AU724849B2 - New Brucella antigens, recombinant polypeptides, nucleic acids coding for the same and use thereof in diagnostic and prophylactic methods and kits - Google Patents

Description

WO 98/08951 PCT/EP97/04668 1 NEW BRUCELL. A.4NTIGENS. RECOMBINANT POLYPEPTIDES, NUCLEIC ACIDS CODING FOR THE SAME AND USE THEREOF IN DIAGNOSTIC AND PROPHYLACTIC METHODS AND KITS The present invention relates to isolated and pure Brucella antigens, to nucleic acids encoding said antigens, as well as to diagnostic methods and kits using said antigens and nucleic acids for detecting Brucella infection in human and cattle. The invention also relates to recombinant polypeptides, a process for preparing the same and their use in methods and kits for the diagnosis of Brucella infection. The invention also relates to the use of said isolated antigens, or said recombinant polypeptides. or said nucleic acids as an active principle of a vaccine composition against Brucella infection. The invention relates also to a vaccine composition comprising a recombinant Brucella strain, specifically deleted for the gene(s) encoding said antigen(s).

Brucellosis is an infection due to a small intracellular gram-negative bacterium which is pathogenic for humans as well as for domestic animals. This infection induces abortions in livestock animals leading to severe economic losses. Within the genus Brucella, six closely related species have been described (Fekete et al.. 1992; Verger et al., 1985; Verstraete Winter, 1984), the most important of which are B. abortus and B. melitensis. Humans and ruminants (sheep, goats and cows) are predominantly infected by these two Brucella strains.

Although the humoral immune response is important in controlling Brucella infection mainly during secondary infection (Cannat et al., 1978), Brucella as a facultative intracellular pathogen elicits primarily a cellular immune response and evidence has been reported that protection from infection and elimination of B. abortus requires the cell-mediated immune response (Araya et al., 1989; Baldwin et al. 1985: Pavlov et al. 1982).

The diagnosis of bovine brucellosis is mainly based on serological tests to detect the presence of anti-lipopolysaccharide (LPS) antibodies (Abs). However, these tests by themselves do not allow the detection of all Brucella-infected animals (Blasco et al. 1994; Dohoo et al. 1986; Diaz-Aparicio et al. 1994). The problems involved include a lack of sensitivity to detect Abs directed against Brucella during early infections or in latently infected carriers and the inability to distinguish, in some circumstances, vaccinated animals from WO 98/08951 PCT/EP97/04668 2 animals infected with virulent strains (Alton, 1978; Nicoletti, 1980). Furthermore, serological cross-reactions between B. abortus and many other bacterial species were described (Corbel, 1985; Garin-Bastuji et al. 1992; Weynants et al. 1995)).

The delayed type hypersensitivity (DTH) test (=intradermic test) has been widely used for the diagnosis of brucellosis in ruminants and various allergens from different Brucella species have been prepared for this purpose (Hoffman et al, 1990, Jones et al. 1973, Smith et al. 1990)). Allergic diagnosis is actually used in a number of countries but the type and specificity of reactions induced in animals and the sensitivity of the methods are variable; indeed, the nature of the allergens differs depending on the strains used, the culture techniques, the method of production and purification (Bhongbhibhat et al., 1970; Jones et al.

1 9 7 3 ;Ottosen et al. 1949).

Among these allergens, Brucellergen® (Rh6ne-Mrieux), consisting of a mixture of to 30 cytoplasmic proteins and prepared from a rough strain of B. melitensis B115 (Jones et al. 1973) has proved to be valuable in detecting Brucella-infected bovines (Fensterbank, 1977) and is a useful diagnostic method complementary to serology (Fensterbank, 1982).

However the intensity of the reaction is weak in comparison with the tuberculin reaction and makes the interpretation of the results difficult. Furthermore, although a R strain of Brucella was used to prepare Brucellergen, this strain is not devoid of LPS-like molecules (Cloeckaert et al. 1992); therefore, in addition to proteins, the different batches may contain various amount of LPS. LPS does not seem to be stimulatory for B. abortus specific bovine lymphocytes in vitro (Brooks-Alder et al. 1988); however, the presence of LPS antigen in the allergen may perturb the DTH reaction and induce an antibody response which may interfere with subsequent serological tests. Furthermore the variation in protein content of the various batches may be one cause of the heterogeneity of the DTH response.

The identification of specific antigens for brucellosis diagnosis is therefore a matter of great interest for the development of a specific serological and/or intradermic test.

In order to produce an allergen made of a limited number of components, the identification of biologically active molecules for the DTH test is a prerequisite. And, although identification of T-immunodominant Brucella cytoplasmic or soluble proteins has 9 been frequently reported (Brooks-Alder et al., 1988; Brooks-Worrel et al. 1992a: 1992b; Stevens et al. 1994a; 1994b; 1995; Zhan et al. 1993a; 1993b), only one brucellin- WO 98/08951 PCT/EP97/04668 3 immunodominant protein, the ribosomal protein L7/L12, has been identified and the activity of the recombinant antigen studied in detail (Bachrach et al. 1994; Oliveira et al. 1994).

Moreover, as Brucella abortus induces a Thl cytokine pattern from human T cells (Ficht et al. 1989a, 1989b), the identification of specific antigens that preferentially induce a Thl subset response would provide a relevant way for the design of candidate molecular vaccines.

For prophylactic vaccination against brucellosis, today two live vaccine strains are being used succesfully. The B 19 abortus) strain is mostly used in cattle and the Rev. 1 strain in small ruminants. The H38 killed vaccine has also been used. Although good protection is generally obtained with these vaccines, the general drawback is the inevitable induction of an immune response in the vaccinated animals, which precludes the distinction between field-infected and vaccinated animals.

In this respect, it may be useful to generate a vaccine strain which is immunologically distinct from the strains responsible for field-infection. This would allow the distinction between vaccinated and field infected individuals if an appropiately designed diagnostic test is used. The vaccine strain can be given an immunological "signature" by genetic engineering techniques.

The aim of the present invention is to provide new Brucella antigens and polynucleic acids encoding the same which are useful for the in vitro diagnosis and/or prevention of Brucella infection brucellosis) in mammals (humans, ruminants).

More particularly it is an aim of the present invention is to provide new Brucella antigens useful in the serodiagnosis of brucellosis in mammals.

Another aim of the present invention is to provide new Brucella antigens useful in an assay for measuring the cellular immune response of Brucella infected individuals.

Another aim of the present invention is to provide new Brucella polynucleic acids which are useful in an assay for detecting the presence of Brucella nucleic acids in a sample.

Another aim of the present invention is to provide new Brucella antigens or polynucleic acids encoding the same which may be used as components of a molecular vaccine for S preventing brucellosis in mammals.

An additional aim of the present invention is to provide Brucella antigens or polynucleic acids encoding the same which are useful for differentiating between field infected and vaccinated individuals.

It is further a specific aim of the present invention is to provide purified and isolated 39kDa and 15kDa antigens of Brucella, and more particularly purified and isolated 39kDa and antigens of B. abortus.

Another specific aim of the present invention is to provide amino acid and corresponding nucleotide sequences of a Brucella abortus 39kDa antigen.

Another specific aim of the present invention is to provide amino acid and corresponding nucleotide sequences of a Brucella abortus 15 kDa antigen.

Another aim of the present invention is to provide antibodies specifically directed against said Brucella 39kDa and 15 kDa antigens.

Another aim of the present invention is to provide primers and probes derived from the nucleotide sequences encoding said Brucella 39 kDa and 15 kDa antigens.

Another aim of the present invention is to provide diagnostic methods and kits for diagnosing brucellosis in mammals, using the above-mentioned 39 kDa and/or 15 kDa antigens as their principle reagents.

More particularly, it is an aim of the present invention to provide diagnostic methods and kits for differentiating between Brucella field-infected and vaccinated individuals, using the above-mentioned 39 kDa and/or 15 kDa antigens as their principle reagents.

I Another aim of the present invention is to provide vaccine compositions conferring protective immunity towards brucellosis in mammals (humans, ruminants), with said vaccine compositions comprising the above-mentioned Brucella 39 kDa and/or 15 kDa antigens as their immunogenic component(s).

Another aim of the present invention is to provide a recombinant Brucella strain in which the gene encoding the Brucella 39kDa antigen and/or the gene encoding the Brucella 15 kDa antigen has been deleted or inactivated.

Another aim of the present invention is to provide a vaccine composition conferring protective immunity towards brucellosis in mammals and allowing subsequent differentiation between field infected and vaccinated individuals, said vaccine composition comprisinm a recombinant Brucella strain in which the gene encoding the Brucella 39kDa antigen and/or the gene encoding the Brucella 15 kDa antigen has been deleted or inactivated.

The present invention relates to an isolated Brucella antigen or a fragment thereof, wherein said antigen or fragment is characterized by an amino acid sequence showing at least 60%, preferably at leat 70%, more preferably at least 80% homology to any of the amino acid sequences as shown in SEQ ID NO: 2 or 4, wherein said fragment consists of at least 9 contiguous amino acids of SEQ ID NO: 2 or 4.

The term "isolated" refers to a purity grade of at least 90%, preferably 95 and more preferably of 98% of the antigen expressed in weight versus contaminants, as determined by one or two dimensional SDS-PAGE. Said purity may be obtained by purification of the naturally occurring polypeptide, or by de novo synthesis of the polypeptide, by chemical methods or by recombinant DNA technology, and subsequent purification. The term "isolated" thus implies that the antigen is in a different state and environment than the naturally occurring antigen.

The word "antigen" refers to a molecule which provokes an immune response (also called "immunogen"), or which can be recognized by the immune system (also called "antigen sensu strictu"). The immune response or the immune recognition reaction can be of the cellular or humoral type.

In what follows, the terms "39kDa antigen" and "15kDa antigen" refer to antigens S having an approximate molecular weight of respectively 39kDa and 15kDa as determined by SDS-PAGE. Said determined molecular weight may vary according to strain to strain variations, or according to methodology variations.

The terms "homologous" and "homology" are used in the current invention as synonyms for "identical" and "identity"; this means that amino acid sequences which are e.g.

said to be 55% homologous, show 55% identical amino acids in the same position upon alignment of the sequences.

According to a preferred embodiment, the invention relates to a polypeptide or peptide comprising in its amino acid sequence part of any of the amino acid sequences as represented in SEQ ID NO 2 or 4, said part consisting of at least 9 contiguous amino acids selected from any of the amino acid sequences represented in SEQ ID NO 2 or 4.

In a very specific embodiment the invention relates to an isolated 39kDa Brucella abortus antigen, also called P39 antigen, characterized by the 401 residue amino acid sequence as shown in figure 3 (SEQ ID NO or a fragment thereof, said fragment consisting of at least 9 contiguous amino acids of the amino acid sequence represented in SEQ ID NO 2.

