CA2085191C - Vaccines based on modified type 1 bovine herpesviruses - Google Patents

Abstract

The present invention relates to vaccines based on type 1 bovine herpesviruses (BHV-1) which contain modifi-cations 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.

Description

The present invention relates to vaccines based on type 1 bovine herpesviruses (BHV-1) which contain modifi-cations 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.
Infection with type 1 bovine herpesvirus (BHV-1) in cattle becomes manifest mainly in the organs of the-respiratory tract and of the genital tract, hut also in the peripheral and central nervous system. The clinical pictures induced thereby are described as infectious bovine rhinotracheitis, infectioua bovine vulvovaginitis and infectious pustulous balanophosthitis. Besides these, conjunctivitis, orchitis, endometritis, mastitis, abor-tions 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 tissue, the lympihocytic tissue, macrophages and lympho-cytes are suggested as sites of virus persistence. As a consequence of reactivations of these latent infections, 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 29 649 - 1 -zo~~a.9~
are strains on the organism with latent infection, such as stress, immunosuppression or secondary infection.
Because of the serious economic damage (such as, for example, rearing losses, decrease in milk yield, reduc-tion 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 inter-nationally in cattle suspected of BHV-1 is allowed.
Available for the prophylactic control of disorders associated with BHV-1 are live vaccines based on attenu-ated BHV-1 strains, inactivated vaccines and so-called subunit vaccines based on purified virus proteins. In the control measures currently in progress for the eradi cation of BHV-1 or for freeing herds infected with BHV-1 of the disease, arid 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-1-infected cattle, which could make measures for eradi-cation and fre:sing of disease considerably more effective, is not at present passible.
This type of differentiation of immunised from infected livestock is possible in other areas. Vaccines for pigs Le A 28 649 _ 2 -for the prophylaxis of the clinical mazaifestation 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 Hublished Specification 316 658 is said to permit differentiation of immunised and infected cattle. A HHV-1 mutant prepared by recom binant technology, which does not, because of a mutation (deletion or insertion) in the gene for the non-essential structural glycoprotein gIII, 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, far example, of glyco-proteins, necessarily determines the absence of biologi-cal functions of these viruses. Thus, the non-essential structural protein gIII of Aujeszky virus is signifi-cantly involved in the immune response to Aujeszky virus.
Specifically cellular defence mechanisms, which have a Le A 28 649 - 3 -predominant role in the immunological r_iefence against herpesviruses, are directed against the protein gIII of Aujeszky virus (Zuckermann et al., 199C), J. Virol. 64, 802-812 ) .
Applied to the BHV-1 system, this means that the absence of the protein gIII, comparable in function, in BHV-1 should bring about a reduction. in th.e immunogenicity.
In order to prepare BHV-1 vaccines with high immunogenicity, the BHV-1 mutants which are employed a~> immunising viruses must therefore have deletions or other- alterations in proteins or protein sections which are not the aim of relevant defence mechani.sm:~ .
According to one aspect of the present invention, there is provided a use of a strain of BHV-1 for the preparation of a vaccine against one or more BHV-1 infections which permit dz.f=ferentiation of vaccinated livestock from field-infected livestock, said strain of BHV-1 containing one or more modified DNA sequences) encoding one or more non-essential regions of glycoprotein gIV of BHV
strain Reg. No. I 1204, said one or more non-essential regions relating to epitopes which correspond to amino acid positions 310 to 338 in said glycoprote:in gIV of said BHV
strain Reg. No. I 1204.
According to another aspect c~f the present invention, there is provided the use as described above, wherein strain Reg. No. I 1204 is emplc.~yed as the BHV-1 strain.
According to still another aspect of the present invention, there is provided a use of one or more peptides which are homologous to one or more amino acid sequences _ 4 _ of one or more non-essential regions of= glycoprotein BHV-1 field viruses which are het:erologous too the modified amino acid sequence positions 310 to 338 in :e=_rological methods for differentiating cattle which are infected with BHV-1 field viruses from cattle which have been immunized with vaccines based upon BHV--1 strain Reg. I: 1204.
The present invention now relates to:
1. Use of strains of BHV-1 which, compared with field viruses, contain, because of a DT~dA sequence which is modified at one or more points, one or more modified amino-acid sequences in non-essential regions of essential proteins for the preparation of vaccines against BHV-1 infections.

2. Use of BHV-1 strains acc°ording to (1), in which the modifications affect the essential glycoprotein gIV.

3. Use of BHV-1 strains according to (1), in which the modifications affect the region of glycoprotein gIV which projects directly out of the merr~bran.e coat of the virus.
4a -~0~~.~~1 4. Use according to (1)-(3), wherein the subtype 3 (BI3V-1.3) is employed as H1HV-1 strain.

5. Use according to (1)-(4), wherein the strain N 569 is employed as HHV-1 strain.

6. Use according to (5), wherein the amino-acid sequence of the glycoprotein gIV in the strain N 569 has been modified compared with a field virus in the region of positions 310 to 338.

7. Use of BHV-1 strains according to (1), in which the amino-acid sequences in the glycoprotein gIV, which--correspond in the region to the positions 310 to 338 of the glycoprotein gIV of the strain N 569, are modified.

8. Process for the preparation of BBV-1 strains for use according to (1)-(7) characterised in that a-1) the DNA sequence of viral genes for essential proteins 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 a-2) the amino-acid sequence of viral essential proteins is determined directly and Le A 28 649 - 5 -z~~~.~9~.
b) the amino-acid sequence determined as in a-1) or a-2) is compared with the amino-acid sequence of the corresponding essential pro-teins of BHV-1 field viruses, or c) immunological methods using BHV-1 specific antibodies against essential proteins of field viruses are used to search for altered amino acid sequences, and the BHZi-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 axe obtained according to a), b) or c), 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 a), b) or c) must be involved in the formation of one or more epitopes which do not occur in the strains obtained according to a), b) or c). 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).
a ) The strains among those identified according to Le A 28 649 -d) which are immunogenic in cattle are selected.
f) As an alternative to a) to c), 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 sequences in non-essential parts of essential proteins are altered in such a way that one or more epitopes for cattle are modified or absent by comparison with the field virus.

9. Use of DNA sequences of BFiV-1 strains which code for essential proteins with non-essential regions forty the identification of BHV-1 strains which can be used according to (1), and for the targeted alte-ration of DNA sequences, which encode non-essential regions of essential proteins, of B8V-1 strains for use according to (1).

10. DNA sequences which code for essential proteins of BHV-1 strains and are altered in their nucleotide sequence in such a way that they code for proteins whose amino-acid sequence is modified in non-essential regions by comparison with field viruses.

11. DNA sequences which code for the protein gIV of BHV-1 strains and are altered in their nucleotide sequence in such a way that the amino-acid sequence of glycoprotein gIV is modified in non-essential Ze A 28 6~9 - °7 -~~8~.~~~
regions by comparison with field viruses.

12. DNA sequence which codes for the essential glyco-protein gIV of the BAV-1 strain N 569 and is charac-terised in that it is modified in its nucleotide sequence in such a way that the amino-acid sequence of the protein gIV is modified in the region of positions 310-338 by comparison with field viruses.

13. Process for the preparation of the DNA sequences according to 11-12, characterised in that the DNA is isolated from the BHV-1 strains prepared according to ( 8 ) 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 according to (1), and this modified BHV-1 DNA is isolated from the vectors in a manner known per se.

14. Use of essential proteins of BHV-1 strains with modifications in non-essential regions for the identification of BHV-1 strains which can be used according to (1), and for the preparation of vaccines, and for the preparation of BHV-1 strains with modified amino-acid sequences.

