AU660741B2

AU660741B2 - Vaccines based on modified type 1 bovine herpesviruses

Info

Publication number: AU660741B2
Application number: AU29688/92A
Authority: AU
Inventors: Georg Floss; Gunther Keil; Walter Strube; Peter Thein
Original assignee: Bayer AG
Current assignee: Bayer AG
Priority date: 1991-12-16
Filing date: 1992-11-26
Publication date: 1995-07-06
Anticipated expiration: 2012-11-26
Also published as: CA2085191A1; EP0547446A3; CA2085191C; ZA929719B; KR930012826A; AU2968892A; HU215537B; KR100265717B1; EP0547446B1; ATE165620T1; DE4141400A1; AR248047A1; ES2116306T3; JPH05276942A; MX9206973A; HUT65487A; EP0547446A2; HU9203976D0; DK0547446T3; DE59209310D1

Description

Our Ref: 449339 6 im P/00/011 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT *0

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.5 C S *5 S ls Applicant(s): Bayer Aktiengesellschaft D-5090 Leverkusen Bayerwerk

GERMANY

DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Address for Service: Invention Title: Vaccines based on modified type 1 bovine herpesviruses The following statement is a full description of this invention, including the best method of performing it known to me:- 5020 The present invention relates to vaccines based on type 1 bovine herpesviruses (BHV-1) which contain modifications in regions of their genome which code for nonessential parts of essential proteins. It is possible with the aid of these vaccines to differentiate immunised from non-immunised cattle. The invention furthermore relates to processes for the isolation and preparation of the modified BHV-1 strains, isolation and preparation of the modified proteins and peptides.

S 10 Infection with type 1 bovine herpesvirus (BHV-1) in cattle becomes manifest mainly in the organs of therespiratory tract and of the genital tract, but also in the peripheral and central nervous system. The clinical pictures induced thereby are described as infectious bovine rhinotracheitis, infectious bovine vulvovaginitis and infectious pustulous balanophosthitis. Besides these, conjunctivitis, orchitis, endometritis, mastitis, abortions or meningoencephalitis are also observed. Following the initial infection there may be latent persistence of BHV-1 throughout the life of the infected animal. The cells of the nervous system, but also the epithelial O tissue, the lymphocytic tissue, macrophages and lymphocytes are suggested as sites of virus persistence. As a consequence of reactivations of these latent infections, 25 phases with clinical manifestation with virus excretion alternate with phases with inapparent symptoms with or without excretion of pathogen. The causes of reactivation Le A 28 649 Le A 28 649 1 are strains on the organism with latent infection, such as stress, inmunosuppression or secondary infection.

Because of the serious economic damage (such as, for example, rearing losses, decrease in milk yield, reduction in fertilisation rate, sterility and abortions) caused world-wide by the disorders associated with BHV-1, prophylactic control measures have great importance.

Besides the damage caused by the disorders, the indirect consequences of BHV-1 infection have great importance.

Thus, only very restricted trade nationally and internationally in cattle suspected of BHV-1 is allowed.

Available for the prophylactic control of disorders" associated with BHV-I are live vaccines based on attenuated BHV-1 strains, inactivated vaccines and so-called subunit vaccines based on purified virus proteins. In the control measures currently in progress for the eradication of BHV-1 or for freeing herds infected with BHV-1 of the disease, and in livestock export, cattle are identified as possible virus carriers on the basis of detecting BHV-1-specific antibodies.

Differentiation of BHV-1-immunised cattle from BHV-1infected cattle, which could make measures for eradication and freeing of disease considerably more effective, is not at present possible.

25 This type of differentiation of immunised from infected livestock is possible in other areas. Vaccines for pigs

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Le A 28 649 2o for the prophylaxis of the clinical manifestation of infection with Aujeszky virus (procine herpesvirus 1) have already been developed and permit differentiation of immunised and infected livestock. These vaccines are based on virus strains which lack, entirely or in parts, proteins which are not necessary for virus replication, so-called non-essential virus proteins, or they are based on individual purified virus proteins. Serological detection methods for detecting specific antibodies directed against the virus proteins missing from the vaccine permit identification of Aujeszky virus-infected pigs and differentiation from livestock immunised against this pathogen.

A vaccine described in European Published Specification 316 658 is said to permit differentiation of immunised and infected cattle. A BHV-1 mutant prepared by recombinant technology, which does not, because of a mutation (deletion or insertion) in the gene for the non-essential structural glycoprotein gill, contain this glycoprotein, is described.

It is possible to assign particular biological functions to individual, viral proteins. The absence of protein components in viruses, such as, for example, of glycoproteins, necessarily determines the absence of biological functions of these viruses. Thus, the non-essential structural protein gill of Aujeszky virus is significantly involved in the immune response to Aujeszky virus.

Specifically cellular defence mechanisms, which have a LeA28 649 -3- -4predominant role in the immunological defence against herpesviruses, are directed against the protein gIII of Aujeszky virus (Zuckermann et al., 1990, J. Virol. 64, 802-812).

Applied to the BHV-1 system, this means that the absence of the protein gIll, comparable in function, in GHV-1 should bring about a reduction in the immunogenicity.

In order to prepare BHV-1 vaccines with high immunogenicity, the BHV-1 mutants which are employed as immunising viruses must therefore have deletions or other alterations in proteins or protein sections which are not the aim of relevant defence mechanisms.

The present invention now relates to:- 1. Vaccines against BHV-1 infections which are based on strains of BHV-1 which, compared with field viruses, contain a DNA sequence which is modified at least in position 310-388 of the region coding for essential glycoprotein IV.

2. Process for the preparation of BHV-1 strains for the preparation of vaccines against BHV-1 infections which are based on strains of BHV-1 characterised in that a-1) The DNA sequence of viral genes for essential glycoprotein IV, at least in position 310-338, is determined for a BHV-1 strain which has been isolated from an infected natural host (for example cattle, pigs, goats) or passaged in cells, and the amino-acid sequence of the encoded protein is derived therefrom or S" 25 a-2) The amino-acid sequence of viral essential glycoprotein IV, at least in position 310-338, is determined directly and b) The amino-acid sequence determined as in a-i) or a-2) is compared with the amino-acid sequence of the corresponding essential glycoprotein IV of 30 BHV-1 field viruses, or o* *•o c) Immunological methods using BHV-1 specific antibodies against essential glycoprotein IV of field viruses are used to search for altered amino-acid sequences, at least in position 310-338, and the BHV-1 strains which are not recognised by one or more antibodies are selected, and d) The strains which have amino-acid sequences altered by comparison with the field virus and which are obtained according to b) or and the field virus are tested for antigenicity in a manner known per se. In this connection, the amino-acid sequence of the field virus which corresponds to the altered amino-acid sequences of the strains according to b) or c) must be involved in the formation of one or more epitopes which do not occur in the strains obtained according to b) or Cattle not infected with BHV-1 must not contain any antibodies against amino-acid sequences of field viruses, which are altered in the strains obtained according to a), b) or c).

e) The strains among those identified according to d) which are immunogenic in cattle are selected.

f) As an alternative to a) to it is possible in a manner known per se to alter nucleotide sequences on the genome of BHV- I1 strains in such a way that the amino-acid sequence at least in position 310-338 of the region coding for essential glycoprotein IV is altered in such a way that one or more epitopes for cattle are modified or absent by comparison with the 25 field virus.

3. DNA sequence which codes for the essential glycoprotein gIV of the BHV-1 strain N 569 and is characterised in that it is modified in its nucleotide sequence in such a way o* that the amino-acid sequence of the protein gIV is modified in the region of positions 310-338 by comparison with field viruses.

*eo -6- 4. Process for the preparation of the DNA sequences according to characterised in that the DNA is isolated from the BHV-1 strains prepared according to in a manner known per se, or the genome of BHV-1 field viruses undergoes molecular cloning in vectors in a manner known per se, and the cloned BHV-1 DNA fragments are altered in their nucleotide sequence in such a way that, after incorporation thereof into the genome of BHV-1 strains, they result in BHV-1 strains which can be used as vaccines against BHV-1 infections, and this modified BHV-1 DNA is isolated from the vectors in a manner known per se.

5. Protein gIV of the BHV-1 strain N 569, whose amino-acid sequence is modified in the region of positions 310-338 by comparison with field viruses.

6. Process for the preparation of proteins according to characterised in that the protein is isolated from the BHV-1 strains prepared according to in a manner known per se, or by cloning and expressing the genes which code for these proteins in pro- or eukaryotic expression systems in a manner known per se.

7. A method for the identification of BHV-1 strains which can be used as vaccines against BHV-1 infection which involves exposure of BHV-1 field strains to antibodies against region 310-338 of glycoprotein IV of BIHP-1 field viruses..

8. Antibodies against non-essential regions, which are modified at least in the region of positions 310-338 by comparison with BHV-1 field viruses, of glycoprotein IV of BHV-1 strains which can be used as vaccines against BHV-1 infections.

9. Process for the preparation of the antibodies according to characterised in that animals (for example rabbits, mice, goats, sheep) are immunised with BHV-1 strains which have been prepared according to or with glycoprotein IV from BHV-1 strains which is modified at least in the region of positions 310-338, and the antibodies which are formed are isolated in a manner known per se.

-7- The method of differentiating cattle which are infected with BHV-1 field viruses from cattle which have been immunised with vaccines based on the BHV-1 strain N 569 which comprises contacting serum from said cattle with peptides which are homologous with amino-acid sequences of non-essential regions in the glycoprotein gIV in BHV-1 field viruses and which are heterologous with the modified amino-acid sequence of position 310-338 in the glycoprotein gIV of the BHV-1 strain N 569.

11. A method of preventing infection by BHV-1 viruses in an animal which consists of treating an animal in need of such treatment with a vaccine according to such that said animal can then be differentiated from non-immunised, field-infected animals.

12. A method for the preparation of vaccines against BHV-1 infections, substantially as herein described with reference to the examples and/or Figures.

13. DNA sequences which code for glycoprotein IV of BHV-1 strains and which have been altered so that said proteins have their amino acid sequence altered in at least position 310-338, said DNA sequences substantially as herein described reference to the examples and/or Figures.

14. Glycoprotein IV of BHV-1 strains which is modified at least in position 310-338 by comparison with field viruses, or the fragments and/or peptides thereof, substantially •o as herein described with reference to the examples and/or Figures.

Antibodies against region 310-338 of glycoprotein IV of BHV-1, substantially as 25 herein described with reference to the examples and/or Figures.

25 herein described with reference to the examples and/or Figures.

*o *o *o• o* *o* o The terms mentioned above have the following meanings:- BHV means bovine herpesvirus BHV-1 means bovine herpesvirus of serotype I according to the terminology in "Virus Disease in Laboratory and Captive Animals" F.J. Conraths, H. Ludwig, G.Darai 1988 published by Martinus Nijhoff, Boston.

0* *t p: wpdocs a\44 93 39\ab BHV-1.3 means bovine herpesvirus of subtype 3 of serotype 1.

Representatives of this subtype were isolated for the first time in Australia in 1962 as strain N569 (French, 1962, Australian Veterinary Journal 38, 216ff) and as strain A663 in Argentina (Carrillo et al. 1983, Zbl.Vet.Med.B 30, 327ff) where there were outbreaks of virus-related encephalitis in cattle.

Both Metzler and coworkers (1986, Archives of Virology 87, 205ff) and Engels and coworkers (1986/87, Virus Research 6, 57ff) described these isolates, whose DNA restriction enzyme fragment pattern was uniform and distinguishable by comparison with other representatives of BHV type 1, as' subtype 3 of serotype 1 of BHV. The strain Na67 which was isolated by Bartha and coworkers in Hungary from a bovine suffering from encephalitis (Bartha et al., 1969, Acta Veterinariae Academiae Scientarium Bungaricae 19, 145ff) showed, according to investigations by Magyar and coworkers (1989, Acta Veteri aria Hungaricae 37, 3ff), the typical BHV-1.3 DNA pattern. Studdert designates this virus group as "Bovine Encephalitis Herpesvirus (BEHV)" (Studdert, 1989, Vet.Rec. 584).

O 25 The representatives of subtype 3 play a completely minor quantitative role compared with the occurrence of the two other subtypes 1 and 2 of BHV-1 (BHV-1.1 and BHV-1.2). Subtypes 1.1 and 1.2 differ by a DNA pattern, which is characteristic of each of them,

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Le A 28 649 after treatment of their genomic DNA with certain restriction enzymes (Engels et al, (1981), Arch.

Virol. 67, 169 ff). Typical representatives which may be mentioned are the Cooper strain for BHV-1.1 and the SchBnb6ken strain for BHV-1.2.

Field viruses are type 1 bovine herpesviruses which occur under natural conditions. Their DNA sequences code for at least one epitope in non-essential parts of essential proteins, which is identical in the overwhelming majority of all variants which occur under natural conditions and is thus to be designated as characteristic of BHV-1 field viruses. The formation of antibodies in cattle against these (this) epitope(s) therefore suggests infection with BHV-1 field viruses.