WO 98/08951 PCT/EP97/04668 6 In a very specific embodiment the invention relates to an isolated 15kDa Brucella abortus antigen, also called Br25 antigen, characterized by the 140 residue amino acid sequence as shown in figure 4 (SEQ ID NO or a fragment thereof, said fragment consisting of at least 9 contiguous amino acids of the amino acid sequence represented in SEQ ID NO 4.

The fragments of the above-mentioned polypeptides are more than 8, preferably more than 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 50, 55 or 60 amino acid residues long, said amino acids being contiguous amino acids selected from the amino acid sequences of any of the polypeptides described above.

The 39kDa Brucella abortus polypeptide (P39) of SEQ ID NO 2, and the corresponding nucleic acid of SEQ ID NO 1, are new. The gene encoding the 39 kDa antigen is also referred to as bru39 gene, or P39 gene.

The 15kDa Brucella abortus polypeptide of SEQ ID NO 4, and the corresponding nucleic acid of SEQ ID NO 3, are new. The gene encoding the 15kDa antigen is also referred to as the br25 gene.

A preferred embodiment of the present invention relates to any of the polypeptides or polypeptide fragments as described above, with said polypeptides or polypeptide fragments having at least one of the following immunological (antigenic) properties: being specifically recognized by sera from Brucella field infected individuals, and/or being specifically recognized by the cellular immune response from Brucella contacted individuals, and/or being able to elicit a Brucella specific immune response when used for vaccination of individuals prone to brucellosis disease.

Of the above-mentioned immunological properties, the specific recognition by sera from field-infected individuals is the most preferential one.

An additional characteristic of some of the polypeptides of the invention is the differential recognition by sera from field-infected individuals as opposed to sera from vaccinated individuals.

The wording "(Brucella) field-infected individuals" as used in the current invention refers to individuals which have been infected by the Brucella pathogen, and in which the infection leads to brucellosis disease.

The wording "(Brucella) vaccinated individuals" as used in the current invention refers WO 98/08951 PCT/EP97/04668 7 to those individuals which have been vaccinated against brucellosis, and in which the vaccination has lead to protection of the individual against active disease and against vertical transmission of the pathogen.

The wording "(Brucella) contacted individuals" as used in the currrent invention refers to individuals which have been in contact with the Brucella pathogen, said contact possibly leading to subsequent disease, or to subsequent protection.

The expression "specifically recognized by sera from Brucella field-infected individuals" refers to the fact that the Brucella antigens of the present invention show preferentially a positive immunological reaction with sera from Brucella field infected individuals, whereas they show preferentially no reaction with sera from "control" individuals (such as healthy individuals or individuals infected by other pathogens). Thus, sera originating from Brucella field infected individuals preferentially contain antibodies reacting with the Brucella antigens of the present invention, while sera originating from control individuals preferentially don't contain antibodies recognizing these Brucella antigens. This will be demonstrated in the Examples section. More particularly, it is shown that at least 30%, more preferably at least 40%, most preferably at least 50% or even at least 60 of the sera from field-infected individuals react with at least one of the polypeptides of the invention, while at most 10%, preferably at most 5 and most preferably at most 1% of the sera from control individuals react with the polypeptides of the invention.

It should be understood that, since the above-mentioned reactivities are usually a result of optical density measurements, e.g. in a standard ELISA assay, positive and negative values have to be considered against a so called "cutoff value", which is not an absolute value, but which can be calculated from the optical density values of the control sera cutoff value mean optical density of the control sera 2 (or 3) standard deviations). This is demonstrated further in the examples section.

The term "individual" refers to an animal or human being liable to be infected by Brucella (species). Brucellosis infects mammals, mostly humans and ruminants.

The expression "to elicit a Brucella specific immune response" refers to the fact tha: the Brucella immunogens according to the invention are able to give rise to the production of antibodies which are specific for Brucella, or that they elicit a cellular immune response specific for Brucella, in individuals liable to be infected by Brucella (species).

WO 98/08951 PCT/EP97/04668 8 The expresssion "specifically recognized by the cellular immune response" throughout the current application refers to the recognition of the polypeptides of the invention by the Tcell population of the Brucella contacted or infected or vaccinated individuals, said recognition being detectable in vitro e.g. by lymphoproliferation assays in presence of the polypeptides of the invention, or in vivo e.g. by a delayed type hypersensitivity reaction upon subcutaneous injection of the polypeptides of the invention in the individual.

As disclosed above, the present invention also relates to antigens characterized by an amino acid sequence showing a homology of at least 60%, preferably at least 70%, and even more preferably at least 80% or 90% to the amino acid sequence of any of the new Brucella abortus proteins as depicted in figures 3 and 4. Said "related" antigens may also be referred to as "analogues" of the proteins shown in SEQ ID NO 2 or 4. The term "analogues" may be defined as proteins containing substitutions and/or deletions and/or additions of one or several amino acids, provided that said analogues have retained the antigenic immunogenic properties of the Brucella proteins of the invention, i.e.: being specifically recognized by sera from Brucella field infected individuals, and/or being specifically recognized by the cellular immune response from Brucella contacted individuals, and/or being able to elicit a Brucella specific immune response when used for vaccination of individuals prone to brucellosis disease.

An overview of the amino acid substitutions which could form the basis of such analogues are given in Table 1.

Said "analogues" may be the result of strain to strain variations of the Brucella (species) antigens of the invention, or may be the result of modifications introduced in the original polypeptide sequences, said modifications bringing about a desirable side effect to the polypeptide molecule better physicochemical properties, more efficient purification, more efficient coating characteristics, more stable The 39kDa antigen of Brucella abortus, represented by SEQ ID NO 2, has homologous counterparts in other Brucella species, like e.g. Brucella melitensis, Brucella ovis, Brucella suis, etc. These homologous genes and proteins are also part of the present invention. The family of proteins homologous to the sequence as represented in SEQ ID NO 2 are called throughout the current specification "the 39kDa Brucella antigens".

WO 98/08951 PCT/EP97/04668 9 The 15kDa antigen ofBrucella abortus, represented by SEQ ID NO 4, has homologous counterparts in other Brucella species, like e.g. Brucella melitensis, Brucella ovis, Brucella suis, etc. These homologous genes and proteins are also part of the present invention. The family of proteins homologous to the sequence represented in SEQ ID NO 4 are called throughout the current specification "the 15kDa Brucella antigens".

It should be evident that said "analogues", although falling within the above-given definitions, may have a molecular weight which is slightly different from 39kDa or 15kDa as determined by SDS-PAGE.

The words "polypeptide" and "peptide" are used interchangeably throughout the specification and designate a linear series of amino acids connected one to the other by peptide bonds between the alpha-amino and carboxy groups of adjacent amino acids. Polypeptides can be in a variety of lengths, either in their natural (uncharged) forms or in forms which are salts.

and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications. It is well understood in the art that amino acid sequences contain acidic and basic groups, and that the particular ionization state exhibited by the peptide is dependent on the pH of the surrounding medium when the protein is in solution, or that of the medium from which it was obtained if the protein is in solid form. Also included in the definition are proteins modified by additional substituents attached to the amino acids side chains, such as glycosyl units, lipids, or inorganic ions such as phosphates, as well as modifications relating to chemical conversions of the chains, such as oxidation of sulfhydrvl groups. Thus, "polypeptide" or its equivalent terms is intended to include the appropriate amino acid sequence referenced, subject to those of the foregoing modifications which do not destroy its functionality.

Functional peptides according to the present embodiment of the invention can be readily determined by the person skilled in the art by applying any of the techniques teached in the Examples sections of the present invention or any other immunological and epitope mapping techniques known in the art.

The polypeptides of the invention, and particularly the shorter peptides, can be prepared by classical chemical synthesis.

The synthesis can be carried out in homogeneous solution or on solid phase.

For instance, the synthesis technique in homogeneous solution which can be used is the WO 98/08951 PCT/EP97/04668 one described by Houbenweyl in the book entitled "Methode der organischen chemie" (Method of organic chemistry) edited by E. Wunsh, vol. 15-1 et II. THIEME, Stuttgart 1974.

The polypeptides of the invention can also be prepared in solid phase according to the methods described by Atherton and Shepard in their book entitled "Solid phase peptide synthesis" (IRL Press, Oxford, 1989).

The polypeptides according to this invention can also be prepared by means of recombinant DNA techniques as described by Maniatis et al. (1982).

According to another embodiment, the invention relates to a polynucleic acid comprising a sequence of at least 10 contiguous nucleotides selected from: the polynucleic acid sequences which code for any of the polypeptides described above, or the polynucleic acid sequences which are degenerate as a result of the genetic code to the polynucleic acid sequences as defined in and which still encode a polypeptide as described above, or the polynucleic acid sequences which hybridize to any of the polynucleic acids as defined in or According to yet another embodiment, the present invention relates to a polynucleic acid sequence, in an isolated form, comprising a contiguous sequence of at least nucleotides, more particularly 11, 12, 13, 14, 15, 20 or more contiguous nucleotides selected from any of the polynucleic acid sequences as described here above.

The term "polynucleic acid" refers to a single stranded or double stranded nucleic acid sequence which may contain from 10 nucleotides to the total number of nucleotides of the polynucleotide sequence (such as for instance 20, 30, 40, 50, 60, 70, 80 or more nucleotides).

A polynucleic acid which is smaller than about 100 nucleotides in length is often also referred to as an oligonucleotide. A polynucleic acid may consist of deoxyribonucleotides or ribonucleotides, nucleotide analogues or modified nucleotides, or may have been adapted for specific purposes, such as for cloning purposes, or for in vivo therapy, or prophylaxis.

The expression "hybridizes to" refers to preferably stringent hybridization conditions (Maniatis et al., 1982), allowing hybridisation between sequences showing at least 70%, 90%, 95% or more homology with each other.

The expression "in isolated form" refers to the fact that said polynucleic acid is WO 98/08951 PCT/EP97/04668 11 preferably 90%, more preferably 95 most preferably 98 pure as measured by its weight versus the weight of possible contaminants.

The Brucella polynucleic acids according to this embodiment of the present invention are preferably more than 55% homologous, more preferably more than 65%, and most preferably more than 75% homologous more than 85%, more than 90%, more than homologous) to any of the nucleic acid sequences represented by SEQ ID NO 1 or 3.

The terms "homologous" and "homology" are used in the current invention as synonyms for "identical" and "identity"; this means that nucleic acid sequences which are e.g.

said to be 55% homologous, show 55% identical basepairs in the same position upon alignment of the sequences.