15. Essential proteins of BHV-1 strains Which are Le A 28 649 - g -modified in their amino-acid sequence in non-essential regions by comparison with field viruses.

16. Protein gIV of B~iV-1 strains, whose amino-acid sequence is modified in non-essential regions by comparison with field viruses.

17. 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.

18. Process for the preparation of proteins according to 15-17, characterised in that the protein is isolated from the BHV-1 strains prepared according to (8) 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.

19. Use of antibodies against non-essential regions of essential proteins of BBV-1 field viruses for the identification of BFIV-1 strains which can be used according to (1).

20. Use according to (19) of antibodies against non-essential r~sgions of the protein gIV of BFiV-1 field viruses.

21. Antibodies against non-essential regions, which are modifiod by comparison with BBV-1 field viruses, of Le A 28 649 - 9 -essential proteins of BF3V-1 strains which can be used according to (1).

22. Process for the preparation of the antibodies according to ( 21 ) characterised in that animals ( for example rabbits, mice, goats, sheep) are immunised with BHV-1 strains which have been prepared accord-ing to (8), or with essential proteins from BHV-1 strains which are modified in non-essential regions, or with peptides whose amino-acid sequences cor-respond to the modified regions non-essential parts of essential proteins, and the antibodies which are formed are isolated in a manner known per se.

23. Use of antibodies according to (21) for the identi fication of BIiV-1 strains which can be used according to (1).

24. Use of peptides which are homologous with amino-acid sequences of non-essential regions in essential proteins o:E BHV-1 field viruses and which are heterologous with the modified amino-acid sequences of the non-essential regions in essential proteins of the BHV-1 strains used according to (1), in serological methods for differentiating cattle which are infected with BHV-1 field viruses from cattle which have keen immunised with vaccines according to (1).

25. Use of peptides which are homologous with amino-acid he A 2B 649 - 10 ~~5~.9.~
sequences of non-essential regions in the glyco-protein gIV in BFiV-1 field viruses and which are heterologous with the modified amino-acid sequences of the non-essential regions in essential proteins of the BHV-1 strains used according to (1), in serological methods for differentiating cattle which are infected with BHV-1 field viruses from cattle which have been immunised with vaccines according to ~l).

26. Use of peptides which are homologous with amino-acid sequences of non-essential regions in the glyco-protein gIV in BHV-1 field viruses and which are heterologous with the modified amino-acid sequence-position 310-338 in the glycoprotein gIV of the BF3V-1 strain N 569, in serological methods for differen-tiating cattle which are infected with BHV-I field viruses from cattle which have beer. immunised with vaccines based on the BHV-I strain N 569.
The terms mentioned above have the following meanings BHV means bovine herpesvirus means bovine herpesvirus of serotype 1 according to the terminology in "Virus Diseases in Laboratory and Captive Animals°' F.J. Conraths, H. Ludwig, G. Darai 1988 published by Martinus Nijhoff, Boston.
Le A 28 649 - 11 -~~Pa~.9~.
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 (1986187, Virus Research 6, 57ff) described these isolates, whose DNA restriction enzyme fragment pattern was uniform and distinguishable by com-parison 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 Hungaricae ~9, 145ff) showed, according to investigations by Magyar and coworkers (1989, Acts Veterinaria 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).
The repreae~ntatives 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, Le A 28 649 - 12 -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 SchBnboken 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 essen-tial proteins, which is identical in the overwhelm-ing majority of all variants which occur under natural conditions and is thus to be designated as characteristic of BFiV-1 field viruses. The formation of antibodies in cattle against these (this) epitope(s) therefore suggests infection with B~iV-1 field viruses.
Essential protein means one or more proteins which are necessary for virus replication of BIiV-1 in cell culture. These proteins arse encoded by the genome of BIiV-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 25. are gB and gD (named in accordance with the termi-nology for ~iSV) (Wyler et al., Infectious Bovine Rhinotracheitis/Vulvovaginitis, in "Developments in Veterinary Virology", Vol. °'Herpesvirus Diseases of Le A 28 6~9 - 13 - .

battle, Horses, and Pigs'°, ed. by Wittmann, ICluwer Academic Publishers, 1969).
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 epitapes which lead in BHV-1-infected cattle to the formation of specific antibodies.
Modified amino-acid sequence means that the amino-acid seguence 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) regions) 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 virus in inactivatedl form or parts thereof thus lack specific antibodies against at least one epitope in the field virus, which are formed by them after infection with a field virus.
Lc A 28 649 - 14 -20~~.~91 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 alte-ration 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 HBV-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 these Le A 28 649 - 15 -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 glycosyl.itic 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; F'luwer Academic Publishers,~-1989). The nucleotide sequence of the gene for gIV
and its location on the genome are described for the Sch~nb8ken and Australia BHV-1 strains (Beninga, diploma thesis in the faculty of biology of the University of Tiibingen, 1989). The mature glyco-protein gIV which is incorporated into the extra-cellular virus particle has, in the typical repre-sentatives of BHV types 1.1 and 1.2, a relative molecular weight, determined by SDS polyacrylamide gel electrophoresis, of about 7lRda (Wyler et al . , InfectiouslBovine Rhinotracheitis/Vulvovaginitis, in "Developments in Veterinary Virology", Vol. "Herpes-virus 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.
Le A 28 649 - 16 -~~~~1~1 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 Tiibingen, 1989) was able to determine, by sequencing the gene for gIV of the BHV-1 Schonboken 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 investi-gations, 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 hydro-philic 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 50 amino acids in the case of the BHV-1 Sch~nb~ken strain projects directly out of the membrane coat of the virus and carries the possible modifications in the amino-acid sequence according to 3 ( above ) . This is followed directly, in the direction of the carboxy tea.~minus, by a very highly hydrophobic region which is designated as membrane domain and anchors the glycoprotein in the membrane.

