PT100810A - Deletion mutants of bovine herpes virus type 1, vaccines based on these mutants, and diagnostic kits for the detection of bovine herpes virus type 1 - Google Patents

Abstract

The present invention relates to a deletion mutant of bovine herpes virus type 1 which presents a deletion in the glycoprotein gE gene. The mutant can also present a deletion in the thymidine kinase gene and/or in the glycoprotein gI gene, or present an insertion of a heterologous gene; to a recombinant nucleic acid which includes the gE gene or a part thereof; to the glycoprotein gE, peptides based thereon and complexes formed by the glycoproteins gE and gI, and antibodies against them; to vaccines and diagnostic kits which include any one of these materials.

Description

DETAILED DESCRIPTION OF THE INVENTION PATENT OF INVENTION No. 100,810 APPLICANT: STICHTI-NG CENTRAAL DIERGENNESKUNDIK INSTITUUT Dutch, industrial, established at EdelHertweg 15, 8219 PH Lelystad, The Netherlands EPIGRAF: " DELIVERY MUTANTS OF HERPES BOVINE TYPE 1 VIRUS, BASIC VACCINES THEREOF MUTANTS AND DIAGNOSTIC EQUIPMENT FOR THE DETECTION OF HERPES BOVINE TYPE 1 VIRUS " INVENTORS: FRANCISCUS ANTHONIUS MARIA RIJSEWIJK, JOHANNES THEODORUS VAN OIRSCHOT, residing in the Netherlands and ROGER KAMIEL MAES, residing in the United States of North America

Claim of the right of priority under Article 4 of the Paris Convention of 20 March 1883. MOO 113 R t 1S73Í "HERPES VIRUS DELETATION MUTANTS OF BOVINE TYPE 1, VACCINES BASED ON THESE MUTANTS AND DIAGNOSTIC EQUIPMENT FOR THE DETECTION OF HERPES BOVINE TYPE 1 VIRUS" "

DESCRIPTIVE MEMORY

SUMMARY The present invention relates to a bovine herpes virus type 1 deletion mutant which exhibits a deletion in the gE glycoprotein gene. The mutant may further exhibit a deletion in the thymidine kinase gene and / or the glycoprotein gene. glutamine, or exhibiting an insertion of a heterologous gene; to a recombinant nucleic acid which includes the gE gene or a part thereof; to glycoprotein gE, peptides based thereon and complexes formed by gE and gl glycoproteins, and antibodies against them; to diagnostic vaccines and equipment (" kits ") which include any of these materials. 2

SCOPE OF THE INVENTION

This invention relates to the fields of vaccination and diagnosis for diseases that are caused by pathogens and involves the use of either the classical methods used to arrive at a live attenuated vaccine or an inactivated vaccine such as modern methods based on DNA recombination technology.

More specifically, the invention relates to live attenuated vaccines and inactivated vaccines for the protection of animals, especially cattle, for bovine herpes virus type 1 (bovine herspesvirus type 1 " or BHV-1), both vaccines designed in such a way that they are not only safe and effective but also create the possibility of distinguishing, in a vaccinated population, infected animals from those that are not infected.

&Quot; kits " which can be used in a test of the above type to distinguish, in a vaccinated population, infected animals from those not infected, are also an aspect of the present invention.

BACKGROUND OF THE INVENTION BHV-1, including infectious bovine rhinotracheitis virus (IBCV) and infectious pustular vulvovaginitis virus (IPC), plays a role development of respiratory diseases and fertility-related conditions in cattle. After acute infection, BHV-1 frequently remains in the host in latent form. A virus present in latent form can be reactivated 3

under the influence of, among other factors, stress. which may or may not be accompanied by clinical phenomena - and subsequently excreted. Consequently, infected cattle should be regarded as constituting throughout life a potential source of BHV-1 spread. It is estimated that BHV-1 infection occurs endemic in 75% of Dutch livestock farms. Especially the older cattle are serologically positive.

There are a number of inactivated (" dead ") " vaccines and a number of attenuated (" live ") vaccines for inoculation against BHV-1 infections. Inactivated vaccines are prepared by killing the BHV-1 virus, for example by heat treatment, irradiation or treatment with ethanol or formalin. However, these vaccines often confer insufficient protection. Attenuated vaccines are prepared by a large number of passages in homologous (bovine) cells or in heterologous cells such as pig or dog cells, and sometimes viruses are also treated by physical or chemical methods at that time. In this way unknown mutations / deletions are developed in the genome of the virus which often reduce the disease producing properties of the virus. Live attenuated vaccines confer better protection than inactivated vaccines, among other reasons because they present to the host immune system a greater amount of viral antigens. Another important advantage of live vaccines is that they can be administered intranasally, i.e. at the site where the first wild type virus (" wild type ") occurs after infection. And yet, live vaccines persist in areas where improvements are possible. Some live vaccines appear to continue to have abortogenic properties, which manifest in particular following intramuscular administration. In addition, all live vaccines are likely to remain dormant in vaccinated cows. Further, there is a possibility of a reversal phenomenon and a return to the virulence state in case the vaccine differs only slightly from the wild-type virus. But a major problem is that BHV-1 vaccines do not prevent wild-type virus infections. Which means that vaccinated cattle can also disseminate wild-type BHV-1.

For a suitable BHV-1 control program, it is necessary to have an effective and safe vaccine that is distinguishable from the wild type virus, since the application of an effective vaccine can greatly reduce the circulation of BHV-1 and a test that distinguishes between a vaccine and a wild-type virus makes it possible to detect (and subsequently remove) infected cattle in a vaccinated population.

However, BHV-1 vaccines have been developed which appear to be safer than conventional vaccines and are distinguished from wild type virus. A thymidine kinase deletion mutant was isolated which is less abortogenic, becomes latent less frequently and can not be reactivated. In addition, and using DNA recombination techniques, a BHV-1 vaccine was created which has a deletion in the glycoprotein GIII gene, which allows the distinction of this wild-type BHV-1 vaccine using serological techniques. However, there remain some objections to these vaccines. On the one hand, the thymidine kinase gene is involved in viral replication and a lesser degree of replication may result in a lower degree of protection. On the other hand, gIII glycoprotein is important for the production of protective antibodies, which makes a GIII deletion vaccine less effective. A practical problem is that intranasal administration, which generally provides the best protection, of recombinant vaccines is not permitted in some countries, therefore, there is a need for a vaccine which is both safe and effective and, however, may be distinguished from wild-type BHV-1, it is still desirable that at least one of these vaccines is based on an attenuated virus following a traditional pathway rather than based on a virus constructed by DNA recombination techniques.

Via passages in cell cultures, a BHV-1 strain now lacking the gE glycoprotein gene has now been obtained. The first results of our research indicate that this gene is very useful for establishing a serological distinction regarding wild-type BHV-1 and that it is involved in the expression of virulence. Therefore, its deletion contributes to safety and may render superfluous the use of thymidine kinase deletions. The gly glycoprotein gE appears to be less important in inducing protection than the glycoprotein gI1. A conventionally attenuated strain of BHV-1 that can be serologically distinguished from wild-type virus is unique. The location and DNA sequence of the gE gene described herein for the first time was not previously known, as oligonucleotides, polypeptides and olpeptides that may be derived from it were not known. A test permitting a serological distinction based on the gE gene is also unique.

A major advantage of this conventional " GE deletion mutant " (the term " conventional " refers to the use of a conventional method for isolating an attenuated virus) is that it will be possible to administer it intranasally in countries where this is prohibited as far as recombinant vaccines are concerned. However, in view of the different views on the safety issue, well defined recombinant versions have also been constructed in addition to this conventional GE depletion vaccine. These recombinant vaccines also exhibit a deletion of gE which may or may not also exhibit a deletion in the thymidine kinase gene - and may also be used as vectors for expression of heterologous genes. All of these recombinant vaccines can be distinguished from the wild type virus by the same gE-specific assay. The use of a standardized test for a set of different vaccines can be a major advantage in combating BHV-1 as an international effort. Such an approach to the problem had not previously been described in the field of BHV-1 vaccines. Serological analysis of the anti-BHV 1 reaction in cattle showed that a major fraction of the anti-gE antibodies is directed against a complex formed by the glycoprotein gE and another BHV-1 glycoprotein: the glycoprotein gl. Serological tests which demonstrate (also) the presence of these specific antibodies to the complex may therefore be more sensitive than the tests which can detect only anti-gE antibodies. Cattle vaccinated with a single gE deletion mutant can produce anti-gl antibodies that may interfere with the detection of anti-gl / gE antibodies. Accordingly, this invention also includes a vaccine with a double deletion of gl / gE.

SUMMARY OF THE INVENTION

First, this invention provides a BHV-1 deletion mutant which exhibits a deletion in the gE glycoprotein gene. The " a deletion in " is intended to encompass a deletion of the entire gene.

A preferred embodiment of this invention is a BHV-1 deletion mutant which has a deletion in the gE glycoprotein gene which has been elicited by an attenuation process, such as the Difivac-1 deletion mutant described below.

Further preferred embodiments of the invention consist of a BHV-1 deletion mutant which includes a deletion in the gE glycoprotein gene that has been constructed by means of DNA recombination techniques, such as the deletion mutants 1B7 or 1B8 described below .

Another preferred embodiment of this invention is a BHV-1 double deletion mutant which includes a deletion in the glycoprotein gE gene and a deletion in the gly glycoprotein gene, such as the double deletion mutant of Difivac-IE gly / ggE which is described below.

In addition, in order to obtain maximum safety, a BHV-1 deletion mutant having a deletion in the gE glycoprotein gene and a deletion in the thymidine kinase gene is preferred according to the invention. This invention also encompasses a BHV-1 deletion mutant which has a deletion in the gE glycoprotein gene, the gly glycoprotein gene and the thymidine kinase gene. The invention provides a vaccine for vaccination of animals, in particular mammals, more specifically bovine animals, for the purpose of protecting them from BHV-1, which includes a deletion mutant as defined above and a suitable carrier or adjuvant. This composition can. be a live or inactivated vaccine. The invention has another of its embodiments in a BHV-1 mutant which has a deletion in the gE glycoprotein gene and contains a heterologous gene introduced by means of DNA recombination techniques. This relates preferably to a BHV-1 mutant containing a heterologous gene introduced by means of DNA recombination techniques at the glycoprotein gE gene site, which heterologous gene is under the control of the gE gene regulatory sequences and is optionally linked to that part of the gE gene encoding a signal peptide. Said heterologous gene may also be under the control of a promoter other than BHV-1, or under the control of a heterologous promoter. When the BHV-1 mutant displays deletions in addition to the deletion in the glycoprotein gE gene, such as a deletion in the thymidine kinase gene and / or a deletion in the glycoprotein gl gene, said heterologous gene may also be inserted into the of this additional deletion (s). Multiple insertions are other possibilities, either together at the site of a deletion, or distributed across the sites of several deletions. The introduced heterologous gene preferably encodes an immunogenic protein or peptide or protein of another pathogen, or a cytokine that promotes the immune reaction. Examples of suitable cytokines are interleukin 2, interferon-alpha and interferon-gamma. The invention also provides a vaccine (live or inactivated) for the vaccination of animals, particularly mammals, more specifically bovine animals, in order to protect them from a (different) pathogen, which includes a BHV-1 mutant having a qen heterologous peptide encoding an immunogenic peptide or protein of said other pathogen, and a suitable carrier or adjuvant. Of course, the protection may relate to more than one pathogen, i.e. a multivalent vaccine in which the mutant has a variety of heterologous genes.

The invention further relates to a composition comprising a recombinant nucleic acid which includes the BHV-1 gE glycoprotein gene, a part of this gE glycoprotein gene or a nucleotide sequence derived from that glycoprotein gE gene. This composition may include a cloning or expression vector displaying an insertion of a recombinant nucleic acid which includes the BHV-1 gE glycoprotein gene, a part of this gE glycoprotein gene or a nucleotide sequence derived from that glycoprotein gE gene. %

This invention also includes a composition which includes the BHV-1 glycoprotein gE, a part of this glycoprotein gE, a peptide derived from this glycoprotein gE, or a complex of the gE and gly glycoproteins, and a composition that includes an antibody which is specific to GH glycoprotein of BHV-1, to a part of this glycoprotein gE, to a peptide derived from this glycoprotein gE or to a complex of gE and gl glycoproteins. The " antibody " should be understood as pertaining to both a polyclonal antibody preparation and a preferred monoclonal antibody for most applications. The terms " a part of the glycoprotein gE " and " a peptide derived from the glycoprotein gE " are to be understood as referring to specific amino acid sequences of gE which will generally have a length of at least 8 amino acids.