Essential protein means one or more proteins which are necessary for virus replication of BHV-1 in cell culture. These proteins are encoded by the genome of BHV-1 and may be a component of the mature virus particle or may contribute to its replication without becoming a component of the mature virus particle. Examples of essential proteins of BHV-1 which may be mentioned are gB and gD (named in accordance with the terminology for HSV) (Wyler et al., Infectious Bovine Rhinotracheitis/Vulvovaginitis, in "Developments in Veterinary Virology", Vol. "Herpesvirus Diseases of .lo Le A 28 649 a L a *r Cattle, Horses, and Pigs", ed. by Wittmann, Kluwer Academic Publishers, 1989).

Non-essential regions of essential proteins designate parts of essential proteins. Alterations of the amino-acid sequence in these parts are possible without blocking the function of the overall protein for virus replication. In connection with the present invention, it is important that these non-essential regions are able, on the basis of their amino-acid sequence, to form epitopes which lead in BHV-1-infected cattle to the formation of specific antibodies.

Modified amino-acid sequerce means that the amino-acid sequence of an essential protein of a BHV-1 strain differs in at least one non-essential region by comparison with the field virus in such a way that the epitope present in the corresponding protein of the field virus in this (these) region(s) is no longer present in the protein of the virus with the altered amino-acid sequence. Cattle which become infected with the virus which contains the modified amino-acid sequence or receive administration of this rirus in inactivated form or parts theraof thus lack specific antibodies against at least one epitope in the field virus, which are formed by them after infection with a field virus.

Le A 28 649 *Tin 9 k A The modified amino-acid sequences may appear in naturally occurring BHV-1 strains, such as, for example, positions 310 to 338 in the essential structural protein gIV (gD) of the BHV-1 strain N569; they may be produced during cell passages of field viruses or be prepared after identification of non-essential regions of essential proteins by altering the DNA sequence in the genome of BHV-1 by genetic engineering methods.

The modification in the amino-acid sequence of a non-essential region in an essential protein may be based on a replacement of one or more aminoacids, absence of one or more aminoacids, the insertion of* one or more amino acids or of any combination of the said possibilities by reason of a DNA sequence of the virus genome which is modified by comparison with the field virus. It is crucial that the alteration does not block the function of the essential protein in virus replication and results in the loss of at least one epitope, it being perfectly possible for one or more new epitopes to be produced therein.

Modified DNA sequence means that the virus genome of a BHV-1 strain has a different nucleotide sequence, by comparison with field viruses, as a consequence of one or more nucleotide replacements, of a deletion of one or more nucleotides, of an insertion of one or more nucleotides or of any combination of thebe Le A 28 649 o•* possibilities. The modification results in each case in the loss of at least one epitope in at least one non-essential region of an essential virus protein, it being perfectly possible for one or more new epitopes to be produced therein although this must not block the function of the affected virus protein for virus replication.

Glycoprotein gIV designates the glycosylitic structural protein of BHV-1, which is designated as homologous gD of HSV (Wyler et al., Developments in Veterinary Virology; Vol. Herpesvirus Diseases of Cattle, Horses, and Pigs; ed. Wittmann; Fluwer Academic Publishers, 1989). The nucleotide sequence of the gene for gIV and its location on the genome are described for the Schbnb6ken and Australia BHV-1 strains (Beninga, diploma thesis in the faculty of biology of the University of TUbingen, 1989). The mature glycoprotein gIV which is incorporated into the extracellular virus particle has, in the typical representatives of BHV types 1.1 and 1.2, a relative molecular weight, determined by SDS polyacrylamide gel electrophoresis, of about 71Kda (Wyler et al., Infectious Bovine Rhinotracheitis/Vulvovaginitis, in 9 25 "Developments in Veterinary Virology", Vol. "Herpesvirus Diseases of Cattle, Horses, and Pigs", ed. by Wittmann, Kluwer Academic Publishers, 1989). The glycoprotein gIV of the BHV-1.3 strains has a relative molecular weight of about 68 KDa.

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Le A 28 649 '7 Region which projects directly out of the virus coat of the virus means the region in the glycoprotein gIV which in the orientation of the protein from the amino terminus in the direction of the carboxy terminus is located shortly before the so-called membrane domain. Beninga (diploma thesis in the faculty of biology of the University of Tibingen, 1989) was able to determine, by sequencing the gene for gIV of the BHV-1 Sch6nbbken and Australia strains, the amino-acid sequence and, from this, the secondary structure as well as the hydrophilic and hydrophobic regions of this protein. According to these investigations, a strongly hydrophobic region is to be found at the amino terminus and is followed by a region of about 300 amino acids which has hydrophilic and hydrophobic regions and is relatively extensively folded. A linear region predominantly hydrophilic in character follows in the direction of the carboxy terminus. This region with approximately amino acids in the case of the BEV-1 Sch6nb6ken strain projects directly out of the membrane coat of the virus and carries the possible modifications in O the amino-acid sequence according to 3 (above). This is followed directly, in the direction of the carboxy terminus, by a very highly hydrophobic region which is designated as membrane domain and anchors the glycoprotein in the membrane.

Q/1h Natural host means any animal which can be infected with BHV-1 and replicates this virus. Natural hosts can be cattle, pigs and goats (Rolle and Mayr, Mikrobiologie, Infektions- und Seuchenlehre [Microbiology, infection and epidemiology textbook] F. Enke Verlag Stuttgart).

BHV-1 strain passaged in cells means that a BHV-1 strain is replicated in eukaryotic tissue culture cells. This entails the harvest from a replication on tissue culture cells S or a BHV-1 isolate from an animal, possibly after storage, being inoculated onto tissue culture cellsand harvested again after virus replication. The number of replications on the tissue culture cells depends in this connection on the aim of the tissue culture passages. If the virus is to be replicated to obtain relatively large amounts for analytical or preparative approaches, a small number, for example 1 to 10 replication cycles suffices. If the virus is to be modified in its biological or biochemical properties, for example virulence, amino-acid sequence and/or DNA sequence, by the tissue culture passages, a larger number of cell passages is advisable. Thus, for example, it was possible to alter the BHV-1 Schinb8ken strain, which was origitnally pathogenic for cattle, to apathogenicity by S. about 200 cell passages.

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g i The occurrence of alterations in the biological or biochemical properties can be influenced by the choice of the cells employed for the cell passages.

Cells which, for example, derive from non-natural hosts of BHV-1, such as, for example, dog kidney cells, may offer advantages for the selection of, for example, apathogenic mutants of BHV-1, which may result in attenuation of an originally pathogenic BHV-1 strain.

Determination of the DNA sequence means that the virus strain to be investigated is replicated, the virus genome is isolated and at O least parts thereof undergo molecular cloning invectors. The clones with viral genes for essential proteins can be identified by hybridisation using DNA probes. Examples of DNA probes which can be employed are DNA fragments or synthetic oligonucleotides with nucleotide sequences of genes for essential proteins of BHV-1 or of other herpesviruses. Nucleotide sequences of BHV-1 genes, or DNA fragments with parts of or with complete BHV-1 genes for essential proteins of BHV-1, for example gD and gB, which can be employed as DNA probes, are known from Beninga (diploma thesis in the faculty of O 25 biology of the University of TUbingen, 1989) and Chase and coworkers (Journal of Tissue Culture Methods, 11, 75ff, 1988, and J.Gen.Virol. 1561ff, 1989). The identified DNA clones which carry DNA sequences of the virus genome for non-essential *9 9 99 Le A 28 649 9 .o proteins can be used to determine the nucleotide sequence of the virus genes by the methods known per se.

To determine the amino-acid sequence directly means that the amino-acid sequence of one or more essential BHV-1 proteins is determined by the biochemical methods which are known per se. The essential proteins are previously purified from BHV- 1 infected cells or from virus particles by, for example, immunoaffinity chromatography using known b monoclonal antibodies against the essential virus proteins such as, for example, gB and gD (Chase and coworkers, Journal of Tissue Culture Methods, 11, 1988; Van Drunen Littel-van den Hurk and coworkers, Vaccine 8, 358ff, 1990; Fitzpatrick and coworkers, Virology 176, 145ff, 1990; Duque and coworkers, Vaccine 7, 513ff, 1989; Marshall and coworkers, Virology 165, 338ff, 1988).

Alternatively, the proteins to be sequenced can also be synthesised in eu- or prokaryotic expression systems and then purified by immunoaffinity chromatography.

Comparison of the amino-acid sequence means that the amino-acid sequence of essential proteins of the investigated BHV-1 strain is compared with the amino-acid sequence of the corresponding essential proteins of one or more BHV-1 field viruses. To do this, the amino-acid sequences *0 a.* 17 eA 28 649 24e* 7* I N6 A8 *.:4U :r.0 to be compared are laid against one another in the same orientation from the amino terminus to the carboxy terminus diagrammatically in such a way that identical amino acids are present at the maximum number of positions in the sequences to be compared.

In order to achieve this it is also perfectly possible for the individual amino-acid sequences to be diagrammatically separated and interrupted.

Epitopes in the essential proteins of BHV-1 field viruses which do not occur in the essential proteins O of the investigated BHV-1 strain are sought. Regions of at least 5 consecutive amino acids in the aminoacid sequence of the essential proteins of the field virus (or of the field viruses) which do not occur in the corresponding proteins of the investigated BHV-1 strain indicate, for example, possible modifications of epitopes in the investigated BHV-1 strain.

Specific antibodies designates sera or serum fractions from animals with antibodies against parts of essential proteins of BHV-1 field viruses, or monoclonal antibodies (in O the form of hybridoma culture media or ascites from mice treated with hybridoma cells) against individual epitopes of essential proteins of BHV-1 field viruses. BHV-1 strains which have a modified aminoacid sequence in the corresponding regions of their essential proteins, which these antibodies recognise S* 2 0* *e Le A 28 649 2

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00 in BHV-1 field viruses, are not recognised by these antibodies.

Sera or serum fractions from cattle are preferred.

To prepare these antibodies, animals, for example cattle, which have no antibodies against BHV-1 are immunised with parts of essential proteins of BHV-1 field viruses, where appropriate with the addition of adjuvants, by administration one or more times.

The sera of the immunised animals can be employed unpurified or as serum fractions which contain only antibodies directed against the antigens employed for the immunisation. Serum fractions of this type are obtained, for example, by purification by affinity chromatography.

The cattle can also be immunised with intact BHV-1 field viruses. It is then necessary in every case to purify the serum fractions from these animals, which contain antibodies only against individual parts of essential proteins of BHV-1 field viruses. This can be carried out by treating the sera with purified parts of essential proteins from BHV-1 field viruses as antigen to adsorb corresponding antibodies. The adsorbed antibodies are then eluted and collected (immune-affinity chromatography).

25 Monoclonal antibodies from, for example, mice which ~have been immunised with intact BHV-1 field viruses e.

•0* e or purified essential proteins from BHV-1 field viruses or with parts of essential proteins of BHV-1 field viruses are very particularly preferred. The immunisation of the animals, fusion of their spleen cells with myeloma cells and obtaining of the antibodies takes place according to the methods known per se for the preparation of monoclonal antibodies ("Antibodies, a Laboratory Manual"; E. Harlow and D.Lane; Cold Spring Harbor Laboratory; 1988). If the mice are immunised with intact BHV-1 field viruses it is necessary to select the hybridoma cultures which form antibodies against essential proteins of BHV-1 field viruses. These hybridoma cultures can be selected, for example, bytesting their cell culture supernatants in an ELISA with essential proteins from BHV-1 field viruses or parts of these proteins as antigen.

Immunological methods mean techniques with whose aid parts of essential proteins of BHV-1 can be investigated using sera, serum fractions or monoclonal antibodies. (For review: "Antibodies, a Laboratory Manual"; E.Harlow and D. Lane; Cold Spring Harbor Laboratory; 1988).

The sera, serum fractions or monoclonal antibodies S 25 used in this case recognise parts of essential proteins from BHV-1 field viruses. BHV-1 strains whose essential proteins are not recognised by these sera, serum fractions or monoclonal antibodies, which thus have different epitopes in essential o* Le A 28 649 proteins by comparison with field viruses, are sought. The choice of the suitable technique can be based on the serum, serum fraction or monoclonal antibody material available.

An example of a technique which may be mentioned is immunoblotting (Western blot). This entails the proteins from BHV-1 being fractionated by the SDS polyacrylamide gel electrophoresis method known per se, and the proteins subsequently being transferred in an electrical field to filter paper, for example O nitrocellulose filter. This filter is then incubated with sera or serum fractions with antibodies against parts of essential proteins of BHV-1 field viruses or with monoclonal antibodies against essential proteins of BHV-1 field viruses. The binding of these antibodies to antigens on the filter is visualised by enzyme-mediated colour reactions known per se ("Antibodies, a Laboratory Manual"; E.Harlow and D.Lane; Cold Spring Harbor Laboratory; 1988).

