals.
8. Immunoaenicity studies on horses
8.1 Vaccine preparation
8.1.1 Preparation of the eluate vaccine
After determination of the protein content
of the glycoprotein ~eluate fractions by
photometry, the latter were adjusted with
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sterile-filtered elution buffer to the
required protein content per vaccine dose of
2 ml. The vaccine formulations were com-
pleted by addition of 0.2 ml of R halogen D
as adjuvant (correspond.s to 10%) and 0.001%
Merthiolate as preservative, and were
subsequently checked for pH and sterility.
The following test vaccines were prepared in
this way:
EHV-1 Al: 12.5 ~g of protein / dose (=2m1)
A2: 75.0 ~g of protein / dose (=2ml)
A3: 225 . 0 ~g of protein / dose (=2m1 )
8.1.2 Preparation of the lect:in vaccine
The suspension of the l.ectin complexed with
glycoproteins was diluted by addition of
sterile-filtered PBS to the required protein
concentration of glycoproteins per dose for
administration. 10% Havlogen D as adjuvant
and 0.001% Merthiolate as preservative
completed the formulations.
The following test vaccines were prepared in
analogy to the concentrations of the eluate
vaccine:
EHV-1: B4: 12.5 ~g of con-A-bound
protein / dose (= 2 ml)
B5: 75.0 ~g of con-A-bound
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protein / dose (= 2 ml)
B6: 225.0 ~g of con-A-bound
protein / dose (= 2 ml)
8.2 Test animals
26 yearlings were available to us for the investiga-
tions. The herd comprised warm-blood horses of the
"Deutsches Reitpferd" breed and thoroughbred Arabs.
8.3 Immunisation schedule
The horses intended for the tests were initially
checked in a serum neutralisation test for their
immune status with respect to ElfiV-1 and EHV-4. After
this, homogeneous groups were made up, and serum
samples of the 0 samples were obtained on the date
of the primary vaccination. In the EHV-1 study, the
basic immunisation of the 6 groups with 4 horses in
each was carried out by vaccination twice at an
interval of 5 weeks. This entailed the animals in
group A1 receiving for primar~r vaccination and as
booster in each case 12.5 gig, those in group A2
75 ~g and those in group A3 2.25 ~g of the eluted
glycoproteins with Havlogen as adjuvant. The horses
in groups B4 to B6 were immunised with the lectin
vaccine correspondingly. After 27 weeks,
revaccination with an eluate formulation (75 ~g of
protein / dose) was carried out uniformly for all
groups.
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The vaccine was administered by deep intramuscular
injection into the pectoral muscle. 2 animals were
used as non-vaccinated controls. Further serum
samples were taken at intervals of 5 to 7 weeks
throughout the course of the teat.
8.4 Investigation of the clinical i:olerability
Initially, the tolerability of the two formulations
was examined in a preliminary test. Elution buffer
(2 ml/dose) on the one hand., and packed con-A
Sepharose (5 ~1/dose) on the other hand, in each
case without viral proteins and adjuvant, was
administered to 3 animals in each case. At hourly
intervals, the body temperature, heart, circulation,
skin trugor, head, mucous membranes, general signs
and any local reactions at the injection site were
investigated. This revealed no findings deviating
from the physiological norm in any of these para-
meters. The tolerability tests carried out during
the course of the immunogenicity studies did not
show, with the exception of one animal in group 85
(with slight local reaction 24 hours after vaccina-
tion), any local or systemic reaction to the
vaccination whatever.
8.5 Serological investigations in 'the SNT
The sera were investigated for neutralising anti-
bodies (serum neutralisation test SNT) against
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EHV-1 and EHV-4. This took place by the serum
dilution method in microtitre plates. The serum
samples to be investigated were, after inactivation
(30 minutes at 56°C), diluted with a reaction amount
of 25 ~1 in powers of two with E-MEM 2.0 g and
placed in 4 wells per dilution stage. Addition of a
virus dose of 100 CIDso in 25 ~1 per well was
followed by incubation at 37 °C for 2 hours . 50 ~1 of
the cell suspension with 300 x :103 cells per ml (PR15
cells in EHV-1 SNT and ED cells in EHV-4 SNT) were
added per well, and the plates were incubated
passing in 5~ COZ at 37 °C for 7 days . The cpE was
read off on day 4 and 7. Evaluation was by the
method of Karber, Naunyn-Schmiedebergs Arch. exp.