This invention further relates to a " kit " for detecting BHV-1 nucleic acid in a sample, in particular in a biological sample such as blood or blood serum, cells in the blood, milk, body fluids such as tears, lung lavage fluid, nasal fluid, sperm , in particular semen, saliva, sputum or tissues, in particular tissues of the nervous system, coming from an animal, in particular from a mammal, more specifically from a bovine, which includes a probe or " nucleic acid sequence having a nucleotide sequence derived from the BHV-1 gE glycoprotein gene, and a detection means suitable for a nucleic acid detection assay.

Further, the invention relates to a " kit " in particular in a biological sample such as blood or blood serum, saliva, sputum, body fluids such as tears, lung lavage fluid, nasal fluid, milk, or tissues derived from an animal, in particular from a mammal, more specifically from a bovine animal, which includes BHV-1 gE glycoprotein, a part of this gE glycoprotein, a peptide derived from that gE glycoprotein, or a complex of gly glycoproteins gE and gl , and a detection means suitable for an antibody detection test. One such " kit " may additionally comprise one or more antibodies specific to the BHV-1 gE glycoprotein or specific to a BHV-1 gE and gl glycoprotein complex.

This invention also relates to a " kit " BHV-1 proteins in a sample, in particular in a biological sample such as blood or blood serum, cells in the blood, milk, body fluids such as tears, lung lavage fluid, nasal fluid, sperm, in particular semen, saliva, sputum or tissues, in particular tissues of the nervous system, from an animal, in particular from a mammal, more specifically from a bovine, which includes one or more specific antibodies to the BHV-1 gE glycoprotein or which are specific for the BHV-1 gE and gl glycoprotein complex, and a detection means suitable for a protein detection test. The invention further provides a method of determining BHV-1 infection in an animal, in particular in a mammal, more specifically a bovine, which includes examination of a sample from the animal, in particular a biological sample such as blood or blood serum , cells in the blood, sperm, in particular semen, saliva, sputum, body fluids such as tears, lung lavage fluid, nasal fluid, milk, or tissues, in particular tissues of the nervous system, for the presence of acid nucleotide sequence comprising the BHV-1 gE glycoprotein gene, or the presence of BHV-1 gE glycoprotein or a BHV-1 gE and gl glycoprotein complex, or the presence of antibodies that are specific for the gE glycoprotein of BHV-1 or specific for a complex of the BHV-1 gE and gl glycoproteins. The sample to be examined may come from an animal which has not previously been vaccinated with a vaccine of according to the invention or of an animal which has been previously vaccinated with a vaccine according to the invention.

DETAILED DESCRIPTION OF THE INVENTION The invention relates to a set of both live and inactivated BHV-1 vaccines which have in common the fact that they do not have the gE glycoprotein gene, in whole or in part. This set includes both a natural gE deletion mutant and constructed gE deletion mutants which may or may not also exhibit a deletion of the thymidine kinase gene and / or the gly glycoprotein gene, and constructed gE deletion mutants which are used as vectors for heterologous genes. This invention further relates to nucleotide sequences encoding the BHV-1 gE glycoprotein gene, to oligonucleotides derived from those sequences, to the gE glycoprotein itself, to peptides derived therefrom and to monoclonal and polyclonal antibodies which are directed against gE glycoprotein and peptides thereof. The invention also relates to complexes of BHV-1 gE and gl glycoproteins and to antibodies directed against such complexes.

These materials according to the invention can be used to: 1) vaccinate cattle against diseases caused by BHV-1 such that it is possible to distinguish between BHV-1 infected animals and vaccinated animals; the conventional vaccine and the vaccine vaccine can be used side by side; 2) vaccinate cattle against diseases caused by BHV-1 and diseases caused by other pathogens, and sequences therefrom encoding the protective antigens may be incorporated into BHV-1 deletion mutants; (3) testing blood, blood serum, milk or other body fluids from livestock in order to determine serologically or using nucleic acid detection techniques (eg PCR) if the animals were infected with wild-type BHV-1 or were vaccinated with a gE deletion mutant. Synthesis of oligopeptides, polypeptides and glycoproteins derived from the coding sequence of the gE glycoprotein gene and the BHV-1 gly glycoprotein gene

The results of the UNA sequence analysis described in the examples of the glycoprotein gE gene (Fig. 3A) and the isolated DNA fragments encoding this gene make it possible, using conventional procedures in molecular biology, not only to synthesize peptides from the protein gE (oligo or polypeptides) as expressing the whole gE protein or a considerable portion via the prokaryotic (in bacteria) or eukaryotic (e.g., murine) pathway. By means of these routes it is possible to obtain specific antigen for gE which may, for example, serve to produce monoclonal antibodies (" monoclonal antibodies " or Mabs) specific to gE. In addition, gE-specific antigens (and specific Mabs relative to gE) can be used in serological tests to enable a distinction to be made between animals vaccinated with a BHV-1 gE deletion vaccine and virus-infected animals BHV-1 wild type.

The results of the partial analysis of the DNA sequence of the glycoprotein gl gene - described in the examples - and of the isolated DNA fragments encoding this gene, together with the eukaryotic cells expressing the glycoprotein gE, allow the expression of the gl / gE complex in eukaryotic cells (see Figures 13 and 14). This glycoprotein complex can be used to produce specific monoclonal antibodies relative to gl / gE. The gl / gE complex can also be used as an antigen in serological tests to allow differentiation between cattle vaccinated with a BHV-1 gE single deletion mutant or with a BHV-1 gl / gE double deletion mutant and infected cattle with wild-type BHV-1 virus. 14

GE specific peptides

Based on a sequence encoding a known protein, and by means of an automatic synthesizer, it is possible to prepare polypeptides having not less than 40-50 amino acids. Now that the GH glycoprotein gE protein coding sequence of the BHV-1 Lam strain (Fig. 3A) has been disclosed, it is possible to synthesize polypeptides from this BHV-1 gE glycoprotein. With such polypeptides, and according to conventional methods, it is possible to immunize experimental animals such as mice or rabbits in order to produce antibodies specific for gE. In addition, using these specific gE peptides, it is possible to more fully specify the sites where the anti-gE antibodies react with the gE protein (the epitopes), using, for example, the PEPSCAN method (Geysen et al., 1984, Proc Natl Acad Sci USA 81, 3998-4002). Specific gE oligopeptides may also be used in serological tests demonstrating anti-gE antibodies.

Prokaryotic expression of gE

For the synthesis of the gE protein in bacteria (i.e., for the prokaryotic expression of gE), the DNA fragments encoding the gE glycoprotein or parts thereof must be cloned into prokaryotic expression vectors. Prokaryotic expression vectors are circular DNA molecules that remain inside a bacterium as separate replicating molecules (plasmids). These expression vectors have one or more marker genes (" marker genes ") that encode a resistance to an antibiotic, thereby allowing the selection of the bacteria containing the expression vector. In addition, the expression vectors include a promoter region (often controllable) behind which DNA fragments can be ligated which are then expressed under the promoter irifluene. In many standard prokaryotic expression vectors, the desired protein is expressed fused to a protein termed carrier protein (" carrier protein "). To this end, the sequence encoding the carrier protein is located in the vector behind the promoter, the DNA fragment may be attached immediately adjacent thereto. The resulting fusion proteins are often more stable and easier to recognize and / or isolate. The state of equilibrium that a specific fusion protein can attain in a particular bacterial strain differs from fusion to fusion and from strain to strain. It is common to experience various combinations.

Eukaryotic expression of gE glycoprotein gene

Although prokaryotic expression of proteins has certain advantages, proteins do not exhibit the modifications, such as glycosylation and the like, which occur in eukaryotic cells. Accordingly, eukaryotic-expressed proteins often constitute more suitable antigens. For heterologous expression of proteins in eukaryotic cells, such as murine cells, eukaryotic expression vectors are used. These vectors are plasmids that not only can multiply not only in E. coli bacteria but also subsist in a stable way in eukaryotic cells. In addition to a prokaryotic selection marker, they also have a eukaryotic selection marker. As with prokaryotic expression vectors, eukaryotic expression vectors have a promoter region behind which the desired genes can be ligated. In all cases, the promoter sequences in the eukaryotic vectors are specific for eukaryotic cells. In addition, fusion with carrier proteins is only rarely used in eukaryotic vectors. These vectors are introduced into the eukaryotic cells by a standard transfection method (F.L. Graham and A.J. van der Eb, 1973, Virology 52, 456-467). In addition to eukaryotic plasmid vectors there are also viral vectors, in which the heterologous gene is introduced into the genome of a virus (e.g., retroviruses, herpesviruses and vaccinia viruses). The eukaryotic cells can then be infected with recombinant viruses.

In general, it is not possible to predict which type of vector and cell are most suitable for a particular gene. In most cases various combinations are tried

Eukaryotic expression of both glycoprotein gE and glycoprotein gl

The final structure of a protein depends on its primary amino acid sequence, how it folds on itself, post-translational modifications, etc. An important factor contributing to the structure of a protein is its interaction with one or more other proteins. We have also found that the glycoprotein gE of BHV-1 forms a complex with at least one other glycoprotein: the BHV-1 gly glycoprotein. The first indication of the existence of such a complex came from our results with candidate anti-gE Mabs: 1, 51, 67, 75 and 78 (see Table 2). These Mabs did not react with Difivac-1, nor with Lam gE, and were also unable to recognize 3T3 cells expressing the gE glycoprotein. However, these Mabs reacted with 3T3 cells expressing gE after infection with Difivac-1, showing that additional factors were required to confer glycoprotein gE on the appropriate antigenic conformation.for these Mabs. In some of our radio immunoprecipitation experiments with Mab 81 we have observed co-precipitation of a protein with an apparent molecular weight of 63 kD. Taking into account that herpes simplex virus gE glycoprotein forms a complex with a comparable molecular weight protein (HSV1 glycoprotein gl), we conclude that the BHV-1 gE glycoprotein forms a complex with the BHV-1 homologue of the glycoprotein gl. In order to be able to study this BHV-1 gE / gl complex and to produce gE antigen with the appropriate antigenic structure we both expressed glycoproteins in a single eukaryotic cell. For this, we applied the same procedures described for eukaryotic expression of gE glycoprotein alone. The only additional prerequisite is the use of expression vectors with different eukaryotic selection markers.