Another example is the enzyme-linked immunosorbent assay which is known per se and which likewise visualises the binding of antibodies to antigens via an enzyme-coupled colour reaction, and in which BHV-1 is employed as antigen ("Antibodies, a 25 Laboratory Manual"; E.Harlow and D.Lane; Cold Spring 44Harbor Laboratory; 1988).

4*4 Another example is the immunofluorescence test known A 28 649 4* 4 Ti A 28 649 V i^ v per se, which detects antibody binding to an antigen by fluorescence.

Testing for antigenicity means that the corresponding amino-acid sequence in BHV-1 field viruses which was unidentifiable in the investigated BHV-1 strain is investigated for its ability to act as antigen, that is to say to induce the formation of antibodies, in the animal. This can take place by, for example, investigating this amino-acid sequence by assessing its solubility in water and the prediction of possible secondary structures for the probability of surface exposure of this sequence in the essential protein of BHV-1 field viruses.

administering to animals, preferably to cattle, a synthetically prepared peptide with the amino-acid sequence of the BHV-1 field viruses which was unidentifiable in the investigated BHV-1 strain.

After this immunisation, blood samples are taken from the immunised animals, for example as weekly intervals, and the sera of these animals are investigated for the content of antibodies against this peptide.

investigating the sera of animals, preferably of cattle, which have been infected with BHV-1 field viruses for the content of antibodies against the 22 Le A28 649 amino-acid sequence in essential proteins of BHV-1 field viruses which was unidentifiable in the investigated BHV-1 strain. To do this, for example, the sera are investigated for their ability to bind a synthetically prepared peptide with the amino-acid sequence from BHV-1 field viruses which was unidentifiable in the investigated BHV-1 strain. A technique which may be mentioned are the enzyme-linked immunoadsorbent assay (ELISA) known per se or the immuno-dot-blot, known per se, with synthetic peptideo as antigen.

Epitope is a specific binding site for antibodies based on an amino-acid sequence or, where appropriate, on a glycosylated amino-acid sequence.

Nucleotide sequences are parts of the genomic DNA of BHV-1.

Alterations of nucleotide sequences comprise all methods which are suitable for modifying the nucleotide sequences which code for essential proteins from BHV-1. The modification may be based on -deletion of one or more nucleotides and/or insertion of one or more nueleotides and/or 0* 25 replacement of one or more nucleotides.

A selection of methods for modifying nucleotide sequences is given in "Molecular Cloning" 2nd edition, 1989, ed. J.Sambrook, E.F.Fritsch, T.Maniatis, Cold Spring Harbor Laboratory Press. The modification of the nucleotide sequence is intended to eliminate epitopes in essential proteins of BHV-1 without in this way blocking the biological function of this protein for virus replication.

Deletion means removal of one or more building blocks from a sequence without replacement and without blocking the function of the protein.

Substitution means that a building block is deleted from a sequence and is replaced by another building block at the same site without blocking the function of the protein.

Isolation of DNA means that the DNA is extracted by molecular genetic methods known per se from a DNA-containing mixture (for example from purified virus particles of BHV- 1) (Virologische Arbeitsmethoden [Methods in virology research] Vol. III, ed. A.Mayr; P.A.Bachmann; B.Mayr-Bibrack; G.Wittmann; Gustav 25 Fischer Verlag).

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Le A 28 649 )(4 T 7 3A Molecular cloning of genome (compare point 14 above) means that the genomic DNA of BHV-1 is isolated and the DNA fragments which contain genes or DNA sequences of genes for essential proteins of BHV-1 are inserted into conventional DNA vectors (for example bacterial plasmids such as pBR322, pUC18/19 inter alia). The identification of DNA fragments from the genome of BHV-1 which contain genes or parts of genes for essential proteins can take place by the DNA sequences for genes of essential O proteins from BHV-1 being known (for example gI described in Chase et al., 1989, J.gen.Virol. 1561-1569) or via homologies of genes for essential proteins from other herpesviruses such as, for example herpes simplex virus, Aujeszky virus, equine herpesvirus 1 and 4, for which the DNA sequences of their genes for essential proteins are known.

It is possible via DNA/DNA hybridisations of these DNA sequences with geiomic DNA fragments of BHV-1 to find DNA fragments of the latter with genes for essential proteins.

A selection for methods for the preparation and 25 cloning of DNA fragments is given by "Molecular Cloning" 2nd edition, 1989, ed. J.Sambrook, E.F.Fritsch, T.Maniatis, Cold Spring Harbor

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Le A 28 649 Laboratory Press. These vectors with the BHV-1 DNA fragments as inserts are used, for example, for preparing identical copies of the originally isolated DNA fragments with genes or DNA sequences of genes for essential proteins of BHV-1.

Vectors which can be employed for the cloning of DNA fragments of the genome of BHV-1 and/or, where appropriate, for the specific alteration of DNA sequences in genes for essential proteins of BHV-1 and which may be mentioned are all conventional bacterial or eukaryotic plasmid vectors (such as, for example, pBR322, puC18/19 inter alia), bacterial phages (such as, for example, lambda, M13 inter alia). Possibilities for the insertion of the BHV-1 DNA fragments into vectors, the replication thereof in host cells and the reisolation of the DNA inserts are described in detail in "Molecular Cloning" 2nd edition, 1989, ed. J.Sambrook, E.F.Fritsch, T.Maniatis, Cold Spring Harbor Laboratory Press.

Use of essential proteins of BHV-1 strains with modifications in non-essential regions for the identification of BHV-1 strains 9 means that a BHV-1 strain which has modified aminoacid sequences in essential proteins and whose ability for use according to has been demonstrated is used to identify other BHV-1 strains utilisable according to Le A 28 649 -29--

S•

i* eo o4U 9 0 a. The identification of modified amino-acid sequences also locates non-essential regions in essential proteins of BHV-1 whose amino-acid sequence can be modified without blocking the biological function of these proteins for virus replication. This simplifies the search for other BHV-1 strains with modified amino-acid sequences in essential proteins, because narrower delimitation of the regions to be investigated in their essential proteins is possible.

O b. Knowledge of non-essential regions in essential proteins of BHV-1 simplifies the preparation of BHV-1 strains according to (9f) because this means that the region which can be modified, via modifications of the DNA sequence in the genome of BHV-1, in essential proteins is known.

c. Antibodies prepared against modified amino-acid sequences in non-essential parts of essential proteins of BHV-1 strains can be utilised for the identification of other BHV-1 strains which have identical modifications.

d. The essential proteins of BHV-1 which have modified amino-acid sequences can be employed as immunogenic components in BHV-1 vaccines. It is 25 necessary for these proteins to be prepared in pure form for this purpose. This can take place, for example, by known biochemical or

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Le A 28 649 0 o t.

immunological purification methods from viruscontaining material or by expression of genes which code for these proteins in bacterial or eukaryotic expression systems. After the immunogenicity of these proteins has been verified they can be employed, where appropriate with the addition of adjuvants, as vaccines for controlling BHV-1 infections.

Isolation of the proteins for preparing proteins according to (16) to (18) comprises the known methods for the isolation of virus proteins from virus-containing material.

Examples which may be mentioned are a. purification of proteins by antibodies which are specific for the protein to be purified, in, for example, immune-affinity chromatography ("Antibodies, a Laboratory Manual"; E.Harlow and D.Lane; Cold Spring Harbor Laboratory; 1988).

b. purification of virus proteins by chromatographic methods such as, for example, ion exchange chromatography based on their specific charge Q relationships or molecular sieve chromatographies based on their molecular weights.

Expression of proteins for preparation according to (16) 25 to (18) in pro- or eukaryotic expression systems indicates the known methods of molecular genetics Le A 28 649 31

S:

L eA"i 2864 s S for preparing proteins with the aid of cloned genes.

A review of the conventional expression systems and instructions for handling them is given in "Molecular Cloning" 2nd edition, 1989, ed.

J.Sambrook, E.F.Fritsch, T.Maniatis, Cold Spring Harbor Laboratory Press. To do this, the DNA fragments from the genome of BHV-1 which contain genes for the essential proteins must be isolated. These DNA fragments are inserted into the conventional expression vectors in such a way that synthesis of the encoded proteins is possible. Expression systems which may be mentioned are a. prokaryotic systems such as, for example, the lambda gt-11 system for E.Coli, b. eukaryotic systems such as, for example, yeast expression systems, viral vector systems such as, for example, vaccinia systems or baculo-virus systems.

Serological methods for differentiating cattle which are infected with BHV-1 field viruses from cattle which have been immunised with vaccines according to and/or have not been infected with BHV-1 comprise investigations on sera from cattle for their content of antibodies 25 against the amino-acid sequences in essential proteins of BHV-1 field viruses which are modified in the BHV-1 strains employed for preparing vaccines .i •Le A 28 649 **u -0i aI according to The detection of antibodies in sera against the amino-acid sequence as occurs in BHV-1 field viruses permits the interpretation that the animals from which these sera have been obtained are or have been infected with a BHV-1 field virus.

Animals whose sera do not recognise this amino-acid sequence from BHV-1 field viruses have possibly been immunised with a BHV-1 vaccine according to (1) based on a BHV-1 strain with a modified amino-acid sequence and, in any case, are not infected with a BHV-1 field virus.

Methods which can be mentioned for the determination of antibodies against amino-acid sequences in BHV-1 field viruses is an ELISA. Employed as antigen in this ELISA is a short-chain, for example chemically synthesised, peptide with the amino-acid sequence from BHV-1 field viruses which is modified in the BHV-1 strains according to If it is additionally intended to investigate whether the same animals which have no antibodies against the amino-acid sequence from BHV-1 field viruses have been immunised with vaccines according to the sera from these animals are investigated for further BHV-1 specific antibodies, for example in an ELISA with complete BHV-1 particles as antigen. Detection of these antibodies, combined with *the abovmentioned ELISA results, proves that the animals donating these sera have been immunised with *Le A 28 649 33 4* Le A 28 649 -33e *l" eo vaccines according to A. Identification of BHV-1 strains with modified aminoacid sequences according to 9 (above) Replication of the BHV-1 strain, genome purification and molecular cloning according to 9a-1 All BHV-1 strains are suitable for replication for identification. Strains of BHV-1 of the subtype 3 (BHV-1.3) are preferred. The BHV-1.3 strain N569 O is particularly preferred.

The strain N569 was deposited on 13th April 1992 in accordance with the Budapest treaty under Reg.No.I 1204 at the Institut Pasteur CNCM.

The viruses were replicated in a conventional way in tissue cultures of animal cells as primary cells or permanent cell lines, for example in bovine cells, monkey cells, pig cells or dog cells, preferably in bovine kidney cells such as, for example, the permanent bovine kidney cell MD BK (ATCC CCL22 or O 20 derivatives thereof) or the primary bovine kidney cell EBK or monkey kidney cells such as the permanent monkey kidney cell Vero (ATCC CRL1586, CRL1587 or derivatives thereof) or in pig kidney cells such as the permanent pig kidney cell PK15 (ATCC CCL33 or 25 derivatives thereof) or in dog kidney cells such as LeA 28 649 -3

U.

9 .o the permanent dog kidney cell MDCK (ATCC CCL34 or derivatives thereof).

Replication takes place in a manner known per se in stationary, roller or carrier cultures in the form of closed cell populations or in suspension cultures. The replication media employed for the cells are all cell culture media known per se, for example described in the product catelogue of Flow Laboratories GmbH, Post 1249, 5309 Meckenheim, such as, in particular, the minimal essential medium (MEM) which contains as essential components amino acids, vitamins, salts and carbohydrates, completed with buffer substances such as, for example, sodium bicarbonate or (hydroxyethylpiperazine-N-2-ethanesulphonic acid (Hepes) and, where appropriate, animal sera such as, for example, sera from cattle, horses or their fetuses. The use of fetal calf serum in a concentration of 1-30% by volume, preferably 2by volume, is particularly preferred.

The cells and cell lawns used for replication of these viruses are grown in a conventional manner virtually to confluence or to optimal cell density.

Before infection thereof with viruses, the cell growth medium is preferably removed and the cells are preferably washed with virus replication medium.

;Employed as virus replication media are, all cell culture media known per se, such as, in particular, the abovmentioned MEM. This is followed by infection

:S

Le A 28 649 t J Lu with a virus suspension. In the virus suspension, the virus is diluted in the virus replication medium in such a way that infection takes place with a MOI multiplicity of infection corresponds to infectious virus particles on existing cells) of 0.01-50, preferably 0.10-10.

The viruses are replicated with or without the addition of animal sera. In the case where serum is employed, the latter is added to the replication medium in a concentration of 1-30% by volume, preferably 2-10% by volume.