Le A 28 649 - 17 -2~~5~.~~
Natural host means any animal which can be infected with BBV-1 and replicates this virus. Natural hosts can be cattle, pigs and goats (Rolls and Mayr, Mikro-biologie, Infektions- and Seuchenlehre [Micro-biology, infection and epidemiology textbook]
F. Enke Verlag Stuttgart).
BHV-1 strain passaged in cells means that a BF~V-1 strain is replicated in eukaryotic tissue culture cells. This entails the harvest from a replication on tissue culture cells or a BHV-1 isolate from an animal, possibly after storage, being inoculated onto tissue culture cells--and 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. Tf 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, fox example, it was possible to alter the BEV-1 Schonb8ken strain, which was origi-nally pathogenic for cattle, to apathogenicity by about 200 cell passages.
Le A 28 649 - 18 -20~~19~
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 B~dV-1, which may result in attenuation of an originally pathogenic BHV-I strain.
Determination of the DNA sequence means that the virus strain to be investigated is replicated, the virus genome is isolated and at least parts thereof undergo molecular cloning in-vectora. The clones with viral genes for essential I5 proteins can be identified by hybridisation using DNA probes. Examples of DNA probes which can be employed are DNA fragments or synthetic oligonucleo-tides with nucleotide sequences of genes for essential proteins of BHV-1 or of other herpes-viruses. Nucleotide sequences of BI~V-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 Benin~ga (diploma thesis in the faculty of biology of the University of Tiibingen, 1989) and Chase and coworkers (Journal of Tissue Culture Methods, ~1, 75ff, I988, and J.Gen.Virol. 70, 1561ff, 1989). The identified DNA clones which carry DNA sequences of the virus genome for non-essential Le A 28 649 - I9 -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 monoclonal antibodies against the essential virus proteins such as, for example, gB and gD (Chase and coworkers, Journal of Tissue Culture Methods, 11, 75ff, 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 than amino-acid sequence means that the amino-acid sequence of essential proteins of the investigated BHV-1 strain is com pared with the amino-acid sequence of the corres ponding essential proteins of ane or more BHV-1 field vixuses. To do this, the amino-acid sequences Le A 28 6~9 - 20 -~08.~.~~~.
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.
Tn 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 BIiV-1 field viruses which do not occur in the essential proteins of the investigated BBV-1 strain are sought. Regions of at least 5 consecutive amino acids in the amino-acid 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 BFIV-1 strain indicate, for example, possible modifi-cations of epitopes in the investigated BBV-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 the form o:E hybridoma culture media or ascites from mice treated with hybridoma cells) against indivi-dual epitopes of essential proteins of BHV-1 field viruses. B1HV-1 strains which have a modified amino-acid sequence in the corresponding regions of their essential proteins, which these antibodies recognise ~e A 28 649 - 21 -~0~19~
in BFIV-I 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 HIiV-1 are immunised with parts of essential proteins of HHV-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 os 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 HHV-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 HFfV-1 field viruses as antigen to adsorb corresponding antibodies. The adsorbed antibodies are then eluted and collected (immune-affinity chromatography).
Monoclonal antibodies from, for example, mice which have been immunised with intact BI~V-1 field viruses Le ~1 28 649 _ 22 -2~~~19~.
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;
ZO 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-l field viruses. These hybridoma cultures can be selected, for example, by-testing 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, eaerum fractions or monoclonal antibodies 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 Le A 28 649 - 23 -~~~~1.91 proteins by comparison with field viruses, are sought. The choice of the suitable techra.ique 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 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).
2o Another example is the enzyme-linked immunosorbent assay which is known per se and which likewise visualises i:he binding of antibodies to antigens via an enzyme-coupled colour reaction, and in which BHV-1 is employed as antigen (..Antibodies, a Laboratory r4anual"; E.Harlow and D.Lane; Cold Spring Harbor Laboratory; 1988).
Another example is the immunofiuorescence test known Le A 28 649 - 24 -208~1~~.
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 BEiV-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 iymnunised animals, far example as weekly intervals, and the sera of these animals are inves-tigated fox' the content of antibodies against this peptide.
investigating the sera of animals, preferably of cattle, which have been infected with BIit1-1 field viruses for the content of ant~.bodies against the Le A 28 649 - 25 -amino-acid sequence in essential proteins of BHV-1 field viruses which was unidentifiable in the investigated BHIV-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 BI3V-1 field viruses which was uniden-tifiable in the investigated BHV-1 strain. A tech-nique which may be mentioned are the enzyme-linked immunoadsorbent assay (ELISA) known per se or the 1~ immuno-dot-blot, known per se, with synthetic peptides 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 alJl methods which are suitable for modify 2~ ing the nucleotide sequences which code for essen tial 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 - replacement of one or more nucleotides.
Le A 28 6~g - 26 -~0~~~01 A selection of methods for modifying nucleotide sequences is given in "Molecular Cloning" 2nd edition, 1989, ed. ,~.Sambrook, E.F.Fritsch, T.Maniatis, Cold Spring Barbor 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 l~ sequence and is replaced by another building block at the same site without blacking 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.Ma~yr-Bibrack; G.Wittmann; Gustav Fischer Verlag).
Le A 28 649 - 27 -Molecular cloning of genome (compare point 14 above) means that the genomic DNA
of BgV-1 is isolated and the DNA fragments which contain genes or DNA sequences of genes for essen-tial proteins of HHV-1 are inserted into conven tional DNA vectors (for example bacterial plasmids such as pHR322, pUClB/19 inter alia) . The identi fication 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 proteins from HHV-1 being known (for example gI
described in Chase et al., 1989, J.gen.Virol. 70,-1561-1569) or - via homologies of genes for essential proteins from other herpesviruses such as, for example herpes simplex virus, Aujeszky virus, equine herpesvirue l 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 genomia DNA fragments of H8V-1 to find DNA fragments of the latter with genes for essential proteins.
A selection for methods for the preparation and cloning of DNA fragments is given by "Molecular Cloning" 2nd edition, 1989, ed. J.Sambrook, E.F.Fritsch, T.Maniatis, Cold Spring Harbor Ire A 28 649 - 28 -~o~~~~~
Laboratory Press. These vectors with the HHV-1 DNA
fragments as inserts are used, for example, for preparing identical copies of the originally iso lated DNA fragments with genes or DNA sequences of genes for essential proteins of BBV-1.
Vectors which can be employed fox the cloning of DNA frag-ments of the gename of BHV-1 and/or, where appropri-ate, for the specific alteration of DNA sequences in genes for essential proteins of BgV-1 and which may be mentioned are all conventional bacterial or eukaryotic plasmid vectors (such as, for example, pBR322, puClB/19 inter alia), bacterial phages (such as, fox example, lambda, M13 inter alia). possibili-ties 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.Sambraok, L.F.Fritsch, T.Maniatis, Cold Spring Harbor Laboratory Press.
Use of essential proteins of BBV-1 strains with modifications in non-essential regions fox the identification of HFiV-1 strains means that a BHV-1 strain which has modified amino acid sequences in essential proteins and whose ability far use according to (1) has been demonstrated is used to identify other BF~V-1 strains utilisable according to (1).
Le A 28 649 - 2g -a. The identification of modified amino-acid sequen-ces also locates non-essential regions in essential proteins of BH'7-1 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 delimi tation of the regions to be investigated in their essential proteins is possible.
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 BHiI-1 vaccines. It is necessary for these proteins to be prepared in pure form for this purpose. This can take place, for example, by known biochemical or Le A 28 649 - 30 immunological purification methods from virus-containing material or by expression of genes which code for these proteins in bacterial or eukaryotic expression systems. After the immuno-genicity of these proteins has been verified they can be employed, where appropriate with the addition of adjuvants, as vaccines for controll-ing HHV-1 infections.
Tsolation 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 relationships or molecular sieve chromatographies based on their molecular weights.
Expression of proteins for preparation according to (16) to (18) in pro- or eukaryotic expression systems indicates the known methods of molecular genetics Le A 28 649 - 31 -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 frag-ments from the genome of BHV-1 which contain genes for the essential proteins must be isolated. These DNA fragments are inserted into the conventional ZO 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 (1) and/or have not been infected with BHV-1 comprise investigations on sera from cattle for their content of antibodies 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 Le A 28 649 ~o~~~o~
according to (1). 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 beers 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-said sequence from BHV-1 field viruses which is modified in the BHV-1 strains according to (1).
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 ( 1 ) , the sera from these animals are investigated for further BHV-1 specific antibodies, for example in an ELTSA with complete BHV-1 particles as anti-gen. 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 -vaccines according to (1).
A~ Identification of BFiV-1 strains with modified amino-acid se~xuences according to 9 (above) Replication of the BHV-1 strain, c~enome 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 (BBV-1.3) are preferred. The BHV~1.3 strain N569 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 derivatives thereof) or the primary bovine kidney cell EBK or monkey kidney cells such as the perms--nent 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 derivatives thereof) or in dog kidney cells such as Le A 28 649 - 34 -20~~1~.~
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 cul-tures. The replication media employed for the cells are all cell culture media known per se, for example described in the product catalogue of Flow Labora-tories GmbH, Fost 1243, 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-ethane-sulphonic 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 2-10% by 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 Le ~ 28 649 - 35 -with a virus suspension. In the virus suspension, the virus i~ diluted in the virus replication medium in such a way that infection takes place with a MCI
(~ 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 at-temperatures between room temperature and 40°C, preferably between 32 and 39°C, particularly prefer-ably at 37°C far 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 ~m and/or centrifugation at up to 10,000 g.
Filtrate or centrifugation supernatant are used for virus concentration and purification. For this, filtrate or supernatant are subjected to high-speed centrifugation until the virus particles have sedimented. It is possible, whets appropriate, to Le A 28 649 - 36 -follow 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 axe preferably employed. Sodium dodecyl sulphate, sodium lauryl sulphate are particularly preferred.
Prateases 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.
Proteases are employed in concentrations of 0.01-10 mg per ml of virus lysate, preferably 0.05-0.5 mg per m1 of virus lysate.
Operations are preferably carried out in aqueous Le A 28 649 -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 pS values at which the DNA is not denatured. pH values of 5-9, particu-larly 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 ethylenediamino-tetraacetic 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, chlorofos~a, isoamyl alcohol ,.....w ... or mixtures thereof. A mixture of phenol and chloroform/isoamyl alcohol is preferably employed initially, with the extraction taking place in one or more stages:
Preferably employed in the last stage of the ~g A 28 649 - 38 -zo~~~o~
extraction is chloroform/isoamyl alcohol.
Alternatively, it is possible to employ first phenol and subsequently chloroformlisoamyl alcohol.
Other methods for the isolation of the virus DNA
are, for exaanple, centrifugation of a virus lysate in a CsCl. density gradient or in gel electrophoresis (Sharp et al. Biochem. 1973 (I2) pages 3055-3063).
The extraction of nucleic acids is described in '°Molecular Cloning'°, A Laboratory Manual, 2nd Edition 1989, ed. ~'. Sambrook, E.F. Fritsch and T. Maniatis, Cold Spring Harbor Laboratory Press.
The DNA extracted in this way is preferably precipi-tated 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, prefer-ably 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 80$