Serological tests

The serological methods used to distinguish between cattle vaccinated with Difivac-1 and cattle infected with wild-type BHV-1 based on anti-gE antibodies are based on the use of monoclonal antibodies directed against gE. These can be used in the following ways: a) According to the principle described by Van Oirschot et al. (Journal of Virological Methods, 22, 191-206, 1988). In this ELISA procedure for the detection of gl antibodies against the Aujeszky's disease virus, the demonstration of the antibodies is done by means of its blocking effect of the reaction of two Mabs presenting two different epitopes in gl. The test is carried out as described below. The microtiter plates are coated with Mab 1, remaining overnight at 37 ° C, after which they are stored, e.g. at 4 ° C or -20 ° C. The serum to be analyzed is preincubated with antigen in different uncoated microtiter plates, e.g. for 2 h at 37 ° C. The plates coated with Mab 1 are washed, e.g. 5 times, after which Mab 2 bound to horseradish peroxidase ("horseradish peroxidase" or "HRPO") is added to these plaques. The pre-incubated serum-antigen mixtures are then transferred to the plates in which the two Mabs are present, which is followed by incubation, e.g. for 1 h at 37 ° C. Plates are washed and substrate is added to each well. After ex. 2 h at room temperature, the plates are analyzed spectrophotometrically. Four negative control sera and four serial dilutions of a positive serum are included on each plate. Serum having an optical density value (" optical density " or OD) of less than 50% of the mean OD value of the four negative control sera examined on the same plate is considered positive. b) According to the principle of " Indirect Double Antibody Sandwich " or IDAS. Here, the microtiter plates are coated with a Mab or a polyclonal serum directed against the gE protein. Incubation with a gE antigen preparation results in the binding of gE to the coating. Antibodies specifically directed against gE in bovine serum to be examined subsequently bind to gE. These bound antibodies are recognized by an anti-bovine immunoglobulin conjugate. Antibodies in this conjugate bind by means of covalent bonds to the enzyme peroxidase. Finally, the bound conjugate is visualized by the addition of a chromogenic substrate. The specificity of the reaction is checked using the same procedure with a gE-negative control preparation in place of the gE antigen preparation. In each microtiter plate are included positive and negative control sera, the test is valid if the positive serum shows a positive result at a given dilution value. A serum is positive if it has a OD value higher than 0.2% of the conventional negative control serum. 19 19

(c) according to the principle described, as described in b), but after incubation of the serum to be examined, an anti-gE / HRPO Mab conjugate is used in place of the anti-bovine immunoglobulin conjugate. An anti-gE peptide serum or an anti-gE polyclonal serum may be used instead of the anti-gE Mab. The plates are washed and to each well is added a chromogenic substrate. After, e.g. 2 h at room temperature, the plates are analyzed spectrophotometrically. Four negative control sera and four serial dilutions of a positive serum are included in each plate. Serum having a lower OD value of 50% at the mean OD value of the four negative control sera examined on the same plate is considered positive. d) According to the principle of a blocking ELISA procedure, in which a virus antigen which may or may not be purified is used to coat the microtiter plate overnight. In these plates, the serum to be examined is incubated for, for example, one hour or longer at 37 ° C. Following a washing procedure, an anti-gE Mab is added to the plates, followed by incubation for, eg, 1 h at 37 ° C. An anti-gE peptide serum or an anti-gE polyclonal serum may be used instead of the anti-gE Mab. The plates are washed and to each well is added a chromogenic substrate. After, for example, 2 h at the temperature, the plates are analyzed spectrophotometrically. Four negative control sera and four serial dilutions of a positive serum are included in each plate. The serum having a OD value of less than 50% at the mean OD value of the four negative control sera examined on the same plate is considered positive.

In all of the procedures described above, conventional antigen of cultured virus containing gE can be used, but the same is true of gE antigen whose 20

expression takes place through prokaryotes or eukaryotes. Alternatively, oligopeptides based on the BHV-1 gE sequence, instead of conventional antigen, could be used in the above diagnostic tests. In addition, such oligopeptides could be used for the development of a so-called " cow-side test " according to the principle described in an article by Kemp et al., Science 241, 1352-1354, 1988. Such a test, would then bind the oligopeptide antigenic sequence to anti-gE antibodies present in infected animals. For such a test, the oligopeptide would have to be bound to a Mab directed against bovine erythrocytes.

Nucleic acid analysis using the PCR technique

Oligonucleotides (probes and " primers ") may, for example, be used in a polymerase chain reaction in order to distinguish between vaccinated and infected animals. Polymerase chain reaction (PCR) is a technique by which the nucleic acids of a pathogen can be multiplied billions of times in a short time (De polymerase kettingreactie, PF Hilderink, JA Wagenaar, JWB van der Giessen and BAM van der Zeijst, 1990, Tijdschrift voor Diergeneeskunde de 115, 1111-1117). The gE oligonucleotides may be chosen such that in a positive gen of gE if a product other than that forms in a negative genome is formed. The advantage of this is that an animal that has been vaccinated with a gE deletion vaccine also gives a positive signal in a PCR test. However, this approach depends on the presence of virus nucleic acids in a sample, for example blood, from the animal to be tested. 21

Following acute infection with DIIV-1, there is a high probability that the demonstration of BHV-1-specific nucleic acids can be made in the blood, but it has not yet been established whether BHV-1 nucleic acids can also be demonstrated in the blood during the latency period. The use of BHV-1 as vector

For the expression of heterologous genes in the BHV-1 genome, accurate information about the area where the heterologous gene is to be inserted is required. No fundamental sequences should be disturbed, and the regulatory sequences must remain available for expression of the heterologous gene. In principle, the gE glycoprotein gene is a suitable site for the expression of heterologous genes. The gE gene is not essential, so there is no objection to the replacement of the gE gene by a heterologous gene. As a consequence, the heterologous gene may be positioned such that it is under the influence of the regulatory sequences of the gE gene. However, it is not necessary to use the regulatory sequences of the gE gene. Expression of heterologous genes can be controlled, alternatively, by other regulatory sequences, e.g. stronger, different genes. It is also possible to link the heterologous gene to the signal peptide (exported) of the gE gene, such that the secretion of the heterologous gene product can be influenced. It is obvious that detailed knowledge of the gE gene and the gE protein confers the possibility of using BHV-1 as a vector in a very controlled manner. The developed vectors may furthermore be serologically serotyped wild type. The construction of mutants of BHV-1 expressing heterologous genes can be carried out in a manner analogous to that which was used for the construction of gE deletion mutants, as described in the examples. However, the deletion fragments are then replaced by a fragment having a heterologous gene at the deletion site.

EXAMPLES 1) Isolation and identification of a mutant with natural gE deletion a) Isolation of a natural mutant

Genomic DNA was isolated from a series of attenuated vaccines in a conventional manner according to known methods, which was analyzed using restriction enzymes. In particular, we searched for genomic shifts that were adequate to distinguish wild-type BHV-1 virus. The attention was directed in particular to the Ug region of the BHV-1 genome, since a region of genes that encode non-essential glycoproteins are probably located in this region - analogously to herpes simplex virus virus 1 glycoprotein gene with a cell cluster dispensable for growth in cell culture, R. Longnecker, S. Chatterjee, RJ Whitley and B. Roizman (1987), Proc. Natl. Acad. Know. 84, 4303-4307].

A batch of a BHV-1 vaccine from the University of Zagreb, Yugoslavia (Lugovic et al., Veterinarski Arhiv 55, 241-245, 1985), following a large number of passages in bovine kidney embryonic cells and embryonic bovine trachea (Ebtr) revealed to present a UQ region presenting

D deviation beyond a normal Ug region. These vaccines also appeared to give rise to the formation of small and large platelets in Etbr cells. From this mixed population a virus was isolated with a region u showing deviation, by means of

The three dilution steps with limitation, each choosing small platelets. The virus isolated by this method was further examined and designated Difivac-1, having been deposited with the Institut Pasteur, Paris, France, on May 27, 1992, with the deposit number 1-1213. b) Identification of the deletion in the gE gene in Difivac-1

For further analysis of this deviation in the Ug region, the genomic DNA of Difivac-1 was isolated according to standard methods and subjected to a " Southern blot " (Fig. 1A). Hybridization of this " blot " with a K fragment of • 32

P-labeled wild type HindIII confirmed that the fragment, located in the central region of the U-region, was about 1.0 kiloba-

The if (kb) shorter in Difivac-1. In addition, it was possible through this analysis to obtain the approximate position of the missing part (Fig. 1B). For further analysis of this deletion, the Ug region of the BHV-1 wild type Lam strain was isolated and cloned into prokaryotic vectors. To this end, and according to conventional methods, genomic DNA of the Lam strain (Fig. 2A) was isolated and cloned into pUC18, pACYC and pBR322 vectors (Fig. 2B). A physical map of the area around the assumed position of the deletion was made (Fig. 2C). From this physical map, suitable subclones were constructed for the determination of the nucleotide sequences of that area in the vectors pKUN19 and pUC18 (Fig. 2D). Using these subclones, the nucleotide sequence of the two strands was determined throughout the area (indicated in Fig. 2C) using the Sanger method. This nucleotide sequence (SEQ ID NO: 1) was analyzed using the PC / Gene program. From conceptual translation, nucleotides (nt) 168 to nt 1893 appeared to encode an open reading frame (" open reading frame ") of 575 amino acids (Fig. 3A). Further analysis revealed that this amino acid sequence exhibits the characteristics of a transmembrane glycoprotein, as shown in Fig. 3B. In fact, the first 26 amino acids (aa) are recognized as constituting a typical eukaryotic export signal (" export signal ") and the area between aa 423 and aa 450 is recognized as constituting a transmembrane region. In addition, there are three potential sites of N-linked glycosylation in this sequence. This predicted amino acid sequence shows clear similarities to the herpes simplex virus (HSV) glycoprotein gE gene; see Figs. 4A and 4B. These and other similarities warrant the conclusion that the gene found constitutes the homologue of BHV-1 gE. For this reason, the gene is designated g £. In order to determine to what extent this BHV-1 gE gene is lacking in Difivac-1, the p318 fragment was isolated. The p301 fragment begins at the 55 nt Alui site before the post-BHV-1 gE open reading frame and ends 133 nt thereafter. The genomic DNA of Difivac-1 was analyzed with this p318 fragment using the " Southern blot hybridization " technique. This procedure revealed that Difivac-1 did not contain any detectable sequences of p318 (Fig. 5). This experiment confirmed that Difivac-1 shows a deletion and clearly demonstrated that such deletion covers the entirety of the gE gene.

To determine the size and position of the deleted region, genomic sequences encompassing the Ug region of Difivac-1 were cloned into prokaryotic vectors. See Figure 11C. The 14.5 kb EcoRI fragment was cloned into the pACYC vector and designated p775. The 7.4 kb HindIII fragment was cloned independently into pUC18 vector and designated p728. From clone p728 two subclones were isolated: the 1.4 kb PstI fragment in clone p737 and the 350 bp Aluyl-PstI fragment in p754 clone. Restriction enzyme analysis and analysis by " Southern blot " of these clones (results not 25

shown) show that the deletion of gE in Difivac-1 has a length of 2.7 kb, starting immediately 5 'from the gE gene and ending at the border of the U-region. These 2.7 kb were replaced by

O tuled by a duplication of a 1 kb segment, located in the U0 region that opposes the gE gene, as extension / aberration of the repeat region. See Figure 11B. To confirm the results of this analysis and to determine the exact point of recombination, the nucleotide sequence of most of the inserted fragment (" insert ") of p754 clone was determined and compared to wild-type sequences. See Figure 12. This analysis showed that the recombination point lies 77 bp upstream of the " start " of the gE gene. c) Evaluation of the safety and efficacy of Difivac-1 Difivac-1 was tested on seven-week calves presenting BHV-1 specific seronegativity and free of pathogens. Seven calves were vaccinated by. 5 intranasal with 10 TCIDα in 2 ml, of which 1 ml was sprayed into each nostril. Eight week-old calves showing seronegativity specific to BHV-1 and free of pathogens maintained in a separate isolation unit were given 2 ml of culture medium intranasally and served as unvaccinated control animals. Five weeks after vaccination, vaccinated and control calves were challenged intranasally with 10 TCID ™ of the Iowa strain, an extremely virulent strain of BHV-1. Six weeks after challenge, all calves were intramuscularly treated with dexamethasone for 5 days in order to reactivate putative latent viruses. The evolution of clinical symptoms, rectal temperatures and growth of their bodies was followed. Virus was isolated from nasal material collections and neutralizing antibody titers were determined in serum.

After vaccination, behavior, appetite, rectal temperature and calf growth rate remained normal, but vaccinated animals had nasal discharges of serum and some hypersalivation. No lesions were observed in the nasal mucosa. The excretion of Difivac-1 was collected in nasal material collections after vaccination (Fig. 17). All vaccinated calves produced neutralizing antibodies to BHV-1.

After the challenge, all unvaccinated calves showed signs of apathy, loss of appetite, ocular and nasal discharges, redder lower jaw glands, severe nasal mucous lesions up to 14 days post challenge and a 4-day growth interruption. The vaccinated calves had small lesions of the nasal mucosae that quickly healed and did not present any interruption of the growth process. In Figs. 18, 19 and 20, the clinical values, rectal temperatures and development of the daily growth process after the challenge are presented. After the challenge, all calves released viruses from the muzzle, although the amount of excretion and the period during which it occurred was markedly reduced in the vaccinated calves (Fig. 21). In the vaccinated calves a secondary antibody reaction took place and the unvaccinated calves produced all antibodies after challenge.