Infection and virus replication are carried out attemperatures between room temperature and preferably between 32 and 39 0 C, particularly praferably at 37 0 C for several days, preferably until the infected cells have been completely destroyed.

The virus-containing medium from the infected cells is worked up further, for example by removing the cell detritus by filtration with pore sizes of, for example, 0.1-0.45 pm and/or centrifugation at up to 10,000 g.

Filtrate or centrifugation supernatant are used for virus concentration and purification. For this, 2 filtrate or supernatant are subjected to high-speed centrifugation until the virus particles have sedimented. It is possible, where appropriate, to S.Le A 28 649 36 .s Le A 28 649 -3follow with other purification steps by, for example, centrifugation in a density gradient.

The genome of the viruses which have been replicated and purified as described above is isolated and purified.

The extraction of native viral DNA is preferably carried out by treating the purified virions with aqueous solutions of detergents and proteases.

Detergents which may be mentioned are anionic, cationic, amphoteric, non-ionic detergents. Ionic detergents are preferably employed. Sodium dodecyl sulphate, sodium lauryl sulphate are particularly preferred.

Proteases which may be mentioned are, all proteases which operate in the presence of detergent, such as, for example, proteinase K and Pronase. Proteinase K may be mentioned as preferred.

Detergents are employed in concentrations of 0.1-10% by volume, preferably 0.5-3% by volume.

20 Proteases are employed in concentrations of 0.01-10 mg per ml of virus lysate, preferably 0.05-0.5 mg per ml of virus lysate.

6.

Operations are preferably carried out in aqueous SA 28 649 a o. buffer solution in the presence of DNase inhibitors.

Buffer substances which may be mentioned are: salts of weak acids with strong bases such as, for example, tris(hydroxymethylaminomethane), salts of strong acids with weak bases such as, for example, primary phosphates or mixtures thereof.

The following buffer system may be mentioned as preferred: tris(hydroxymethylaminomethane).

The buffer substances or buffer systems are employed in concentrations which ensure pH values at which the DNA is not denatured. pH values of 5-9, particularly preferably 6-8.5, very particularly preferably 7-8, are preferred, and particular mention may be made of operation in the neutral range.

Examples of DNase inhibitors are ethylenediaminotetraacetic acid in concentrations of 0.1-10 mMol, preferably about 1 mM.

The lipophilic constituents of the virus lysate are subsequently extracted. Used as extractants are solvents such as phenol, chloroform, isoamyl alcohol or mixtures thereof. A mixture of phenol and O chloroform/isoamyl alcohol is preferably employed initially, with the extraction taking place in one or more stages.

25 Preferably employed in the last stage of the 0..o Le A 28 649 38 0 Le A 28 649 -38oo 4 extraction is chloroform/isoamyl alcohol.

Alternatively, it is possible to employ first phenol and subsequently chloroform/isoamyl alcohol.

Other methods for the isolation of the virus DNA are, for example, centrifugation of a virus lysate in a CsCl. density gradient or in gel electrophoresis (Sharp et al. Biochem. 1973 (12) pages 3055-3063).

The extraction of nucleic acids is described in "Molecular Cloning", A Laboratory Manual, 2nd Edition 1989, ed. J. Sambrook, E.F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press.

The DNA extracted in this way is preferably precipitated from the aqueous solution with, for example, alcohol, preferably with ethanol or isopropanol and with the addition of monovalent salts such as, for example, alkali metal chlorides or acetates, preferably lithium chloride, sodium chloride or sodium acetate, potassium acetate.

The concentration of alcohol in this case is between 40 and 100% by volume, preferably between 60 and by volume, particularly preferably at about 70% by O volume.

a. The chloride or acetate concentration is between .0.01 or 1 molar, preferably between 0.1 and 0.8 25 molar, If LiCI is employed, its concentration is Le A 28 649 -39n 4 between 0.1 and 1 molar, preferably between 0.4 and 0.8 molar.

Methods for the precipitation of nucleic acids are described in detailed in "Molecular Cloning" loc.

cit.. The precipitated DNA is isolated from the aqueous suspension by, for example, centrifugation, preferably washed with alcohol, for example 70% by volume ethanol, and finally resolubilised in aqueous buffer solution.

A buffer substance which may be mentioned is tris- (hydroxymethyl)aminomethane in concentrations of 1-100 mM, preferably 10-50 mM. Preferred pH values are 6-8.5, particularly preferably 7-8.

Examples of further additives which may be mentioned are EDTA (ethylenediaminotetraacetic acid) in concentration of 0.1 to 10 mM, preferably 1 to mM.

Alternatively, the precipitated DNA can also be resolubilised in 0.1 x SSC buffer (Molecular Cloning, loc. cit.) or in ammonium carbonate buffer.

d The viral DNA purified in this way is treated with restriction enzyme in accordance with the manufacturer's instruction. Suitable restriction enzymes are those which recognise at least one cleavage site specific for them on the virus genome.

37 Le A 28 649 e o•* The genome fragments produced in this way are isolated, after the electrophoretic fractionation thereof, for example, from the separating gel (for example agarose), and the fragment which contains the DNA sequence to be investigated is inserted into a plasmid or a phage vector.

Suitable methods for the fractionation of the DNA fragments are electrophoretic and chromatographic methods.

Gel filtration may be mentioned among the chromatographic methods.

Supports which may be mentioned in the electrophoretic methods are agarose or polyacrylamide.

Examples of electrophoresis buffers which may be mentioned are ethylenediaminetetraacetic acid, phosphate/buffer (EPP) tris(hydroxymethyl)aminomethane borate-ethylenediaminetetraacetic acid buffer (TBE) which has the following composition: tris 10-100 mM, preferably 40-90 mM, particu- 20 larly preferably 80-90 mM, Boric acid 10-100 mM, preferably 40-90 mM, particularly preferably 80-90 mM, EDTA 1-10 mM, preferably 1-2.5 mM pH 7-9, preferably 8-8.5

A

(eo V tu tris-(hydroxymethyl) aminomethane acetate-ethylenediaminetetraacetic acid buffer (TAE) which has the following composition: tris 10-100 mM, preferably 30-90 mM, particularly preferably 40 mM, Sodium acetate 1-100 mM, preferably 5-50 mM, EDTA 1-10 mM, preferably 1-2.5 pH 7-9, preferably 7.5-8.5.

A detailed list and description of electrophoresis buffers is described in Current Protocols in Molecular Biology 1987-1988, published by Wiley-Interscience, 1987 A Practical Guide to Molecular Cloning, B, Perbal, 2nd edition published by Wiley-Interscience, 1988 Molecular Cloning, loc. cit.

Virologische Arbeitsmethoden (Methods in Virology O Research), Volume III Gustav Fischer Verlag, 1989.

The procedure for the method is described in "Molecular Cloning" loc. cit., or in Virolog.

e..

:Le A 28 649 -42l f N IBU0 Arbeitsmethoden Volume loc. cit..

The DNA fragment to be investigated is isolated from the support for example by electroelution of the support region containing the fragment. Alternatively by low-melting agarose method (Molecular cloning loc. cit.) or by adsorption of the DNA fragment on to glass surfaces (Gene-clean method).

For the insertion of the DNA fragment, doublestranded plasmid or phage vector DNA molecules are treated with restriction enzymes so that ends suitable for the insertion are produced.

Examples of plasmids used are pAT153, pACYC184, pUC18/19, pBR322, pSP64/65.

Used as phage vectors are lambda phage variants such as, for example, -ZAP, -gtlO/11 or phage M13mpl8/19.

The restriction enzymes which can be employed are known per se, for example from Gene volume 92 (1989) Elsevier Science Publishers BV Amsterdam.

O The plasmid treated with restriction enzyme, or the phage vector is mixed with an excess of the DNA fragment to be inserted, for example approximately ~in the ratio 5 to 1, and treated with DNA ligases in order to bond the DNA fragment end-to-end covalently into the vector.

9 T L6 4: <a/ Ligases are enzymes which are able to join two DNA -molecules via 3'-OH-5' radicals.

The ligation mixture is, for replication of the plasmids or phages, introduced into pro- or eukaryotic cells, preferably into bacteria.

Cells or bacteria are propagated.

Examples of bacteria used are Escherichia coli strain K-12 and its derivatives, for example SK 12-600 (Molecular cloning loc. cit.).

The preparation of the ligation mixture and of the bacterial culture is carried out in a manner known per se as described in Molecular Cloning loc. cit..

The bacteria which contain plasmids with inserted foreign DNA, or phages with foreign DNA, are selected.

Determinations of the DNA sequence of viral genes and deduction of the amino-acid sequence, resulting from the sequence determined, of the corresponding proteins according to 8a-1 20 The nucleotide sequence of the cloned virus DNA fragments prepared above, or subclones thereof are determined by the conventional methods as are described, for example, in Molecular Cloning, loc.

*I

g 8* 4.

4 Le A 28 649 -4r cit.; A Practical Guide to Molecular Cloning, ed. B.

Perbal, A Wiley-Interscience Publication or Viroogische Arbeitsmethoden volume III, ed. A. Mayr, P.A. Bachmann, B. Mayr-Bibrack and G. Wittmann, Gustav Fischer Verlag.

The amino-acid sequences of the viral proteins are then determined from the nucleotide sequences via the genetic code known per se (Biochemie (Biochemistry), A.L. Lehninger Verlag Chemie 1977).

Direct amino-acid sequencing of viral proteins according to 8a-2 As an alternative to the determination of the aminoacid sequence by DNA sequencing, the sequencing of the amino-acid building blocks of a protein can also be carried out directly on the purified protein. A detailed description of various methods is described in Virologischen Arbeitsmethoden, volume III, ed.

A. Mayr, P.A. Bachmann, B. Mayr-Bibrack and G. Wittmann, Gustav Fischer Verlag.

Identification of altered amino-acid sequences in )O viral proteins according to 8b 25 Altered regions in proteins are identified by 'comparing the amino-acid sequence of the investigated BHV-1 strain with the corresponding amino-acid sequence of other BHV-1 strains and/or isolates or Le A 28 649 L e A 8 69 -^7 t field viruses. In this connection, an altered region is regarded as suitable when at least one Sepitope is lost by the alteration, for example when the amino-acid sequence differs in at least 5 consecutive amino acids from the comparison strain. It is possible that one or more new epitopes are produced by thh alteration.

Immunological method for the identification of BHV-1 strains with altered regions in virus proteins according to 8 c As an alternative to DNA sequencing or direct aminoacid sequencing, it is possible for altered regions in virus proteins to be identified by poly- or mcaoclonal antibodies which specifically recognise only parts of a single protein of BHV-1 field viruses. For this purpose, the ability of these antibodies to bind to the BHV-1 strain to be investigated is tested. Examples of methods which may be mentioned for this are: immunoflorescence test, ELISA, immunoblotting. If, in contrast to BHV-1 field viruses, the antibody does not bind to the BHV-1 strain to be investigated, then the latter has an altered amino-acid sequence in the part of the protein for which the antibody is specific.

Le A 28 649 *l *A IU B. Testing for antigenicity according to 8d B. 1. Theoretical evaluation of the antigenicity of an amino-acid sequence The physicochemical nature of an amino-acid sequence (for example the extent of the hydrophilic or hydrophobic behaviour) provides information on a possible antigenicity of this amino-acid sequence. It is possible in this way, using conventional computer programs, to identify potentially antigenic sequences.

B.2 Investigations of the antiqenicity in vivo The peptide with the amino-acid sequence of BHV-1 field viruses which corresponds to that identified as altered in the BHV-1 strain investigated is chemically synthesised or biochemically purified.

This peptide is tested for antigenicity in the animal, preferably in cattle. If the peptide is in the form of a hapten, it is advisable to couple this peptide to a carrier before administration. Carriers which may be mentioned are bovine serum albumin, O ovalbumin or KLH (keyhole limpet haemocyanine). The method of linking the synthetic peptide to a carrier is carried out by the methods known per se.

The animals receive single or multiple parenteral or enteral, preferably parenteral, administration of Le A 28 649 7 o 5 the peptide. It is preferable for various dosages of the peptide to be administered to various groups of animals. Dosages which may be mentioned for the peptide are 0.01 pg to 100 mg, preferably 0.1 pg to 1 mg per administration. Serum samples are obtained from the animals at weekly to monthly intervals and are investigated for antibodies against the peptide administered. Methods which may be mentioned for investigating for antibodies are the conventional immunological test methods such as, for example, virus neutralisation tests, enzyme-linked immuno- O assays or immunoblots. Preferably employed are enzyme-linked imunosorbent assays (ELISA) in which the peptide administered, the protein from which thepeptide derives, or intact or defective BHV-1 particles are employed as antigen.

The administered peptide can be designated as an antigen when animals measurably form antibodies after administration of the peptide. This provides information on the suitability of the investigated strain as immunising strain according to 1 (above).