According to a subsequent embodiment, the invention relates to a polynucleic acid as described above comprising a sequence of at least 10 contiguous nucleotides selected from: the polynucleic acid sequences as represented by SEQ ID NO 1, or, the polynucleic acid sequences which are degenerate as a result of the genetic code to the polynucleic acid sequences as shown in SEQ ID NO 1, and which still encode a Brucella abortus 39kDa antigen as shown in SEQ ID NO 2, the polynucleic acid sequences which hybridize to the polynucleic acid sequences as defined in or According to a subsequent embodiment, the invention relates to a polynucleic acid as described above comprising a sequence of at least 10 contiguous nucleotides selected from: the polynucleic acid sequence as shown in SEQ ID NO 3, or, the polynucleic acid sequences which are degenerate as a result of the genetic code to the polynucleic acid sequences as shown in SEQ ID NO 3, and which still encode a Brucella abortus 15kDa antigen as shown in SEQ ID NO 4, the polynucleic acid sequences which hybridize to the polynucleic acid sequences as defined in or According to a preferred embodiment, the present invention relates to any nucleotide sequence which, upon expression, gives rise to a polypeptide sequence as represented in SEQ ID NO 2 or 4, or part thereof.

Another embodiment of the invention refers to a polynucleic acid as described above being comprised in a cDNA clone or a genomic clone, with said clone being obtainable by a WO 98/08951 PCTIEP97/04668 12 process comprising essentially the following steps: preparing a Brucella species genomic library and/or expression library, by digestion of chromosomal DNA of said Brucella species and ligation in an appropiate cloning and/or expression vector, and.

immunoscreening said Brucella species expression library with Brucella field infected individuals sera, or with antibodies raised against any of the polypeptides of the invention described above, and/or screening said Brucella species genomic library with a probe hybridizing specifically with a polynucleic acid of the invention as described above, and characterizing the positive clones obtained in the screening steps of and/or by means of sequence analysis, after the insert of the obtained clones has been isolated and possibly amplified by means of PCR, and possibly repeating step and using the obtained clone, or part thereof, as a probe to isolate the complete gene.

The term "clone" refers to a population of cells or organisms formed by repeated asexual division from a common cell or organism. To "clone a gene" means to produce many copies of a gene by repeated cycles of replication.

The term "genomic library" refers to a collection of clones, each clone containing a different insert of (c)DNA in a cloning vector, said insert corresponding to a different part of the genome of the respective organism. A "(genomic) expression library" enables the expression of the (c)DNA inserts into polypeptide fragments.

Another embodiment of the invention provides for an oligonucleotide probe comprising part of any of the polynucleic acid sequences as described above, with said probe being able to act as a specific hybridization probe for detecting the presence in a sample of a Brucella polynucleic acid according to the invention, or part thereof.

Preferably said oligonucleotide probe comprises at least 10 contiguous nucleotides, more preferably 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 30 or 50 contiguous nucleotides selected from any of the above-mentioned polynucleic acid sequences.

The term "probe" refers to single stranded sequence-specific oligonucleotides which have a sequence which is sufficiently complementary to hybridize to the target sequence to be detected.

WO 98/08951 PCT/EP97/04668 13 Probes according to this aspect of the present invention may be chosen according to any of the techniques known in the art. Probes may be provided hybridizing to Brucella species which cause brucellosis in different kinds of animals and in humans abortus, B. melitensis, B. ovis, B. More particularly, probes specifically hybridizing to certain species of Brucella may be provided. Under appropriate hybridization conditions, such probes may allow to distinguish different Brucella species present in a sample to be analyzed.

According to the hybridization solution (SSC, SSPE, etc.), these probes should be stringently hybridized at their appropriate temperature in order to attain sufficient specificity.

However, by slightly modifying the DNA probes, either by adding or deleting one or a few nucleotides at their extremities (either 3' or or substituting some non-essential nucleotides nucleotides not essential to discriminate between types) by others (including modified nucleotides or inosine) these probes or variants thereof can be caused to hybridize specifically at the same hybridization conditions the same temperature and the same hybridization solution). Also changing the amount (concentration) of probe used may be beneficial to obtain more specific hybridization results. It should be noted in this context, that probes of the same length, regardless of their GC content, will hybridize specifically at approximately the same temperature in TMACI solutions (Jacobs et al., 1988).

The latter implies that "variant probes" contemplated within this aspect of the present invention can be defined as probes hybridizing with the same specificity as the probe they are derived from, possibly under different, but stringent, hybridization and wash conditions (different solutions, different concentrations of buffer, different concentrations of probe, different temperatures).

The oligonucleotides used as primers or probes may also contain or consist of nucleotide analoges such as phosphorothioates (Matsukura et al., 1987), alkylphosphoro-thiates (Miller et al., 1979) or peptide nucleic acids (Nielsen et al., 1991; Nielsen et al., 1993) or may contain intercalating agents (Asseline et al., 1984).

As most other variations or modifications introduced into the original DNA sequences of the invention, these variations will necessitate adaptions with respect to the conditions under which the oligonucleotide should be used to obtain the required specificity and sensitivity.

However, the eventual results of hybridization will be essentially the same as those obtained with the unmodified oligonucleotides.

WO 98/08951 PCT/EP97/04668 14 The introduction of these modifications may be advantageous in order to positively influence characteristics such as hybridization kinetics, reversibility of the hybrid-formation, biological stability of the oligonucleotide molecules, etc.

The term "complement" refers to a nucleotide sequence which is exactly complementary to an indicated sequence and which is able to hybridize to the indicated sequences. It should be clear that all polynucleic acids of the invention, although only represented by one strand, also encompass the other complementary strand. This implies that all probes and primers specified may also be used in their complementary form, be it under different hybridization or amplification conditions.

The invention also relates to an oligonucleotide primer comprising part of any of the polynucleic acid sequences as described above, with said primer being able to initiate specific amplification of a Brucella polynucleic acid encoding any of the polypeptides of the invention as described above, or part thereof.

Preferably said oligonucleotide primer contains at least 10, more preferably at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 contiguous nucleotides of any of the polynucleic acid sequences as described above.

The term "primer" refers to a single stranded DNA oligonucleotide sequence capable of acting as a point of initiation for synthesis of an extension product which is complementary to the nucleic acid strand to be copied. The length and the sequence of the primer must be such that they allow to prime the synthesis of the extension products. Preferably the primer is about 5-50 nucleotides long, more preferably 10-30 nucleotides long. Specific length and sequence will depend on the complexity of the required DNA or RNA targets, as well as on the conditions of primer use such as temperature and ionic strength.

The fact that amplification primers do not have to match exactly with the corresponding template sequence to warrant proper amplification is amply documented in the literature (Kwok et al., 1990).

The amplification method used can be either polymerase chain reaction (PCR; Saiki et al., 1988), ligase chain reaction (LCR; Landgren et al., 1988; Wu Wallace, 1989; Barany, 1991), nucleic acid sequence-based amplification (NASBA; Guatelli et al., 1990; Compton, 1991), transcription-based amplification system (TAS; Kwoh et al., 1989), strand displacement amplification (SDA; Duck, 1990; Walker et al., 1992) or amplification by means of QB WO 98/08951 PCT/EP97/04668 replicase (Lizardi et al., 1988; Lomeli et al., 1989) or any other suitable method to amplify nucleic acid molecules using primer extension. During amplification, the amplified products can be conveniently labelled either using labelled primers or by incorporating labelled nucleotides. Labels may be isotopic C 2 P, etc.) or non-isotopic (biotin, digoxigenin, etc.).

The amplification reaction is repeated between 20 and 80 times, advantageously between and 50 times.

According to a further embodiment, the invention relates to a recombinant vector particularly for cloning and/or expression of any of the polynucleic acids of the invention as described above, with said recombinant vector comprising a vector sequence and at least part of any of the polynucleic acid sequences as described above, and wherein, in case of an expression vector, the coding sequence of said polynucleic acid sequence is operably linked to a control sequence capable of providing for the expression of the coding sequence by the specific host.

The expression "operably linked" refers to a juxtaposition wherein the components are configured so as to perform their usual function.. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of the coding gene.

The term "control sequences" refers to those sequences which control the transcription and/or translation of the coding sequences; these may include but are not limited to the promoter sequences, transcriptional and translational initiation (ribosome binding sites) and termination sequences. In addition, control sequences refer to sequences which control the processing of the polypeptide encoded within the coding sequence; these may include, but are not limited to sequences controling secretion, protease cleavage, and glycosylation of the polypeptide. The control sequences are usually provided by the vector but may also be comprised in the polynucleic acid to be expressed. The control sequences comprised in the polynucleic acids of the invention are located in the region upstream of the sequences coding for the polypeptides of the invention.

The term "recombinant vector" may include a plasmid, a phage, a cosmid or a virus.

A variety of vectors may be used to obtain recombinant expression of antigenic proteins. Bacteria are most often transformed by plasmids or bacteriophages. Lower eukaryotes such as yeasts are typically transformed with plasmids, or are transformed with a recombinant virus. The vectors may replicate within the host independently, or may integrate WO 98/08951 PCT/EP97/04668 16 into the host cell genome. Higher eukaryotes may be transformed with vectors, or may be infected with a recombinant virus, for example a recombinant vaccinia virus.

The invention further relates to a host cell transformed by any of the recombinant vectors described above, with said host cell being preferably a prokaryotic organism, and more preferably E. coli, a Salmonella species or a lactic acid bacterium.

Host cells suitable for the expression of the polynucleic acids of the invention may also include lower eukaryotic cells (like yeasts) or higher eukaryotic cells.

Another embodiment of the invention provides for a recombinant polypeptide encoded by at least part of any of the polynucleic acids of the invention described above, and being expressed in a transformed cellular host as described here above.

Said recombinant polypeptide is also called an "expression product".

According to an alternative embodiment, the current invention also provides for a recombinant vector allowing expression of the polypeptides of the invention as fusion proteins, i.e. whereby the amino acid chain of the polypeptide of the invention is linked to a heterologous amino acid sequence at the amino-terminal or carboxy-terminal end.

The term "heterologous sequence" as used in the current invention signifies any sequences different from the 39kDa and 15kDa Brucella antigen sequences of the present invention. Said heterologous sequences may also be called "foreign" sequences. The heterologous sequences are provided by the vector, and are fused in frame with the coding sequence of the polynucleic acid of the invention to be expressed.

The invention thus also relates to a recombinant polypeptide as described above, with said recombinant polypeptide consisting of a heterologous sequence, provided by the vector, fused in frame to the amino acid sequence of any of the polypeptides of the invention described above or part thereof.

The heterologous sequence may bring about any desired side effect to the resulting fusion protein, e.g. it may optimize the expression, the purification, the immobilization on a surface etc.

The invention also relates to a method for production of a recombinant polypeptide as described above, comprising: transformation of an appropiate cellular host with a recombinant expression vector as described above, wherein any of the polynucleic acids of the invention, or part WO 98/08951 PCT/EP97/04668 17 thereof, has been inserted under the control of the appropiate regulatory elements, culturing said transformed cellular host under conditions enabling expression of said insert, and harvesting and purifying said polypeptide.

