AU649728B2 - Vaccines against equine herpesviruses and the preparation thereof - Google Patents

Abstract

The present invention relates to vaccines 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.

Description

p Our Ref: 407341 P/00/011 Regulation 3:2

AUSTRALIA

Patents Act 1990 Ad& 549/28

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT

S

5.5.

S S OS S es. S S

S

*5 5

S

S.

S S 55 so Applicant(s): Address for Service: Bayer Aktiengesellschaft D-5090 Leverkusen Bayerwerk

GERMANY

ARTHUR S. CAVE CO.

Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Vaccines against equine herpesviruses and the prepar:ation thereof S9 0 55 5s5555 S S Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me:rin2f The present inventic. 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 dise ses 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.

e e EHV-1 (Equine abortion virus), a pathogen belonging to the alphaherpesviruses, previously called EHV-1 subtype 1 (rhinopneumonitis virus), EHV-2 (Equine cytomegalo-like virus), a betaherpesvirus, EHV-3 (Equine coital exanthema virus), belonging to the alphaherpesviruses and

O.

EHV-4 likewise an alphaherpesvirus, previously called 20 EHV-1 subtype 2.

The equine herpesviruses of serotypes 1 to 4 persistently cause economic damage in horsebreeding throughout the

C

Le A 27 917 1 world.

This damage arises, in particular, from recurrent epidemics 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 respiratory form of the infection with equine herpesviruses of types 1, 2 and 4, should not be underestimated.

0 10 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, 15 parturition, castration or -transport may lead to reactivation of latent EHV infections. Because such reactivation may, although associated with virus excretion, take a clinically inapparent course, every horse once infected must be regarded as a potential excretor and thus as a 20 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 25 immune response.

nvestigations into the immunogenic structural copoents Investigations into the immunogenic structural components Le A 27 917 2 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 immunity 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 O infection with a lethal dose of HSV (Balachandran et al., Infection and Immunity 37 (1982) pages 1132 to 1137).

In experiments with equine EHV-1 herpesviruses in the hamster model, protection from homologous loading infections was achieved with live and inactivated, complete virions and with purified complete coat proteins. By 66 contrast, administration of fractions of the coat proteins 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 S structural proteins which, apart from the 6 polypeptides sof 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.

Le A 27 917 3 I 4 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 to 46 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 immunogenicity of purified glycoproteins in horses. On the one hand, the monoclonal antibodies were prepared in mice 15 and, on the other hand, preparation started from the complete viruses.

Currently employed in practice are monovalent EHV-1 live vaccines or inactivated vaccines in combination with other antigens. Despite the use of these vaccines, 20 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 satisfactory, in particular there is no immunity to EHV-4.

overall, the practical situation with the vaccines Thus, overall, the practical situation with the vaccines Le A 27 917 4 5 employed at present is unsatisfactory.

It was desirable to find an EHV vaccine which is straightforward to use and has high efficacy.

The present invention discloses: 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.

o e o o «o l.

20 2. Process for the preparation of the vaccines according to 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, Le A 27 917 5 f) subsequently the lectin-ligand complex and/or the isolated glycoprotein mixture is formulated in a customary manner.

The present invention also discloses: 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.

r 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 15 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 Rhinopneumonisis (Stutenabort) der Pferde" [Investigations into the development of a live vaccine against equine rhinopneumonitis (abortion in mares)] (Mayer et.al., 1968; Zbl.Vet.Med. Vol. 5, 406-408).

2. The strain DA8 (EHV-1) was deposited in compliance Le A 27 917 6 I I 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 the Institut Pasteur CNCM Paris, France.

4. The strain Thein 166 (EHV-3) The strain Thein 252/1 (EHV-4) was deposited in compliance with the Budapest Treaty on 24.7.1990 O: 1under the designation J-982 at the Institut Pasteur CNCM Paris, France.

0 0 0 0 0 6. The strain MAR 87 (EHV-1) was deposited in compliance with the Budapest Treaty on 24.7.1990 *0* under the designation J-980 at the Institut Pasteur CNCM Paris, France.

The glycoproteins for preparing the vaccines according to the invention can be obtained from extracellular virus or from infected cells. The virus is grown in a manner known 20 per s for this purpose.

DOOU

too*&: The growing of the viruses is cdrried 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 derm cells such as the permanent Le A 27 917 7 equine derm cell ED (ATCC 57 or its derivatives) or porcine kidney cells such as th 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 stitionary, roller or carrier cultures in the form of united cell assemblages or in suspension cultures.

Employed as growing media are all cell culture media 0 known per se, for example described in the product catalogue of Flow Laboratories GmbH Post 1249, 5309 Meckenheim, such as, in particular, the minimal essential medium (MEM) which contains as essential consistuents •a s. 15 amino acids, vitamins, salts and carbohydrates, completed S with buffer substances such as, for example, sodium bicarbonate or (hydroxyethylpiperazine-N-2ethanesulphonic 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.