The crucial point is whether the appropriate amino- O acid sequence, which has been identified as modified in the investigated BHV-1 strain, in the field virus S* 25 is recognised as antigen in cattle.

For this purpose, cattle are infected with a BHV-1 whose amino-acid sequence is not altered and, after A 28 649 4

OU

the infection, investigated for the formation of antibodies. Serum samples are taken from the animals at weekly or monthly intervals and tested immunologically. If, after the infection, the animals form antibodies against the amino-acid sequence of the BHV-1 employed for the infection, corresponding to that sequence which was identified as modified in the BHV-1 strain investigated, then the antigenicity of the unmodified sequence is regarded as proven. An immunological detection method which may be mentioned for the determination of the antibodies against the unmodified amino-acid sequence is an ELISA in which a peptide with the complete or with parts of the unmodified amino-acid sequence is used as antigen.

Cattle which are infected one or more times with the investigated BHV-1 strain which contains the modified amino-acid sequence, and/or receive administration of the latter as inactivated virus, must not form any antibodies against the amino-acid sequence of field viruses which corresponds to that which has been identified as modified in the BHV-1 strain investigated.

B.3. Investigation of bovine sera with known BHV-1 antibody status Bovine sera whose content of BHV-1-specific antibodies is known are investigated for the content Le A 28 649 49of antibodies against the complete or parts of the corresponding amino-acid sequence of BHV-1 field -viruses, which corresponds to that sequence which has been identified as altered in the BHV-1 strain investigated.

An investigated BHV-1 strain with altered amino-acid sequence can be employed as immunising strain according to 1) according to the invention when 1) antibodies against the unmodified amino-acid sequence are also detectable in bovine sera which contain antibodies against BHV-1, determined, for example, in the virus neutralisation test or in the ELISA based on BHV-1 as antigen, and 2) sera from cattle which contain no detectable antibodies against BHV-1 also contain no detectable antibodies against the unmodified amino-acid sequence.

C. Testing for immunoqenicity according to 8e O 20 Identification of a BHV-1 strain which is suitable according to the invention as virus strain of a BHV-1 marker vaccine includes testing for immunogenicity of this strain.

*For this purpose, the investigated BIV-1 strain is e A 28 649 All 11 administered to cattle either as virus capable of replication (live vaccine) or else as inactivated virus, in both cases possibly formulated with an adjuvant. Administration can take place, as required, intramuscularly, subcutaneously, intratracheally, intranasally, intravaginally, intrapreputially or into the conjunctival sac.

The success of immunisation can S be tested serologically via the induction of S 10 BHV-1-specific antibodies after immunisation, determined via neutralisation test or ELISA, or be determined by an experimental infection test with virulent BHV-1. This entails comparison of the clinical manifestation between immunised and non-immunised cattle after a BHV-1 infection produced experimentally.

Suitable strains prevent or reduce distinctly the clinical manifestation of BHV-1 infection after immunisation of cattle once or twice.

D. Genetic engineering preparation according to 8f of BHV-1 strains for use according to 1 *Le A 28 649 D.1 Identification of non-essential regions in essential proteins of BHV-1 Identification of regions which are non-essential for virus replication in essential proteins can take place by a) identification of modified amino-acid sequences in existent BHV-1 strains. As described above, BHV-1 strains which have modified amino-acid sequences are identified by DNA or amino-acid analyses. After identification, the corresponding genome sequences undergo molecular cloning, if this has not already taken place.

b) Modification of genome sequences which code for proteins, for example by replacement of nucleotides, deletion of nucleotides or insertion of nucleotides, and subsequent examination of the significance of the modification for the ability of the modified virus to replicate, in which case the investigated genome sequence and the amino-acid sequence 'resulting there from are to be regarded as nonessential for virus replication when an alteration in the amino-acid sequence does not prevent replication of the modified virus.

*M

ex *e oe D.2 Detection of the antigenicity, of the identified non-essential regions for cattle after infection with BHV-1 a) Theoretical evaluation of the antiqenicity of an amino-acid sequence Theoretical evaluation of the amino-acid sequence identified in D.1 takes place as described above b) Investigation of the antigenicity in vivo 10 After chemical synthesis of the peptide with-.

the corresponding amino-acid sequence of BHV-1 which corresponds to that sequence which has been identified as non-essential for virus replication, or after biochemical purification of this peptide from BHV-1, this peptide is tested for antigenicity in the animal, preferably in cattle. The testing in the animal takes place as described above.

The crucial point is that the amino-acid sequence which has been identified in BHV-1 as non-essential is recognised as antigen after an infection with BHV-1 field virus.

For this purpose, cattle are infected with BHV-1 and, after the infection, investigated for the

L

*o

S.

*e Le A 28 649 -53-

S

;1 97r formation of antibodies. For this purpose, serum samples are obtained from the animals in weekly or i monthly intervals and tested immunologically. If, after the infection, the animals form antibodies against the amino-acid sequence, identified above, in BHV-1 field viruses, then the antigenicity of this sequence is regarded as proven. An immunological detection method which may be mentioned for the determination of antibodies against this aminoacid sequence is an ELISA which has as antigen this amino-acid sequence from BHV-1 field viruses as peptide. The peptide can, for example, be chemically synthesised or biochemically purified from BHV-1 field viruses.

Cattle which are infected with BHV-1 or contain the latter as inactivated antigen do not develop, even after multiple administration, any antibodies against the amino-acid sequence identified in BHV-1 as non-essential.

D.3 Preparation of a BHV-1 mutant by alteration of the identified amino-acid sequence by insertion, substitution or deletion The identified non-essential region in BHV-1 can be altered by deletion, insertion or substitution. For this purpose, the genome fragments which have the DNA sequence coding for this non-essential amino- 0: acid sequence undergo molecular cloning.

e A 28 649 4 Subsequently, the DNA sequences which code for the non-essential amino-acid sequences are removed, partly or for their entire length, from these DNA sequences, which are preferably cloned in vectors, by the conventional methods of molecular genetics and/or, where appropriate, a different nucleotide sequence is inserted. If only parts of a protein essential for virus replication are removed, this deletion must not block the function of the protein for virus replication. The effect of the alteration in the amino-acid sequence on virus replication is checked in tissue culture. BHV-1 strains whose amino-acid sequence have been altered in non-essential parts of essential proteins and which are to be.employed according to must be able to replicate in tissue culture.

The inserted nucleotide sequence must likewise not block the biological function of the corresponding protein into which it has been inserted, because it is a protein essential for virus replication, and it can code for an amino-sequence by means of which identification of the virus altered in this way is possible. The DNA sequence altered in this way by deletion and/or insertion can be recombined, for O 25 example by cotransfection, with the genome of BHV-i in order to obtain recombinant BHV-i which has deletion of the non-essential amino-acid sequence in whole or in parts or contains in place of the latter a new, altered amino-acid sequence.

S2 ••Le A 28 649

*I

*e D.4. Establishment of the correlation of the occurrence of antibodies against BHV-1 and against the identified amino-acid sequence a) Investigation of bovine sera with known BHV-1 antibody status Bovine sera whose content of BHV-1-specific antibodies is known are investigated for the content of antibodies against the amino-acid sequence identified above. A method which can be employed is the abovementioned ELISA based on a peptide with the amino-acid sequence identified above as antigen.

An amino-acid sequence identified as nonessential can be employed as BHV-1 vaccine marker according to the invention, when 1) antibodies against the identified aminoacid sequence can also be detected in bovine sera which contain antibodies against BHV-1, determined, for example, in a neutralisation test or in an ELISA based O on BHV-1 as antigen, and 2) sera from cattle which contain no detectable antibodies against BHV-1 also contain no detectable antibodies against the Le A 28 649 56 4 4 4 4 4.

*eo ooT" identified amino-acid sequence.

It is possible to employ as test once again the ELISA described above and based on a peptide with the identified amino-acid sequence as antigen.

Testing for immunoqenicity The testing is carried out, as described above.

I. Material and methods 1. Cell and virus material A permanent bovine kidney cell (MDBK cell) was used as cell system.

The following BHV-1 strains were employed in the investigations: BHV-1.1 Schleswig-Holstein (SB) BHV-1.2a Sch8nbbken (SB) BHV-1.2b Australia 12 (Ausl2) BHV-1.3 N569 SB, SB and Ausl2 strains were used as examples of BHV-1 field viruses.

4* too.

0* 9 9 99 .04.

6 9. 4 a.

Le A 28 649 2. Cell growth and virus replication For cell growth, 100 ml of MDBK cells were inoculated with 50x10 3 cells/ml per Roux dish (175 cm 2 and grown in E-MEM 0.85 g of bicarbonate/litre (Mayr et al. (1974). Virologische Arbeitsmethoden volume II. Gustav Fischer Verlag, Stuttgart) FCS (foetal calf serum).

For virus replication, 3-5 days after cell inoculation the culture medium was removed from the confluent cell lawn (about 500x10 6 cells/Roux dish) and replaced by E-MEM 2.0 g of bicarbonate/litre (Mayr et al. (1974). Virologische Arbeitsmethoden volume II. Gustav Fischer Verlag, Stuttgart). Virus infection took place with a multiplicity of infection (MOI) of 0.01 to 0.5. The virus was harvested 3-4 days after virus inoculation, at 100% cpE (cytopathogenic effect).

3. Determination of the virus titre 96-Well plates (Nunc) with a confluent MDBK cell lawn were used for determination of the titre of virus suspensions. Serial dilutions in loglO steps 1 to 100 of the virus, prepared in E-MEM 2.0 g of bicarbonate, were applied to the cell lawns at 200 pl/well. 8-Fold determination was carried out 25 for the titration. The virus titre obtained was calculated by the method of Spearman and Karber 8 6 o•* (Mayr et al. (1974). Virologische Arbeitsmethoden volume I. Gustav Fischer Verlag, Stuttgart).

4. Visualisation of the BHV-1 structural proteins For the visualisation of viral proteins, confluent MDBK cells were infected and metabolically labelled with 35 S-methionine from 6 to 20 h after infection.

The viral gIV was precipitated from infected cells with the aid of a monospecific anti-gIV serum, fractionated in a 10% SDS polyacrylamide gel via the method of Lammli and, after fluorography, visualised O by autoradiography. In this design of experiment, gIV was already predominantly glycosylated. Only "mature" forms of gIV were precipitated.

Purification of viral DNA Roux dishes with a confluent MDBK cell lawn were inoculated with virus and incubated at +370C for 3-4 days. At 100% cpE, the complete virus suspension was centrifuged at 5000 xg for 20 minutes, the resulting pellet was resuspended in PBS (140 mM NaCI; 2.7 mM KCl; 6.5 mM Na 2

HPO

4 0.7 mM CaCl 2 0.5 mM MgCl 2 mM KH 2

PO

4 and again spun down (10000 xg; O 20 minutes). The supernatants were combined and then centrifuged at 100000 xg for 1 h and the virus pellet was resuspended in 100 ul overnight. The virus pellet was then resuspended ad 5 ml of PBS *with 5 mM MgC1 2 homogenised, subsequently mixed with Le A 28 649 9 9 o DNAseI (final concentration 100 pg/ml) and incubated at +37 0 C for 1 h. After the DNAse treatment the virus particles were centrifuged through a strength sucrose cushion at 100000 xg for 1 h.

Subsequently the pellet was resuspended in 1.8 ml of mM tris buffer pH 8.0 and, after addition of 200 ul of 20% strength sarcosyl, incubated at +56°C for 1 h. The viral DNA was centrifuged in a CsCl equilibrium gradient (5.7 mM CsC12; 10 mM tris pH 7; 100 mM EDTA) in a fixed angle rotor at 300000 xg at 0 C for 48 h, the gradient was fractionated and aliquots were investigated for the DNA content in a f 0.6% agarose gel. DNA-containing fractions were combined, again centrifuged in a CsC1 equilibrium" gradient and dialysed against 20 mM tris buffer pH 8.0. The DNA concentration was determined in a photometer at 260 nm.

6. Cloning and sequencing of gIV 6.1 Cloning The purified viral DNA from the BHV-1 SB strain was restricted with HindIII (Boehringer-Mannheim, in accordance with the manufacturer's instructions).

Subsequently the resulting DNA fragments were separated by electrophoresis in a 0.6% agarose gel 25 in TA buffer (33 mM tris, 66 mM potassium acetate, 10 mM MG acetate, pH 7.9 with glacial acetic acid, 0.1 mg/ml BSA, 0.5 mM DDT), and the HindIII L

T

7 57 Le A 28 649 fragment with 6.6 kbp was electroeluted (Sambrook et al. (1989). Molecular Cloning. Cold Spring Harbor Laboratory Press.). Using T4 ligase, the HindIII L fragment was inserted into the HindIII cleavage site .of the vector pUC12. For this purpose, the DNA of the vector was treated with HindIII, recircularisation was prevented by eliminating the group using alkaline phosphatase, and the linearised DNA of the vector pUC12 was incubated with the isolated HindIII fragment and T4 ligase.