by volume, particularly preferably at about 70$ by volume.
The chlorieie or acetate concentration is between 0.01 or 1 molar, preferably between 0.1 and 0.8 molar, zf LiCl is employed, its concentration is Le A 28 649 - 3g -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 mentianed is tris-(hydroxymethyl)aminomethane in concentrations of 1-100 mM, preferably 10-SO mM. Preferred pFi values are 6-8.5, particularly preferably 7-8.
Examples of further additives which may be mentioned 1S are EDTA (ethylenediaminotetraacetic acid) in concentration of 0.1 to 10 mM, preferably 1 to 10 mM.
Alternatively, the precipitated DNA can also be resolubilised in 0.1 x SSC buffer (Molecular Clon-ing, loc. cit.) or in ammonium carbonate buffer.
The viral DNA purified in this way is treated with restriction enzyme in accordance with the manu facturer's instruction. Suitable restriction enzymes are those which recognise at least one cleavage site specific for them on the virus genome.
Le A 28 649 - 40 -The genome fragments produced in this way are isolated, after the electrophoretic fractionation thereof, fox 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 chromato-graphic methods.
Supports which may be mentioned in the electro-phoretic methods are agarose or polyacrylamide.
Examples of electrophoresis buffers which may be mentioned are ethylenediaminetetraacetic acid, phosphate/buffer (EPP) tris(hydroxymethyl)amino-methane borate-ethylenediaminetetraacetic acid buffer (TBE) which has the following composition:
tris 10-100 mM, preferably 40-90 ml~, particu-larly preferably 80-90 mM, Boric acid 10-100 mM, preferably 40-90 mM, particu larly preferably 80-90 mM, EDTA 1-10 mM, preferably 1-2.5 mM
pH 7-9, preferably 8-8.5 Le A 28 649 - 41 -or tris-(hydroxymethyl)aminomethane acetate-ethylenedi-aminetetraacetic acid buffer (TAE) which has the following composition:
tris 10-100 mM, preferably 30-90 mM, particularly preferably 40 mM, Sodium acetate l-100 mM, preferably 5-50 mM, EDTA 1-lO 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, 15, perbal, 2nd edition published by Wiley-Inter science, 1988 - Molecular Cloning, loc. cit.
Virologische Arbeitsmethoden (Methods in Virology Research), Volume III Gustav Fischer Verlag, 1989.
The procedure for the method is described in "Molecular Cloning" loc. cit., or in Virolog.
Le A 28 649 - 42 -~~8~~9~.
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. Altern-atively by low-melting agarose method (Molecular cloning loc. cit.) or by adsorption of the DNA
fragment on to glass surfaces (Gene-cleans method).
For the insertion of the DNA fragment, double-stranded plasmid or phage vector DNA molecules are treated with restriction enzymes so that ends suitable far the insertion are produced.
Examples of plasmids used are pAT153, pACYC184, pUClB/19, pBR322, pSP64/65.
Used as phage vectors are lambda phage variants such as, fox example, -ZAP, -gtl0/11 or phage M13mp18/19.
The restriction enzymes which can be employed are known per se, for example from Gene volume 92 (1989) Elsevier Science Publishers BV Amsterdam.
The plasmidi 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.
Le A 28 649 - 43 -Ligases are enzymes which are able to loin 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 K 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 seguence of viral genes az~d deduction of the amino-acid sequences resulting from the sequence determined of the correspondinct proteins accordincLto 8 a-1 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.
Le A 28 649 -~~~~19~.
cit.; A Practical Guide to Molecular Cloning, ed. B.
Perbal, A Wiley-Interscience Publication or Viro lagische 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 (Bio-chemistryy, A.L. Lehninger Verlag Chemie 1977).
Direct amino-acid sequencing of viral ,proteins according to 8 a-2 As an alternative to the determination of the amino-acid sequence by D~tA 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, H. Mayr-Bibrack and G. Wittmann, Gustav Fischer Verlag.
Identification of altered amino-acid- seguences in viral proteins according to 8b Altered regions in proteins are identified by comparing the amino-acid sequence of the inveati-gated BHV-1 strain with the corresponding amino-acid sequence of other BHV-1 strains and/or isolates or Le A 28 649 - 45 -field viruses. In this connection, an altered region is regarded as suitable when at least one epitope 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 th~ alteration.
Immunoloaical 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 amino-acid sequencing, it is possible for altered regions in virus proteins to be identified by poly- or~
monoclonal 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 invest-igated 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 investi gated, then the latter has an altered amino-acid sequence in the part of the protein for which the antibody iav specific.
I~e A 2g 649 - 46 -~~~~1~3.
B. Testina~ for antiuenicity according to 8d B. ~ 1. Theoretical evaluation of the antictenicitv of an amino-acid seguence The physicochemical nature of an amino-acid sequence (for example the extent of the hydrophilic or hydro phobic 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 antigenicity in vivo The peptide with the amino-said 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, ovalbumin oz: 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 - 47 -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 ~g to 100 mg, preferably 0.1 ~g 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-assays or immunoblots. Preferably employed are enzyme-linked imunosorbent assays (ELISA) in which the peptide administered, the protein from which the-peptide derives, or intact or defective HHV-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 l (above).
The crucial point is whether the appropriate amino acid sequence, which has bean identified ae modified in the investigated HHV-1 strain, in the field virus is recognised as antigen in cattle.
For this purpose, cattle are infected with a HHV-1 whose amino-acid sequence is not altered and, after Le A 28 649 - 48 -2Q~~1~~
the infection, investigated for the formation of antibodies. Serum samples are taken from the animals at weekly or monthly intervals and tested iaununo-logically. 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 men tioned for the deterzninatian 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 modi-fied amino-acid sequence, and/or receive admini-stration 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 B~iV-1 strain inve:atigated.
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 - 4g -of 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 HHV-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, deter-mined, 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. Testina for immunoge c'ryr accordina to 8e 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 immuno-genicity of this strain.
For this purpose, the investigated BHV-1 strain is Le A 28 649 - 50 -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, intramuscularl y, subcutaneously, intra-tracheally, intranasally, intravaginally, intra-preputially or into the conjunctiva) sac.
The success of immunisation can - be tested serologically via the induction of BI3~l-1-specific antibodies after immunisation, determined via neutralisation test or ELISA, or -- be determined by an experimental infection test with virulent BH'V-1. This entails comparison of the clinical manifestation between immunised and non-immunised cattle after a BHV-1 infec tion produced experimentally.
Suitable strains prevent or reduce distinctly the clinical manifestation of BFiV-1 infection after immunisation of cattle once or twice.
D. Genetic engineering preparation according to 8f of BFiV-1 strains for use according to 1 Le A 28 f49 - 51 -D.1 identification of non essential regions in essential proteins of 8HV-1 Identification of regions which are non-essential fox virus replication in essential proteins can take place by a) identification of modified amino-acid sequences in existent eHV-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 non-essential for virus replication when an alteration in the amino-acid sequence does not prevent replication of the modified virus.
Le A 28 649 - 52 -D.2 ~tection of the antiaenici~, of the identi :ed non-essential r~aions for cattle after in1°ection with BHV-1 a ) Theoretical evaluation of the antiyenicit3r of an amino-acid seouence Theoretical evaluation of the amino-acid sequence identified in D.l takes place as described above b) ~vestiaation of the antigenicityWin vivo After chemical synthesis of the peptide with--the corresponding amino-acid sequence of 8aV-1 which corresponds to that sequence which has been identified as non-essential for virus replication, or after biochemical purification of this peptide from HHV-1, this peptide is tested for antigenicity in the animal, prefer-ably in cattle. The besting 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 8HV-1 field virus:
F'or this purpose, cattle are infected with 8HV-1 and, after the infection, investigated for the Le A 28 ø49 - 53 -zo~~~.~~
formation of antibodies. For this purpose, serum samples are obtained from the animals in wee7cly or monthly intervals and tested immunologically. If, after the infection, the animals form antibodies against the amino-acid sequence, identified above, in BFiV-1 field viruses, then the antigenicity of this sequence is regarded as proven. An immuno-logical detection method which may be mentioned for the determination of antibodies against this amino-acid sequence is an ELISA which has as antigen this amino-acid sequence from B~-1 field viruses as peptide. The pegtide can, for example, be chemically synthesised or biochemically purified from BFiV-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-said sequence identified in BHV-1 as non-essential.
D.3 Preparations of a BBV-1 mutant by alteration of the identified amino-acid seguence by insertion, substitution or deletion The identified non-essential region in BHV-1 can be altered by deletion, insertion or substitution. For this purpoe;e, the genome fragments which have the DNA sequence coding for this non-essential amino-acid sequence undergo molecular cloning.
Le A 28 649 - 54 -2085.~9.~
Subsequently, the DNA sequences which code for the non-essential amino-acid sequences are removed, partly or fox 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. BFiV-1 strains whose amino-acid sequence have been altered in nonessen-tial parts of essential proteins and which are to be..
employed according to (1) 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. 3~he DNA sequence altered in this way by deletion and/or insertion can be recombined, for example by cotransfection, with the genome of HHV-1 in order t:o obtain recombinant HF3V-1 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.
Le A 2g 649 - 55 -D.4. Establishment of the correlation of the occurrence - of antibodies against BHV-1 and against the identi fied amino-acid seauence a) Investication 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 non essential can be employed as BIiV-1 vaccine marker according to the invention, when 1) antibodies against the identified amino-acid 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 on BFiV-1 as antigen, and 2) sera from cattle which contain no detect-able antibodies against BHV-1 also contain no detectable antibodies against the Le A 28 649 - 56 -identified amino-acid sequence.
- Tt is possible to employ as test once again the ELTSA described above and based on a peptide with the identified amino-acid sequence as antigen.
D.5. Testincr for immunogenicitv The testing is carried out, as described above.
I. Material and methods 1. Cell and virus material A perananent bovine kidney cell (MDB~C cell) was used as cell system.
The following BHV-1 strains were employed in the investigations:
BHV-1.1 Schleswig-Holstein (SH) Z5 - BHV-1.2a SchonbSken (SB) - BHV-1.2b Australia 12 (Ausl2) - BHA-1.3 N569 SH, SB and Ausl2 strains were used as examples of BHV-1 field viruses.
Le A 28 649 - 57 -2. Cell growth and virus replication For cell growth, 100 ml of MDBK cells were inocu-lated with 50x10' cells/ml per Roux dish ( 175 cm2) and grown in E-MEM + 0.85 g of bicarbonate/litre (Mayr et al. (1974). Virologische Arbeitsmethoden volume II. Gustav Fischer Verlag, Stuttgart) + 10%
FCS (foetal calf serum).
For virus replication, 3-5 days after sell inocu-lation the culture medium was removed from the confluent cell lawn (about 500x10 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 infec-tion (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 lA~ were-used for determination of the titre of virus suspensions. Serial dilutions in 1og10 steps 10-1 to 10-~ of the virus, prepared in E-MEM + 2.0 g of bicarbonate, were applied to the cell lawns at 200 ~1/well. 8-Fold determination was carried out for the titration: The virus titre obtained was calculated by the method of Spearman and K~Irber Le A 28 649 - 58 -(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 35S-methionine from 6 to 20 h after infection.
The viral gIV was precipitated from infected cells with the aid of a monospecific anti._gIy serum, fractionated in a 10~ SDS polyacrylamide gel via the method of L~mmli and, after fluorography, visualised by autoradiography. In this design of experiment, gIV was already predominantly glycosylated. Only "mature" forms of gIV were precipitated.
5. Purification of viral DNA
Roux dishes with a confluent MDBK cell lawn were inoculated with virus and incubated at t37°C for 3-4 days. At 100 cpF, 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
KC1; 6.5 mM NaiHP04; 0.7 mM CaCl2; 0.5 mM MgClZi 1.5 mM KI~zPiDw) and again spun down ( 10000 xg;
20 minutes). The supernatants were combined and then centrifuged at 100000 xg for 1 h and the virus pellet was resuspended in 100 ~1 overnight. The virus pellet was then resuspended ad 5 ml of PBS
with 5 mM MgCl2, homogenised, subsequently mixed with Le A 28 649 - 5~ ..