I. The cells and cell lawns used for growing the viruses are grown in a customary manner almost to confluence or to 25 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 Le A 27 917 8 culture media known per se, such as, in particular, the abovemertioned MEM, Infection with a virus suspension is ther. carried out. The virus is present in the virus suspension diluted in the virus growing medium in such a way tihat infection is with a MOI multiplicity of infection corresponds to infectious virus particles for each cell present) of 0.001 50, preferably 0.10 The viruses are grown with or without addition of aminal sera. In the case whose serum is used, it is addr2 to the growing medium in a concentration of 1 30% by volume, Spreferably 2-10% by volume.

Infection and virus growing is carried out at tempera- S" 1t-res between room temperature and 40"C, preferably between 32 and 39"C, particularly preferably at 37"C.

15 In the case where the intention is to work subsequently

S.

wiVni 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 20 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.

*e In the case where the intention is to work with extracellular virus, the virus is worked up by removing the Le A 27 917 9 cell detritus and isoliLing 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 viruscontaining growing medium is, after removal of the cell j 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 15 centrifugation for pelleting the virus particles is carried out through an aqueous solution with higher O. 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 been, for example, prepurified by a low-form centrifugation at 5,000-15,000 x g and sedimented by a subsequent 25 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 fo4xml,1000t Le A 27 917 10 I, i I 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. Joncentration is carried out by filtration or centrifugation. Centrifugation is preferably carried out at 300 to 2,000 g. The centrifugation is carried out at a volume of about 1 1 for about 15-45 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.

0* Suitable detergents are: anionic surfactants such as Ka lauryl sulphate, fatty 15 alcohol ether sulphates, mono/dialkyl polyglycol ether orthophosphoric ester monoethanolamine salt, calcium alkylarylsulphonate, sodium deoxycholate, cationic surfactants such as cetyltrL ethylammonium chloride, ampholytic surfactants such as di-Na N-lauryl-:-iminodi- 20 propiont or lecithin, non-ionic surfactants, for example .9 polyoxyethylated castor oil, polyoxyethylated sorbitan monooleate, sorbitan monostearate, glycerol monostearate, polyoxyethylene stearate, alkylphenol polyglycol ether.

Non-ionic detergents may be particularly mentioned: Le A 27 917 1i S. non-ionic, water-soluble emulsifiers with an HLB (hydrophilic/lipophilic/balance value) greater than 10, for example Emulgator NP 400 (Bayer AG), alkylaryl polyglycol ether; Renex 678 6 (Atlas Chemical Industries), polyoxyethylene alkylaryl ether; Tween 20® (Atlas), polyoxyethylene sorbitan monopalmitate; Myri 530 (Atlas), polyoxyethylene stearate; Atlas G 37070, polyoxyethylene lauryl ether; Atlas G 3920", polyoxyethylene oleyl ether; Atlas G 9046 To, polyoxyethylene mannitan monolaurate; Emulgator 1371 B (Bayer AG), alkyl polyglycol ether; Emulgator 17360 (Bayer AG), alkyl polyglycol ether (oleyl polyglycol ether); Emulgator OXO (Bayer AG), alkyl polyglycol ether (dodecyl polyglycol ether); Ninox BM-2 O (Stepan Chemical ethoxylated nonylphenol; Triton X- 15 1000 (Rohm an Haas isooctylphenol polyethoxyethanol; Cremophor ELO, Nonidet P 400 (Shell), glycopyranoside e. (Sugena), N-decantoyl-N-methyl-glycoside.

The detergents are used in the form of dilute aqueous solutions. Solutions which may be mentioned are those containing 0.1 to 10 percent by volume, preferably 0.5 to percent by volume, particularly preferably about 1 percent by volume of detergent.

0 The detergent solution is added in the ratio of about 1 1 to about 10 1 by volume to the cell or virus 25 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.

Le A 27 917 12 I 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 examlie.

The centrifugation supernatant obtained in this way or o. the filtrate can be stored at low temperatures (0 to 70 0 C) before it is processed further.

To isolate the glycoproteins contained in the solution, it is treated with lectins. For this, it is previously 15 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 20 lectins to be employed. The concentration is preferably 9 adjusted by dialysis of the lysates.

Lectins are proteins or glycoproteins from plants, 4 specifically their seeds, microorganisms, vertebrates and invertebrates which specifically bind sugars and their conjugates. Used according to the invention are lectins which recognise and bind glycoproteins from EHV-infected Le A 27 917 13 v, I 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.

00

O

6e O 060 9.*15 *9* 9 20 000

S

6 90.SS9 6 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 Le A 27 917 14 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 addition to the known salts which are customary and necessary for the stability and reactivity of the lectins. The salt and detergent solution of the lectins 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 15 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 lect1,n 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.

009 so 0 sees 0 S4B

OS

S

4 .5 g..

25

S.

a 0 The lectin treatment is carried out at -5 to +30 0

C,

preferably at 2 to 8 0 C for about 10 min to 3 days, preferably at 1 hour to 2 days.

Le A 27 917 15 ir 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 to 20 mg, of lectin are employed per ml of packed gel.

The glycoprotein-lectin complex is removed from the complete solution or suspension in a customary manner.

This can be carried out by centrifugation, filtration or, O in the case of chromatography, by washing.