In the case of the Ausl2 strain, the 8.0 kbp HindIII DNA fragment which cross-hybridises with the HindIII L fragment of the SB strain was isolated in the..

manner described above and likewise incubated with the linearised and HindIII-treated DNA of the vector pUC12 and the T4 ligase.

The plasmids were subsequently transvected into E.coli C600 (Sambrook et al. (1989). Molecular Cloning. Cold Spring Harbor Laboratory Press.) and bacterial clones with recombinant plasmids were selected on the basis of their ampicillin resistance and lack of lacZ activity (Sambrook et al. (1989).

O Molecular Cloning. Cold Spring Harbor Laboratory Press.).

25 For sequencing, the bacteria with the recombinant plasmids were grown, and the plasmid DNA was purified (Sambrook et al. (1989). Molecular Cloning.

e A 28 649 Le A 28 649 e« Cold Spring Harbor Laboratory Press.). Subsequently the DNA was cleaved with PstI, and the cleavage products were again separated by electrophoresis in a 0.6% agarose gel and electroeluted (Sambrook et al. (1989). Molecular Cloning. Cold Spring Harbor Laboratory Press). The isolated DNA fragments were inserted into the sequencing vector pEMBL 19 and again replicated in E.coli C600 (Sambrook et al.

(1989). Molecular Cloning. Cold Spring Harbor Laboratory Press.).

To identify the gIV gene in the strain N569, the O genomic DNA thereof was cleaved with PstI, and the fragments were separated by electrophoresis in a 0.6% agarose gel in TA buffer. The DNA fragments were subsequently transferred to a nitrocellulose membrane by Southern blotting (Sambrook et al.

(1989). Molecular Cloning. Cold Spring Harbor Laboratory Press.) and hybridised with the radioactively labelled HindIII L fragment of the SB strain (Sambrook et al. (1989). Molecular Cloning.

Cold Spring Harbor Laboratory Press.). A fragment 10.1 kbp in size was identified by means of the hybridisation reaction as carrier of the gIV gene in f N569. After identification of the gIV gene, the corresponding 10.1 kbp fragment was isolated by gel electrophoresis and electroelution and inserted into the PstI cleavage site of the vector pUC12 using T4 ligase in analogy to the procedure for the SB and Ausl2 strains. For this purpose, the DNA of the 9• eLe A 28 649 -62- .9T 0. 9 A *so^ vector was treated with PstI, recircularisation was prevented by elimination of the 5'-phosphate group using alkaline phosphatase, and the linearised DNA of the vector pUC12 was incubated with the isolated 10.1 kbp fragment and T4 ligase.

6.2 Sequencing The sequencing of the PstI fragments was carried out by a method which is also called "chromosome walking". It is very suitable for the rapid sequencing of large inserts from an existent clone, without further subcloning. This entailed employing as primers synthetic oligonucleotides which hybridised at different points on the insert and starting the Sanger sequencing reaction there. For this, the fragments were sequenced starting from the flank of the PstI cleavage site. Towards the end of this sequence a hybridising oligonucleotide was then prepared and used in place of the universal primer.

This new primer was then used for further sequencing into the fragments, and then the abovementioned procedure was repeated. Since the resulting sequences did not overlap in some regions, the frag- Q ments were additionally specifically truncated with the ExoIII/S1 system, ligated and cloned in E.coli 25 C600. These subclones were sequenced and the sequences yielded the required overlaps with the sequences already present.

9 6i j#4k. *99 7. Determination of serum-neutralising antibodies against BHV-1 The serum neutralisation test for determining neutralising BHV-l antibodies was carried out in accordance with the instructions in Virologischen Arbeitsmethoden volume II (Mayr et al. (1974).

Gustave Fischer Verlag, Stuttgart).

8. Coupling of the Peptide to BSA as carrier protein 8.1 Pep2 10 The synt )tically prepared Pep2 has the amino-acid sequence of the peptide segment AA317 to AA333 of gIV of the BHV-1 Schb5nbbken strain: gly-gly-ala-glu-gly-pro-lys -pro-gly-pro-ser-pro-aspala.

8.2 Pep3 Pep3, which was likewise chemically synthesised, has, besides the 15 amin.) acids of Pep2, another amino acids of the Schbnbbiken gIV. It comprises the sequence of the peptide segment from AA307 to AA338 in gIV of the BHV-i SB strain: asp-gly-glu-ser-gln-thr-pro-glu-ala-asn-gly-gly-alaglu-gly-glu-pro-lys-pro-gly-pro-ser-pro-asp-ala-asparg-pro-glu-gly.

e A 28696 .7

N

8.3 Coupling of snythetic peptide to BSA mg of synthetically prepared peptide (Pep2 or Pep3) were dissolved in 1 ml of PBS pH=7.4 and adjusted to pH 7.0. 11 mg of BSA in the case of Pep2, and 6.2 mg of BSA in the case of Pep3, were dissolved in 1 ml of PBS and added to the vigorously stirred peptide solution. Subsequently 2 ml of a 0.2% glutaraldehyde solution in PBS were added dropwise to the continuously stirred peptide-BSA mixture. The complete mixture was incubated at room temperature overnight and, the next day, 0.8 ml of I a 1M glycine solution in PBS was added by pipette, stirred for 1 hour, and then the conjugate wasdialysed against PBS (pH=7.4) for 5 hours, divided into portions and frozen at -20 0

C.

9. ELISA for BHV-1 antibody determination (CV ELISA) in bovine sera The antigen employed in the complete virus ELISA was gradient-purified virus particles of the BHV-1 SH strain. The Immulon 96-well plate (F form, Dynatech) was coated with 50 ng of antigen in 50 pl of coating O buffer (1.06 g of Na 2 C0 3 2.93 g of NaHCO 3 ad 1.01 distilled water, pH=9.6) per well. The plates were then incubated at +370C in a CO 2 incubator for 16 hours. 3 Washes with washing buffer (PBS 0.05% Tween 20 IM NaC1) were followed by charging the plates, apart from the blank, with blocking buffer o2 .i Le A 28 649

XI

.ij v

C

(200 pl PBS/Tween 1M NaCl 2% ovalbumin). Both the plates and the test sera diluted 1:100 in blocking buffer were incubated at 37 0 C for 1 hour.

3 Washes with washing buffer followed by 100 pl of serum being pipetted per well (diluted 1:100 to 1:51200 in 2-fold series) in the plate for duplicate determinations, and the plates were subsequently incubated at +37 0 C in a humidity chamber for 2 hours. The plates were thoroughly washed and then charged with 50 pl of POD-coupled rabbit anti-bovine conjugate per well (diluted 1:2000 in PBS 0.05% Tween 20). The plates were incubated again at +37 0

C

for 2 hours, washed, charged with 100 pl substrate solution (11 mg of ABTS dissolved in 2.1 g citric acid to 100 ml of distilled water, pH 4.2) and incubated in the dark for 60'. The optical density was measured at 405 nm. The serum dilution which led in the ELISA to an extinction of 0.1 was defined as antibody titre. Sera with a titra 1:1000 were assessed as BHV-1 positive.

The following controls were made up for each ELISA: Control Antigen 1st Ab 2nd Ab Substrate Blank no no no yes Antigen control yes no yes yes SAntibody control no yes yes yes Conjugate control no no yes yes Le A 28 649 .i LeA 28 649 a A* ELISA with synthetic peptide (Pep2) as antigen for bovine sera to quantify antibodies against gD of BHV-1 (Pep E1ISA) The Immulon 96-well plate in the F form, supplied by Dynatech, was employed in the Pep ELISA. Pep2 coupled to BSA (see point 6) 100 ng/wel, in 50 pl of coating buffer, was employed as antigen.

The coated plates were incubated at +37 0 C in a humidity chamber for 16 hours. 3 Washes with washing buffer (PBS 0.05% Tween 20) were followed by all wells being charged with 200 pl of blocking buffer (PBS 0.05%'Tween 20 1% skimmed milk powder, orthe test sera being diluted 1:100 in blocking buffer 1.5M NaCl. Both the blocked plates and the diluted sera were incubated at +370C for 1 hour. Then 200 pl of each of the sera to be tested, diluted 1:100 to 1:3200 in blocking buffer 1.5M NaC1, were pipetted into the plates, and the plates were incubated at +370C for 2 hours. Subsequently the plates were washed 3 x with washing buffer, and 50 pl of rabbit anti-bovine-POD conjugate (diluted 1:2000 in PBS 0.05% Tween 20) were added to each well, incubated at +37 0 C for 2 hours, the plates were again washed 3 x with washing buffer, and 100 ul of substrate solution (11 mg of ABTS dissolved in 98 ml of substrate buffer (2.1 g of citric acid ad 98 ml with distilled water, pH=4.2)) were pipetted into each well. The colour reaction was stopped with 100 pl of Le A 28 649 -4 1% strength SDS solution after incubation at +37 0

C

in the dark for one hour. The measurement was carried out at 405 nm.

The following controls were made up for each ELISA: Control Antigen 1st Ab 2nd Ab Substrate Blank no no no yes Antigen control yes no yes yes Antibody control no yes yes yes Conjugate control no no yes yes Evaluation: Sera for which the extinction of the 1:100 dilution less the extinction of the antibody control was 2 0.100 O.D were assessed as BHV-1 positive.

Sera for which the extinction of the 1:100 dilution less the antibody control was 0.100 Ob, were assessed as BHV-1 negative.

11. Dot-blot Before protein loading, the carrier membrane (Immobilien P, Millipore) waa briefly wetted with *methanol and subsequently washed thoroughly with distilled water and stored in PBS (pH 7.4) until used. 0.5 pg and 1.0 pg of synthetic peptide (Pep3 e.

SLe A 28 649 -6- R .i C 06 .c coupled to BSA), or 1.0 pg of BSA each in 100 pl PBS were loaded on to the membrane filter.

The free binding sites on the Immobilien membrane were subsequently blocked with blocking buffer (PBS 0.5% skimmed milk powder 0.05% Tween) for 2 hours. The membrane was then incubated with the first antibody (diluted 1:50 in PBS 0.05% Tween) for 2 hours, washed 3 x (PBS 0.05% Tween), and incubated with the 2nd antibody (anti-bovine-POD conjugate; Sigma) (diluted 1:2000 in PBS 0.05% Tween) for a further 2 hours and again washed 3 x in I PBS 0.05% Tween. All the incubation steps were carried out at room temperature. After the lastwashing, freshly made up.substrate mixture: 20 mM NaCH 3 COO solution, pH 5.0-5.5 10 mg of 3--amino-9-ethylcarbazole dissolved in 3 ml of DMSO 0.04 ml of a 30% strength H 2 0 2 solution was added and the coloration was evaluated after minutes. Binding of Pep3-specific antibodies to Pep3 was thus signalled by a colour reaction on the membrane.

12. Bovine sera Bovine sera from animals which had been infected 25 with the following BHV-1 strains were available for

O

Tg7 e A 28 649 69 the serological investigations in the Pep ELISA, CV ELISA, SNT and dot-blot: Australial2 N569 Schbnbbken Schleswig-Holstein In addition, field sera from Northern Germany with a known content of BHV-1 specific antibodies, tested in the ELISA, were employed in Pep ELISA, CV ELISA and SNT.

II Results 1. Heterogeneity of size in gIV In the following investigations, the gIV from BHV-1 SB, Ausl2 and N569 strains, prepared as described under point 4, was investigated for its size in gel electrophoresis. The molecular weights of the proteins were deduced from their migration behaviour in the gel.

The gIV in the samples investigated by gel electrophoresis showed distinct differences in size in the Ausl2, SB and N569 strains. The size of gIV was 84KD in the case of Ausl2, 72KD for SB and 68KD for N569.

A heterogeneity of size can in theory be caused by e 67 R eA 28 649 'a 4U4U 7" T$ open reading frames (ORF) of different size in the gIV gene of the various BHV-1 strains. Other possi- -bilities are different co- and post-translational modifications of gIV.

To characterise the ORF of gIV in various BHV-1 strains, the complete sequence of the gIV gene was determined for the Ausl2, N569 and SB strains, compared with one another, and the amino-acid sequence of the protein was deduced therefrom.

Fig. 1 shows for comparison the amino-acid sequence of gIV for the Ausl2 and SB virus strains, Fig. 2 I for the N569 and SB strains.

In the case of Ausl2, the ORF of gIV comprises 1524 bp which code for 507 amino acids, in the case of SB 1254 bp which code for 417 amino acids. A repetetive sequence is responsible for the heterogeneity in size of the ORF in the gIV genes. This repeat sequence 90 bp in size is present as direct repetition in the Ausl2 strain in a copy number of 4, in the SB strain in a copy number of 1. In the SB strain, this repeat sequence is located from bp 1022 to bp 1111 of the ORF and codes for amino acids 306-335 with the following sequence:

P-E-G-D-G-E-S-Q-T-P-E-A-N-G-G-A-E-G-E-P-K-P-G-P-S-

25 P-D-A-D-R.

o e• In the case of Ausl2, this repeat sequence extends

L

.o 9

C*

*9 e Le A 28 649 o

SA

T Z from bp 1318 to bp 1408 of the ORF and codes for amino acids 306-425, a 4-fold copy of the sequence ih the SB strain.