Path. Pharm 162 (1931) page 480 or of Reed and
Muench, Am. J. Hyg. 2? (1938) page 493.
Results
1. Kinetics of the expression of membrane antigens of
EHV-1 and EHV-4 infected cells
In order to determine the optimal time of harvesting
for preparing virus-specific antigens from infected
cell cultures, the time course of the incorporation
of virus-specific proteins into the cell membranes
was investigated kinetically. For this, infected
cells were harvested at various times after infec-
tion and lysed with NP40. Following this, the
detergent-soluble proteins of these lysates were
fractionated in parallel with the cells uninfected
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by cell lysates in a reducing SDS page. Electro-
transfer of these proteins to nitrocellulose strips
was followed by immunostaining in order to analyse
their antigenicity.
The dependence of the antigen content on the time of
harvesting was verified by immunostaining of the
lysate proteins of EHV-1-infected cells when harves-
ted at 35%, 75% and 90% cpE with the "Maienschein"
serum. Whereas only two antigens with 82 and 44 kDa
were weakly detectable in the: immunoblot of the
lysate preparation harvested at 35% cpE, seven
virus-specific, antigenic proteins were detectable
in the lysate obtained at 75'~ cpE. The relative
molecular weights of these proteins were at 137, 96,
86 to 78, 56, 44, 35 and 2T kDa. The dominant
staining was of the bands with 86 to 78 and 44 kDa.
The same antigens, although in reduced amount, were
also demonstrated in the membrane lysate harvested
at 90% cpE. Also evident in a:ll preparations were
protein bands with 60 and 48 kDa, which were also
detectable in the negative control.
The result of these kinetics revealed that the yield
of viral antigens was highest" both qualitatively
and quantitatively, in the lysat~es of EHV-1-infected
cells harvested at 75% cpE. At a later time in the
infection (90% cpE), the antigen content in the
preparations decreased again. I:n this way, the time
of harvesting of the infected cells determined from
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these reproduced kinetic investigations was about
80% cpE in order to obtain an optimal yield of
EHV-1 membrane antigens.
The lysate of EHV-4-infected cells harvested at a
cpE of 10% still contained. no virus-specific
antigens detectable by the mon.ospecific EHV-4 goat
serum. By contrast, two virus-specific proteins with
molecular weights of 62 and 44 kDa were detectable
as antigens in lysates prepared at 40% cpE. In
addition to these dominant antigens, further EHV
antigen bands with molecular weights of 58, 56, 37
and 25 kDa, and only weakly detectable at 140 kDa,
were found in lysates with progressive cpE of 60%
and 90%. On the basis of this investigation, har-
vesting at about 80% cpE was also chosen for obtain-
ing EFiV-4 membrane antigen routinely.
A large proportion of the virus antigens which had
already been identified in extracellular virions of
EHV-1 were also detectable in i:he lysates of infec-
ted cells. In this connection, the antigens with
molecular weights of 140, 98 tc~ 88, 84 to 78, 44 and
27 kDa were present in both antigen preparations.
By contrast, in the case of EHV-4, the antigen
pattern differed depending on the antigen prepara-
tion. The membrane proteins obtained from infected
cells represented virus-specific antigens with
molecular weights of 27 to 62 )cDa. Thus, the higher
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molecular weight antigens (140 to 70 kDa) which were
recognised by the monospecific EHV-4 serum in the
extracellular virus were absent in these membrane
protein preparations.