The suspensions or gels which contain the lectin-glycoprotein complexes and which are obtained in this process step are washed to reduce the salt and detergent concen- 15 tration. For this, the suspension or the gel is mixed with a solution which contains 0.1 to 0.01 times the «r *a 0. detergent concentration and sodium chloride 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-glyc( rotein 0,6, complexes.

'.ft The suspensions or gels of the lectin-glycoprotein complexes obtained in this way can be employed directly 25 or, where appropriate, after addition of adjuvant for immunisation of horses. Depending on the content of lectin-bound glycoprotein, the suspensions or gels wJ Le A 27 917 16

I'

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, preferably 10 to 100 mg, particularly preferably 25 to 50 mg, of glycoprotein.

In the resulting suspensions or gels of the lectinglycoprotein complexes, the concentration of the detergent can be altered, as far as eliminating it, by filtration, centrifugation, dialysis or other washing pro- S 10 cesses. The same methods can also be used to alter the salt concentration in the physiologically tolerated range.

dew* 206 a s 2

VO&GO

*4 It o s 15 4'* a..

0s 0 0 o *6bs 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 glycoprotein-lectin complexes to prepare the vaccine. For this, the suspensions or gels are treated with a sugar-containing, aqueous salt solution.

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 concentration and composition of the salt content and detergent content of the sugar solution corresponds to that of the Te T n Ig 1 17 *1 gels or suspensions containing the glycoprotein-lectin complexes.

The treatment with the sugar solution is carried out at to +30"C, preferably between 2 and 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 chromatography).

0 em..

0* 0 4400 S 04 4' 15 04 0

S

pr 20 0000 9 0 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 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.

Le A 27 917 aI, 4 The concentrations of detergent and salts in the preparations can be altered as described 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 glycoproteins 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, aluminium hydroxides or ®Havlogen.

Administration is carried out in a customary parenteral manner such as intramuscularly, subcutaneously, intranasally.

Examples 1. Preparation of the cell cultures a a S 06 a. 0

US

a. a 0 IS a Ore a '0 1.1 Media and solutions a O900 20 Culture medium: E-MEM 0.85 g (Earle minimal essential medium (cf. Virol.

Arbeitsmethoden [Virol. Working Methods] Vol. 1, Gustav Fischer- Verlag, Stuttgart 1974, A.

Mayer, P.A. Bachxaann, B. Bibrack G. Wittmann) 0.85 g NaHCO/l 6

S

Le A 27 917 19 I Trypsin EDTA: 0.2 g of EDTA-ethylenediaminotetraacetic acid sodium salt; 1 g of glucose; 0.4 g of KC1; 8 g of NaCl; 0.58 g of NaHC03; g of trypsin to 1 1 distilled water.

Fetal calf serum (FCS): supplied by "Gibco" 1.2 Cell culturing Vp 10 a s~ 0 Thb 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 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

S

20 2.1 Media and solutions Maintenance medium:

S

Se e 5 05 5 5

S

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) Le A 27 917 20 PBS: 8 g of NaCI, 0.2 g of KH 2

PO

4 1.44 g of Na 2

HPO

4 0.2 XC1 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 maintenance 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. (multiplicity of infection) 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 15 24 hours after infection, while the EHV-4-infected cells required at least 48 hours.

3. Preparation of the cell lysates 3.1 Chemicals and solutions Extraction buffer: 25 mM tris HC1 (tris(hydroxy- 20 methyl)-aminomethane HC1), 1% pH 8.6 NP40: Nonident P 40 (ethylphenyl polyethylene glycol) Storage buffer: 0.1 M NaCH 3 COO, 1 mM CaCl 2 1 mM Le A 27 917 21 A Elution buffer: MgCl 2 1 mM MnClz, 1 M NaCI, pH 6 0.1 M haCHlCOO, 1 mM CaCl 2 1 mM MgCI 2 1 mM MnC1 2 0.1 M NaCI, 0.1% NP40, 0.3 M methyl mannopyranoside, pH 6 3.2 Detergent treatment *s 15 a 20 4.

The ED monolayer cell cultures cultured in roller bottles (850 cm 2 Falcon) or dish stacks (6000 cm 2 Nunc) were inoculated at 90 to 100% confluence with EHV-1 or EHV-4. According to the results of kinetic investigations (harvesting at a cpE of 10 to 100%), the infected cells were harvested at a cpE of for the preparative experiments. 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, cr were suspended in the culture medium by vigorously shaking the dish stacxs. The suspension was subsequently centrifuged at 3,000 g for '0 min, and the cell pellet obtained in this way was washed 2 x in PBS before it was finally taken up 1:2 in PBS v/v and stored at -70"C. Extraction was carried out by adding 3 ial of extraction buffer per 1 ml of cell suspension and subsequently treating with ultrasound (3 bursts of sec each at 7 microns). This cell lysate was stirred at 4 C for 24 hours before insoluble Le A 27 917 22 particles were removed by sedimentation by centrifugation in an SW41 rotor (38,000 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 glycoproteins The purification of glycoproteins on con A-Sepharose by affinity chromatography was described by TODD et w 10 al., Arch. Virol 96 (1987), pages 215 224, for the preparation of glycoproteins from Aujeszky virus.