20~~~.9.~
DNAseI (final concentration 100 ~g/ml) and incubated at +37°C for 1 h. After the DNAse treatment the _ va.rus particles were centrifuged through a 15%
strength sucrose cushion at 100000 xg for 1 h.
Subsequently the pellet was resuspended in 1.8 ml of 20 mM tris buffer pH 8.0 and, after addition of 200 ~,1 of 20% strength sarcosyl, incubated at +56°C
for 1 h. The viral DNA was centrifuged in a CsCl equilibrium gradient (5.7 mM CsCIZp 10 mM tris pH 7;
100 mM EDTA) in a fixed angle rotor at 300000 xg at 20°C for 48 h, the gradient was fractionated and aliquots were investigated for the DNA content in a 0.6% agarose gel. DNA-containing fractions were combined, again centrifuged in a CsCI equilibrium gradient and dialysed against 20 mM tris buffer pH 8Ø The DNA concentration was determined in a photometer at 260 nm.
6. Cloning arid 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 br~r electrophoresis in a 0.6% agarose gel 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
Le A 28 649 - 6p -~~8~~.~~
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 pUCl2. For this purpose, the DNA of the vector was treated with HindIII, recircular-isation was prevented by eliminating the 5'-phosphate group using alkaline phosphatase, and the linearised DNA of the vector pUCl2 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 pUCl2 and the T4 ligase.
The plasmids were subsequently transvected into B.coli 0600 (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).
Molecular Cloning. Cold Spring Harbor Laboratory Press.).
For sequencing, the bacteria with the recombinant plasmids were grown, and the plasmid DNA was puri-fied (Sambrook et al. (1989). Molecular Cloning.
Le A 28 649 - 61 -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~ agarase 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 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 radio-actively labelled HindIII L fragment of the SH
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 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 pUCl2 using T4 ligase in analogy to the procedure for the SB and Ausl2 strains. For this purpose, tha DNA of the Le A 28 649 - 62 vector was treated with Pstl, recircularisation was prevented by elimination of the 5'-phosphate group using alkaline phosphatase, and the linearised DNA
of the vector pUCl2 was incubated with the isolated 10.1 kbp fragment and T4 ligase.
6.2 Sequencing The sequencing of the Pstl fragments was carried out by a method which is also called "chromosome walk-ing". 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 Pstl 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 i:ragments, and then the abovementioned procedure was repeated. Since the resulting sequences did not overlap in some regions, the frag-ments were additionally specifically truncated with the ExoITIfSI system, ligated and cloned in E.coli C600. Thee~e subclones were sequenced and the sequences yielded the required overlaps with the sequences already present.
Le A 28 549 -zo~~~.~~
7. Determination of serum-neutralising antibodies against BHV-1 The serum neutralisation test for determining neutralising BIiV-1 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 "
The synthetically prepared Pep2 has the amino-acid sequence of the peptide segment AA317 to AA333 of~
gIV of the BHV-1 Schonboken strain:
gly-gly-ala-glu-gly-pro-lys-pro-gly-pro-ser-pro-asp-ala.
8.2 Pep3 Pep3, which was likewise chemically synthesised, has, besides the 15 amino acids of Pep2, another 15 amino acids of the Schonb8ken gIV. Tt comprises the sequence of the peptide segment from AA307 to AA338 in gIV of the BHV-1 SB strain;
asp-gly-glu-ser-gln-thr-pro-glu-ala-asn-gly-gly-ala-glu-gly-glu-pro-lys-pro-gly-pro-ser-pro-asp-ala-asp-arg-pro-glu-gly.
he A 28 649 - 64 -8.3 Coupling of snythetic peptide to BSA
5_ mg of synthetically prepared peptide (Pep2 or Pep3) were dissolved in 1 ml of PBS pH=7.4 and adjusted to pH 7Ø 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 PB5 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 a 1M glycine solution in PBS was added by pipette, stirred for 1 hour, and then the conjugate wasw dialysed against PBS (pH=7.4) for 5 hours, divided into portions and frozen at -20°C.
9. ELISA for BHV-1 antibody determination (CV ELISA) in bovine sera The antigen employed in the complete virus ELTSA was gradient-purified virus particles of the BHV-1 SH
strain. The Tmmulon 96-well plate (F form, Dynateeh) was coated with 50 ng of antigen in 50 ~1 of coating buffer ( 1. 06 g of NazC03, 2. 93 g of NaHC03 ad 1. O1 distilled water, pHm9.6) per well. The plates were then incubated at +37°C in a COZ incubator for 16 hours. 3 Washes with washing buffer (PBS + 0.05$
Tween 20 + 1M NaCI) were followed by charging the plates, apart from the blank, with blocking buffer Le A 28 649 - 65 -{200 ~1 PHS/Tweeri + 1M NaCl + 2% ovalbumin). Hoth the plates and the test sera diluted 1:100 in - blocking buffer were incubated at 37C for 1 hour.