In order to carry out the expression of the polypeptides of the invention in bacteria, like E. coli, the above-mentioned steps can be followed according to principles known in the art, as exemplified in the Examples section.

The techniques for carrying out the expression of recombinant polypeptides in any of the other hosts as specified above, are also well known in the art of recombinant expression technology.

The invention further relates to an isolated antibody recognizing specifically the 39kDa polypeptide of the invention as described above, with said antibody being possibly a polyclonal antibody, and preferably a monoclonal antibody.

A further embodiment of the present invention relates to an isolated antibody, more particularly a monoclonal antibody, characterized in that it is specifically raised against an S' antigenic determinant of an isolated 39kDa Brucella polypeptide; more particularly against San antigenic determinant of the 39kDa Brucella abortus polypeptide as represented in SEQ ID NO: 2.

In a preferred embodiment, the invention, relates to the monoclonal antibody "i 5E1E8 recognizing the 39kDa Brucella antigen of the invention, prepared as described further in the examples section. The hybridoma cell line secreting this monoclonal antibody, called A435E1E8 has been deposited with the ECACC on July 31, 1996 under the (provisional) accession number 96073115.

It should be understood that the invention also relates to the 39kDa Brucella polypeptides, or fragments thereof, recognized by the above-mentioned antibodies, more specifically recognized by the monoclonal antibody 5E1E8.

It should be understood that the invention also relates to the 15kDa Brucella WO 98/08951 PCT/EP97/04668 18 polypeptides, or fragments thereof, recognized by the above-mentioned antibodies.

According to an alternative embodiment, the present invention also relates to an antigen-binding fragment of the antibody (=antibody combining site), said fragment being of the Fab or single chain Fv type, or any type of recombinant antibody derived from said specific antibodies or monoclonal antibodies.

The terms "antigenic determinant" or "epitope" refer to that portion of a molecule that is specifically bound by an antibody combining site. Antigenic determinants may be determined by any of the techniques known in the art or may be predicted by a variety of computer prediction models known in the art.

The expression "antibody recognizing specifically" means that binding between the antigen as a ligand and the antibody combining site is specific, signifying that no cross-reaction occurs.

The expression "antibody specifically raised against a compound" means that the sole immunogen used to produce said antibody was said compound.

Antibodies according to a preferred embodiment of the invention include specific polyclonal antisera raised against the Brucella polypeptides of the invention, and having no cross-reactivity to others proteins, and monoclonal antibodies raised against the Brucella polypeptides of the invention.

The possible crossreactivity of the polyclonal antisera may be eliminated by preabsorption of the polyclonal antiserum against the cross-reacting antigenic determinants.

The monoclonal antibodies of the invention can be produced by any hybridoma liable to be formed according to classical methods from the fusion of splenic cells of an animal, particularly of a mouse or rat, immunized against the Brucella polypeptides of the invention defined above on the one hand, and of cells of a myeloma cell line on the other hand, and to be selected by the ability of the hybridoma to produce the monoclonal antibodies recognizing the polypeptides according to the invention, or fragments thereof.

The monoclonal antibodies according to this preferred embodiment of the invention may be humanized versions of the mouse monoclonal antibodies made by means of recombinant DNA technology, departing from the mouse and/or human genomic DNA sequences coding for H and L chains or from cDNA clones coding for H and L chains.

Also fragments derived from these monoclonal antibodies such as Fab, and WO 98/08951 PCT/EP97/04668 19 scFv ("single chain variable fragment"), providing they have retained the original binding properties, form part of the present invention. Such fragments are commonly generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases. It is well known to the person skilled in the art that monoclonal antibodies, or fragments thereof, can be modified for various uses.

The antibodies involved in the invention can be labelled by an appropriate label of the enzymatic, fluorescent, or radioactive type.

The invention also relates to the use of the proteins of the invention, analogues thereof, or fragments thereof, for the selection of recombinant antibodies by the process of repertoire cloning (Perrson et al., 1991).

According to a preferred embodiment of the present invention, an antibody, or an antigen-binding fragment Fab, single chain Fv and all types of recombinant antibodies, as defined above are further characterized in that they show at least one of the following properties: they bind to Brucella cells or Brucella antigens, in solid or soluble phase, and/or they inhibit the infection ofBrucella strains to the specific cell type which they infect in vivo.

According to another embodiment, the present invention relates to a monoclonal antibody as defined above, obtainable by a process comprising at least the following steps: fusing the splenocytes from mice infected with Brucella species together with myeloma cells, and selecting the anti-Brucella hybridomas by means of ELISA and subsequent limiting dilution, selecting the hybridomas producing a monoclonal antibody, specifically directed against any of the 39kDa or 15kDa Brucella polypeptides of the current invention by means of ELISA, and, recovering the monoclonal antibodies from ascites fluid or from a culture of the selected hybridomas.

The present invention also relates to a hybridoma producing any of the monoclonal antibodies as defined above.

The present invention further relates to an anti-idiotype antibody raised against any of the antibodies as defined above.

WO 98/08951 PCT/EP97/04668 The term "anti-idiotype antibodies" refers to monoclonal antibodies raised against the antigenic determinants of the variable region of monoclonal antibodies themselves raised against the Brucella polypeptides of the invention. These antigenic determinants of immunoglobulins are known as idiotypes (sets of idiotopes) and can therefore be considered to be the "fingerprint" of an antibody (for review see de Preval, 1978; Fleishmann and Davie, 1984). The methods for production of monoclonal anti-idiotypic antibodies have been described by Gheuens and McFarlin (1982). Monoclonal anti-idiotypic antibodies have the property of forming an immunological complex with the idiotype of the monoclonal antibody against which they were raised. In this respect the monoclonal antibody is often referred to as Abl, and the anti-idiotypic antibody is referred to as Ab2. These anti-idiotype Ab2s may be used as substitutes for the polypeptides of the invention or as competitors for binding of the polypeptides of the invention to their target.

The present invention further relates to antisense peptides derived from the Brucella polypeptides as defined above.

More particularly, the term "antisense peptide" is reviewed by Blalock (1990) and by Roubos (1990). In this respect, the molecular recognition theory (Blalock, 1990) states that not only the complementary nucleic acid sequences interact but that, in addition, interacting sites in proteins are composed of complementary amino acid sequences (sense ligand with receptor or sense ligand with antisense peptides). Thus, two peptides derived from complementary nucleic acid sequences in the same reading frame will show a total interchange of their hydrophobic and hydrophilic amino acids when the amino terminus of one is aligned with the carboxy terminus of the other. This inverted hydropathic pattern might allow two such peptides to assume complementary conformations responsible for specific interaction.

The antisense peptides can be prepared as described in Ghiso et al. (1990). By means of this technology it is possible to logically construct a peptide having a physiologically relevant interaction with a known peptide by simple nucleotide sequence analysis for complementarity, and synthesize the peptide complementary to the binding site.

The present invention still further relates to a method for in vitro diagnosis of Brucella (species) infection in an individual comprising the step of contacting a sample taken from said individual, said sample possibly containing anti-Brucella (species) antibodies, Brucella (species) antigens and/or Brucella (species) nucleic acids, with: WO 98/08951 PCT/EP97/04668 21 a 39kDa and/or 15kDa Brucella polypeptide or peptide as defined above, under conditions allowing the formation of an immunological complex, or, a Brucella oligonucleotide probe as defined above, under conditions allowing the formation of a hybridization complex, with said nucleic acids of said sample being possibly amplified prior to hybridization, or, an antibody specifically directed against a 39kDa and/or 15kDa Brucella antigen as defined above, under conditions allowing the formation of an immunological complex, or, an anti-idiotype antibody as defined above, under conditions allowing the formation of an antibody-anti-idiotypic complex, or, an antisense peptide as defined above, under conditions allowing the formation of an antigen-antisense peptide complex, and subsequently detecting the complexes formed.

The Brucella species which may be diagnosed by the above method include B. abortus, B. melitensis, B. ovis, B. suis, and preferably include B. abortus and B. melitensis.

The term "sample" may refer to any biological sample (tissue or fluid) possibly containing Brucella nucleic acid sequences, antibodies or polypeptides.

In a preferred embodiment, the detection method is the detection of antibodies against Brucella (species), and the preferred sample in that case is serum or plasma.

The invention thus preferably relates to a method for detecting antibodies to Brucella (species) present in a biological sample, comprising: contacting the biological sample to be analysed with at least one of the polypeptides as described above, under conditions allowing the formation of an immunological complex, and detecting the immunological complex formed between said antibodies of the sample and said polypeptide.

Conditions allowing the formation of an immunological complex are known to the person skilled in the art.

As described further in the Examples section, the 39 kDa and 15 kDa Brucella antigens are complementary in serological detection assays, i.e. both antigens together detect more positive inidividuals compared to the detection with each of the individual antigens on its own.

WO 98/08951 PCT/EP97/04668 22 Therefore, a preferred embodiment of a method to detect antibodies to Brucella species present in a sample comprises contacting the sample with both antigens (or fragments thereof) of the invention, i.e. with the Brucella 39 kDa antigen and the Brucella 15 kDa antigen or fragments thereof.

In a special embodiment, the polypeptides used in the above-described method for detection of anti-Brucella species antibodies, can be replaced by anti-idiotype antibodies as described above, acting as their equivalents.

Conditions allowing the formation of an antibody-anti-idiotypic complex are known in the art.

The invention further relates to a method for detecting the presence of Brucella (species) antigens in a biological sample, comprising: contacting the biological sample to be analysed with an antibody according to the invention, under conditions allowing the formation of an immunological complex, and detecting the immunological complex formed between said antigens and said antibody.

In a special embodiment, the antibodies used in the above-described method for detection of Brucella antigens, may be replaced by anti-sense peptides as described above, acting as their equivalents.

Conditions allowing the formation of an antigen-antisense peptide complex are known in the art.

Design of immunoassays, suitable for both detection of antibodies or detection of antigens in a sample, is subject to a great deal of variation, and many formats are known in the art. Protocols may, for example, use solid supports, or immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide; the labels may be, for example, enzymatic, fluorescent, chemiluminescent, radioactive, or dye molecules. Assays which amplify the signals from the immune complex are also known, examples of which are assays which utilize biotin and avidin or streptavidin, and enzyme-labeled and mediated immunoassays, such as ELISA assays.

An advantageous embodiment provides for a method for detection of anti-Brucella (species) antibodies in a sample, whereby the polypeptides of the invention are immobilized WO 98/08951 PCT/EP97/04668 23 on a solid support, eventually on a membrane strip. Different polypeptides of the invention may be immobilized together or next to each other in the form of parallel lines which is the case in the Line Immuno Assay The polypeptides of the invention may also be combined with other antigens from other organisms, belonging to the genus Brucella or to other genera.