The cell lysates were initially dialysed at for 0 24 hours against the storage buffer of the con A- Sepharose 4B completed with the detergent NP40 (1% 15 final concentration). The concanavalin A-Sepharose 4B (Sigma C9017) was introduced bubble-fre~ into a column (Phanmacia 1.5 cm diameter) and equilibrated with 20 times the bed volume of the storage buffer described above NP40) at 4°C. The glycoproteins were subsequently adsorbed onto the lectin by applying the cell lysates to the column (15 mg of protein/ml of packed gel). For this, the cell lysate (average protein content 2.1 mg/ml) was introduced, by means of a peristaltic pump with a pumping rate 25 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- Le A 27 917 23 I I I graphic operations were carried out at about 4 0 C. To reduce the detergent and salt concentration, the gel adsorbed with glycoproteins was reequilibrated with times the column volume of a modified equilibration buffer with reduced NaCI (0.1 M) and content 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 5 ml/hour, and the column was subsequently incubated 6 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- 15 nopyranoside concentrations the adsorbed glycoproteins 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 elution 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 NaC1, 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 a Le A 27 917 24 i I determination of proteins, it was necessary to dilute the NP40 in the sample by preceding ultrafiltration. For this, an aliquot of 50 pl (in the case of lysates containing 1% NP40) or 500 pl (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 S Bio-Rad protein assay. The protein content in the initial sample could be calculated from this by simple computation.

*6446e a 6. Preparation of -lctin-bound clycoproteins *e.

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 NP40). To reduce the salt content and to eliminate the detergent, the lectin was repeatedly washed, **o Le A 27 917 25 I I first with the modified storage buffer (containing 0.1M NaCI and 0.1% NP40) and subsequently in the same buffer without NP40. This suspension of the lectin adsorbed with glycoproteins was stored at 4 0 C. The proteins bound to con-A were quanti...- as follows: an aliquot of 200 pl of the lectin bound with glycoproteins was incubated twice with 500 pl each time of elution buffer (storage buffer containing 0.1M 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. Antiaenicity analyses Transfer 50 mM tris(hydroxymethyl)aminomethane buffer: 40 mM glycine 1.3 mM Na dodecyl sulphate methanol pH 9.3 20 Sample 15 mM tris(hydroxymethyl)aminomethane buffer: 2% w/v Na dodecyl sulphate w/v p-mercaptoethanol 6 M urea 10% glycerol 0.02% w/v bromophenol blue pH 6.8 S

SO:

Le A 27 917 26

'I

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 pl of a denaturing sample buffer in each case. After ultrasound treatment for 5 min, they were boiled for 4 min and loaded into the sample recesses.

Goes*: The marker proteins used were Bio-Rad "Prestained SDS-Page low range standards" with protein weights *15 of 110, 84, 47, 33, 24 and 16 kDa.

*o SAfter the electrophoresis, the SDS polyacrylamide gels were stained for 16 hours in a Coomassie staining solution, shaking gently at room temperature. 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 snitrocellulose Le A 27 917 27 t I 0r .m.

S

000S 0 *000 After evaluation of the stained gel, proteins fractionated by SDS Page were transferred to nitrocellulose membranes in accordance with the principle described by Burnette 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/cm 2 of gel.

7.3 Immunostaining The nitrocellulose strips to be stained were initially agitated for 1 hour in the blocking buffer (3% 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'.50 to 1:10,000) in the same buffer at room temperature for 2 hours. After thorough washing with PBS, the peroxidase-labelled anti-specieL 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 Te 1 "7 917 28 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 immunostained proteins.

The horse sera used for the immunological investigations were sera from the following animals: 0 "Maienschein" a warm-blood horse immunised 15 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 "Arfe" a horse from the test described hereinafter with a high EHV-1 SN titre (1:80) and low, cross-reacting see EHV-4 SN titre "Ellis" a mare which was experimentally 25 infected in the USA with EHV-1 and aborted during this infection, with Le A 27 917 29 a high EHV-1 SN titre (1:144) and a low EHV-4 SN titre "Feldserum" a saddle horse with clinical signs of respiratory disorder and with moderately high EHV-1 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 4 this, 100 pg of dissolved virus protein/dose were homogeneously formulated in each case with Freund's incomplete adjuvant 3 x at 14-day intervals and 0 administered to the rabbit subcutaneously. Serum was obtained likewise at 2-week intervals.

8. Immunoqenicity studies on horses 8.1 Vaccine preparation 8.1.1 Preparation of the eluate vaccine s After determination of the protein content of the qlycoprotein eluate fractions by S. photometry, the latter were adjusted with Le A 27 917 30 sterile-filtered elution buffer to the required protein content per vaccine dose of 2 ml. The vaccine formulations were completed by addition of 0.2 ml of R halogen D as adjuvant (corresponds 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 pg of protein dose (=2ml) J A2: 75.0 pg of protein dose (=2ml) A3: 225.0 pg of protein dose (=2ml) 8.1.2 Preparation of the lectin vaccine *.e The suspension of the lectin 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 pg of con-A-bound 25 protein dose 2 ml) 75.0 pg of con-A-bound Le A 27 917 31 protein dose 2 ml) B6: 225.0 pg of con-A-bound protein dose 2 ml) 8.2 Test animals owls 255 so so 0* a..