3 Washes With washing buffer followed by 100 ~1 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 +37C in a humidity chamber for 2 hours. The plates were thoroughly washed and then charged with 50 ~l of POD-coupled rabbit anti-bovine conjugate per well (diluted 1:2000 in PBS + 0.05%

Tween 20). The plates were incubated again at +37C

for 2 hours, washed, charged with 100 ~1 substrate solution (11 mg of ARTS dissolved in 2.1 g citric acid to 100 ml of distilled water, p8 ~ 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 titre > 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 Antibody control no yes yes yes Conjugate control no no yes yes 10. ELISA with synthetic peptide (Pep2) as antigen for bovine sera to quantify antibodies against gD of _ HHV-1 (Pep ElISA) The Immulori"' 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/well, in 50 ~1 of coating buffer, was employed as antigen.
The coated plates were incubated at +37C 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 ~1 of blocking buffer (PBS + 0.05% Tween 20 + 1% skimmed milk powder, orw the test sera being diluted 1:100 in blocking buffer + 1.5M NaCl. Both the blocked plates and the diluted sera were incubated at +37C for 1 hour. Then 200 ~1 of each of the sera to be tested, diluted 1:100 to 1:3200 in blocking buffer + 1.5M NaCl, were pipetted into the plates, and the plates were incubated at +37C for 2 hours. Subsequently the plates were washed 3 x with washing buffer, and 50 ~1 of rabbit anti-bovine-POD conjugate (diluted 1:2000 in PBS +