The combination of different antigens in one single detection method as described may have certain advantages, such as: achieving a higher test sensitivity: e.g. by combining several antigenic determinants from Brucella species, the number of positively reacting sera from field infected individuals may be greater, and/or enabling differentiation between Brucella (species) field-infected and vaccinated individuals, and/or enabling differentiation between individuals infected by different pathogens, belonging to the genus Brucella or to other genera.

The invention thus also relates to a solid support onto which at least one of the polypeptides of the invention, possibly in combination with other polypeptides, have been immobilized.

Another embodiment of the invention provides for a method for detecting the presenze of Brucella (species) polynucleic acids present in a biological sample, comprising: possibly extracting the polynucleic acids contained in the sample, amplifying the Brucella (species) polynucleic acids with at least one oligonucleotide primer as described above, detecting the amplified nucleic acids, possibly after hybridization with an oligonucleotide probe as described above.

Conditions allowing hybridization are known in the art and e.g. exemplified in Maniais et al. (1982). However, according to the hybridization solution (SSC, SSPE, etc.), the probes used should be hybridized at their appropriate temperature in order to attain sufficient specificity (in some cases differences at the level of one nucleotide mutation are to be discriminated).

Amplification of nucleic acids present in a sample prior to detection in vitro may be accomplished by preferably first extracting the nucleic acids present in the sample according WO 98/08951 PCT/EP97/04668 24 to any of the techniques known in the art, followed by amplification of the extracted nucleic acids. In case of extraction of RNA, generation of cDNA is necessary; otherwise cDNA or genomic DNA is extracted.

The amplification methods are detailed above.

Suitable assay methods for purposes of the present invention to detect hybrids formed between oligonucleotide probes according to the invention and the Brucella nucleic acids in a sample may comprise any of the assay formats known in the art. For example, the detection can be accomplished using a dot blot format, the unlabelled amplified sample being bound to a membrane, the membrane being incubated with at least one labelled probe under suitable hybridization and wash conditions, and the presence of bound probe being monitored. Probes can be labelled with radioisotopes or with labels allowing chromogenic or chemiluminescent detection such as horse-radish peroxidase coupled probes.

An alternative is a "reverse" dot-blot format, in which the amplified sequence contains a label. In this format, the unlabelled oligonucleotide probes are bound to a solid support and exposed to the labelled nucleic acids from the sample, under appropriate stringent hybridization and subsequent wash conditions. It is to be understood that also any other assay method which relies on the formation of a hybrid between the nucleic acids of the sample and the oligonucleotide probes according to the present invention may be used.

According to an advantageous embodiment, the process of detecting Brucella polynucleic acid sequences contained in a biological sample comprises the steps of contacting amplified copies derived from the Brucella genetic material, with a solid support on which probes as defined above, have been previously immobilized.

In a very specific embodiment, the probes have been immobilized on a membrane strip in the form of parallel lines. This type of reverse hybridization method is specified further as a Line Probe Assay (LiPA).

The invention thus also relates to a solid support onto which at least one the oligonucleotide probes of the invention have been immobilized.

The invention further relates to a method for detecting individuals having been in contact with Brucella (species), comprising: contacting a polypeptide according to the invention with the cellular immune system of the individual, either in vitro or in vivo, and WO 98/08951 PCT/EP97/04668 detecting and/or quantifying the cellular immune response raised against said polypeptides.

The above-said cellular immune response can be measured either in vivo, such as a delayed type hypersensitivity reaction upon subcutaneous injection of the polypeptides of the invention (=intradermic test), or in vitro, such as stimulation of periferal blood lymphocytes or secretion of certain cytokines, such as interferon-gamma, upon addition of the polypeptides of the invention to a sample of periferal blood lymphocytes under conditions allowing recognition of the polypeptides by the cells responsive for the immune response, conditions which are known to the person skilled in the art.

The invention thus also relates to a method for detecting and/or quantifying the cellular immune response of an individual against the polypeptides of the invention, said method comprising: measurement of a delayed type hypersensitivity reaction after subcutaneous injection of at least one of the polypeptides of the invention, or measurement of the stimulation of periferal blood lymphocytes isolated from the individual to be tested, upon addition of at least one of the polypeptides of the invention to a sample of the periferal blood lymphocytes.

The invention further relates to a diagnostic kit for the detection of antibodies to Brucella (species) present in a biological sample, said kit comprising at least one of the polypeptides according to the invention, with said polypeptides being preferably bound to a solid support.

The present invention relates more particularly to a kit for determining the presence of anti-Brucella (species) antibodies as defined above present in a biological sample comprising: at least one polypeptide or peptide as defined above, preferentially in combination with other polypeptides or peptides from Brucella, with said polypeptides being preferably immobilized on a solid substrate, a buffer or components necessary for producing the buffer enabling a binding reaction to occur between these polypeptides and the anti-Brucella antibodies possibly present in the biological sample, means for detecting the immune complexes formed in the preceding binding WO 98/08951 PCT/EP97/04668 26 reaction, possibly also including an automated scanning and interpretation device for inferring the presence of anti-Brucella antibodies in the sample from the observed binding pattern.

The kit according to this aspect of the invention comprises at least a 39 kDa Brucella polypeptide or peptide as described above, or a 15 kDa Brucella polypeptide or peptide as described above. Preferably, the kit according to this aspect of the invention comprises both Brucella antigens (39 kDa and 15 kDa) of the invention, or fragments thereof. Furthermore, the kit according to this aspect of the invention may comprise in addition to peptide or polypeptide antigens according to the invention, also other Brucella antigenic proteins or peptides known in the art (such as outer membrane protein (OMP) proteins, or the 17 kDa Brucella antigen (International application W096/17065 published on 06.06.96) or other bacterial antigenic proteins or peptides in general.

In a very specific embodiment the invention relates to a kit for the detection of anti- Brucella species antibodies in a biological sample as described above, whereby the polypeptides of the invention are replaced by the anti-idiotype antibodies as described above.

The invention further relates to a diagnostic kit for the detection of antigens of Brucella (species) present in a biological sample, said kit comprising an antibody as described above, with said antibody being preferably bound to a solid support.

In a very specific embodiment, the invention relates to a diagnostic kit for the detection of antigens of Brucella species present in a biological sample, whereby the antibody as described above is replaced by an antisense peptide.

The invention further also relates to a diagnostic kit for the detection of Brucella (species) polynucleic acids present in a sample, said kit comprising a probe as described above and/or a primer as described above.

According to a preferred embodiment, the present invention relates to a kit for determining the presence of Brucella polynucleic acids as defined above present in a biological sample liable to contain them, comprising: possibly at least one primer or a set of primers as defined above, at least one oligonucleotide probe as defined above, with said probe(s) being preferentially immobilized on a solid substrate, and more preferentially on a WO 98/08951 PCT/EP97/04668 27 membrane strip, a buffer or components necessary for producing the buffer enabling a hybridization reaction to occur between the above-mentioned probe(s) and the possibly amplified Brucella polynucleic acids from the sample, a solution or components necessary for producing the solution, enabling washing of the hybrids formed under the appropiate wash conditions, means for detecting the hybrids resulting from the preceding hybridization, and possibly also including an automated scanning and interpretation device for inferring the Brucella (strain) polynucleic acids present in the sample from the observed hybridization pattern.

According to an advantageous method, the probes are immobilized in a Line Probe Assay (LiPA) format. This is a reverse hybridization format (Saiki et al., 1989) using membrane strips onto which several oligonucleotide probes (including negative or positive control oligonucleotides) can be conveniently applied as parallel lines.

A LiPA support may contain on its surface different oligonucleotide probes derived from the polynucleic acid sequences according to the invention which hybridize specifically with certain strains of Brucella (such as B. abortus, B. melitensis, B. ovis, B. suis) or may contain at least one Brucella oligonucleotide probe derived from a polynucleic acid sequence according to the present invention in addition to other Brucella probes or probes derived from other bacterial and/or viral organisms, as long as all probes are functional under the same hybridization and wash conditions.

The LiPA, as described by Stuyver et al. (1993) and in international application W094/12670 provides a very rapid and user-friendly hybridization test. Results can be read within approximately 4 h after the start of the amplification. After amplification during which usually a non-isotopic label is incorporated in the amplified product, and alkaline denaturation, the amplified product is contacted with the probes on the membrane and the hybridization is carried out for about 0.5 to 1,5 h. Consequently, the hybrids formed are detected by an enzymatic process resulting in a visual purple brown precipitate. From the hybridization pattern generated, the results can be deduced either visually, but preferably using dedicated software. The LiPA format is completely compatible with commercially available scanning devices, thus rendering automatic interpretation of the results very reliable. All those WO 98/08951 PCT/EP97/04668 28 advantages make the LiPA format liable for the use of Brucella detection in a routine setting.

The LiPA format should be particularly advantageous for detecting the presence of different Brucella strains, or for detecting simultaneously Brucella strains and other pathogens possibly present in the same sample.

The invention further relates to a kit for the detection and quantification of the cellular immune response against Brucella (species) in an individual, said kit comprising any of the polypeptides according to the invention as described above.

A method and kit for brucellosis diagnosis, based on the quantification of the cellular immune response, as specified above, enables the identification of individuals (humans or ruminants) which have been in contact with the Brucella pathogen. Said contact may subsequently lead to a disease state (=field infected individuals) or to a protected state of the individual (=vaccinated individuals).

According to a preferred embodiment, the present invention relates to a method or a kit for diagnosis of Brucella infection as defined above, further characterized in that said polypeptides, peptides, polynucleic acids, antibodies, anti-idiotypic antibodies or anti-sense peptides are particularly useful for differentiating Brucella (species) field infected individuals from Brucella vaccinated individuals.

The invention further relates to a vaccine composition which provides protective immunity against Brucella (species) infection in a mammal (human, ruminants) comprising as an active principle at least one of the polypeptides according to the invention, or at least one of the polynucleic acid sequences or recombinant vectors according to the invention, said active principle being combined with a pharmaceutically acceptable carrier.

According to a special embodiment, the vaccine composition as described above may comprise as an active principle one of the anti-idiotype antibodies as described above.

Besides the Brucella 39kDa and 15kDa proteins according to the invention, said vaccine composition may comprise also other Brucella immunogenic components (such as outer membrane proteins ((OMP) Cloeckaert et al. 1991) or other bacterial or immunogenic components in general.

In a specific embodiment, polynucleic acid sequences coding for any of the polypeptides as defined above, are used as a vaccine, either as naked DNA or as part of recombinant vectors. In this case, it is the aim that said nucleic acids are injected into the WO 98/08951 PCTIEP97/04668 29 individual and that they express in situ the immunogenic protein peptide which they are encoding, thus conferring in vivo protection to the vaccinated host Ulmer et al., 1993).

The active ingredients of such a vaccine composition may be administered orally, subcutaneously, conjunctivally, intramuscularly, intranasally, or via any other route known in the art including for instance via the binding to carriers, via incorporation into liposomes, by adding adjuvants known in the art, etc.