I

The N569 strain has a different amino-acid sequence than the SB and Ausl2 strains in the region of the repeat sequence, which is present only in one copy in it, exactly as in SB.

In this region there are only 19% homology between the two strains, SB and N569, investigated, compared O 10 with 81.5% homology based on the complete gIV protein in these two BHV-1 strains. The sequence in the case of N569 in the region of the repeat reads:

E-G-P-A-A-A-G-P-D-G-P-P-P-G-E-P-R-P-G-P-G-G-P-G-A-

D-V-D-R.

Since the gIV both in SB and in N569 comprises 417 amino acids it is possible that the heterogeneity in size observed in samples, investigated by electrophoresis, in gIV of these two strains is not based on ORFs of different size. On the contrary, in the N569 strain there is substitution of the amino acid 309 in position 44 by the amino acid isoleucine. The loss of a glycosilation site which is associated with this is presumably the reason for the heterogeneity in size, observed in the gel electrophoresis, of gIV in SB and N569.

.i A 28 649 7 A" Lu 6 2. Computer analysis The repeat region was identified as potential antigen (hydrophilic region) by means of the Chou-Fosman prediction for the gIV protein of the SB strain.

3. Antigenicity in cattle Bovine sera taken before and after infection with the BHV-1 Ausl2, SB, SH and N569 strains, or field sera from cattle with known BHV-1 antibody content, 10 were investigated in the Pep ELISA for Pep2-specific antibodies, in the CV ELISA and SNT for BHV-1-.specific antibodies and in the dot-blot for Pep3specific antibodies.

3.1 Australia 12 sera Tab. 1 shows for comparison the results of 18 bovine sera from 7 animals before and after infection with the BHV-1 Ausl2 strain. For the characterisation, the sera were investigated in the Pep ELISA, CV ELISA, SNT and dot-blot for Pep- and BHV-1-specific antibodies. No antibodies directed against BHV-1 virus particles, Pep2 and Pep3 were detectable in any of the bovine sera before loading infection in the SNT, CV ElISA, Pep ELISA and dot-blot.

Se..

It was possible with the SNT to detect in all sera Le A 28 649 73 I W after infection, except in the serum of OM 25123 after first infection and in the serum of OM 25126 after booster infection, virus-neutralising BHV-1 antibodies. In the case of OM 25123, neutralising BHV-1 antibodies were detectable only after booster infection.

It was likewise possible with the CV ELISA to detect BHV-1-specific antibodies in the same sera after infection in which virus-neutralising antibodies were detectable, with the exception of the serum of OM 25124 after first infection.

Antibodies directed against Pep2 were likewise detectable in the ELISA for sera which showed serumneutralising antibodies. An exception was the serum of OM 87 after infection, which indeed had virusneutralising BHV-1 antibodies in the SNT but had no Pep2 antibodies in the ELISA.

No BHV-1 and Pep2-specific antibodies were detectable in the serum of the bovine OM 25123 after the first infection and in the serum of the bovine OM 25126 after reinfection, both in the serum O neutralisation test and in the Pep ELISA.

Antibodies directed against Pep3 were detectable with the aid of the dot-blot in sera which were 25 taken after the first infection with BHV-1 and which had virus-neutralising antibodies. In addition, it *0* o as Le A 28 649 7- A I was possible to detect with the dot-blot antibodies directed against Pep3 in the serum of OM 25123 which was taken after infection (on 21.09.90) and which contained no serum-neutralising and ELISA antibodies.

3.2 N569 sera Tab.2 shows, in analogy to Tab.l, for comparison the results of bovine sera before and after infection with the BHV-1 N569 strain. For the characterisation, the sera were investigated for Pep2- and BHVvirus particle specific antibodies in the Pep ELISA, CV ELISA and SNT.

Before infection with the BHV-1 N569 strain, no antibodies directed against BHV-1 or Pep2 were detectable in any of the bovine sera both in the SNT, CV ELISA and in the Pep ELISA. Following the infection, BHV-1-specific antibodies were detectable in both sera in the SNT and CV ELISA. Antibodies directed against Pep2 were, however, not to be found in the Pep ELISA even after loading infection.

Thus, although the cattle infected with N569 pro- O duced BHV-1-specific antibodies which react in the CV ELISA and SNT, did not recognise as antigen in the Pep ELISA after infection the Pep2 which was 25 detected in the gIV of the SB and Ausl2 strains but not identified in the case of N569.

72 Le A 28 649 3.3 Schleswig-Holstein and Sch6nb6ken sera STab. 3 shows for comparison the results of bovine sera before and after infection with the BHV-1 SB and SH strains. These sera were also investigated for Pep- and BHV-1-specific antibodies in the Pep ELISA, CV ELISA, SNT and dot-blot for the characterisation. Antibodies directed against BHV-1, Pep2 and Pep3 were detectable in no tested serum before infection with the BHV-1 SH and SB strains, and in all bovine sera after infection, in the SNT, CV O ELISA and Pep ELISA.

3.4 Field sera Tab. 4 shows the results of the investigations of bovine sera from controlled stocks whose BHV-1 antibody status had been tested in the ELISA. The sera were investigated for BHV-1 and Pep2-specific antibodies in the Pep2 ELISA and CV ELISA.

BHV-1-specific antibodies were detectable in 29 of the 31 tested positive sera with the CV ELISA.

Antibodies directed against Pep2 were found in all 31 positive sera in the Pep ELISA.

No BHV-1- and Pep2-specific antibodies were detectable in the negative sera in the case of the 6 sera tested in the CV ELISA and in the case of 5 of the 25 6 sera tested in the Pep ELISA.

o 73 Le A 28 649

S.

o *5 Le A 28 649 6-- 4. Immunogenicity testing of the N569 strain Tab. 5 shows the results of the investigations of bovine sera in the SNT after immunisation with the N569 strain. The 4 cattle were immunised with N569 strain in different ways.

Serum-neutralising antibodies were detectable in serum from the immunised animals no later than 13 days after immunisation. The highest antibody titre was reached by the bovine immunised intravenously with a titre of 1:152. Antibody levels of 1:45 and 1:27 were reached after intramuscular and intranasal administration of the N569 strain, and the antibody level reached after intravaginal (i.vag.) administration was 1:9.

5. Conclusion It was possible by combined comparison, of the antibodies directed against the modified amino-acid sequence of the N569 strain of antibodies directed against BHV-1 virus particles, to differentiate between cattle free of BHV-1 antibodies, animals immunised with the N569 strain, and cattle infected with field strains.

o* The BHV-1 N569 strain proved to be immunogenic for cattle after i.n. and i.vag.

Lo *o o* 74- •Le A 28 649 -4- X

W

administration.

Describtion of the figures: Fig. 1 shows for comparison the amino-acid sequences of the gIV of the BHV-1 Ausl2 and SchbnbBken strains. The amino-acid sequence and homologies were identified by the PC program PC-Gen. The upper sequence belongs to the Ausl2 strain, and the lower sequence to the Sch6nb6ken strain. The sequences start at the 5' end (N terminus) of gIV and end at the 3' end (C terminus). Lines between the two strands indicate homologies. The arrows drawn in identify the amino acid cysteine.

Fig. 2 shows for comparison the amino-acid sequences of the gIV of the BHV-1 W569 and Sch8nbbken strains. The amino-acid sequence and homologies were identified by the PC program PC-Gen. The upper sequence belongs to the N569 strain, and the lower sequence to the Schbnbbken strain.

The sequences start at the 5' end (N terminus) of gIV and end at the 3' end (C terminus). Lines between the two strands indicate homologies. The sequence of the synthesised Pep3 is underlined. The box in position AA41-43 identifies the absent glycosilation site of the BHV-1 N569 strain.

Tab. 1 shows for comparison the results of bovine sera Sbefore and after infection with the BHV-1 Ausl2 strain.

The sera were investigated for Pep- and BHV-1-specific antibodies in the Pep ELISA, CV ELISA, SNT and dot-blot o Le A 28 649 78 -1

•P~

for the characterisation.

I

S

C*

S.

C*

C C *0

C.

C

*YCC

C C *5 C C. S C S S. S

S*

S.

S C C. C 76 eA28 649

C

jv1~

LU

0

S

C 00 :0.

0. 1 a am

C

S.

Ca a Tab. 1 5 ma a *ma m.

Ca me a maa**. ma 1 rr rr\r 1 CM I Date I Infect.

BSA Pep Pep LU.4U MnJ I cv ELISA I Pep2- "lite Dot- I Ab C jPep Elisa ircipro4v Elisak-cKgal1)1 -blot Virus Ibefore after inf. Ab C

I

86 86 87 87 25123 25123 25123 25124 25124 25124 25125 25125 25125 25126 25126 25126 111.07.90 108.08.90 111.07.90 108.08.90 i U.07.90 108.08.90 131.08.90 121.09.90 130.10.90 131.08.90 121.09.90 I i.v. I Austr.12 I i.v. I Austr.12 I i.vag. I Aumtr.12 I ivag. I Austr.12 ICont. Contri Auatr.12 ICont. ContrJ Autr.12 I i.n. I Austr.12 I i.n. I Ii.n. (Boater) I I Lvag.

I i.vag.

Ni 130.10.90 i.vag. (Bost.) 1 131.08.90 1 i.M. I 121.09.90 1 I 130.10.90 Ii.. (oaoster) I 131.08.90 1 i.v. I 121.09.90 I i.v. I 130.10.90 1i.v. (Booster) I Austr.12 Austr.12 Arwtr.12 Arutr.12 Austr.12 Autr.12 kAutr.12 Austr.12 hAmtr.12 iAutr.12 hAutr.12 1 0,073 I I 0,116 I 1 0,135 I 0,118 I 0,234 I

S-

1 0,156 I I 1 0,168 I I I 0,121 I I 0,960 1,015 0,235 0,810 0,117 0,373 0,558 0,595 0,385 0,09 0,000 0,000 0,129 0,129 0,120 0,120 0,185 0185 0,185 0.088 0,088 0,088 0,094 0,094 0,094 0,099 0,099 0,099 0.073 0,960 I -0.013i 0,886 1 0,015 1 -0,002 1 0,049 1 0,050 1 0,625 0,068 1 0,029 I 0,285 1 0,074 i 0,464 1 0,5011 0,022 0,286 I -0,009' neg.

neg.

negnegneg.

poS-

POO.

neg.

nag.

nag. Poo rM (100 4109 236 1085 350 1486 250 956 4289 284 452 2852 366 1532 1565 2T71 3607 (100 nag.

neg.

POO.

nag.

neg.

nag.

neag.

neg.

IXD.

Deg.

ne.

POO.

neg.

Poo- W.e neg.

neg.

n.d.

neg.

Pas.

neg.

nag.

i.v.

i.vag.

i.n.

i.m.

Ab C intravenous intravaginal intranasal intrantscular antibody control Serum fran 06.11.90 wplqoyad in the Dot-blot because no more serum from 30.10.90 available.

06.11.90 Sm' 1:54 Tab. 2 -shows f or comparison the results of bovine sera before and after infection with the BHV-1 N569 strain.

The sera were investigated for Pep- and BHV-1-specific antibodies in the Pep ELISA, CV ELISA and SNT f or the characterisation.

S.

S

S.

S S

S

'N~

-8 -1- Le A 28 649 a~ a as I I V.

Tab. 2 CM I Date I Infect. I 81 111.07.90 1 ia. 1 81 108.08,90 1 1 89 111.0790 1 i.vaq. I 89 108.03.90 1 i.va. I Virus 1569 4569 N569 N569 lbefore 1 0,186

S-

1 0,11 I Pep ELISA [o.D.405 IIl BSA Pep2 Pep2- Pep afterinf. Ab CI Ab C 1 Eisa CV ELISA .>PTtre Cv 1trcprtxa1 Elisa 1 181 1 neg.

2262 1 ps.

1 738 1 neg.

I 2560 I pas.

SuJr 1 (2 1 27 1 (2 1 152 0,217 0,161 0,174 1 0,012 0,174 1 0,043 0,060 1 0,051 0,070 1 0,091 neg.

meg.

neg.

-J

Tab. 3 shows f or comparison the results of bovine sera before and after infection with the BHV-1 SB and SH strains. The sera were investigated for Pep- and BHV-1specific antibodies in the Pep ELISA, CV ELISA, SNT and dot-blot for the characterisation.

S

S. S S. S

SS

S.

SS

S.