After reactivation, challenge virus was isolated from a vaccinated calf and 5 unvaccinated calves. Could not rcactivate Difivac-1. 27

The above results demonstrated that Difivac-1 practically did not induce any disease symptoms in young calves and could not be reactivated. Difivac-1 markedly reduced the severity of the disease and the amount of virus excretion after challenge.

In conclusion, Difivac-1 is a safe and effective vaccine for use in cattle against infections caused by BHV-1.

2) Construction of recombinant BHV-1 gE deletion mutants In order to provide differentiable BHV-1 vaccines which, from a molecular point of view, are better defined than Difivac-1 and which, if In addition to a deletion in the gE gene, apart from Difivac-1, recombinant gE deletion mutants were constructed, for example, in the thymidine kinase gene. Starting from the determined position of the gE glycoprotein gene and using the cloned DNA fragments flanking the gE gene, it was possible to construct a gE deletion fragment. Using a standard technique (FL Graham and AJ van der Eb, 1973, Virology 52, 456-467), it was possible to recombine this deletion fragment in the genome of a wild type BHV-1 strain, which resulted in a deletion mutant of gE. The)

The construction of the gE deletion fragment

For the construction of the gE deletion fragment, the aim was to obtain a fragment which, on the one hand, lacked the entire gE sequence and which, on the other hand, had a flanking sequence of sufficient length 28 28

to allow recombination with the wild-type genome. On the 5 'side (upstream), the 1.2 kb PstI-AsuII fragment terminating 18 nt prior to the " start " codon was chosen. of the gE gene. For the 3 'fragment (downstream) the 1.2 kb EcoNI-Dral fragment was chosen, starting 2 nt before the " stop " of the gE gene (Figure 6).

For construction of the gE deletion fragment, the 1.4 kb PstI-Smal fragment from the 8.4 kb HindIII K fragment of the BHV-1 Lam strain, located on the 5 'side of the gE gene, was subcloned into the Smal and PstI sites of plasmid pUC18. This clone was designated p515. The EcoNI-Smal fragment located on the 3 'side of gE and from the 4.1 kb HindIII-EcoRI clone was cloned into the single AsuII site of p515. Thus, the construction of the gE deletion fragment was completed, and the clone thus constructed was designated p519. Although, in principle, the entire PstI-SmaI insertion fragment (" insert ") of p519 could be used as a gE deletion fragment, this was not advisable. In fact, the PstI-Smal fragment extends by about 100-150 base pairs (bp) into the repeat sequence (" repeat sequence ") flanking the Ug region. This 100-150 bp fragment may, in principle, recombine with the repeat sequence on the other side of the Ug area where the gE gene is not located, thereby giving rise to undesired recombination products. For this reason, the PstI-Dral fragment was chosen for the recombination experiment, whereby 100 bp of the repeat sequence is removed. b) Recombination of the gE deletion fragment with the wild-type BHV-1 genome

In order to carry out the recombination between the constructed gE deletion fragment and the wild-type BHV-1 genome, microgram quantities of the two DNA molecules were cotransfected into bovine tracheal embryonic cells (Ebtr ) according to the conventional FL technique Graham and A.J. van der Eb (1973, Virology 52, 456-467). The mechanisms of cellular recombination provide recombination of a small percentage of the DNA molecules (2-4%) that were incorporated by the cells. For the purpose of selecting recombinant gE deletion mutants, the virus mixture that forms after transfection is disseminated in a fresh culture of Ebtr cells. In most cases, the different virus populations thus developing (platelets) originate from a virus. For the isolation of the GE deletion mutants of the Lam strain of BHV-1, 230 of these platelets were isolated and examined according to standard immunological methods using BHV-1 specific monoclonal antibodies (Mabs) that do not react with cells infected with Difivac-1. These Mabs are directed against the gE glycoprotein. Five of the 230 platelets did not react with these Mabs. Additional DNA analysis of these 5 platelets was performed. c) DNA analysis of the gE deletion mutants of the BHV-1 Lam strain constructed

DNA preparations of 3 (1B7, 1B8 and 2H10) of the above 5 candidate gE deletion mutants were further analyzed using the standard " Southern blot " (Sambrook et al., 1989). Double digests of these PstI and Dral DNA preparations, followed by gel electrophoresis and hybridization by " Southern blot " with the 2.3 Kb PstI-Dral deletion fragment acting as a probe revealed that the gE gene of the 1B7 and 1B8 virus population genome had been removed in the desired manner; see Figs. 30 30

7Α and 7B. The 2H10 population had a PstI-Dral fragment that deviated from this situation. Hybridizations by " Southern blot " with a specific probe relative to gE revealed no gE sequences located in any of the three DNA preparations (results not shown). BHV-11 virus populations 1B7 and 1B8 were considered populations of recombinant gE deletion mutants. The BHV-11B7 virus population was tested for its possible vaccine properties. d) Construction of double deletion mutants of

thymidine / gE

Since recombinant deletion mutates of BHV-1 with a deletion in only one gene may not have a sufficiently reduced virulence, deletions in the thymidine kinase gene (" thymidine kinase " or TK) of the Lam strains and Harberink of BHV-1. These mutants were constructed in a manner analogous to that used for the aforementioned gE deletion mutants (results not shown). These TK deletion mutants were used to construct TK / gE double deletion mutants. glycoprotein e) Construction of double deletion mutants

gl / glycoprotein gE

Since cattle vaccinated with a single deletion mutant gE can produce anti-gl antibodies that may interfere with the detection of anti-gl / gE antibodies (situation analyzed below), we have also devised a vaccine with a double gl / gE deletion. Such a double deletion mutant gT / gE can. be constructed using the same procedures that were used for the construction of the single deletion mutant gE. Partial analysis of the sequence of 31 31

nucleotides of the upstream end of the 1.8 kb PstI fragment - which covers the 5 'end of the gE gene - revealed an open reading frame exhibiting significant homology relative to gl homologues found in other herpes viruses. See Figures 13 and 14. Using the 350 bp Smal-PstI fragment which includes the putative 5 'end of the gl gene and the EcoNI-Smal fragment, located downstream of the gE gene, it is possible to construct a gE / gI deletion fragment . This fragment can be recombined with the wild-type wild-type genome to provide a BHV-1 deletion mutant of gl / gE. See Figure 16. 80-90 amino acids which in theory can still be produced will be unable to promote the onset of antibodies which may in some way interfere with the detection of anti-gl / gE antibodies. Further analysis of the nucleotide sequence of the gl gene will allow to construct a deletion of gl covering the entire gl coding region. This double-deletion mutant gl / gE was renamed Difivac-IE.

f) Evaluation of the safety and efficacy of Lam gE mutants

and Lam gE, TK

Common immunogenic properties of BHV-1 Lam gE- and Lam gE-, TK- mutant strains were tested on seven-week calves, seronegative relative to BHV-1, and free of specific pathogens. Each of the mutant strains was administered to 6 calves by intranasal spray. At 5 each calf was given a total dose of 10 TCID 2 in 2 ml culture medium, 1 ml was sprayed into each nostril. Six other calves were sprayed intranasally with virus-free culture medium, and served as unvaccinated control animals. Five weeks after vaccination, all calves, both those that had been vaccinated and the controls, were

submitted to a challenge intranasally with 10 TCID 50 of 32

strain Iowa, an extremely virulent strain of BHV-1. After vaccination and after challenge, the evolution of clinical symptoms, rectal temperatures and body weights was followed. Nasal material was collected in order to determine the number of nasal virus delivery days.

After vaccination, the behavior, appetite, rectal temperature and calf growth rate remained normal. In all vaccinated calves nasal discharge of serum and small lesions of the nasal mucosa were observed. Virus could be isolated from the muzzles of vaccinated calves for about 7 days (Table 1).

After the challenge, all unvaccinated calves showed apathy, lack of appetite, ocular and nasal discharges, reddish lower jaw gums, severe nasal mucous lesions, and reduced growth. The calves vaccinated with Lam gE, TK all developed some nasal discharge and showed slight lesions of the nasal mucosa. Not all calves vaccinated with Lam gE have developed nasal discharges or lesions of the nasal mucosae. In vaccinated calves there was no apathy, lack of appetite or other clinical symptoms of the disease. Rectal temperatures, growth and clinical values after challenge are shown in Figs. 22, 23 and 24. Unvaccinated calves released viruses from the muzzle for twice the time of vaccinated calves (Table 1).

The above results demonstrate that mutant strains of BHV-1 Lam gE- and Lam gE, TK practically do not induce any clinical symptoms of the disease in young calves. Both strains avoided the disease after the challenge and reduced the period of nasal release of the virus by 50%. 33 33 3)

BHV-1 mutant strains Lam gE and Lam gE, TK are safe and effective and can be used in the vaccination of bovine cattle against infections caused by BHV-1.

Expression proc.ari expression of gE

For the prokaryotic expression of the BHV-1 gE glycoprotein gene, pGEX expression vectors (D.B. Smith and K.S. Johnson, Gene 67 (1988) 31-40) have heretofore been used. The pGEX vectors encode the glutathione carrier protein S-transferase (" glutathione S-transferase " or GTS) from Schistosoma japonicum which is under the influence of tac promoter which can be induced to be expressed by Isopropylthiogalactoside " Isopropylthiogalactoside "; or IPTG). It is an example of a GST-fusion protein and the product of the constructed material pGEX-2T600s3 (Fig. 8A). In this material constructed using standard molecular biology techniques (Sambrook et al., 1989), a 600 bp Smal fragment encoding a 200 amino acid N-terminal region of the gE protein was ligated behind the GST gene. This constructed material was designed in triplicate, a reading frame other than the 600 bp fragment being bound to the GST each time. The three materials constructed were introduced into the IPTG-induced Escherichia coli DH5a strain and the proteins that formed were transferred to nitrocellulose after electrophoresis on polyacrylamide gel by " Western blotting " technique. Immunological detection with anti-GST antibodies revealed that only the appropriate reading frame (Ns 3) encoding the gE protein area results in the expression of a prominent fusion protein with the predicted size of 27k (GST) + 20k (gE ) = 47k. Three of the Mabs isolated by us not reacting with Difivac-1 recognize the 47 kD GST-gE fusion protein in a " Western blot " see Figure 8B. 34 34

4) Eukaryotic expression of the gE glycoprotein gene

For the eukaryotic expression of the gE glycoprotein gene, the pRVHis vector was chosen, among others, among others. The pEVHio vector exhibits the HisD gene encoding histidinol dehydrogenase (EC 1.1.1.23) (C. Hartmann and R. Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85, 8047-8051) which leads to cells surviving the toxic concentration of 2.5 mM histidinol. This vector further includes the immediate early gene promoter region (" human cytomegalovirus " or HCMV) with unique restriction enzyme sites located behind it. For the construction of a pEVHis / gE expression vector a fragment was used which included the entire coding region of the gE glycoprotein gene. This starts at the local Alui 55 bp before the open reading frame of gE that was postulated and ends 133 bp behind it. This region was cloned behind the HCMV promoter of the pEVHis vector, thereby obtaining the constructed material (" construct ") pEVHis / gE (Fig. 9). PEVHis / gE was amplified in E. coli DH5α cells and purified by a cesium chloride gradient (Sambrook et al., 1989). This purified DNA was transfected into Balb / C-3T3 cells according to the method of Graham and Van der Eb. The transformed cells were selected with histidinol, which allowed the isolation of twenty colonies resistant to histidinol. These colonies were examined with Mab 81 by an Immuno Peroxidase Monolayer Assay type assay " (IPMA). Four colonies revealed to express the gE protein. Of these four colonies, the 3T3 gE clone 9 was used to isolate a subclone exhibiting a high expression of gE. The clone isolated by this method (termed 3T3gE 9.5) was used for the characterization of candidate anti-gE monoclonal antibodies. 35 35