The invention also provides for recombinant Brucella (species) strains in which at least one of the genes of the invention have been deleted. More particularly, the current invention provides for Brucella vaccinal strains, such as B. abortus B19 or B. melitensis Rev. 1, deleted in at least one of the genes of the invention.

According to the above-mentioned embodiment, the current invention provides for a recombinant Brucella (species) strain in which the gene encoding a Brucella 39kDa antigen as described above has been deleted or inactivated. More particularely, the invention provides for B. abortus B19 and B. melitensis rev. strains deleted in the gene encoding the 39 kDa antigen of the invention (P39 gene). Two examples of such deleted vaccine strains, i.e. B.

abortus B19AP39 and B. melitensis Rev. 1AP39 have been deposited at the NCTC on July 31, 1996 under accession numbers NCTC 12944 and NCTC 12942 respectively.

According to another embodiment, the current invention also provides for a recombinant Brucella (species) strain in which the gene encoding a Brucella 15kDa antigen as described above has been deleted or inactivated. More particularely, the invention provides for B. abortus B19 and B. melitensis rev.1 strains deleted in the gene encoding the antigen of the invention (Br25 gene). An example of such a deleted vaccine strain, i.e. B.

melitensis Rev.lABr25 has been deposited at the NCTC on July 31, 1996 under accession number NCTC 12943.

According to still another embodiment, the current invention provides for a recombinant Brucella (species) strain in which both genes of the invention, i.e. the gene encoding the 39 kDa antigen and the gene encoding the 15 kDa antigen, have been deleted or inactivated.

The above-mentioned embodiments are further illustrated in example 8 of the description.

The invention also relates to a vaccine composition which provides protective immunity WO 98/08951 PCT/EP97/04668 against Brucella (species) infection in a mammal (human, ruminants) and which still allows the subsequent differentiation between field infected and vaccinated individuals, said vaccine composition comprising as an active principle a recombinant Brucella (species) strain in which the gene encoding the Brucella 39kDa antigen of the invention and/or the gene encoding the Brucella 15 kDa antigen of the invention has been deleted or inactivated.

Preferably, the Brucella strain in which the gene(s) of the invention is (are) deleted is the B. melitensis Revl strain (typically used for vaccination of sheep and goat) or the B.

abortus B19 strain (typically used for vaccination of bovine).

The above-described recombinant Brucella strains are interesting components of a vaccine composition, providing protective immunity against brucellosis, yet permitting differentiation between Brucella field infected and vaccinated individuals, because of their distinctive immunological signature. For example, if such deleted Brucella strains are used for vaccination, a diagnostic screening test a serological assay) based on an antigen encoded by one of the deleted genes more particularely the 15 kDa antigen and/or 39 kDa antigen) as described above would allow to detect specifically the field infected individuals and not the vaccinated individuals.

Vaccination and protection experiments in the mouse model with the above-described deleted Brucella vaccine strains, are described in Example 9 of the description.

From the above, it follows that the current invention also relates to a combined method of vaccination against and detection of brucellosis, said method comprising vaccinating an individual liable to become infected by brucellosis with a recombinant Brucella vaccine strain, in which the gene encoding the Brucella 39 kDa antigen and/or the gene encoding the Brucella 15 kDa antigen has been deleted, and detecting in an individual liable to be infected by brucellosis anti-Brucella antibodies possibly present in the serum or plasma with a detection method comprising contacting the sample with the 39 kDa and/or 15 kDa polypeptide or peptide of the invention under conditions allowing the formation of an immunological complex, and subsequent detection of the complex formed.

Alternatively to the serological detection of antibodies against the polypeptides of the invention, the above-described combined method may also use a method to detect the cellular immune response against the polypeptides of the invention, such as described above.

WO 98/08951 PCT/EP97/04668 31 The above-described combined method allows the differentiation between vaccinated and field-infected animals, a differentiation which is not always straightforward, and sometimes not even possible, with the existing methods for vaccination and diagnosis (Alton, 1978; Nicoletti, 1980).

The invention also relates to a combined kit for the vaccination against and the detection of brucellosis, said kit comprising at least the following components: a vaccine composition comprising as an active principle a recombinant Brucella vaccine strain, in which the gene encoding the Brucella 39 kDa antigen and/or the gene encoding the Brucella 15 kDa antigen has been deleted, and a polypeptide or peptide as described above, or a recombinant polypeptide as described above, with said combined kit further characterized by the fact that its components enable the differentiation between vaccinated and field-infected individuals.

The invention also relates to any of the above-mentioned substances (polypeptides, antibodies, polynucleic acids, anti-idiotype antibodies, antisense peptides) for use as a medicament, more particularly for any of the medical (diagnostic or prophylactic) applications as mentioned above.

Furthermore, the invention relates to the use of any of the above-mentioned substances (polypeptides, antibodies, polynucleic acids, anti-idiotype antibodies, antisense peptides) for the manufacture of a medicament, more particularly for the preparation af a vaccine or for the preparation of a diagnostic composition.

In a particular embodiment, the invention relates to the combined use of a recombinant Brucella vaccine strain, in which the gene encoding the Brucella 39 kDa antigen and/or the gene encoding the Brucella 15 kDa antigen has been deleted or inactivated, for use in vaccination, on the one hand, with at least one of the (recombinant) polypeptides or peptides of the invention, more particularly the 39 kDa Brucella antigen (poly)peptides and/or the 15 kDa Brucella antigen (poly)peptides, for use in a diagnostic method, on the other hand.

WO 98/08951 PCT/EP97/04668 32 FIGURE LEGEND Fig. 1. Purification of the 39 kDa antigen of B. abortus starting from brucellergen.

Elution profile after chromatographic separation of brucellergen (batch 8A083) proteins mg) on a Mono Q Sepharose Fast Flow column HR 5/5 with a bed volume of 60 ml. For elution, 10 mM Tris-HCI buffer (pH 8.5) with a linear gradient of 0 to 0.3 M NaCI was used.

Eluate fractions were pooled into 5 major protein fractions which were subjected to SDS- PAGE in comparison with brucellergen (Bruc); the gel was stained with Coomassie brilliant blue). Immunoblot analysis of the same fractions as in with MAb 5E1E8. (D) Analysis by SDS-PAGE and silverstaining of peaks 2 and 3 from the chromatographic separation of F4 on a phenyl Superose column.

Fig. 2. Restriction endonuclease maps of the cloned 3.9-kb and 1.6-kb DNA fragments of B.

abortus. Subclones are presented and the size of the bru39 gene products detected by immunoblot are indicated on the right of each clone (E=EcoRI, H=HindIII, S=Sphl, X =Xbal).

Fig. 3. Nucleotide sequence (SEQ ID NO 1) and deduced amino acid sequence (SEQ ID NO 2) of the gene encoding the 39kDa antigen of B. abortus (=bru39 gene or P39 gene) and of the corresponding protein. The putative ribosome binding site is indicated in bold and underlined. The dyad symetry of the putative terminator stem loop structure is indicated The asterisk denotes the termination codon. The sequence of the internal peptide obtained by automated Edman degradation is underlined. Relevant restriction sites used during cloning procedures are indicated. Protein translation starts from the putative start codon GTG at position 241. Other putative start codons ATG/GTG are indicated in italics.

Fig. 4. Nucleotide sequence (SEQ ID NO 3) and deduced amino acid sequence (SEQ ID NO 4) of the gene coding for the 15 kDa antigen of B. abortus (=br25 gene) and the corresponding protein. The asterisk denotes the termination codon. Relevant restriction sites are indicated.

WO 98/08951 PCT/EP97/04668 33 Fig. 5. Vectors used for expression of the Brucella antigens of the invention in E. coli Prokarvotic expression vector plGALMPH. Heterologous gene expression is under control of the early lambda promoter Pr. The crolacl leader fused to the hexahistidine gene fragment is indicated upstream of the BamHI restriction site. The fd terminators of transcription are also shown (Tfd). Unique restriction sites are also indicated. Proteins expressed in this vector contain the crolacl-His6 tag at their N-terminal end.

Figure 5b: Prokarvotic expression vector pIGRHISA. Heterologous expression is under control of the early lambda P1 promoter. The hexahistidine coding gene fragment is indicated upstream of the Nsil site. The detailed sequence of this region is shown below the figure. The pIGRHISG vector has as second codon GGT (encoding glycin) instead of GCT. All fusion proteins expressed in this vector start with the ATG codon included in the Ncol site. The ribosomal RNA terminator is also indicated (rrnBTlT2). Some unique restriction sites are also indicated.

Figure Prokarvotic expression vector plGFHIO. Heterologous expression is under control of the early lambda promoter PI. The mTNF-hexahistidine gene fragment encoding the fusion partner is indicated upstream from the BamHI site. All fusion proteins expressed in this vector contain the mTNF-His6 tag at their N-terminal end. The ribosomal RNA terminator is also indicated, as well as some unique restriction sites.

Fig. 6. Expression and purification of the 15 kDa Brucella antigen Figure 6a: Induction of the 15 kDa Brucella protein in E.coli. Lysate from uninduced or induced cells was analysed on SDS PAGE and detected with coomassie blue staining. In each lane the equivalent of 0.1 OD of cell culture measured at 600 nm was loaded. Lane 1, uninduced culture; lanes 2, 3, 4 and 5 induction for 4, 3, 2 and lh respectively. Size of marker proteins are indicated in kDa. The arrow indicates the position of the induced protein.

WO 98/08951 PCT/EP97/04668 34 Egulre Analysis on SDS PAGE of purified 15 kDa fusion protein and detected by coomassie blue staining. Lane 1, 10 jg protein; lane 2, 3 ig protein. Lane 3, marker proteins indicated in kDa.

Fig. 7. Expression and purification of the 39 kDa Brucella antigen Fiure 7a: Induction of the 39 kDa Brucella protein in E.coli. Lysate from uninduced cells was analysed on SDS PAGE and detected with coomassie blue staining. In each lane the esuivalent of 0.1 OD of cell culture measured at 600 nm was loaded. Lanes 1, 2 and 3, induction for 1, 2 and 3h respectively. The arrow indicates the 39 kDa fusion protein.

Figure 7h: Purified 39 kDa fusion protein analysed on SDS PAGE and detected by coomassie blue staining. Lane 1, 10 /g protein; lane 2, 3 4g protein. Lane 3 contains marker proteins indicated in kDa.

Fig. 8. Serological reaction of 10 individual goat sera (CH1-CH10) on plates coated with the recombinant antigens or with the combination of both antigens (combi). The mean of the negatives for the 15 kDa antigen was 0.130, for the 39 kDa antigen it was 0.073 and for the combined ELISA it was 0.156. The S/N was calculated by dividing the signal for each serum by the mean of the negatives. The cut off (co) was placed at S/N=2.