2. Isolation of glycoproteins from membrane lysates of
EHV-1 and EHV-4 infected cells
In a preliminary screening, concanavalin-A (con-A),
Helix pomatia and Phytolacca americana were examined
in affinity chromatography to select the lectin
suitable for these investigations. H. pomatia and P.
americana adsorbed only a small proportion of the
membrane proteins recognised as antigenic. By
contrast, con-A produced the highest yield of virus-
specific antigens, both qualitatively and quantita-
tively. Accordingly, con-A was subsequently used in
the lectin affinity chromatography.
In order to verify the effectiveness of the purifi-
cation process and to analyse the antigenicity of
the purified glycoprotein fraci:ion, the proteins of
the membrane lysates, of the flow-through from the
chromatography (proteins not adsorbed onto the
lectin) and of the eluates of EHV-1-infected cells
were tested by immunoblotting.
Despite identical amounts of protein in the samples,
the staining of the particular antigens with the
"Maienschein'~ serum was more intense in the eluates
Le A 27 917 - 37 -
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than in the corresponding lysa.tes. This means that
the relative content of EHV-1-:specific antigens was
higher in the eluate preparations than in the
lysates. Moreover, there were particular reactions
by 4 proteins with molecular weights of 96 to 88, 78
to 76, 62 and 46 to 42 kDa in this immunoblot.
Additionally recognised in they eluate samples were
weakly detectable antigens at 135 and 27 kDa which
were undetectable in the lysates, and thus were
likewise enriched in the eluate. It also emerged
that the only antigen containe<i in the flow-through
from the chromatography was a protein of 62 kDa
which reacted with the antibodies in the "Maien-
schein" serum. However, this protein was also
detectable in the lysate, the flow-through and the
eluate of uninfected cells, which indicates that
this was a cell membrane protein.
On comparison of the membrane antigens in the
lysates and eluates of EHV-4-infected cells by
immunoblotting, the same proteins in both prepara-
tions were recognised as antigen by the anti-EHV-4
goat serum employed. These were proteins with
molecular weights of 62, 56, 54, 44 and 27 kDa, with
the antigen at 62 kDa dominating in quantity.
Additionally detectable in the eluate preparation
were very weakly represented bands at 76 and 35 kDa.
These tests show that it was possible by lectin
affinity chromatography with concanavalin-A to
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achieve purification of membrane glycoproteins from
lysates of EHV-infected cells without altering the
antigenic structures thereof. The antigen patterns
of the eluates were essentially identical to those
of the lysates. In addition, the distinctly more
intense colour reaction of the antigenic proteins in
the eluates by comparison with 'the lysates when used
in immunostaining demonstrated an increased content
and thus an enrichment of these antigens in the
eluate preparations.
The protein patterns, ignoring the antigenicity of
the individual preparations before and after the
purification, were analysed by the protein-specific
Coomassie staining of the proteins fractionated by
electrophoresis. Whereas there were still at least
30 proteins in the membrane lysates, the number of
proteins detectable with Cooma.asie decreased in the
eluates to about 12. This resu7Lt applies equally to
the particular preparations of EHV-1- and of EHV-4
infected cells.
It was thus possible to show that it was possible to
achieve by this described method an increase in the
antigen content in the eluates" with a reduction in
the total number of different proteins. Table 1
summarises diagrammatically ithe balance of the
content of individual glycoproteins during the
purification.
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Table 1
Diagrammatic representation of the antigens from lysates
and, obtained therefrom, the glycoprot~in fractions of
EHV-1 infected cells
Membrane Membrane Purified Purified
lysate of lysate of glycoprot:eins glycoproteins
uninfected infected of uninfE~cted of infected
cells cells cells cells
,~ 135-140 kDa
i ~ ~ ; - 88-96 kDa
78-86 kDa
~ ; - 62 kDa
56 kDa
i ~ i ~ 48 kDa
- 44-46 kDa
35 kDa
~"~ 2 7 k
D
a
3. Specificity of the purified glycoproteins
Initial analysis was of the extent to which the
antigens, which have been recognised to date by the
"Maienschein" standard serum, of the purified
glycoproteins of EHV-1 infected cells are also
recognised as antigen by immunostaining with other
immune sera.