S

S S. S S 55 S 873-- Le A 28 649 5* e :1 C *5 *5*

V

S *5 Tab. 3 C14 I DatelI Infect. I Virus I before PeP ELISA[O.D.405 m]n BSA~ Pep2 Pep2- Pep after inf Ab CI Ab C I Elisa CV ELISA jEite Cv (A~mnrccaj) Elisa iDct- (recipxxm)I blot 2878 2878 2873 2873 82 82 82 25079 25079 25079 25084 25084 27I.01.87 I I27.02.87 I I27.01.87 I I27.02.87 I 111.07.90 I 101A0.90 I 108.08.90 1 111.07.90 1 i29.11.90 I 126.03.91 1 111.07.90 129.11.90 1 126.03.91 1 iLtrad..

i. trach.

i. tradi.

i. traci.

iLtradi.

i.tradi.

iLtradi.

i tradi.

iLtradi.

iLtrach.

iLtradi.

i. tradi.

iLtrach.

0,100 0,167 n.d.

n.d.

0,392 0,963 n.d.

n.d.

0,075 0,070 0,162 0,162 n.d.

n.d.

0,025 0,322 0,00~ n.d.

n.d.

negneg.

Poon.d.

n.d.

n.Z.

492 1543 195 16457 n.d.

n.d.

657 23948 25417 386 22400 24048 negneg.

POS.

n.d.

Meg.

neg- IxB.

(2 (2 56 (2 27 (2 90* n.d.

(2 13* n.d.

negnegneg.

POS.

neg.

Pos.** Serum fran 29.11.90 **Serum fran 26.03.91 Tab. 4 shows the results of Pep2 ELISA, CV ELISA and SNT with field sera of known BHV-1 status. 31 Positive sera and 6 negative sera whose BHV-1 antibodies status had been determined by ELISA by the Oldenburg Animal Health Service were available for the investigations. All the sera were characterised in the Pep ELISA and CV ELISA. 3 Positive sera were tested in the SNT.

a a.

a.

as a.

a a a a a a. a *4 a.4 131 Le A 28 649 Tab. 4 Tab 4 1 1 Pep ELISA 5 ml I CV LISA I S W I I BSA Pep2 Pep2 Pep I CV I Titre CM IVius i O.D. Ab C IAb C IEElisa ji~r,~iElsa I(ripr a1)I

O

r, N*1 POe. FS* 1 0,293 N*2 pos. FS* 1 0,518 N13 POe. I FS* 1 0,330 N*4 Pxs. I FS* 1 0,364 NM6 POe. I FS* 1 0,520 W7 pus. I FS* 1 0,468 ?M poe. I FS* 1 0,537 N*9 po.I FS* 1 0,219 pOS. I FS* 1 0,366 MIlpoe. I FS I 0,272 N1*2 pos. I FS* I 0,251 N1*3 poe. I F*I1 0,522 N114 poe. I F* 1 0,361 poe. I FS* 1 0,395 N1*6 pos. I FS 0,353 NK17 pos. I FS 1 0,351 N1*8 poe. I FS* 1 0,320 NM19 poe. I FS* 1 0,359 POS I FS* 1 0,425 NM21 poO. I FS* 1 0,576 N122 pos. I ES* 1 0,599 W2 pOS. I FS 1 0,474 NK24 poe. I FS* I 0,582 poe. I FS 1 0,348 1*26 poe. I FS* 1 0,264 MV poe. I 1S I 0,325 NM28 posi. I FS* 0,348 29 poe. I 1 0,368 pos. I FS* i 0,21 NM31 poe. I 1S I 0,493 *n neg. I FS* 1 0,092 *2 neg. I FS* 1 0,097 M3 neg. I FS 0,212 N*4 neg. I FS" I 0,204 N*6 neg. I FS 0,096 NM6 neg. I FS* 1 0,030 0,000 I 0,293 0,049 1 0,469 0,000 1 0,330 0,117 1 0,247 0,228 1 0,292 0,152 1 0,316 0,218 1 0,319 0,004 1 0,215 0,215 I 0,151 0,169 1 0,103 0,113 1 0,138 0,141 1 0,381 0,165 1 0,196 0,225 1 0,170 0,115 1 0,238 0,111 i 0,240 0,137 1 0,183 0,172 1 0,187 0,226 1 0,199 0,135 1 0,441 0,151 1 0,448 0,118 1 0,356 0,118 i 0,464 0,092 1 0,256 0,129 1 0,135 0,086 1 0,239 0,154 1 0,194 0,117 1 0,251 0,136 1 0,125 0,152 1 0,341 0,008 1 0,084 0,004 1 0,093 0,007 1 0,205 0,105 1 0,099 0,048 1 0,048 0,043 1 -0,013 poe.

Ipoe.

Ips.

poe.

I pos.

Ipos.

Ipos. POS.

poe.

Ipoe.

poe.

Ipos.

Ips.

Ipoe.

po.

Ipos.

Ipoe.

Ips.

poe.

IO.

IOS

POS.

meg.

IPOS.

meg.

Ineg 1 3769 1 11487 12468 1 3634 1 2349 1 2480 1 1720 1 6583 1 5760 1 1482 15404 12677 1 8873 1 1363 1 1376 1368 1 1511 '947 11060 1142 11264 1 1391 12133 11805 11295 1 1481 1 2355 1 2967 6568 4416 1 310 1390 1281 1 748 1560 1 562 Ipos.

Ipos.

pos.

poe.

I poe.

poe.

Ipos.

poe.

Ips.

Ipos.

beg.

Ips.

meg.

poe.

Ioe Ips.

Ipos.

Ia.

poe.

Ipos.

P;BS

Ipos.

POS.

Ipos.

Poe.

Ineg.

beg.

meg.

Ineg.

n.d.

384 n.d.

n.d.

448 224 n.d.

n.d.

FS field sera with known BHV-1 status, tested in the ELISA.

Le A 28 649 Tab. 5 shows the reciprocal antibody titres of bovine sera in the SNT after immunisation with the N569 strain.

Tab. Mode of immunisation I 1 m i.n. Ii.vag. Ii-v.

Date I OM 81 OM M83 1 OM90 OM 089 11.07 1 <2 1 (2 1 (2 1 <2 1 12.07 1 (2 1 (2 1 (2 1 <2 13.07 1 <2 1 <2 1 <2 1 (2 1 14.07 1 2 1 <2 1 <2 1 <2 :15.07 1 (2 1 <2 1 (2 1 <2 1 *16.07 1 <2 I (2 1 <2 1 (2 17.07 1 <2 1 <2 1 <2 I (2 18.07 1 3 1 <2 1 <2 1 <2 *19.07 1 2 1 <2 1 (2 1 3 20.07 1 2 1 (2 1 <2 1 6 21.07 1 4 1 2 1 <2 1 23 1 22.07 1 3 1 2 1 (2 I 45 1 23.07 1 4 1 6 1 2 1 90 1 24.07 1 5 1 11 1 2 1 128 **:25.07 1 8 1 16 1 3 I 64 27.07 1 3 1 13 1 <2 1 152 30.07 1 9 1 16 1 7 1 152 01.08 1 11 1 11 1 6 1 128 03.08 1 13 1 27 1 6 1 181 06.08 1 27 1 11 1 8 1 107 08.08 1 45 1 27 1 9 1 152 Le A 28 649 8

Claims

1. Vaccines against BHV-1 infections which are based on strains of BHV-1 which, compared with field viruses, contain a DNA sequence which is modified at least in position 310-388 of the region coding for essential glycoprotein IV.

2. Process for the preparation of BHV-1 strains for the preparation of vaccines against BHV-1 infections which are based on strains of BHV-1 characterised in that a-1) The DNA sequence of viral genes for essential glycoprotein IV, at least in position 310-338, is determined for a BHV-1 strain which has been isolated from an infected natural host (for example cattle, pigs, goats) or passaged in cells, and the amino-acid sequence of the encoded protein is derived therefiom or a-2) The amino-acid sequence of viral essential glycoprotein IV, at least in position 310-338, is determined directly and b) The amino-acid sequence determined as in a-1) or a-2) is compared with the amino-acid sequence of the corresponding essential glycoprotein IV of BHV-1 field viruses, or 25 c) Immunological methods using BHV-1 specific antibodies against essential glycoprotein IV of field viruses are used to search for altered amino-acid ":sequences, at least in position 310-338, and the BHV-1 strains which are not recognised by one or more antibodies are selected, and 30 d) The strains which have amino-acid sequences altered by comparison with the field virus and which are obtained according to b) or and the -89-- field virus are tested for antigenicity in a manner know 7er se. In this connection, the amino-acid sequence of the field virus which corresponds to the altered amino-acid sequences of the strains according to b) or c) must be involved in the formation of one or more epitopes which do not occur in the strains obtained according to b) or Cattle not infected with BHV-1 must not contain any antibodies against amino-acid sequences of field viruses, which are altered in the strains obtained according to a), b) or c). e) The strains among those identified according to d) which are immunogenic in cattle are selected. f) As an alternative to a) to it is possible in a manner known per se to alter nucleotide sequences on the genome of BHV-1 strains in such a way that the amino-acid sequence at least in position 310-338 of the region coding for essential glycoprotein IV is altered in such a way that one or more epitopes for cattle are modified or absent by comparison with the field virus. 20

3. DNA sequence which codes for the essential glycoprotein gIV of the BHV-1 o strain N 569 and is characterised in that it is modified in its nucleotide sequence in such a way that the amino-acid sequence of the protein glV is modified in the region of positions 310-338 by comparison with field viruses.

4. Process for the preparation of the DNA sequences according to claim 3, characterised in that the DNA is isolated from the BHV-1 strains prepared according to claim 2 in a manner known per se, or the genome of BHV-1 field viruses undergoes molecular cloning in vectors in a manner known per se, and the cloned BHV-1 DNA fragments are altered in their nucleotide sequence in such a 30 way that, after incorporation thereof into the genome of BHV-1 strains, they result in BHV-1 strains which can be used as vaccines against BHV-1 infections, and this modified BHV-1 DNA is isolated from the vectors in a manner known per se.

Protein gIV of the BHV-1 strain N 569, whose amino-acid sequence is modified in the region of positions 310-338 by comparison with field viruses.

6. Process for the preparation of proteins according to claim 5, characterised in that the protein is isolated from the BHV-1 strains prepared according to claim 2 in a manner known per se, or by cloning and expressing the genes which code for these proteins in pro- or eukaryotic expression systems in a manner known per se.

7. A method for the identification of BHV-1 strains which can be used as vaccines against BHV-1 infection which involves exposure of BHV-1 field strains to antibodies against region 310-338 of glycoprotein IV of BHV-1 field viruses..

8. Antibodies against non-essential regions, which are modified at least in the region of positions 310-338 by comparison with BHV-1 field viruses, of glycoprotein IV of BHV-1 strains which can be used as vaccines against BHV-1 infections. 20

9. Process for the preparation of the antibodies according to claim 8 characterised in that animals (for example rabbits, mice, goats, sheep) are immunised with BHV-1 strains which have been prepared according to claim 2, or with glycoprotein IV from BHV-1 strains which is modified at least in the region of positions 310-338, and the antibodies which are formed are isolated in a manner known per se. *4

10. The method of differentiating cattle which are infected with BHV-1 field viruses from cattle which have been immunised with vaccines based on the BHV-1 strain N 569 which comprises contacting serum from said cattle with peptides which are homologous with amino-acid sequences of non-essential regions in the 30 glycoprotein gIV in BHV-1 field viruses and which are heterologous with the modified amino-acid sequence of position 310-338 in the glycoprotein gIV of the 4- BHV-1 strain N 569.

11. A method of preventing infection by BHV-1 viruses in an animal which consists of treating an animal in need of such treatment with a vaccine according to claim 1, such that said animal can then be differentiated from non-immunised, field- infected animals.

13. DNA sequences which code for glycoprotein IV of BHV-1 strains and which have been altered so that said proteins have their amino acid sequence altered in at least position 310-338, said DNA sequences substantially as herein described with reference to the examples and/or Figures.

14. Glycoprotein IV of BHV-1 strains which is modified at least in position 310-338 by comparison with field viruses, or the fragments and/or peptides thereof, substantially as herein described with reference to the examples and/or Figures. 00 0. 20

15. Antibodies against region 310-338 of glycoprotein IV of BHV-1, substantially as herein described with reference to the examples and/or Figures. S DATED this 21st day of February, 1995. BAYER AKTIENGESELLSCHAFT By Its Patent Attorneys DAVIES COLLISON CAVE ABSTRACT The present invention relates to vaccines based on type 1 bovine herpesviruses (BHV-1) which contain modifications in regions of their genome which code for non-essential parts of essential proteins. It is possible with the aid of these vaccines to differentiate immunised from non-immunised cattle. The invention furthermore relates to processes for the isolation and preparation of the modified BHV-1 strains, isolation and preparation of the modified proteins and peptides. .o 4094U/PT