Fig. 9. Proliferative response of PBMC from Brucella-infected cows to fractions of brucellergen (batch 8A083). PBMC (2 x 10/well) were cultured during 4, 6 and 8 days with 1 /g of antigen per well, radio labelled for 18 h, harvested and counted as described in materials and methods (SI) =stimulation index.

Fig. 10. Deletion plasmids used to carry out the deletion experiments as described in example 8. Outline of the plasmid structures prepared for the deletion of the P39 gene and the gene. Only the plasmid part containing the Brucella genes is shown. The relevant restriction sites are indicated. A fusion between two restriction sites without restoration of either of them WO 98/08951 PCT/EP97/04668 is indicated by both sites used, separated with a slash. The sizes of the different Brucella gene fragments are indicated in bp. KanR indicates the kanamycin resistance gene.

Fig. 11. Southern blot analysis of vaccinal deletants and their parental strains. DNA is cut with HindIII. Genes P39 (fig. 11A) and kan (fig. 11B) were used as probes. Lanej: DNA molecular weight marker, fragment sizes were 23130, 9416, 6557, 4361, 2322, 2027, 1353, 1078, 872 and 603 basepairs; l1ge_2: B. melitensis Rev. 1 DNA; lane 3: B. melitensis RevlAP39 DNA; lane4: B. abortus B19 DNA; jani: B. abortus B19AP39 DNA.

Fig. 12. Southern blot analysis of DNA's cut with HindIII of B. melitensis strain 16M and 2 clones of the same strain which have been deleted for the br25 gene. Genes br25 (fig. 12A) and kan (fig. 12B) were used as probes. Lane 1: DNA molecular weight marker, fragment sizes were 23130, 9416, 6557, 4361, 2322, 2027, 1353, 1078, 872 and 603 basepairs; lane 2: B. melitensis 16M DNA; lane3: B. melitensis 16MAbr25 (clone 2) DNA; lane 4: B.

melitensis 16MAbr25 (clone 19) DNA.

Fig. 13. Immune protection conferred in CD-1 mice by Brucella melitensis Revl strains against a B. melitensis H38 challenge. Spleen counts in vaccinated mice are significantly different from the control mice: p <0.05; <0.01; <0.001 (minimal detection level 5 c.f.u. per spleen). DelRev Revl strain deleted in P39 gene.

Fig. 14. Immune protection conferred in CD-1 mice by Brucella abortus B19 strains against a B. abortus 544 challenge. Spleen counts in vaccinated mice are significantly different from the control mice: p<0.05; <0.01; <0.001 (minimal detection level 5 c.f.u. per spleen). DelB19= B19 strain deleted in P39 gene.

WO 98/08951 PCT/EP97/04668 36 TABLE

LEGEND

Table 1. Amino acid substitutions which may form the basis of the analogues according to the present invention.

Table 2. Brucella species and bacteria presenting cross-reactivity with Brucella have been tested for the presence or absence of the 39 kDa protein. SDS-bacterial lysates (108 cells) were loaded on a 12% SDS-PAGE gel; after Western blotting, the blots were probed with MAb 5E1E8 and PRA 39 specific antiserum.

Table 3. Reactivities of ovine sera with the Brucella antigens of the invention in ELISA.

Table 4. Serologic responses and DTH tests of 15 Brucella-infected cows. ELISA anti-LPS Ab titers are expressed in U/ml. A value equal or greater than 2.5 U/ml is considered positive (cut-off value). Skin tests were done with 0.14 mg of allergen and reactions were measured after 72 h. Results are expressed as the difference in skin thickness before and after the injection. A reaction is considered positive when the increase in skin thickness is 1 mm or greater.

Table 5. Serologic responses and DTH tests of cows infected with bacteria presumed to crossreact with Brucella in serological tests. ELISA anti-LPS Abs titers are expressed in U/ml and skin tests were done as described in Table 4.

Table 6. Proliferative response of PBMC from 4 Brucella-infected cows to 2 batches of brucellergen (96G0091, 8A083), and 39 kDa antigen. PBMC (2 10'/well) were cultured during 8 days with 1 lg of antigen per well, radio labelled for 18 h, harvested and counted as described in materials and methods. Results are expressed as stimulation index (SI).

Table 7. Phenotype of possible recombinant strains resulting from recombination events with different selection methods.

WO 98/08951 PCTIEP97/04668 37 Table 8. Brucella strains with confirmed deletions and the genes affected Table 9. Number of deletion strains isolated for each parental strain and per gene studied.

WO 98/08951 PCT/EP97/04668 38 Iablel: Amino acid substitutions which may form the basis of the analogues according to the present invention.

Amino acids Synonymous groups Ser Ser, Thr, Gly, Asn Arg Arg, His, Lys, Glu, Gin Leu Leu, Ile, Met, Phe, Val, Tyr Pro Pro, Ala, Thr, Gly Thr Thr, Pro, Ser, Ala, Gly, His. Gin Ala Ala, Pro, Gly, Thr Val Val, Met, Ile, Tyr, Phe, Leu Gly Gly, Ala, Thr, Pro, Ser Ile Ile, Met, Leu, Phe, Val, Tyr Phe Phe, Met, Tyr, Ile, Leu, Trp, Val Tyr Tyr, Phe, Trp, Met, Ile, Val, Leu Cys Cys, Ser, Thr, Met His His, Gin, Arg, Lys, Glu, Thr Gin Gin, Glu, His, Lys, Asn, Thr. Arg Asn Asn, Asp, Ser, Gin Lys Lys, Arg, Glu, Gin, His Asp Asp, Asn, Glu, Gin Glu Glu, Gin, Asp, Lys, Asn, His. Arg Met Met, Ile, Leu, Phe, Val WO 98/08951 PCT/EP97/04668 39

EXAMPLES

Example 1: Material and methods Chemicals and Reagents. All reagents were of analytical grade and obtained from Merck (Darmstadt, Germany), Sigma (St. Louis, or Biorad Laboratories (Richmond, Calif.). Restriction enzymes and DNA modifying enzymes were purchased from Boehringer Mannheim (Brussels, Belgium) and were used according to the manufacturer's instructions.

Brucellin and anti-brucellin polyserum were obtained from the Institut National de la Recherche Agronomique Station de la Pathologie Infectieuse et Immunologie, Nouzilly, France. Brucellergen was kindly provided by Rhbne-M6rieux, France. Protein Ahorseradish peroxydase (protein A-perox), phytohemagglutinin (PHA) were purchased from Sigma Chemicals Co. (St. Louis, ethyl-[ 3 H] thymidine and [a- 3 S]-dATP from Amersham (Gent, Belgium) and 4-chloro-l-naphtol from Biorad (Richmond, California). The protein concentration was measured by the bicinchoninic acid method (Smith et al. 1985)) by using micro BCA protein assay reagent (Pierce, Rockford, Illinois) with bovin serum albumin (BSA) as a standard.

Bacterial strains and vectors. B. abortus biovar 3, S. urbana, P. maltophilia, and E.

coli 0:157 strain were from the Institut National de Reserche V6t&rinaire Bruxelles). The laboratory strain Y. enterocolitica 0:9 was cultured in the Microbiology Unit at the U.C.L. (Bruxelles). P. multocida was isolated by Dr. S. Bercovich at the C.D.I.

(Lelystad, the Netherlands). The other whole cell extracts of Brucella strains were prepared by J-M. Verger, M. Grayon and G. Bezard Nouzilly, France). Bacteriophage Xgtl 1, E. coli Y1089 and Y1090 were provided by Amersham E. coli DH5a E. coli XL-1 blue, E. coli MC1061 and plasmid vectors pUC19, pBluescript and pTZ19R were purchased from Stratagene (La Jolla, Calif.). E. coli strains used for heterologous expression were SG4044 [C1-857] as described by Gottesman et al. 1981.

Two prototypes of expression vectors are used for expression of fusion proteins in E.

coli. The expression vector plGALMPH (see fig. 5a, and described by Van Gelder et al. 1993) drives the transcription of the heterologous gene by the rightward promoter of phage lambda.

WO 98/08951 PCT/EP97/04668 Transcription can thus be contolled by the lambda C1 repressor, which is provided from a compatible plasmid present in the host cell. When the C1 8 57 allel is used, a temperature induction of the transcription can be achieved. The vector also encodes a 50 amino acid peptide (crolac tag) to which the N-terminal end of the ORF to be expressed is fused in frame.

This allows the detection of any fusion protein expressed in this system with the monoclonal directed to the crolac tag.

The expression vector pmTNFMPH, as described by Gilot et al. 1993, provides the expression of fusion proteins with a mTNF mouse Tumor Necrosis Factor) tag. Derivatives of the latter vector, used in the current invention, are described in Fig. 5b (pIGRHISA/G) and Fig. 5c (pIGFHIO).

Immunizations and production of monoclonal and polyclonal antibodies.

Hybridomas were produced as described elsewhere (Cloeckaert et al. 1990). Briefly, BALB/c mice between 8 and 12 weeks of age, were infected by intraperitoneal injection of 109 B. abortus B3 smooth cells. After 4 months, mice were boosted i.p. with 1 mg of Bruzellin.

Hybridomas secreting antibodies against one of the brucellin proteins were screened by a capture ELISA and cloned by the limiting-dilution technique. Thirteen hybridoma's producing MAbs which reacted with brucellin proteins in ELISA were obtained. Immunoblot analysis with brucellin proteins separated by SDS-PAGE revealed that 7 of these MAbs recognized a protein of an apparent molecular mass of 39-kDa (P39). In Western blot they did no: react with cell extracts from Y. enterocolitica 0:9 and E. coli 0:157 strains (data not shown). Since the reactivity of MAb 5E1E8 was high, this IgG was used further. Ascitic fluids were prepared in pristane conditioned BALB/c mice by i.p. injection of 3 106 cells/mouse.

Rabbit anti-mouse immunoglobulin antiserum (RAM) and anti-P-galactosidase goat serum were produced as described previously (Tibor et al. 1994).

To produce monospecific polyclonal rabbit antisera against the P39 (PRA 39), 0.5 mg of brucellin proteins were resolved on a 12% SDS-PAGE by the method of Laemmli (1970).

The gel was stained with Coomassie brilliant blue and the 39-kDa zone was cut out of the gel.

The gel slice was ground and diluted in phosphate-buffered saline (PBS, pH Rabbits were immunized with approximately 0.15 mg of P39, initially in the presenceof complete Freund's adjuvant (FA) and on day 15 and 30 with incomplete FA (IFA).

The Br25 monoclonal antibody was obtained from Silenus (Victoria, Australia) as WO 98/08951 PCT/EP97/04668 41 (cat. No. 12VET03).