0 00* 26 yearlings were available to us for the investigations. 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 EHV-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 Al receiving for primary vaccination and as booster in each case 12.5 pg, those in group A2 75 pg and those in group A3 225 pg 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 pg of protein dose) was carried out uniformly for all groups.

Le A 27 917 32 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 test.

8.4 Investigation of the clinical tolerability 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 pl/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 S 15 and any local reactions at the injection site were investigated. This revealed no findings deviating from the physiological norm in any of these parameters. The tolerability tests carried out during e.

the course of the immunogenicity studies did not show, with the exception of one animal in group (with slight local reaction 24 hours after vaccination), any local or systemic reaction to the 9 vaccination whatever.

0 8.5 Serological investigations in the SNT 25 The sera were investigated for neutralising antibodies (serum neutralisation test SNT) against Le A 27 917 33 F 15 9, 0 &i a 4 004 0 0

S.

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 minutes at 56 0 diluted with a reaction amount of 25 ul 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 CIDo 0 in 25 il per well was followed by incubation at 37 C f r 2 hours. 50 il of the cell suspension with 30C x 103 cells per ml 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 0 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. 27 (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 infection and lysed with NP40. Following this, the detergent-soluble proteins of these lysates were fractionated in parallel with the cells uninfected

.O.

al o 04 Le A 27 917 34

II

by cell lysates in a re acing SDS page. Electrotransfer 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-l-infected cells when harvested 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 O 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, :15 86 to 78, 44, 35 and 27 kDa. The dominant staining was of the bands with 86 to 78 and 44 kDa.

The same antigens, although in reduced amount, were N also demonstrated in the membrane lysate harvested 9, at 90% cpE. Also evident in all preparations were 20 protein bands with 60 and 48 kDa, which were also detectable in the negative control.

The result of these kinetics revealed that the yield f* of viral antigens was highest, both qualitatively and quantitatively, in the lysates 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. In this way, the time h of harvesting of the infected cells determined from 0 a 4 0.

Le A 27 917 35 15 6S 20

S

'S.

(i e. 25

S

S

these reproduced kinetic investigations was about cpE in order to fb'tain 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 monospecific 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 and 90%. On the basis of this investigation, harvesting at about 80% cpE was also chosen for obtaining EHV-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 the lysates of infected cells. In this connection, the antigens with molecular weights of 140, 98 to 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 preparation. The membrane proteins obtained from 4nfected cells represented virus-specific antigens with molecular weights of 27 to 62 kDa. Thus, the higher Le A 27 917 36 molecular weight antigens (140 to 70 kDa) which were recognised by the monospacif ic EHV-4 serum in the extracellular virus wire absont in these membrane protein prepaeLations.

2. Isolation of glycoproteins from membrane lysates of EHiV-l awl EHV-4 infected cells a of a44 so4 G 25 In a preliminary screening, concanavalin-A (con-A), Helix pomatia and Pbs %lacca americana were examined in affti~nity chromarography to select the lectin suitable for these ivestigations, H. Eomatia and P, americana adsorbed only a small proportion of the imeibraine ptoteirks recogntsed as antigenic. ny contrast, con-A produced the highest yield ol virusopecific antigens, both qualitatI.ively and quantitatively. Accordingly, .con-A was subiiequently used i the lectin affinity chromatography.

In orreer to verify the effectiveness of the pur cation process and to anayse the antigenicity of the purified glycoproteir fraction, this proteins of the melmbrane lys-tes, of,' the flow-th--ough from the chroiate-dphy (proteing not adsorbed onto the lectin) and of the eluates of EHV-X--infected_' cells were tested by imnunoblotting.

Despite identical amounts of pzotein in the samples, the staining of the particular antigens with the "Maienschein" serum was more intense in the eluates Le -A 27 917 37 1 I Ose* C S Se 0

C

20 gee.r U S sce

S

SR

0

C

than in the corresponding lysates. 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 the 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 contained in the flow-through from the chromatography was a protein of 62 kDa which reacted with the antibodies in the "Maienschein" 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 preparations were recognised as antigen by the anti-EHV-4 goat serum employed. Thrse 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 Le A 27 917 38 Do* aR S of S. S *5 S S .55 S 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 &ntigenicity 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 proteins in the membrane lysates, the number of proteins detectable with Coomassie decreased in the eluates to about 12. This result applies equally to the particular preparations of EHV-1- and of EHV-4infected 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 the balance of the content of individual glycoproteins during the purification.

S S5

S

Le A 27 917 39 Y, 0 I Table 1 Diagrammatic representation of the antigens from lysates and, obtained therefrom, the glycoprctein fractions of EHV-1 infected cells Membrane lysate of uninfected cells Membrana lysat. of infected cells Purified glycoproteins of uninfected cells Purified glycoproteins of infected cells e 9 99

I

I

I

I

f

I

t

I

I

t nI i

I

I

a pi 1~11 1%11 8laar~a sagsll ~asa Imaar h, a m 1 135-140 kDa 88-96 kDa 78-86 kDa S 62 kDa 56 kDa 48 kDa 44-46 kDa 35 kDa 27 kDa 09* O 99 S. 9 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.