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For this, in each case eluat:es from lysates of
uninfected cells were fractionated besides the
EHV-1 infected cells from lysat:es in a reducing SDS
Page and tested with different: sera in an immuno-
blot. In this, the antigens stained by the homolog-
ous immunostaining with the serum of the horse
"Arfe" from the EHV-1 immunogenicity study were the
same as with the standard serum. The "Feldserum",
"Ellis" serum and the monospecific goat sera against
EHV-1 strains DA35, DA8 and MSU also recognised in
principle the same antigen pattern as the
"Maienschein" serum. Specifically, antigens with
relative molecular weights of 140, 118, 94 to 88,
78, 62 and the antigens with ~48 and 44 kDa, which
are difficult to differentiate, were stained in this
design of experiment. Moreover,, the antigens of the
EHV-1 eluates recognised by th,e immune sera varied
purely quantitatively, not qualitatively, depending
on the sera used. This is preswnably attributable to
the identical dilutions of all the sera in the
immunoblot, despite different SN titres.
An exception is the EHV-1 MSU serum which, instead
of the 188 kDa antigen, recognised a protein with
105 kDa in the glycoprotein fraction. In the nega-
tive controls which were also stained in each case
there were bands of weakly visible proteins only on
staining with the "Maienschein"' serum at 62 kDa and
with the "Arfe" serum at 62 and 48 kDa.
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To investigate the cross-react;ivity of antigens of
various EHV-1 strains, ED cel7.s were infected with
seven different EHV-1 strains, and the relevant
lysates were purified by lectin affinity chromato-
graphy. In order to compare the antigen patterns of
these preparations, the antigenic glycoproteins
were, after separation in SDS page and transfer to
nitrocellulose, immunostained with various sera in
a Western blot. The results are compiled in Table 2
and 3.
The individual immunostaining;s yielded an antigen
pattern uniform for all investigated EHV-1 prepara-
tions. However, once again, the antigens dominating
in each case varied depending on the serum employed.
Whereas the "Feldserum" and the EHV-1 rabbit serum
generally reacted especially distinctly with the 78
to 86 kDa antigen, the 44 to 46 kDa protein domina-
ted in the staining with the "M.aienschein" serum for
all the strains investigated. An almost uniform
staining of the antigenic glyco~proteins was achieved
with the serum from the horse "Arfe".
The glycoproteins of the individual EHV-1
preparations reacted different7ly with the antibodies
of the EHV-4 goat serum. In this case, two proteins
with 60 and 42 kDa were stained in the case of the
EHV-1 strains AB69 and DA35. In the case of the EHV-
1 strain DA 8, only the 60 kDa protein was
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recognised as antigen by tlhe EHV-4 serum. By
contrast, with all seven EHV-1 strains there was an
antigen band at 84 and at 140 kDa in each case.
However, the latter was also stained in the negative
control in this immunoblot.
A protein with 62 kDa was stained with the three
horse sera in the controls of the eluate from
uninfected cell lysates which were investigated in
parallel in each case. In addition, a protein of
64 kDa reacted with the "Mai~enschein" serum, and
proteins with molecular weighi~s of 25 and 110 kDa
reacted with the "Arfe" serum.
Thus, it was possible to verif:Y the presence of the
antigens with the molecular weights 140, 96 to 88,
86 to 78, 46 to 44 and 27 kI)a in all the inves-
tigated EHV-1 strains in the glycoprotein fraction
from membrane lysates of infecaed cells by various
immune sera.