5) Eukaryotic expression of BHV-1 gE glycoprotein and BHV-1 gly glycoprotein in the same cell In order to express the BHV-1 gly glycoprotein in the same cell as the BHV-1 gE glycoprotein we first determined the putative position of the gl gene of BHV-1. Since the herpes simplex virus gly glycoprotein gene is situated immediately upstream of the gE glycoprotein gene, it has been inferred that the gl gene of BHV-1 would be placed in a corresponding position. To test this hypothesis, the sequence of a region of 283 nucleotides, located about 1 kb upstream of the start of the BHV-1 gE gene, was determined. The conceptual translation of this region revealed that the second reading frame encodes a sequence of 94 amino acids that is homologous to the glycoprotein gl of the herpes simplex virus (Figures 13 and 14). Since the homologous segment is at about 80 amino acids of the " start " codon, it is estimated that " start " putative gene of the open reading frame of the BHV-1 gene is about 250 nt upstream of the region whose sequence has been determined. The 1.7 kb SmaI fragment starting 400 nt upstream of the region whose sequence was determined terminating within the gE gene should contain the entire coding region of the BHV-1 gl gene. This 1.7 kb Smal fragment was cloned into the MSV-neo eukaryotic vector (See Figure 15). This vector includes the strong promoter of the Murine Sarcoma Virus (" Murine Sarcoma Virus " or MSV) and the neo selector gene which encodes the antibiotic resistance " G-418 sulphate Geneticin ". The resulting constructed MSVneo Gl material was amplified in E. coli DH5a cells and transfected into 3T3gE 9.5 cells using the method of Graham and Van der Eb. The transfected cells were selected with 400 μg of GeneLiciii / ml culture medium and the resistant colonies were isolated and tested with candidate anti-gE Mabs that did not react with 3T3gE 9.5 cells. From here we selected the 3T3gE / gI R20 clone reacting with, for example, Mab 66, as well as wild-type BHV-1. 6) Characterization of candidate anti-gE Mabs

Mabs were produced relative to wild type BHV-1, which were selected for their inability to react with Difivac-1-infected bovine tracheal embryonic cells (" embryonic bovine trachea cells " or Ebtr). These Mabs are examined for their reactivity with respect to a) the Lam gE deletion mutant; b) the prokaryotic expression product described above in a " Western blot "; c) the Balb / c-3T3 expressing gE cells described above; d) cells referred to in c) and infected with Difivac-1, and e) Balb / c-3T3 cells expressing the gE / gl complex.

For the reactivity tests referred to in a, c, d and e, an "Immuno Peroxidase Monolayer Assay" type test was used " (IPMA). The results presented in Table 2 show that we produced Mabs directed against gE (ns 2, 3, 4, 52, 66, 68, 72 and 81) and Mabs (n.1, 51, 53, 67, 75 and 78) that can be directed against antigenic conformation domains in the gE / gl complex. A competitive mapping of the antigenic domains recognized by the different Mabs revealed the presence of at least 4 antigenic domains on the gE glycoprotein and the fact that the gE / gl complex probably formed a domain (Table 2 ).

Detection of anti-gE antibodies in cattle infected with BHV-1

To investigate whether anti-gE antibodies are present in the serum of infected cattle, an indirect blocking IPMA (" indirect blocking IPMA ") test was run on the 16 candidate gE-Mabs and the following 8 sera : 2 bovine sera vaccinated with Difivac-1 and challenged with the virulent strain Iowa, which were collected 14 days post challenge; - 2 bovine sera experimentally infected with BHV-1 virus subtype 1, which were collected 20 months after infection. One of the cattle was infected through contact; 2 bovine sera experimentally infected with BHV-1 subtype 2b virus, which were collected 20 months after infection. One of the cattle was infected through contact; a serum of a specific pathogen free calf vaccinated with a ts mutant vaccine and subjected 3 weeks later to challenge with the BHV-1 subtype 2b virus, which was collected 7 weeks post challenge; a serum of a gnotobiotic calf vaccinated with a ts mutant vaccine and subjected 3 weeks later to a challenge with BHV-1 virus subtype 2b, which was collected 7 weeks post challenge. Table 2 shows that all of these sera contained antibodies against the antigenic domains III and IV in gE, and against the antigenic domain I that is probably located in the gE / gl complex. We conclude that gE appears to be a suitable serological marker for distinguishing between cattle infected with BHV-1 and vaccinated cattle. 38 38

7) Detection of BHV-1 nucleic acids by the PCR technique using " primers " specific relative to BHV-1 gE

Starting from the nucleotide sequence of the BHV-1 gE gene that was determined, a pair of " primers " suitable for use in a PCR procedure using the " primer " selection program of Lowe et al. (T. Lowe, J. Sharefkin, S. Qi Yang and C.W. Dieffenbach, 1990, Nucleic Acids Res. 18, 1757-1761). These " primers " were designated P3 and P4 and are shown in Fig. 10. The " primers " are separated from each other by 159 nt and give rise to the amplification of a 200 nt fragment. Conditions for use of the PCR technique were optimized using the " primers " p ^ and P4 and BHV-1 DNA alone. This in particular implied the variation of the concentration of MgCl 2, the concentration of glycerol and the conditions of the cycles. The optimum buffer solution found for P? And P? Use in BHV-1 DNA amplification consists of 10 mM Tris pH 8.0, 50 mM KCl, 0.1% gelatin, 2.6 mM MgCl 2 and glycerol a 20%. The optimum cycle conditions (Perkin Elmer Cetus DNA Thermal Cycler) are, for cycles 1-5, 1 min. 98 ° C, 30 sec. 55 ° C and 45 sec. 72 ° C, and for cycles 6-35, 30 sec. 96 ° C, 30 sec. 55 ° C and 45 sec. 72 ° C. After amplification by the PCR technique, the obtained 200 nt DNA fragment was electrophoresed on a 2% agarose gel, " blotted " on nitrocellulose and subsequently subjected to a " Southern blot " The P dCTP-labeled probe used for the analysis by " Southern blot " is the 137 bp TaqI fragment, which is located between the " primer " (Fig. 10). Following an autoradiography of the hybridized filters, a 200 bp band was observed. Using this pathway, amplification of only 10 genomes of BHV-1 (about 1.5 x 10-15 μg of DNA) still provides a signal 39

which can be properly detected (result not shown). In an analogous manner, a PCR procedure was developed using " primers " based on the glycoprotein GIII coding sequence of BHV-1 (D.R. Fitzpatrick, L.A. Babiuk and T. Zamb, 1989, Virology 173, 46-57). In order to make a distinction between wild-type BHV-1 DNA and a gE deletion mutant vaccine, DNA samples were subjected to both specific PCR analysis relative to gE and specific PCR analysis for gIII. In such a test, a Difivac-1 DNA preparation was found to be positive for gIII and negative for gE.

Since detection of BHV-1 DNA in bovine semen will constitute an important use of the specific PCR procedure for BHV-1, the specific PCR technique for gE in bovine semen infected with BHV-1 was attempted . However, there are unknown components in the semen which exert a strong inhibiting effect on the polymerase chain reaction. A protocol for isolating BHV-1 DNA from bovine semen was therefore developed. To isolate DNA from bovine semen, 30 μΐ of semen were incubated with 1 mg / ml proteinase K (pK) in a total volume of 300 μΐ 0.15 M NaCl, 0.5% Na-Sarkosyl, and DTT 40 mM, at 60 ° C. After 1 hour, the sample was allowed to cool to room temperature, 300 μΐ of 6 M Nal was added and the whole was incubated for 5 minutes. The DNA was isolated from this mixture by conventional extraction with chloroform / isoamylethanol, and precipitated with 1 volume of isopropanol. The precipitate was washed with 2.5 M NH 4 Ac / 70% ethanol and resuspended in 10 mM Tris pH 7.4, 1 mM EDTA, 0.5% Tween 80 and 0.1 mg / ml pK for a fresh incubation for 1 hour at < 30 ° C. This DNA preparation can be subjected directly to the PCR technique. DESCRIPTION OF THE FIGURES Figure 1 40 Analysis

Iowa by the Southern blot technique BHV-1 " Southern blot " of Difivac strains and

A. Image of an auto-radiogram resulting from a " Southern blot " of Difivac-1 and Iowa genomic DNA. In lanes 1 and 3, Difivac-1 DNA was applied after digestion with restriction enzymes HindIII and PstI, respectively. In Lanes 2 and 4, Iowa DNA was applied after digestion with restriction enzymes HindIII and PstI, respectively. Fragment sizes are given in kilobases (kb).

The viral DNA was isolated by centrifuging the culture medium (70 ml / roller bottle) of about 450 μl.

cm) were incubated with virus infected Ebtr cells for 2 hours through a 25% (w / w) sucrose pad in 10 mM Tris pH 7.4, 150 mM NaCl and 1 mM EDTA at 20 krpm in the ultracen SW27 rotor - Beckman L5-65 centrifugal pump. The DNA was isolated from the virus pellet thus obtained according to conventional techniques (J. Sambrook, EF Fritsch and T. Maniatis, 1989, Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, Nova York). This DNA was subjected to restriction enzyme digestions from Boehringer Manheim in " SuRE / cut buffers " supplied by the manufacturer.

After separation on a 0.7% agarose gel for horizontal electrophoresis and " blotting " on a nitrocellulose filter (Schleioher & Schuoll, Inc.), the filter was prehybridized for 6 hours at 42øC in 50% formamide, 3x SSC (1x SSC = 0.15 M NaCl and Na 0.015 M, pH 7.4), 50 41 41

μg of denatured salmon sperm (Sigma) DNA / ml and 0.02% bovine serum albumin, 0.02% polyvinyl pyrrolidone and 0.02% ficoll and Na dodecylsulfate (" sodium-dodecylsulphate " or SDS) at 0.1%. The hybridization was then carried out by adding to the solution the P dCTP-labeled HindIII K fragment (Amersham) (The choice of the HindIII K fragment is based on: cloning and mapping of DNA cleavage sites of virus of bovine herpes 1 (Cooper strain), John F. Mayfield, Peter J. Good, Holly J. VanOort, Alphonso R. Campbell and David A. Reed, Journal of Virology (1983) 259-264). After 12-14 hours of hybridization, the filter was washed for 2 hours in 0.1% SDS and 0.1 x SSC at 60 ° C. The HindIII K fragment was cloned into the pUC18 vector according to standard hybridization procedures (J. Sambrook, E.F. Fritsch and T. Maniatis, 1989, Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory

Press, New York). After HindIII digestion of the pUC / 8.4 HindIIIK clone the vector pUC18 was again separated from the 8.4 kb HindIII K fragment by means of Low Melting Point Agarose gel electrophoresis " (BRL, Life Technologies, Inc.) at 0.7%, and isolated from the agarose by conventional phenol extraction and ethanol precipitation procedures. The isolated HindIII K fragment was labeled " Random Primed DNA labeling Kit 1004.760 " of Boehringer Mannheim. Auto-radiography of the hybridized filters was obtained by exposure for 36 hours of a Kodak XAR film at -70 ° C using a reflecting screen. B. Physical maps of the 8.4 kb HindIII K fragment from Iowa and the 7.4 kb HindIII fragment from Difivac-1. Taking into account the co-migration of the 6 kb PstI fragments and the absence of the 1.8 kb PstI fragment in Difivao-1, the deletion is postulated in the dashed area.

Figure 2

Subcloning of wild-type BHV-1 fragments around the missing region in Difivac-1

In A the components of the BHV-1 genome are shown: the Single Long region (" Unique Long " or U); the single short region

Li (" Unique Short " or Ug) to the two repeating sequences (" repeats ") (Ir and Tr). This map is based on published analysis of the Cooper strain (John F. Mayfield, Peter J. Good, Holly J. VanOort, Alphonso R. Campbell and David A. Reed, Journal of Virology (1983) 259-264).

B fragments of the Ug region that were cloned into prokaryotic vectors are shown: a 15.2 kb EcoRI fragment in pACYC, an 8.4 kb HindIII fragment in pUC18 and a 2.7 kb EcoRI-HindIII fragment and 4.1 kb in pBR322. Isolation of the viral DNA fragments was carried out according to procedures which are reported in the legend of Fig. 1A. Cloning of these fragments into the various vectors was performed according to standard procedures (J. Sambrook, E. F. Fritsch and T. Maniatis, 1989, Molecular cloning: a laboratory manual, 2nd edition, Cold Spring Harbor Laboratory Press, New York).

In C there is a physical map of the region where the deletion postulated in Difivac-1 is located.