15 II11 Le A 27 917 40 0

O..O

**o 15 5 e** so *25 is." 2 For this, in each case eluates from lysates of uninfected cells were fractionated besides the EHV-1 infected cells from lysates in a reducing SDS Page and tested .th different sera in an immunoblot. In this, the antigens stained by the homologous 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 the immune sera varied purely quantitatively, not qualitatively, depending on the sera used. This is presumably attributable to the identical dilutions of all the sera in the irmnunoblot, 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 negative 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.

Le A 27 917 41 V VI

S.

:20 5 55.5

S

25

S..

S

To investigate the cross-reactivity of antigens of various EHV-1 strains, ED cells were infected with seven different EHV-1 strains, and the relevant lysates were purified by lectin affinity chromatography. 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 immunostainings yielded an antigen pattern uniform for all investigated EHV-1 preparations. 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 dominated in the staining with the "Maienschein" serum for all the strains investigated. An almost uniform staining of the antigenic glycoproteins was achieved with the serum from the horse "Arfe".

The glycoproteins of the individual EHV-1 preparations reacted differently 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 Le A 27 917 42 recognised as antigen by the 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 "Maienschein" serum, and proteins with molecular weights of 25 and 110 kDa reacted with the "Arfe" serum., 0 Thus, it was possible to verify the presence of the antigens with the molecular weights 140, 96 to 88, 15 86 to 78, 46 to 44 and 27 kDa in all the investigated EHV-1 strains in the glycoprotein fraction from membrane lysates of infected cells by various immune sera.

*se a a 0*o* 0 a a Le A 27 917 43 sa lip.

Selo*S 6 W: Table 2 Summary of the antigenic membrane glycoproteins of the investigated EHV-1 strains i~n the irnmunostaining with the "Maienschein" serum.

MoI. unin- EH-V-1 strains Wt,. in ted.

kDa b c d e if 180 U U 140 118 C+ 96-88 86-78 64 1;2 46-44 27 I(4) I(4) I Le A 27 917 -4 44 SS I.t s o to Table 3 Summary of the antigenic membrane glycoproteins Df the investigated EHV-1 strains in iinmunoblots with five immune sera IMol. Sera W-11.

in Maien- Arfe Feld- EHV- 1 EHV-4 kDa schein serum serum serum 180() 140 118 U 105 96-88 U 86-78 62 46-44 +41 27 U)+ only weak staining, immunoblots or bands nIot detectable in all 00 9 00

C

SO 50 0 0 0 to intensity of the antigen-antibody reaction Le A 27 917 45 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 two 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- 0..o tion was slightly weaker after primary vaccination, the horses vaccinated with the con-A formulations showed a tendency for the booster effect to be 15 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 revaccinated uniformly with 75 pg of glycoprotein/dose of the eluate 25 formulation. This boosting vaccination again resulted in a distinct increase in the titre by 1 to 2 powers of two in groups Al to A3 and 2 to 3 powers Le A 27 917 46 of two in groups B4 to B6.

a 0 to 15 to 20 6 0 o2 25 t Moreover, the titres in groups Al to A3 were, at values up to 1:170, nevertheless still distinctly above those in groups B4 to B6 (1:120). The horses in groups Al to A3 which received basic immunisation with vaccines with the lowest antigen doses icted 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 booste were still detectable 25 weeks after revaccination (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 observation 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 antibodies were the consequence of the experimental vaccinations.

Le A 27 917 47 4' Table 4 shows the results of the serological invest.tationsi on the individual animals, and Table reproduces the group results as geometric mean of the individual values.

4.2 Investigations on cross-reacting antibodies against EHV-4 i S. 4.

4O S Q 15

S.

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 dilutions 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 revaccination.