0.05% Tween 20) were added to each well, incubated at +37C for 2 hours, the plates were again Washed 3 x with washing buffer, and 100 ~1 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 ~1 of - 67 _ 1% strength SDS solution after incubation at +37°C
in the dark for one hour. The measurement was w 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 z 0.100 O.D were assessed as HHV-1 positive.
Sera for which the extinction of the 1:100 dilution less the antibody control was < 0.100 O.D were assessed as BeV-1 negative.
11. Dot-blot Before protein loading, the carrier membrane (ImmobilieriMP, Millipore) was briefly wetted with methanol and subsequently washed thoroughly with distilled water and stored in PBS (pH 7.4) until used. 0.5 ~g and 1.0 ug of synthetic peptide (Pep3 208~~.9.~
coupled to BSA), or 1.0 ~cg of BSA each in 100 ~1 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 PgS + 0.05%
Tween) for a further 2 hours and again washed 3 x in PBS + 0.05% Tween. All the incubation steps were carried out at room temperature. After the last--washing, freshly made up substrate mixture:
- 20 mM NaCH3C00 solution, pH 5.0-5.5 - 10 mg of 3-amino-9-ethylcarbazole dissolved in 3 ml of DMSO
- 0.04 ml of a ~0% strength HZOZ solution was added and the coloration was evaluated after 15 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 with the following BHV-1 strains were available for Le A 28 649 - 69 -the serological investigations in the Pep ELISA, CV ELISA, SNT and dot-blot:
- Australial2 - Sch~nbciken - Schleswig-Holstein In addition, field sera frown Northeraa ~eranany 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 frown BHV-1 SB, Ausl2 and N569 strains, prepared as described under point 4, was investigated far its size in gel electrophoresis. The molecular weights of the proteins were deduced frown their migration behaviour in the gel.
The gIV in the samples investigated by gel electro-phoresis showed distinct differences in size in the Ausl2, SB and N569 strains. The size of gIV was 84KD
in the case of Ausl2, 72K1D for SB and 68KD for N569.
A heterogeneity of size can in theory be caused by Le A 28 649 - 70 -2Q~5~9~
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 for the N569 and SB strains.
In the case of Ausl2, the ORF of gIV comprises 1524 by which code for 507 amino acids, in the case of SB 1254 by which code for 417 amino acids. A
repetetive sequence is responsible for the hetero-geneity in size of the ORF in the gIV genes. This repeat sequence 90 by 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 by 1022 to by 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-IC-P-G-P-S-P-D-A-D-R.
In the case of Ausl2, this repeat sequence extends Le A 28 649 - 71 -from by 1318 to by 1408 of the ORF and codes for amino acids 306-425, a 4-fold copy of the sequence " is the SB strain.
The N569 strain has a different amino-acid sequence than the SH 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 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 electro-phoresis, 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 Bite which is associated with this is presumably the reason for the hetero geneity in size, observed in the gel electrophoresis, of gIV in SB and N569.
Ls A 28 649 - 72 -2~~~191 2. Computer analysis The repeat region was identified as potential antigen (hydrophilic region) by means of the Chou-Forman 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 BH'V-1 antibody content, 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 Pep3-specific antibodies.
3.1 Australia 12 sera Tab. 1 shows for comparison the results of 18 bovine sera Pram 7 animals before and after infection with the BBV-1 Ausl2 strain. For the characterisation, the sera were investigated in the Pep ELISA, CV
ELISA, SNT and dot-blot for Pep- and BHV-I-specific antibodies. No antibodies directed against B13V-1 virus particles, Pep2 and Pep3 were detectable in any of the lbovine sera before loading infection in the SNT, CV ElISA, Pep ELISA and dot-blot.
It was possible with the SNT to detect in all sera Le A 28 649 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 BgiV-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 fox sera which showed serum-neutralising antibodies. An exception was the serum of OM 87 after infection, which indeed had virus-neutralising BBV-1 antibodies in the SNT but had no Pep2 antibodies in the ELISA.
No BBV-1 and Pep2-specific antibodies were detect-able in the serum of the bovine OM 25123 after the first infecwtion and in the serum of the bovine OM 25126 after reinfection, both in the serum neutralisation teat and in the Pep ELISA.
Antibodies directed against Pep3 were detectable with the aid of the dot-blot in sera which were taken after the first infection with BBV-1 and which had virus-neutralising antibodies. In addition, it Le A 28 649 ~ 74 -was possible to detect with the dot-blot antibodies directed against Pep3 in the serum of 0M 25123 which was taken after infection (on 21.09.90) and which contained no serum-neutralising and ELISA anti-s bodies.
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 HHV-1 N569 strain. For the characterisa-tion, the sera were investigated for Pep2- and B6IV-virus particle specific antibodies in the Pep ELISA, CV ELISA and SNT. _-Before infection with the BFiV-1 N569 strain, no antibodies directed against B13V-1 or Pep2 were detectable in any of the bovine sera both in the SNT, CV ELISA and in the Peg ELISA. Following the infection, BBV-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-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 detected in the gIV of the SB and Ausl2 strains but not identified in the case of N569.
Lc A 2a 649 ~ - 75 ~o~~~~~
3.3 Schleswig-Holstein and Schonb~ken sera - Tab. 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-I-specific antibodies in the Pep ELISA, CV ELISA, SNT and dot-blot for the character-isation. 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
ELISA and Pep ELISA.
3.4 Field sera Tab. 4 shows the results of the investigations of bovine aera 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- a;nd Pep2-specific antibodies were detect able in the negative sera in the case of the 6 sera tested in the CV ELISA and in the case of 5 of the 6 sera tested in the Pep ELISA.
Le A 28 649 - 7g -~~8~~~1 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 (i.v,), with a titre of 1:152. Antibody levels of 1:45 and 1:27 were reached after intramuscular (i.m.) and intranasal (i.n.) 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.
The BHV-1 N569 strain proved to be immunogenic for cattle after i.v., i.m., i.n. and i.vag.
Le A 28 649 - 77 -administration.
Description of the figures~
Fig. 1 shows for comparison the amino-acid sequences of the gIV of the BHV-1 Ausl2 and SchonbSken 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 Sch~nboken 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 BfiV-1 N569 and Schonboken 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 Schonboken 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 synthe-sired Pep3 is underlined. The box in position AA41-43 identifies the absent glycosilation site of the BHV-1 N569 strain.
Tab. 1 shows fox' comparison the results of bovine sera before and after infection with the BHV-1 Ausl2 strain.
The sera were investigated for Pep- and BI3V-1-specific antibodies in the Pep ELISA, CV ELISA, SNT and dot-blot Le A 28 649 - 7g _ ___ _~._________.___~._~._ c7.'~0 ~~~~~ a ~~
a N N~N~NQ'~N~~N ydlv7 r r~d f°~ ~ ~ 'p~

-., _ _ ..e .- ~.. _ _ e. . . o _ _ ~ _ . _ ~.
~ a _-.. _ _. .~ _ _ .- _ _ _ _ .- _ _ u9 N eCf __ ~°~'~ o~°~o~$~~~~~~~~~~~
cwcQoo~o~oooooocas'o~
U' ~~~~~~~~~a~~~~~~~~~
A~~~oo~~~
o .~ o ~o~ a a ..- ~~':~~'.~'H,NH vN~i~~~..1~'d'~~.N.~'.~~vNH
H Y f.i H Ii 4 t~. 1r y y v ~ ~ d ~ ~ a~d ___ ____u ~_____~______ a , ~ c ~ ~ ,.., o°v ~
~~4 -r~ y .N ~.i ~ .r; .,~ .re ..~ ..r r -.a..w ~ ~ ~,r ~.a ~ II
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b~s~~~ ~ $..~r ~~ w~ $~ ~~ ~ aJ
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Le A 28 649 - 8p Tab. 2 shows for 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 for the characterisation.
Le A 28 649 - 81 -zo~~.~~~
- N --- EN
N
v v~
W -H

N

- --_a E

- ----~r1 _ V N

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1 OOOCi c~ooo ~

o , o c o o, o, sn o~~

i ~~

-'~
.~i ~i.ro ~ s~sa N
Le A 28 649 _ 82 _ Tab. 3 shows for comparison the results of bovine sera before and after infection with the BHV-1 S19 and SH
strains. The sera were investigated for Pep- and HHV-1 specific antibodies in the Pep ELISA, ACV EI,~SA, SNT and S dot-blot for the characterisation.
Le A 28 X49 - 83 -~~~~~9.~
-__ ~ _____.~_______ _-_ ~ __--~.-___~~~__ N Pi N ~ N G N ~ L1 D C B
-. _ _.~. _ _ _ _ _ _ _ _ _ ~0 b 'A P ~~.1 "" ~ ~ ~~ B B G ~
~ °~ 'B 'A R9 'd '~ '~9 °~
C G B C O f, B C.
.._ _ _ __ _ _ _ _ _ _ _ _ _ O
~7 N ~ ~ ~ ~ ro 't7 'A '~ '~ '~ 'G b b d ~ ~ i C Q 04 ~ C a G A C C C 4 _ _____ __..~..a____ ~ O NN
t~ e0 s0 b "D 'v "~ '~ b b 'r! 'O i ~ . ~ s ~~
~~4~~=CCC6~'Cf:
o ~ Q c a a a a c ~i a a w b w b w °a w ~ ~e9 a a ~ c c a c ~ ~
-.- ~~~i3~a3~~~~~~3~~

w .~ .~ orer yr O .-1 e w w a~
,ar v sr a nr r1 ~,.1 r ~.l w1 r1 wi w~l wH
~t r1 ~ni ~.1 O