In D, some subclones of this region are indicated, which were used for further analysis. The two PstI fragments were cloned into pI < UN19 and the remaining fragments into pUC18. 43

Figure 3A: Nucleotide sequence comprising 2027 nucleotides of the Ug region of the BHV-1 Lam strain around the postulated site that was deleted on Difivac-1, as indicated in Fig. 2C [of the recognition site of Alui in the far left to the HincII recognition site at the far right]. The nucleotide sequence in the insertion fragments (" inserts ") of the subclones shown in Fig. 2D was determined by analysis of the two strands using the dideoxy sequencing method of Sanger et al. (F. Sanger, S. Nicklen and A.R. Coulson, 1977, Proc Natl Acad Sci USA 74, 5463-5467). To this end, the " T7 sequence kit " according to the procedure specified by the manufacturer. For the purposes of radioactive labeling, [S] dATP (Amersham) was used. The sequence analysis of the GC rich regions with compression artifacts was repeated with the 7-deaza-dGTP variant of " kit " of Pharmacia. Underneath the nucleotide sequence is indicated, using the three-letter code, the amino acid sequence (aa) of the open reading frame of 575 residues aa, which was found after conceptual translation of the nucleotide sequence. This translation is based on the universal code and was determined using the computer program PC / gene (PC / gene version 1.03, November 1987). This open reading frame of 575 aa starts with methionine at nt 168 and ends with the " stop " in nucleotides 1893. Structural analysis of the open reading frame of 575 residues aa was also carried out with the PC / gene computer program. The first 26 aa form a eukaryotic export signal identified in the figure by the designation " signal peptide " (" signal peptide "). With a value of 6.2, the cleavage of this signal sequence is predicted between aa 26 and aa 27. The 575 aa sequence shows three possible N-linked glycosylation sites (" N-bound glycosylation sites " or NXT / S ) indicated by a line under the amino acid residues. According to the method of Rao and Argos, there is a transmerabran region between aa 423 and aa 450, denoted in the figure by the designation " transmembrane helix " (" transmembrane helix "). The (locus) recognition sequences for the restriction enzymes AsuII, SmaI, HindIII and EconNI are underlined. The calculated molecular weight of this polypeptide is 61212. B: Schematic representation of the structural characteristics of the aforementioned open reading frame of 575 aa.

Figure 4

Comparison of amino acids between the amino acid sequence of the BHV-1 gE gene and the amino acid sequence of the herpes simplex virus gE gene (HSV) and other homologous gE genes [pseudo-rabies virus gl (" pseudo-rabies virus " or PRV) and virus gpl " varicella-zoster " (VZV))

The sequences used in this comparison come from the following publications; HSV: Sequence determination and genetic content of the short region in the genome of herpes simplex virus type l. D.J. McGeoch, A. Dolan, S. Donald and F.J. Rixon (1985) Journal Mol. Biol 181, 1-13. VZV: DNA sequence of the Ug component of the varicella-zoster virus genome. A.J. Davidson (1983), EMBO Journal 2, 2203-2209. PRV: Use of lambdagtll to isolate genes for two pseudorabies virus glycoproteins with homology to herpes simplex virus and varicella-zoster virus glycoproteins. E.A. Petrovskis, J.G. Timmins and L.E. Post (1986) Journal of Virology 60, 185-193].

These sequences were compared using the 45 45

Multalin sequences (F. Corpet, 1988, Nucl Acids Res. 16, 10881-10890).

In A there is shown a diagram in which the four amino acid sequences are shown schematically. Here, the transmembrane parts (TM) provided are shown one below the other. In addition to the predicted export signal sequences (SP) and possible N-linked glycosylation sites (I), there are two conserved areas in which the relative position of the cysteine residues remains unchanged (CCC).

In B the results of the comparison, through the Multialin program, of the cysteine rich regions located centrally in the four versions of gE are presented. Asterisks indicate identical amino acids and two points indicate analogous amino acids.

Figure 5

Drawing photos taken from a " Southern blot " of Difivac-1 and Iowa

Image A: Difivac-1 and Iowa genomic DNA resulting from restriction enzyme digestions Bst (1,2), EcoRI (3,4) and

HindIII (5,6), separated on a 0.7% agarose gel, " blotted " on nitrocellulose and hybridized with a P-labeled HindIII K32 fragment of the Lam strain of BHV-1 according to the procedures specified in the legends of Fig.

Image B: " Blot " on nitrocellulose from the same gel that was used in A, hybridized with probe p318 specific for BHV-1 gE. This probe includes the entire AluI-HincII region indicated in Fig. 2C. 46

Figure 6

Construction of the BHV-1 gE deletion fragment

In A is shown the position of the gE gene and the clones used. The components of the BHV-1 genome are: the Single Long region (" Unique Long " or UL); the Single Short region (" Unique Short " or U) and the two repeat sequences (IR and TR). For

O

if the 5 'side of the gE gene was obtained, the 1.4 kb PstI-Smal fragment of the 8.4 kb HindIII k fragment of the BHV-1 Lam strain was subcloned into the SmaI and PstI site of the plasmid pUC18. This clone was designated p515 and is shown in B. The EcoNI-Smal fragment located on the 3 'side of gE, from the 4.1 kb HindIII-EcoRI clone, was cloned into the single AsuII site of p515. For the binding of the EcoNI part to the AsuII part, the p515 clone was digested with AsuII, and then treated with Klenow enzyme (Boehringer Mannheim) and dCTP, to provide a cytosine residue on the AsuII part according to standard methods (Sambrook et al., 1989). This additional cytisine is identified by an asterisk in D. Then, p515 was also digested with the enzyme Smal, after which the EcoNI fragment could be ligated to this vector. The clone so constructed was designated p519.

Figure 7 A. Drawing of a photograph obtained in a " Southern blot " of DNA preparations of 1B7, 1B8 and 2H10. DNA isolation, restriction enzyme digestion, " blotting " and hybridization were performed according to the procedures described in the legends of Fig. 1A. After a double digestion with PstI-Dral from the DNA preparations 1B7, 1B8 and 2H10, the fragments were separated on 0.7% agarose gel and subsequently subjected to " blotting " on a nitrocellulose filter. This filter was hybridized with the P dCTP-labeled 2.3 kb PstI-Dral deletion fragment used as a probe. In lanes 1 to 3, samples 1B7, 1B8 and 2H10, respectively, were separated. Lane 4 DNA was applied from the wild type BHV-1 Lam strain and on lane 5 the 2.3 kb deletion fragment was applied.

B. Physical map of the 15.2 kb EcoRI fragment of the Lam strain of BHV-1. The map shows the position of the PstI, Dral and HindIII recognition sites and the position of the hybridization probe referred to in 7A.

Figure 8

Prokaryotic expression of BHV-1 gE

For the prokaryotic expression of BHV-1 gE, the 600 bp Sma fragment of the gE gene was fused in three reading frames to the coding region of the Schistosoma japonicum glutathione-S-transferase gene in the pGEX-2T vector ( DB Smith and KS Johnson, Gene 67 (1988) 31-40). Recombinant molecules with the appropriate (syn) orientation of the SmaI fragment were identified by restriction enzyme analysis using standard methods. E. coli DH5a clones with this fusion-constructed structure were designated pGEX-2T600sl, pGEX-2T-600s2 and pGEX-2T600s3. A. Diagram of one of the constructed structures pGEX-2T600s. On the NH2 side of the GST-gE fusion product coding region is located the tac promoter region that can be induced by Isopropylthiogalactoside (IPTG) (" Isopropylthiogalactoside (IPTG) inducible tac promoter region "). B. Design of Photographs Obtained in a Western Blot " of total protein preparations from pGEX-2T600s transformed DH5α cells. Cultures of DH5a cells maintained overnight and transfected with the constructed constructs pGEX-2T600sl, pGEX-2T600s2 and pGEX-2T600s3 were continued 1/10 in Luria-Bertani medium (LB) with 50 μg / ιη1 ampicillin and after 1 hour growth induced with IPTG for 5 hours. These induced cultures were centrifuged for 5 minutes at 6,000 x g and incorporated in 1x layermix (2% SDS, 10% glycerol, 5% mercaptoethanol and 0.01% bromophenol blue) [1.5 ml of culture are incorporated into 500 μΐ layermix] and heated at 95 ° C for 5 minutes. Then 50μl per lane were separated on a vertical 12.5% polyacrylamide gel according to standard procedures, and subsequently subjected to a " semi-dry blotting " to a nitrocellulose filter using the LKB-multiphor II New Blot system under conditions specified by the manufacturer.

Protein-labeled protein ("BRL Life Technologies, Inc. 236k, 112k, 71k, 44k, 28k, 18k and 15k) was applied on lanes M and lanes 1, 2 and 3 were prepared total proteins of the DH5a cells transfected with the respective three frames: pGEX-2T600sl, pGEX-2T600s2 and pGEX-2T600s3.

In image A, the result of the analysis can be seen by " Western blot " with anti-GST serum. To obtain this, the filter was incubated according to standard procedures (E. Harlow and D. Lane, 1988, Antibodies: a laboratory manual, Cold Spring Harbor Laboratory, New York) in blocking buffer solution (PBS + 2% and 0.05% Tween 20), and subsequently as rabbit anti-GST polyclonal serum. Then the filter was washed and incubated with horseradish peroxidase (HRPO) conjugated goat-anti-rabbit immunoglobulin serum conjugated goat anti-rabbit serum. Thereafter, the bound goat antibodies were detected immuno-chemically with chromogen (diaminobenzidine, chloronaphthol and H2 O2). The GST-fusion product which is indicated by an arrow has the calculated size of approximately 47 k only in the table (" frame ") 3.

In image B, the result of the analysis can be seen by " Western blot " with monoclonal antibody Mab 4, which recognizes the gE protein. To that end, a double filter as in image A was blocked, incubated with Mab, washed and incubated with HRPO conjugated rabbit anti-mouse serum (" HRPO conjugated rabbit-anti-mouse serum "). Then, the bound rabbit antibodies were detected immunochemically with chromogen. The band that is visible on lane 3 (frame 3) has the dimension of 47 k and is indicated by an arrow.

Figure 9

Construction of plasmid pEVHisgE for the eukaryotic expression of the BHV-1 gE gene

For the eukaryotic expression of the gE gene, the entire gE coding region was cloned in the appropriate orientation behind the HCMV promoter region of the pEVHis expression vector, using standard procedures (Sambrook et al., 1989). To that end, the 394 bp Alui fragment starting 55 bp prior to the open reading frame of gE was cloned into pUC18 and designated p201. Then, after HincII digestion of p201, the 1740 bp HincII fragment, which includes most of the gE gene, was 50 50

cloned into p201. This resulted in the plasmid p318 which in " polylinker " of pUC18 includes the entire gE coding area, from the Alui 55 bp site before the " start " gE to the HincII site 133 bp behind the " stop " of gE. Using the restriction enzyme sites in " polylinker " of the vector, this fragment was separated from p318 with the BamHI and SphI enzymes. First, p318 was digested with SphI and then the SphI site was filled using Klenow polymerase and dNTPs. After digestion with BamHI, the 1.9 kb insert was separated from the pUC18 vector was Low Melting Point Agarose (Low Melting Point Agarose) and ligated into the pEVHis vector which had been digested with BamHI and EcoRV for this purpose. The plasmid thus formed was designated pEVHis / gE.

Figure 10

Location of " primers " specific for gE and probe for the BHV-1 DNA detection PCR procedure

In the figure is shown the nucleic acid sequence of the BHV-1 gE glycoprotein gene, from nucleotide 1272 to 2027 [the sequence was extracted from that of Fig. 3]. &Quot; primers " for the specific PCR procedure relative to gE were designated P3 and P4. The " primer " for P3 and P4 are underlined. The nucleotide sequence of P2 is 5'-ACG-TGG-TGG-TGC-CAG-TTA-GC-3 '(SEQ ID NO: 2). The nucleotide sequence of P4 is (complementary to the " primer " primer referred to above) 5'-ACC-AAA-CTT-TGA-ACC-CAG-AGC-G-3 '(SEQ ID NO: 3). The probe that was used for hybridization by " Southern blot " for the detection of DNA amplified by the PCR technique is the 137 bp Taq1 fragment located between " primer " binding sites, the ends of which are indicated. For the purposes of comparison with Fig. 3, also the HindIII and EcoNI sites.