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Table 2
Summary of the antigenic membrane glycoproteins of the
investigated EHV-1 strains in the immunostaining with the
"Maienschein" serum
Mol. unin EHV-1
wt. fec- ;strains
in ted _____________________.____________________
kDd
a
b
c
d
a
f
g
180 (+) + + (+) (+) (+) (+)
140 (+) (+) (+) (+) (+) (+) (t)
118 + (+) + (+)~ (+) (+) (+)
96-88 + + t + + ++ +
g6-~8 t+ + + + + + ++
64-62 + + + + + + +
46-44 +++ t+t +++ +tir +++ +++ ++
27 (+) (+) (+) (+;I (+) (+) (+)
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Table 3
Summary of the antigenic membrane glycoproteins of the
investigated EHV-1 strains in im~munoblots with five
immune sera
Mol. S era
wt. Maien- EHV-1 EHV-4
in Arfe Feld-
kDa schein ~ serum serum serum
180 (+)
140 (+) + (+) (+) +
118 + + (+)
105 (+) (t)
96-88 ++ ++ + + (+)
86-7g + t +++ +++ +
62 + + + (+) (+)
46-44 +++ ++ ++ ++ (+)
27 (+) + (+)
(+) only weak staining, or bands not detectable in all
immunoblots
+ to +++ intensity of the antigen-antibody reaction
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4. Controlled immunogenicity studies with purified
glycoproteins of EHV-1 infected cells
4.1 Antibody reaction against homologous EHV-1
The primary vaccination led to the induction of
virus-neutralising antibodies within 5 weeks after
vaccination in all 24 immunised horses with the
three dosages employed of the l.wo formulations. The
booster vaccination, which was carried out 5 weeks
after the primary vaccination resulted in a steep
increase in the serum antibody titres up to values
of 1:100. In this case, while the antibody produc-
tion was slightly weaker after primary vaccination,
the horses vaccinated with the con-A formulations
showed a tendency for the booster effect to be
better than the horses vaccinated with eluate
formulations . A distinct fall in the titre was found
in all groups, but especially in groups B4 to B6
(con-A vaccine), 10 weeks after the booster, and
resulted in the titres in groups B4 to B6 22 weeks
after the booster again corresponding almost to
those of the zero sample.
27 weeks after the primary vaccination, all the
horses in the experiment were r~avaccinated uniformly
with 75 ~g of glycoprotein/dose of the eluate
formulation. This boosting vaccination again result-
ed in a distinct increase in the titre by 1 to 2
powers of two in groups A1 to A3 and 2 to 3 powers
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of two in groups B4 to B6.
Moreover, the titres in groups A1 to A3 were, at
values up to 1:170, nevertheless still distinctly
above those in groups 84 to B6 (1:120). The horses
in groups A1 to A3 which received basic immunisation
with vaccines with the lowest antigen doses reacted
to the revaccination with the largest increase in
the titre.
The antibodies persisted considerably longer after
this boosting vaccination; titres approximately
corresponding to those of the maxima after the
booster were still detectable :?5 weeks after revac-
cination (corresponds to 1 year after the start of
the study). Then, a further 10 weeks after this
sampling date, the antibodies had diminished again
so much that the titres were in the range of the
unvaccinated control animals.
Seroconversion was not detectable in any of the
unvaccinated control horses throughout the observa-
tion period. Furthermore, neither respiratory
disorders nor viral abortions occurred in the stud
during the year of observation. This makes it
certain that neither manifest nor intercurrent EHV
infections occurred, and that the described anti-
bodies were the consequence of the experimental
vaccinations.
Le A 27 917 - 47 -
~0~3829
Table 4 shows the results of the serological inves-
tigations on the individual animals, and Table 5
reproduces the group results as geometric mean of
the individual values.
4.2 Investigations on cross-reacting antibodies against
EHV-4
The results of titration of the sera from the
described EHV-1 immunogenicity study against EHV-4,
strain T252 on ED cells, are reproduced in Table 6.
In some cases, clearly readable titres were not
found until the serum dilution:. were 1:8, because of
a cytotoxic effect of the sera in the low dilution
stages on the ED cells employed in the SN test.
The antibodies induced in the horses by immunisation
with membrane glycoproteins from EHV-1 infected
cells also cross-reacted with EHV-4 in the virus
neutralisation. EHV-4 neutralising titres up to 1:20
were detectable at the sampling times 5 weeks after
primary vaccination, but especially 5 weeks after
the booster and 5 and 10 weeks after the revaccina-
tion.
Le A 27 917 - 4g -
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