__ ~

N N

H

__ ~t~N~Vi~ IN
~~
~~
t~s~~f~

*

L~ A 26 649 - ~4 -Tab. 4 shows the results of pep2 ELISA, CV ELISA and SNT
with field sera of known HHV-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.
Le A 28 649 - 85 -20~~~.~.~
Tab. 4 1 Pep I ~ I SNT
I F~,L.ISA ~~ I
Cd.D.405 ~n~

I HvA Pep2 Pep I CV I Titre I Pep2 I

I O.De Ab I I El..~.sa/~1 ;[ . 1~~~1 p.xua C Ab ' ~~

Nu pas.1 0,293 0,000I 1 per.1 37691 pas. ( n.d.
F~* 0,293 1 I

I~t2 ( 0,518 0,049( 1 poe.I 11487I per. 1 n.d.
pos. FS* 0,469 I

Id~f3 1 0,330 0,0(70I I pos.I 24681 p~s I 384 poe. FS* 0,330 I
I

PM4 I 0,364 0,117I ( pos.I 3b34( pas. 8 n.d.
pas. hS* 0,247 I
I

Ar96 I 0,520 0,228I 1 poe.I 2399I pos. I n.d.
pos. FS* 0,292 I
I

N~fl I 0,458 0.1521 I ~. I 2480I pas. 1 n.d.
pas. F'S* 0,316 I

I~ pas.I 0,537 0,218I I pas.I 17~ I pas. 1 n.d.
F'S* 0,319 I
I

td~ I 0.219 0,004I I pas.I 6583I pas. I n.d.
per. E:S* 0,215 1 I

lJ~ilO 1 0,366 0,215I ( pos.i 5760I pas. I n.d.
p08. FS* 0,151 .I
I

I~fill I 0,272 0,169I I pos.I 14821 pos. I n.d.
pos. FS* 0,103 I
I

MI12 I 0,251 0,113I I pos.I 5404i pos. I n.d.
pos. F'S* 0,138 I
I

f~tl3 1 0,522 0,1411 I pos.I 2677I pas. I r~.d.
pos. FS* 0,381 I
I

Mdl4 I 0.361 0,165I 1 per.I 88?3I pos. I n.d.
poa. FS* 0,196 1 I

PM15 I 0,395 0,225I I pos.I 13b3I pos. I 448 pas. F5* 0,170 1 i~116 I 0,353 0,115I I pos.I 1376I Pos. I 224 pos. F5* 0,238 I
I

1017 I 0,351 0,111I I pas.I 3b8 I toeg.I n.d.
pos. F:s* 0,240 .1 I

N418 I 0,3~ 0,1371 1 pays.I 1511I pes. I n.d.
mss. FS* 0,183 I
I

IdRl9 I 0,359 0,172I I p08.I 947 I neg. I n.d.
pas. FS* 0,187 I
I

t4~0 i 0,425 0,226I I pos.I 1060I poe. I n.d.
pos. FS* 0,199 1 I

M121 I 0,576 0.135I 1 poe.I 1842I poe. 1 n.d.
pas. FS* 0,441 I
I

1122 I 0,599 0,1511 I poe.I 1264I pos. I n.d.
pos. FS* 0,448 I
I

1123 I 0,474 0,118I I pae.I 1391I pas. 1 n.d.
pae. FS* 0,356 I

NP129 I 0,582 0,118I 1 pos.I 2133I poa. I n.d.
poa. FS* 0,464 I

I~i25 I 0.348 0,092I I ~. I 1805I pas. 1 n.d.
pas. F8* 0,256 I
I

I~126 I 0,264 0,129I I pos.I 1295I pae. 1 n.d.
pos. FS* 0,135 I
I

N~fL7 1 0,325 0,086I I per.I 14811 pos. I n.d.
pae. F'S* 0,239 I
I

NM28 I 0,348 0,154I 1 pos.I 2355I pos. I n.d.
pas. FS* 0,194 I

N~29 1 0,368 0,1171 I poa.I 2967( pos. ( n.d.
pog. FS* 0,251 I
I

t~C~O 1 0,261 0,1361 I poa.I 6568I pas. I n.d.
pas. FS* 0,125 I
I

Mt31 I 0.493 0,152I I pos.I 4416I pos. I n.d.
pos. FS* 0,341 I
I

Neil I 0,092 0,008I I aeeg.I 310 I r~e9.1 n.d.
neg. FS* 0,084 I
( id~l2 I 0,097 0,0041 1 neg.I 390 I n~eg.I n.d.
neg. F5* 0,093 I
I

N13 I 0,212 0,007I I poe.1 281 I neg. I n.d.
peg. FS* 0,2D5 I
( M!4 I 0,204 0.105I I neg.1 748 I neg. 1 n.d.
neg. FS* 0.099 I
I

DDS I 0,096 0,048I I neg.I 560 I rs~g I n.d.
neg. E'S* 0,048 i I

t~16 I 0,030 0,043I 1 neg.( 562 1 n~. 1 n.d.
nag. FS* -0,013 J
I

* P'S ~ field sera with )gym 81:1<J-1 status, tested iaa the ELISA.
Le A 28 649 _ 86 _ Tab. 5 shows the reciprocal antibody titres of bovine sera in the SNT after immunisation with the N569 strain.
Tab. 5 I Mode of immunisation $

I L a ~ l . n Z a V ~ s V
a I . ( a ~ ~ o I
I

nets I oM e1 oM 83 oM 9o oM e9 __ i $ I I
____ __ _ _ 11.07 j ' _C2____; _______ ~___'2____i ~2 12.07 I <2 I <2 I <2 $ <2 I

13.07 $ <2 I <2 I c2 I e2 I

14.07 I 2 I <2 I <2 1 <2 $

15.07 I <2 I <2 1 <2 I <2 I

16.07 $ <2 I t2 I <2 I <2 $

17.07 I <2 I <2 I <2 I <2 $

18.07 $ 3 I <2 $ e2 $ <2 I

19.07 I 2 I <2 I <2 1 3 $

20.07 I 2 $ <2 $ <2 I 6 i 21.07 I 4 $ 2 I <2 I 23 I

22.07 I 3 $ 2 I <2 I 45 $

23.07 I 4 1 6 I 2 I 90 I

24.07 I 5 I 11 I 2 $ 128 $

25.07 I 8 I 16 I 3 I 64 $

27.07 $ 3 I 13 I <2 $ 152 30.07 $ 9 I 16 I 7 I 152 I

01.08 $ 11 1 11 I 6 I 128 $

03.08 I 13 $ 27 $ 6 1 181 $

06.08 $ 27 $ 11 I 8 I 107 I

08.08 I 45 I 27 I 9 $ 152 I
_________$ _________.I_________I_________I_________I

Le A 28 649 - 87

Claims (3)

1. A use of a strain of BHV-1 for the preparation of a vaccine against one or more BHV-1 infections which permit differentiation of vaccinated livestock from field-infected livestock, said strain of BHV-1 containing one or more modified DNA sequence(s) encoding one or more non-essential regions of glycoprotein gIV of BHV strain Reg. No. I 1204, said one or more non-essential regions relating to epitopes which correspond too amino acid position; 310 to 338 in said glycoprotein gIV of said BHV strain Reg. No. I 1204.

2. The use according to claim 1, wherein strain Reg.
No. I 1204 is employed as the BHV-1 strain.

3. A use of one or more peptides which are homologous to one or more amino acid sequences of one or more non-essential regions of glycoprotein BHV-1 field viruses which are heterologous to the modified amino acid sequence positions 310 to 338 in serological methods for differentiating cattle which are infected with BHV-1 field viruses from cattle which have been immunized with vaccines based upon BHV-1 strain Reg. I 1204.