Figure li

Mapping of the Difivac-1 A deletion map represents the physical map of the 15.5 kb EcoRI fragment of the wild type BHV-1 Lam strain. B represents the physical map of the 14.5 kb EcoRI fragment of Difivac-1. Both EcoRI fragments cover all of the Short Single regions (" Unique Short ") of the respective virus genomes. The position of the gE gene and the putative position of the gl gene are indicated by open boxes. Maps A and B are arranged in such a way that the 6 kb PstI fragments within each map are aligned. In both maps, internal and terminal repeat sequences (" internal repeat & terminal repeat ") were indicated by dashed boxes. The arrows below the repeat sequences indicate the orientation of those sequences.

In A the part of the Ug region lacking in strain Difivac-1 is indicated. C represents the position of the cloned Difivac-1 fragments that were used to establish the map of the gE deletion and to obtain the physical map shown in B. The arrows below the inserted fragments of clones p728, p737 and p754 indicate regions whose sequences were established to determine the recombination point. 52

Abbreviations: A = Alui, E = EcoRI, P = PstI, H = HindIII, r = recombination point, IR = internal repeat sequence (" internal repeat "), TR = terminal repeat sequence " ).

Figure 12

Determination of the exact recombination point in the Us region of Dif ivac-1

To determine the exact boundaries of the deletion of gE found in the Difivac-1 strain, the p754 clone sequences and the clones of the p728 and p737 clones were determined. The fragments inserted into these clones were indicated in Figure 11. The procedures used to establish the sequences were described in the captions of Figure 3.

In A the sequence of most of the Alui-PstI fragment is shown. This sequence starts at the promoter region of the gE gene. A putative TATA box was underlined. At point r (= recombination point), this promoter region fuses with a sequence also found at the opposite site of the U region, b designated: " inverted repeat " The exact recombination point was determined by comparing the repeat sequence found in the gE promoter region with the copy of the repeat sequence found at the opposite site of the Ug region. The point where these sequences diverge has been identified in B (I) by means of an 'r'. A similar comparison was made with the gE promoter sequence found in Difivac-1 and the gE promoter found in the wild type Lam strain. The point at which these sequences diverge has been identified in B (li) 53 also by means of an 'r'. The recombination points found are the same.

Figure 13

Partial analysis of the BHV-1 gl gene sequence The 284 nucleotide sequence within the gl coding region of BHV-1 was determined using the 1.8 kb PstI clone of the BHV-1 Lam strain that penetrates both the gene gl as in the BHV-1 gE gene (see Figure 11). The procedures used to establish the sequence were described in the captions of Figure 3. The sequence was translated based on the universal code by means of the computer program PC / gene version 1.03 (November, 1987). The amino acid sequence encoded by the second reading frame is indicated by means of the single letter code under the nucleotide sequence. This amino acid sequence is homologous to the coding region of other herpes virus gl homologs (see Figure 14).

Figure 14

Comparison of amino acids between the partial amino acid sequence of the putative BHV-1 gl gene and the corresponding parts of the coding regions of the herpes simplex virus gl gene (" herpes simplex virus " or HSV1), pseudo virus gp63 gene (" pseudorabies virus " or PRV) and the gpIV gene of the " varicella-zoster virus " (VZV). The PRV sequence starts at amino acid 82, the HSV1 sequence begins at aa 80 and the sequence VZV begins at αα 76 of its respective coding regions. The sequences used were published in the articles referenced in the legends of Figure 4. The comparison was carried out using the Multalin computer program. Asterisks indicate identical amino acids and are indicated by similar amino acids.

Figure 15

Construction of plasmid MSVneoGI for eukaryotic expression of the BHV-1 gl gene The putative position of the BHV-1 gl gene was determined based on the amino acid comparison of the partial sequence of the BHV-1 gl gene. Based on this determination, it was inferred that the 1.7 kb SmaI fragment should contain the entire coding region of the BHV-1 gE gene. The position of this 1.7 kb SmaI fragment is indicated in A. blunt ends (" blunt ends ") of this 1.7 kb SmaI fragment were ligated " BamHI " linkers, according to standard procedures. The resulting product was digested with BamHI and ligated into the MSV-neo eukaryotic expression vector. The MSV-neo vector has a unique BamHI site behind MSV-LTR, which exhibits intense promoter activity. This vector was described in Rijsewijk et al., 1987 EMBO J. 6, 127-131.

Figure 16

Construction of a BHV-1 double gl / gE deletion fragment The position of the gE glycoprotein gene and the putative position of the gly glycoprotein gene in the Ug region of BHV-1 are plotted in diagram A. The dotted blocks indicate the sequences of repetition lining the Ug region. B represents the physical map of some essential restriction enzyme sites relative to the position of both genes. To construct the deletion fragment gl / gE clone pl.7-SmaI / o containing 55 55

The 1.7 kb SmaI fragment spanning the gl gene must be digested with PstI. The ends of the Pstl site of the remaining 350 bp Smal-PstI insert insert are blunted using standard molecular biology techniques. The ends of the EcoNI-SmaT fragment (see Figure 6B), isolated from the 4.1 kb HindIII-EcoRI fragment depicted in Figure 6A are also blunt and the fragment is ligated to the modified Pstl site. This process is shown diagrammatically in C and D. The 1.4 kb Smal-Dral fragment can be isolated from the resulting p5IE clone to be recombined with wild-type BHV-1 DNA.

Abbreviations: E = EcoRI, H = HindIII, S = Smal, P = Pstl, ENI = EcoNI, D = Dral, kb = kilobase and Ug = Unique Short (" Unique Short ").

Figure 17

Mean virus release by nasal route in calves after vaccination. = vaccinated with Difvac-1, 0 = unvaccinated controls

Figure 18

Mean daily clinical values were calves after challenge with a virulent strain of BHV-1, key as in Fig.

Figure 19

Mean rectal temperature in calves after challenge with a virulent strain of BHV-1, key as in Fig.

Figure 20

Average growth of calves after challenge with a virulent BHV-1 strain, key as in Fig.

Figure 21

Mean virus release of nasal virus in calves after challenge with a virulent strain of BHV-1, key as in Fig.

Figure 22

Mean rectal temperature in calves after challenge with a virulent strain of BHV-1. = vaccinated with Lam gE, 0 = vaccinated with Lam gE / TK, x = unvaccinated controls.

Figure 23

Average growth of calves after challenge with a virulent BHV-1 strain, key as in Fig. 22.

Figure 24

Mean daily clinical values in calves after challenge with a virulent strain of BHV-1, key as in Fig.

TABLE 1

Virus delivery by nasal route in calves after vaccination with Lam gE or Lam gE / TK and after challenge with a virulent BHV-1 strain of the vaccinated and control calves

Mean number of days of nasal virus release Group After vaccination After challenge Control 0 10.33 ± 1.51 Lam gE ~ 7.00 ± 0.89 4,83 ± 1,17 Lam gE ~ / TK '7, 17 ± 1.33 5.17 ± 0.98 TABLE 2

Characterization of gE-Mabs

Mab REACTIVITY OF GE-MABS CANDIDATES WITH Difivac-1 3T3 / EBTR Lam gE &quot; Prok. 3T3 gE · 3T3 gE Difivac-1 3T3 gE / gl Ag Ag Group Ab 1 - - nd - + 7 I + 2 - - - + + + II - 3 - - + + + + 7 - 4 + - + + + + 7 - 42 - - nd - - ·? V? - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - + + + + - + - + - + - + + + + + - + - + - +: All 8 sera tested have a percentage blocking value of> 50% in an indirect blocking IPMA (&quot; indirect blocking IPMA &quot;) analysis. ±: Sera have a blocking percentage value of + 50%. -: Serums have a percentage blocking value < 50%. SEQUENCE LISTING SEQ ID NO: 1 LENGTH: 2027 nucleotides, 575 amino acids TYPE: nucleotide and amino acid STRANDEDNESS: single

60 &gt; deleted in Difivacl 120

iGGCA TTTGGCA 157

gg cgg cgg rrc- c

C ctg cr: 215

10 == PEPTIDEO SIGN

60

%

PvuII CTC GCC GAC CCS CAC GTC TCG GCG CAG CTG GGT CGC AAC GCG ACC GGG 599 Leu Ala Aso Pro K's Val Se 'Ala Gin Leu Glv Aro, 2. C r-> Î ± 3 Thr G1 γ 130 135 140 GTG CTG ATC GCG GCC GCA GCC GAG GAG GAC GGC GGC GTG TAC TTC CTG 647 Val Leu Xle Ara A.iâ Ala Ara G-u. Glu Asp Glv Glv Val Tyr Phe Leu 145 150 155 160 TAC GAC CGG CTC ATC GGC GAC GCC GGC GAC GAG GAG ACG CAG TTG GCG 695 Tyr Asp Arg Leu lie Giy Asp Ala Giy Asp Glu Glu Thr Gin Leu Ala 165 170 175 CTG ACG CTG CAG GTC GCG ACG GCC GGC GCG CAG GGC GCC GCG CGG GAC 743 Leu Thr Leu Gin Val Ala Thr Ala Giy Ala Gin Giy Ala Ala Arg Asp 180 185 190 GAG GAG AGvj GAA CCA GCG ACC GvG CCC ACC CCC GGC CCG CCG CCC CAC 791 Glu Glu Ara Glu Pro Ala Thr C-I and Pro Thr Pro Glv Pro Pro Pro Kis 195 200 205 CGC ACG ACG ACA CGC GCG CCC CCG CGG CGG CAC GGC GCG CGC TTC CGC 839 Ar Thr Thr Thr Arg Al Pro Pro Arg Arg Kis Giy Ala Arg Phe Arg 210 215 220 Srrval GTG CTG Val Leu CCG? ro TAC CAC TCC CAC C-TA Tyr Kis Ser Kis Val TAC Tyr ACC CCG GGC Thr Pro Glv GAT Asp TCC Ser TCT CTG Phe Leu 887 225 230 235 240 CTA TCG GTC- CGT CTG CAG TCT GAG rrrnrf TTC GAC C-AG GCT CCC TTC TCG 935 Leu Ser Val Arç Leu Gin Ser Glu Phe Phe Asp Glu Ala Pro Phe Ser 245 250 255 GCC AGC ATC GAC TGG TAC TTC CTG CGG ACG GCC GGC GAC TGC GCG CTC 983 Ala Serine Asp Tro Tvr Phe Leu Arg Thr Ala Giy Asp Cys Ala Leu 250 265 270 ATC CGC ATA TAC GAC-ACG TCC ATC TTC CAC CCC GAG GCA GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCCGGGGGGGGGGGGGGCCGGGGGGGCCGGGGGGGGGGGGGGGGGGGGGGGGGCGGGGGGGGGGCGGGGGGGGGGGGGGPGGGGGGGGGCCGGGGGGGGGGGCCGGGGGGGGGGGGGGCCGGGGGGGGGGGGGGGGGGGGGGC ccc G «-w GA.C GCG CAG TGC AGC TCC GCG TCG CCG TAC 1079 Leu Kis Pro A-â A.sp Ala Gir. Cvs Ser Phe Ala Ser Pro Tyr Arg Ser 290 295 3C0 GAG ACC GTG TAC AGC CGG CTG TAC GAG CAG TC-C CGC CCG GAC CCT GCC 1127 Glu Thr Val Tyr Ser Arç Leu Tyr Glu Gin Cy3 Arg Pro Asp Pro Ala 305 310 315 320 61

ggc Gly CoC Arg CGG Trp CCG Pro CAC GAG His Glu 325 Cys GAG GGC GCC Glu GIv Ala 330 GCG Ala TAC Tyr GCG GCG CCC Ala Ala Pro 335 GCC Va 1175 GCG Ala CAC K 3 ccc-Leu CGC Arg CGC GCC Pro Ala AAC a AAC AGC GTA GCC GAC GCC CCG TAC GAC G P A A GAC Aso 1223 340 345 350 GCG GCC Wing GCC Wing 355 GCG Wing GCC TCC Wing GGG Gly CCC TAC GCC Leu Tyr Vai 360 CCC Phe GCG. Λν, Leu Glr. Tyr 365 AAC Asn 1271 GGC CAC C-TG HindiII GAA GCC GG TAC AGC CTA GCC GCC ACC TCG GAC CGC 1319 Gly Ki 370 Go Glu Ala Trp Aso 375 Cyr Ser Leu Vai Go 380 Thr Ser Asp Arg CCG Leu 385 G7G Go to CGC Arg GCG Wing GCC ACC Go Thr 390 GAC Asp CAC ACG CGC His Thr Arg CCC Pro 395 GAG Glu GCC GCA GCC Ala Ala Ala GuC Ala 400 1367 GAC GCC CCC GAG CCA GGC CCA CCC-CCC ACC AGGGG CCG GCGG-sjC GCG Asp Ala Pro Glu Pro 405 Gly Pro Pro Leu Thr 410 Ser Glu Pro Ala Gly Ala 415 1415 CCC ACC GC-G CCC GCG CCC CGC-CCC C-CG C-CG CTG GCG GGC GCG CCC GGA 1453 Pro Thr Gly Pro 420 Ala Pro Trp Leu Go 425 Go Leu Go C-iy Ara 430 Leu Gly