Le A 27 917 48 *9 saw 9 9 9 a 09* 9 .8 89* r, Table 4 Immunogenicity study of cell-associated antigens of EHV-1-infected cells in the horse (Titration against ERV-I on PK 15 cells) SN titre; Individual results vaoccrne 11oraf value 15 weeks weeks 110 weeks j16 weeks 122 weeks -5 weeks 110 weeks 117 weeks (25 weeks 135 weeks ,No.*latter vacc ftVr aciner after [after atter 'after latter atter 1after '10.01.89 114.-02-89. er 1oostr oater rvabO~ostr irevacc. ,revacc. Ireacc_ rov-, ,rrvcc- 'PrL=.yacc Booster 1 23.03.89 (25.04.89 105.06.89 107. 22.8.89 25.9.89 189 i23.03.90 r- 9aI 7 :3O 1 140 1:48 1 132 1:48 i 1:28 1 1:224 1:192 1 :160 1:80 1:16 ;luate :31 1:35 1:4 1 1:6 1 t M132 1 1:28 1:4 21 1:160 2 1 :2 106 :12.5u 3: 1:3:51 :1:4 IM 1 1:2 11:2f 1:8 1 1:160 1:160 1:80 1:56 1:16 1 4 1:4 INO4 1:56 1 1:80 I 1:40 1:40 1 1:224 1 1:128 1:128 1 128 I 1:64 I. S 1:5 1:14 1:80 1 1:48 1 1:20 1:16 1:80 1:96 1: 6 1:20 vg/lA 6 1:7 1:16 1:80 1:A8 I 1:12 1:40 1 1:160 196 1:96 1:112 1:48 ,Eluate 7 1:3 I1.64 1:96 1 1181) 1 156 1:28 1:128 M28 1:961 8 1:3.r I1:48 1 1:96 132 1:64 1:32 1 1:160 1 112 1112 1:56 1:24 :A3 1:4 1 2811:64 1 1:5611:16( 1:24 I 1:80 1:3211:28 1:321 1:14 I22SgI/el 1 10 1 1:2.5 1132 1:40 1 140 I 1:40 I 1:96 1:80 1:96 1:48 1:24 :Eluate 1 1 1:3.5 1 1:192 I 1:9 1 1 4 1:28 1:80 1:80 1:96 1:56 1:14 12 1:5 1:24 1:56 1 1:4 1:56 132 1 1:40 1 1:56 1:28 1:40 1:24 :a 4. 113 1:7 1 1:28 1 1:48 1 1:48 1 1:20 1 1:14 1 1:128 1 1:112 1180 156 1112 :12.5sgla: 141 1:10 1:40 1164 1:32 1 1:28 I 1:24 1:80 1:56 :56 IM6 1:32 :Con-A :151 1:10 1:20 1:96 1:64 1:32 It 16 11:14 I 1:32 1:32 1 1:7 1:12 1:96 i 1:48 1t96 1:80 1 1:24 :8 5. 17: 1:2 1:28 1:64 1:20 1 1:14 1 1:80 1 1:64 1 1:641--48 1:14 175AglwI1o 18 1:5 1:20 106 1:24 1 1:12 1 1:12 1 1:96 1 1:80 I 1:56 1:56 I 1:7 "Con-A :19: 1:48 1:128 1 1:46 1:10 I 1:16 1 1:28 1 1:64 164 I 1:80 1:20 1 20 3 1 1:16 1 i:9f i 1:32 1 1:40 A 16 1 1:64 1 1:64 1:24 1:16 1 1:12

I-

1B 6. 121: 1:5 1 1:16 1 96 1 1:28 1:28 j32 1:80 1:40 1:48 1:28 I 1:8 :1225.glm 1 22 1:12 1 1:14 I 1:128 1:28 i 1:15 1:10 1:80 1:96 1:80 I164 1:20 (Con-A :23 1:10 1 1:40 1 1:56 1:40 1:24 I192 1:128 1:128 1160 1:64 1 1 24 1:3 1 1:20 W:12 1:56 I 1:24 1:20 1:224 1:192 1:128 It128 12 :Control 25 1 1:2,5 1 1:6 16 I6 1:10 1 1:6 1:8 1 1:10 I 1:8 1:10 1'.28 1:14 11, 261 1:6 1 i:4 1:12 1:8 1 1:8 1:10 1:8 1:6 1:0 1:8 1:6 I U S So 0 S 0* C S 0*b U U 05 550 455 a 555 0 p. U 0 5 4 5 0 0 o h& @06 I- Table llnmufogenicity study of cell-associated antigens of EHV-1-iflfected cells in the horse (Titrat-4n against EIIV-l on PK 15 cells) SN titre; Geometric group mean Ivaccine 60 value 1,5 weeks 105 -im e k 1 ?iftear vacc4af ter 1 10.018 114.02.819. bocister W S .vaccl Boster 23.03.89 ,A.1 It5.6 l1.~/1 3.48 136.8 576 :Eluate 175 qil1M 4.38 1 33.13 87 .64 t-10 wek -1 after lbooster 1 25 .04-.89 16 weeks I atter I boom ter '05.06.89 122 weeks I after ,1007.89 trevac'.

I iweeks I af ter Irevacc.

122-09-89 1 10 weeks j'11 weeks ,after lafter Irevace. I revacc.

o 25.09.89 14.I1.89 125 weeks after I evacc.

09.01.90 135 weeks latter 23. 0 -1 11 44.53 32.20 36.22 173.16 139.39 116.39 71.83 I Z3.93 49.28 30.45 27.52 127.24 105.91 99.77 68.86 28.34 :A.3 :1 1 1 I 1 95 :225 pqgII 3.64 1 37.9 724 54.16 28.0 so 30.45 '70.41 55.19 51.5 4.5 16.33 Eluate 91 724I "B.4 II j !12.5PgimII 7.27 1 23.66 55.34 42.111 18.82 151.95 9 4.17 1 66.25 70.05 58. 1 22M5 ICon-A 1I I I I I I I I :as 3.811 61 13.24 43.607.3 Itg/ml 1 3.1 I25.6 93.21 28.00 1 61 .6 67.5) 48.44 43.07 1ICon-A 61 I1 I I :22 pgliJI 6.51 1 20.5 10 I3396 25.61 119.80 1 128.50 98.56 189.06 S 5.04 14 l~nrl 3.87 1 9.17 100 14.9 W.1.L3 I I

I

Table 6 Ilnmunogenlicity Ejtudy of cell-associated antigens of EHV-1-infected cells in the horse (Titration against EHV-4 on ED cells) SN titre; Individual results fore[ value 1,5 weeks J' Wea .ka 16 weks 2 We 5 weeks 10 w~eeks 1'17 wek 125 w~eeks 135 weeks iac. No. ls4rvacate !ter after latter I attar latter ljofter *latter latter 1 1. 1 '14.02.89. booster booster booster jbooster Irvc. Iaac rvc. Ieac 1roac 05.06..9 250. j4.18 23.03.90 I PriM'vAccf,%oomter 230. S0_9 0.68 rvc. 122.08.89 25 98 11.9 0 9 .0 3. .g 1:12 1 6 1 1 1 2 1 42 '2 2 44 1 1:4186 1 Ia- 2 1 1:6 C 4 4 4 11 1018 1 11 1 :Eluate 1 3 1 42 -2 1'64 4 1 :24 4 4 1 41 42 1 v2 1 1:6 1 44 c 4 1 <2 1 4 54 'A 2. 15 42 4 1 :6 94 A A4 i I1 45 '6 1:4 e3 17 1&/116 41717.4 4 1 12 0C 44 :11uate 7 4 12 I13 1:6 -4 C4 '4 W:2, I t 1:6 114 '4 68 42 1 42 116 '94 '4 44 1 .:12 Its C5 6 4 '4 191 '2 1 '2 1 1:351 e4 4 4 1 :4 1:6 1 '2 1 103 4 3 25tQ4 1 101 C2 1 I 1:3 j 4 4 4 1 '3 I '3 D 23 1: j 1 -lEiuate lii '21 1 '2 1 1:10 C4 '4 4 4 4 '2 '412 4 c6 4 III 1: 0 4 C4 4 1 1:6 1 1:3 11 I B, 1 131 '2 1 '2 1 '46 '4 1 4 11 !12.5SA91al 14 1 '2 1:2 I 1:2 '4 e4 '4 1 1:4 10:3 14c ICon-A 1 151 '2 1:2 1 <s 4 I 4 '4 1 C3 1. 1:6% s 1: 1 4 .4 -14 1 1:6 1 t 1161 '42 1:3 '2 141 toS. g 1m 17 1 421 <21 <2 '4 -4 I6 46 41 Con-Alm S 2 2 13 C I '4 'd '4 1:4 1 1 6 64 1201 1:3 1 1 :3 1 '4 I '4 <4 1' :86. 121 1 '2 10 1 '2 1 44 4 1 4 1 1:8 1 '3 C 4 <4 .4 :225&g/ml1 22 1 '2 I 1:7 j 1 w 1 '4 4 1 1:3 1 3 'c4 1 0 1.4 i I'Con-A .123 1 '2 11:31 1:6 1 '4 c4 -0 I 1:6 1:4 1:6 1:8 f 16 1 1 24 1 '2 1 42 1 1110 0 1 4 C 4 I 1 :M6 16 4 4 '4

I

,Control. 1 25 1 '2 1 C2 2 '2 <2 1 -2 2 1:2 d 43I 1,'26 1 '2 1 42 C 2 C2 4 C 2 t '3 I :2 4 I '3 1 46

I

Claims (8)

1. Vaccines for horses against infections with equine herpesviruses (EHV), characterised in that they contain isolated glycoproteins and/or their com- plexes with lectins from EHV-infected cells or extracellular EHV virions,

2. Vaccines according to Claim 1, characterised in that the glycoproteins have been isolated by complex formation on lectins.

S3. Vaccines according to Claim 1, characterised i.i that lectins which bind mannose and/or glucose and their conjugates are used for the complex formation with the glycoproteins.

4. Vaccines according to Claim 1, characterised in that lectins from Canavalia ensiformis or Helix pomatia and their coupling products on inert matrices are employed for the complex formation. ***ac

5. Vaccines according to Claim 1, characterised in that the glycoproteins are isolated from equine herpes- viruses of serotypes 1 to 4.

6. Process for preparing the vaccines according to *6 Claim 1, characterised in that a) EHV viruses are grown, a) EHV viruses are grown, Le A 27 917 52 53 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 ,'.xture is iso- lated, f) subsequently the lectin-ligand complex and/or the isolated glycoprotein mixture is formulated in a customary manner.

7. Complexes of glycoproteins from equine herpesviruses or f:om cells which have been infected with equine herpesviruses, with iectins and their coupling products on inert matrices.

8. Glycoproteins from equine herpesviruses isolated and purified with lectins. ass* a a 600 S 55o *r *r S *5 5 *o S 0 S S *555 S DATED this 17th day of Marcl4b994. BAYER AKTIENGESELLSCHAFT By Its Patent Attorneys DAVIES COLLISON CAVE Le A 27 917 53 vaccines against eqLine herpesviruses and the preparation thereof Abs tract The present invention relates to vaccines 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. 0 00 00 00 00 Le A 27 917