==== TRANSMEMBRAN HELIX ====== CTC GCG CCA CTC-CC read Al Gly leu V'e 4 35 GCA GCC CTC GCC GCC CGG C- 'Ala A- to Leu Ala Vai Arg V 440 445 Cy2 Ala L511 CGC CGC GCA AsjC CAG AAG CGC * T. &quot; and Arg Arg Ala Ser Glr. Lys Λ-Tr. r Tyr 450 455 CCC GTA ACC AoC 7Tj CCG AGC AAC? - 2 Go Tv r T'r. r Ser Leu Pro Asr. 1 &quot; j 473 CCA GCC AG ^ - G.A-w GAC G.AA TCC CCC CCC Pro Will Be Asp Asp 455 Glu Phe Ser Leu GAC ATG CTG AAC CCC CCC GGG 1559 Λ 9 I Le Leu Asn 460 P r Ghe Gly G.n. CGG CTC GAC GCG GCG 1607 G 'u Pro 4 7 5 Leu A.sp Go Go Go 430 GAC gc CCAC C-CG GAC 1555 A cr 450 Glu A.sp Ser Pr.e A_a Asp 4 55 62 GAC GAC AGC C-AC GAC GAC GGG CCC GCC AGC AAC CCC CCC GCG GAC GC ^ Asp Asp Asp Asp Asp Asp Gly Pro Ala 505 Ser Asn Pro Pro Ala 510 Asp Ala TAC GAC CCC GCC GGC GCC CCA GAG CCA ACT AGC GGG TCC GCG CGA GCC • Tyr Asp Leu 512 Ala Gly Ala Pro Glu £ 20 Pro Thr Ser Gly Phe 525 Ala Arg Ala CCC CC AAC GGC ACG CGC TCG AGC CGC TCC GGG TCC AAA GCC TGG TTT Pro Ala 530 Se · * - Arg Ser 535 Ser Arg Ser Gly Phe Lys 540 Val Trp Phe AGG GAC CCG CTC GAA GAC GAC GCC GCG CCA GCG CGG ACC CCG GCC GCA \ Arg 545 Asp? Ro Asp 550 Asp A5 Asp 550 Ala Ala Pro Ala 555 Arg Th-Pro Ala EcoNI Ala 560 CCA GAC CAC ACC GCG GTA GOA GCG CAG AAG rpr - - - - - - - - - - TAG Pro Asp Tyr Thr Val 565 Val Ala Ala Arg Leu 570 Ly3 Ser Xle Leu Arg 575 * 1703 1751 1759 1847 1895

GCGCCCi ACA.CAA. CGCAGC

CGGAAA GCACCCGC37 GTAGGGCTC-C IGCCGCT TCGAAGGCAC GGAGAC-CCCA 1955 20152027

SEQ ID NO: 2 LENGTH: 20 nucleotides TYPE: nucleotide STRANDEDNESS: single ACGTGGTGGT GCCAGTTAGC 20

SEQ ID NO: 3 LENGTH: 22 nucleotides TYPE: nucleotide STRANDEDNESS: single ACCAAACTTT GAACCCAGAG CG 22

Lisbon, August 25, 1992

J. PEREIRA DA CRUZ

Official Agent of Industrial Property RUA VICTOR CORDON, 10.A 3 * 1200 LISBOA

Claims (7)

Is a deletion mutant of bovine herpes virus type 1, characterized by a deletion in the gE glycoprotein gene. 22 - Defective mutant of bovine herpes virus type 1, characterized by a deletion in the gE glycoprotein gene, which deletion was provoked by an attenuation procedure. 32 - Difivac-1 deletion mutant of bovine herpes virus type 1, characterized by a deletion in the gE glycoprotein gene, which deletion was provoked by an attenuation procedure. 4 § - Defective mutant of bovine herpes virus type 1, characterized by a deletion in the gE glycoprotein gene, a deletion that was constructed by means of DNA recombination techniques. 5a - 1B7 or 1B8 deletion mutant of bovine herpes virus type 1, characterized by a deletion in the gE glycoprotein gene, which deletion was constructed using DNA recombination techniques. A deletion mutant of bovine herpes virus type 1, characterized in that it has a deletion in the gE glycoprotein gene and a deletion in the thymidine kinase gene. 2 A deletion mutant of bovine herpes virus type 1, characterized by a deletion in the glycoprotein gE gene and a deletion in the gly glycoprotein gene. 8a A bovine herpes virus type 1 deletion mutant characterized by a deletion in the gE glycoprotein gene, a deletion in the thymidine kinase gene and a deletion in the glycoprotein gl gene. 9a - Bovine herpes virus type 1 mutant, characterized as having a deletion in the gE glycoprotein gene and containing a heterologous gene introduced by means of DNA recombination techniques. 10. A bovine herpes virus type 1 mutant, characterized in that, at the position of the gE glycoprotein gene, it contains a heterologous gene introduced by DNA recombination techniques, which is under the control of regulatory sequences, of the gE gene or a heterologous gene, and is optionally linked to that part of the gE gene encoding a signal peptide. 11a - A bovine herpes virus type 1 mutant, characterized in that, in addition to a deletion in the gE glycoprotein gene, there is a deletion in the thymidine kinase gene, a deletion in the glycoprotein gl gene, or both, and containing a heterologous gene introduced by means of DNA recombination techniques in the position of at least one of these deletions. 12. A bovine herpes virus type 1 mutant, characterized by having a deletion in the gE glycoprotein gene and containing a heterologous gene which has been introduced by means of DNA recombination techniques and which encodes a protein or immunogenic peptide of another pathogen or which encodes a cytokine. A composition comprising a recombinant nucleic acid containing the bovine herpesvirus type 1 glycoprotein gE gene, a part of this gE glycoprotein gene, or a nucleotide sequence derived from this gE glycoprotein gene. A composition comprising a cloning or expression vector having an insertion of a recombinant nucleic acid containing the bovine herpes virus type 1 gE glycoprotein gene, a part of this gE glycoprotein gene or a derived nucleotide sequence of this gE glycoprotein gene. A composition comprising bovine herpes virus type 1 GE glycoprotein, a part of this glycoprotein gE, a peptide derived from this glycoprotein gE, or a complex formed by the gE and gl glycoproteins of bovine herpes virus type 1. - A composition comprising an antibody which is specific to the bovine herpes virus type 1 glycoprotein gE, to a part of this glycoprotein gE, to a peptide derived from that glycoprotein gE, or to a complex formed by the gE and gl glycoproteins of the virus of bovine herpes type 1. 17a - composition, characterized in that it comprises a monoclonal antibody which is specific to the bovine herpes virus type gE glycoprotein type 1, to a part of said glycoprotein gE, to a peptide derived from this glycoprotein gE, 4 or to a complex formed by the glycoproteins gE and gl of bovine herpes virus type 1. Composition comprising a polyclonal antibody which is specific to the glycoprotein gE of bovine herpes virus type 1, a part of this glycoprotein gE, a peptide derived from this glycoprotein gE, or a complex formed by the glycoproteins gE and gl of bovine herpes virus type 1. 19A - A vaccine composition for the vaccination of animals, particularly mammals, more especially bovine, for the purpose of protecting them from bovine herpes virus type 1, characterized in that it comprises a mutant of the bovine herpes virus type 1. bovine herpes virus type 1 according to any one of Claims 1-12, and a suitable carrier or adjuvant. A vaccine composition for the vaccination of animals, in particular mammals, more especially bovine, for the purpose of protecting them from a pathogenic agent, characterized in that it comprises a mutant of bovine herpes virus type 1 which has a deletion in the gE glycoprotein and contains a heterologous gene that has been introduced by means of DNA recombination techniques and encoding a protein or peptide immunogenic of the pathogen, and a suitable vehicle or adjuvant. A diagnostic kit for the detection of bovine herpes virus type 1 nucleic acid in a sample, in particular in a biological sample such as blood or blood serum, cells in blood, milk, blood body fluid such as tears, washing fluid from the lungs, nasal fluid, sperm, in particular semen, saliva, sputum or in particular tissues of the nervous system, from an animal, in particular from a mammal, more especially from a bovine animal, characterized in that it comprises a nucleic acid probe or primer having a nucleotide sequence derived from the gE glycoprotein gene of bovine herpes virus type 1, and a detection medium suitable for a nucleic acid detection test. 223 - Diagnostic equipment for the detection of specific antibodies to bovine herpes virus type 1, in a sample, in particular in a biological sample such as blood or blood serum, saliva, sputum, body fluids such as tears, lungs, nasal fluid, milk or tissues, from an animal, in particular from a mammal, more particularly from a bovine animal, characterized in that it comprises bovine herpes virus type gE glycoprotein, a part of this glycoprotein gE, a peptide derived from that glycoprotein gE, or a complex formed by bovine herpes virus type 1 gE and gl glycoproteins, and a detection means suitable for an antibody detection test. A diagnostic kit according to claim 22, further comprising one or more specific antibodies to the bovine herpes virus type gE glycoprotein type 1, or to a complex formed by the bovine herpes virus type 1 gE and gl glycoproteins Diagnostic equipment for the detection of bovine herpes virus type 1 virus in a sample, in particular in a biological sample such as blood or blood serum, cells in the blood, milk, body fluids such as tears, washing of the lungs, nasal fluid, sperm, in particular semen, saliva, sputum or tissues, in particular tissues of the nervous system, coming from an animal, in particular from a mammal, more especially from a bovine, characterized in that it comprises a to bovine herpes virus type 1 GE glycoprotein, or to a complex formed by the gE and gl glycoproteins of the virus of bovine herpes type 1, and a detection medium suitable for a protein detection test. 25. A method of determining the infection of bovine herpes virus type 1 in an animal, in particular in a mammal, more especially in a bovine animal, comprising examining a sample from the animal, in particular a biological sample such as blood or blood serum , blood cells, sperm, in particular semen, saliva, sputum, body fluids such as tears, lung lavage fluid, nasal fluid, milk or tissues, in particular tissues of the nervous system, relative to the presence of nucleic acid which include the bovine herpes virus type 1 gE glycoprotein gene or the presence of bovine herpes virus type 1 glycoprotein gE or a complex formed by the bovine herpes virus type 1 gE and gl glycoproteins or the presence of specific antibodies relative to bovine herpes virus type 1 glycoprotein gE, or to a complex formed by herpes virus gE and gl glycoproteins bovine type 1. A method of determining the infection of bovine herpes virus type 1 in an animal, in particular in a mammal, more especially in a bovine animal, comprising examining a sample from the animal, in particular a biological sample such as blood or blood serum, cells in the blood, sperm, in particular semen, saliva, sputum, body fluids such as tears, lung lavage fluid, nasal fluid, milk or tissues, in particular tissues of the nervous system, relative to the blood. presence of nucleic acid comprising the bovine herpes virus type 1 gE glycoprotein gene or the presence of the bovine herpes virus type 1 gE glycoprotein gene or a complex formed by bovine herpes virus type 1 gE and gl glycoproteins , or to the presence of specific antibodies to bovine herpes virus type 1 glycoprotein gE or to a complex formed by the glycoprotein s gE and gl of bovine herpes virus type 1, the sample to be analyzed coming from an animal that has been vaccinated with a vaccine preparation according to claim 19. Lisboa, August 25, 1992 J. PEREIRA DA CRUZ Official Agent of Industrial Property RUA VtCTOR CORDON. 10-A 3. »1200 USBOA