ELISA. Supernatants of hybridoma cultures or ascitic fluids were assayed for antibody activity by solid-phase capture ELISA using protein A-coated plates as described elsewhere (Cloeckaert et al. 1991). Protein A was at 4 mg/ml in five-fold diluted GBS-EDTA (170 mM NaCI, 100 mM glycine, 6mM NaN 50 mM EDTA) pH 9.2. Hybridoma supernatants were added diluted in GBS-EDTA-0.1% Tween 20 (GBS-EDTA-Tw). MAbs specific for brucellin proteins were revealed with peroxidase-conjugated brucellin (bru-perox.) prepared by a modification of the method of Nakane et al. (Nakane et al. 1974).

To determine the titer of anti-LPS Abs, indirect ELISA was performed as described elsewhere (Limet et al. 1988).

Immunoscreening of a Xgtll library. The construction of a B. abortus genomic library has been previously described (Tibor et al. 1994). Recombinant phages were plated at a density of approximately 3 104 PFU per 150-mm plate on E. coli Y1090. Phages were allowed to grow at 42 0 C for 5 h and subsequently overlaid with a 0.45 tm nitrocellulose filter saturated with 10 mM of isopropyl-p-D-thiogalactopyranoside (IPTG). The plates were incubated at 37 0 C for an additional 18 h. The filters were removed and washed in TBST mM Tris-HCI pH 8.0, 0.5 M NaCI, 0.05% Tween 20) and immunoscreening was done as described previously (Tibor et al. 1994) with anti-39kDa antigen monoclonal antibody 5E1E8 or anti-15kDa antigen monoclonal antibody Br25, diluted in TBST-1% BSA. To identify immunoreactive plaques, the filters were successively incubated with RAM diluted 1/250 and then with protein A-perox. diluted 1/1000 in TBST. Filters were developed by incubation in a solution of a TBS containing 0.06% 4-chloro-l-naphtol (Biorad) and 5 mM HO,. The reaction was stopped by washing in distilled water. Positive plaques were picked and eluted into phage suspension medium (20 mM Tris-HCI pH 7.5, 100 mM NaC1, 10 mM MgSO 4 2% gelatine), replated and screened until all plaques were positive.

Lysogen production and preparation of whole-cell extracts. To determine whether the putative recombinant phages produced P-galactosidase fusion proteins, lysogens of each of the phages were generated in E. coli Y 1089. Crude extracts were prepared as described by Hyunh et al. (1985), boiled for 5 min in SDS-PAGE sample buffer (Laemmli 1970). resolved on a 12% SDS polyacrylamide gel and immunoblotted with Mab 5E1E8 or BR25 and the anti- B-galactosidase polyserum by using the procedure described above. Controls, consisting of WO 98/08951 PCT/EP97/04668 42 lysates of E. coli Y1089, infected by a non recombinant lambda gtll were included in each experiment.

Gel electrophoresis and Western blottingwere performed as described by Tibor et al.

(1994).

Amino acid sequencing. The micro sequencing of the P39 peptides resulting from hydrolysis of the purified protein was performed by automated Edman degradation as previously described (Van Fleteren et al. 1992).

Nucleic acid sequencing. Sequence analysis of the DNA fragments cloned in SK+ plasmids was performed using the chain termination procedure (Sanger et al., 1977), adapted to allow analysis on an automated DNA sequencer (Applied Biosystems, Foster city, Calif.).

Sequencing reactions were carried out using the dye-terminator technology, as described by the manufacturer, using the universal or reverse M13 primers. Sequence manipulations were performed using the Intelligenetics software package (Calif., USA).

Oligonucleotides. M13 universal and reverse primers were purchased from Pharmacia.

All other oligonucleotides were synthesized in the laboratory of Neurochemistry,

U.C.L.,

Bruxelles, Belgium.

DNA sequencing. DNA sequencing was performed by the primer extension dideoxy chain termination sequencing method of Sanger (Sanger et al.. 1977)) with Sequenase Version Biochemicals) or T7 Sequencing kit (Pharmacia).

DNA and protein sequence analysis. DNA sequence data obtained from sequencing gels were compiled and analyzed by the DNA Strider 1.2 program (Marck, 1988). FastA, TFastA, Terminator and Isoelectric programs were used with the Genetics Computer Group Sequence Analysis Software Package version 8.0-Open VMS or 7.3-UNIX. The GenBank, EMBL and SWISS-PROT nucleic acid and protein sequences databases were used for homology searches.

Preparation of PBMC and lymphocyte blastogenic assay. Blood was collected from bovines by jugular venipuncture in a 1/10 volume of PBS containing 1.5% EDTA and centrifuged at 1500 g for 15 min. PBMC were prepared on Ficoll-Hypaque density gradient (Mager et al. 1994) and resuspended in RPMI 1640 supplemented with 2 mM L-glutamine, 100 IU/ml penicillen-streptomycin and 10% decomplemented FCS (Gibco-BRL, Belgium).

PBMC were then cultured (105 cells/well) in 9 6-well-round-bottom plates (NUNC, Rockslide, WO 98/08951 PCT/EP97/04668 43 DK.) at 37 0 C in humidified air with 5% CO,. Brucellergen and purified P39 (1 xg/well) were added at the initiation of the cultures; PHA (1 pg/well) was used as a control of proliferation.

After 4, 6 and 8 days of incubation, the cultures were pulsed with 0.8 JLci [3H] thymidine/well for 18 h and harvested onto glass fibber filters with a cell harvester (Titertek 550, Flow Laboratories). Incorporation of radioactivity into the DNA was measured by liquid scintillation counting (1205 betaplate, Wallac, Pharmacia). All the tests were set up in quintuplet cultures and performed at least twice. Results were expressed either as mean counts per minute (CPM) standard deviation (SD) or as stimulating index (SI) calculated by dividing the mean CPM of stimulated cultures by the mean CPM of non-stimulated cultures. Data were statistically analyzed by student's t-test with p 0.05.

Experimentally infected-cows. All cows were from a certified brucellosis-free herd (Morbihan and Finistere, Bretagne, France). Prior to entry into the experiment, serological assays (Complement fixation test, Agglutination test, Rose Bengale, ELISA) and DTH tests with brucellergen (batch 96G091) were performed to confirm that they had not been previously exposed to B. abortus. Ten animals were infected per os with different doses of bacteria: 2 cows with 1.8 1012 E. coli 0:157,2 with 1.05 1012 P. maltophilia, 2 with 0.72 1012 S. urbana and 4 with 1012 Y. enterocolitica 0:9 KNG 1024; one cow was intranasally infected with 0.24 1012 P. multocida. Blood samples and skin testing were performed 35 days after infection.

Naturally infected-cows. Non-pregnant cows from Brucella- infected herds were housed in a fattening centre; a DTH test with brucellergen (batch 96G091) and anti-LPS Ab detection by ELISA were performed as described above.

Caprine and ovine field sera. Sera from field infected goat and sheep were obtained from J. Blasco (Servicio de Investigaciones Agraria, Deputacion General de Aragon.

Zaragoza, Spain) or from J. Salinas (Dept. Pathologia Animal, Facultad Veterinaria, Campus de Espinardo, Murcia, Spain). All sera have been tested in classical serology assays (Rose Bengale test and/or complement fixation). Negative control sera were obtained from a Brucella free sheep flock (INRA, Tours) or from goats roaming in a brucellosis free area (CNEVA.

Lyon).

Delayed-type hypersensitivity test. Intradermal tests in cattle were performed with brucellergen (batch 96G091) and P39 by using a classical tuberculin syringe with a 4 mm needle. A dose of 0.14 mg of allergen in 0.1 ml of PBS was injected. The increase in skin WO 98/08951 PCT/EP97/04668 44 thickness, expressed in mm, was determined with a spring skin meter (Aesculap, Germany) before and 72 h after the injection. A variation in skin thickness greater than 1 mm was considered positive.

Example 2: Purification, characterization and natural occurence of B. abortus P39 Purification of the 39-kDa protein from brucellergen. Protein concentrate (30 mg) from brucellergen was applied to a pre equilibrated Mono Q Sepharose Fast Flow column HR (Pharmacia, Belgium). The column was washed with 60 ml linear gradient of 500 mM NaCI in the same buffer at a flow rate of 5 ml/min and the absorbance at 280 nm was monitored. The fractions of interest were pooled, dialysed against equilibrating buffer, and concentrated in an Amicon stirred cell (Amicon division, Beverly, MA). For further purification, 5 mg of protein from the fraction 4, enriched in P39, was equilibrated in Tris- HCI 20 mM pH 7.4 with 1.2 M (NH 4 SO and applied to a phenyl Superose column HR (Pharmacia). Proteins were eluted with 60 ml of the same buffer with a decreasing gradient in (NH 4 2

SO,.

Fractionation of brucellergen (batch 8A083) by anion exchange chromatography and purification of the 39-kDa protein. Individual fractions of the eluate from anion exchange chromatography of the brucellergen (Fig. 1A) were analyzed by SDS-PAGE and fractions presenting a similar pattern in SDS-PAGE were pooled to obtain 5 major fractions (Fig. 1B). Fraction 4 (F4) which was eluted at 0.15 M NaCI contained a major band at a molecular mass of 39-kDa and additional minor bands around 50-60-kDa. Immunoblot analysis with MAb 5E1E8 indicated that the P39 protein was mainly present in F4 and to a lesser extent in F5(Fig. 1C).

To further purify the P39 protein, proteins from F4 were loaded on a phenyl Superose column. A peak recorded in the flow-through displayed a band of 50-kDa in SDS-PAGE, two peaks appeared at 0.9 M and 0.6 M (NH 4 2 SO, and displayed a single band of 39-kDa (Fig.

1D). In immunoblot analysis MAb 5E1E8 reacted with the P39 in both peaks 2 and 3 and the anti-brucellin polyserum gave a single band corresponding to the P39 molecular mass (data not shown) which demonstrates the high purity of the protein.

WO 98/08951 PCT/FP97/04668 Natural occurence of P39 in organisms of the genus Brucella and bacteria eliciting cross-reaction in serology. Thirty-four Brucella strains, including all six of the known species and all reported biovars, were examined for expression of the P39 on the basis of antigenic recognition. Western blots of the total SDS-bacterial lysates were tested with MAb 5E1E8 and PRA 39. Both MAb 5E1E8 and PRA 39 reacted with a protein of an apparent molecular mass of 39-kDa present in all Brucella strains tested except for B. abortus biovars 5, 6, 9, B. ovis and B. neotomae (Table MAb 5E1E8 and PRA 39 gave no reaction with whole-cell lysates prepared from Y. enterocolitica 0:9, P. naltophilia, P. multocida, S. urbana and E. coli 0:157 strains.

Previous studies demonstrate variation in protein expression among Brucella species: indeed the BCSP 31-kDa periplasmic protein is not expressed in B. ovis (Bricker et al. 1988) as well as the Cu-Zn superoxide dismutase in B. neotomae (Bricker et al. 1990). The absence of the P39 protein in the 3 B. abortus strains is somewhat unexpected because of the relatively homogeneous expression of the P39 protein in the other B. abortus strains. Moreover.

Southern hybridization studies showed that the P39 gene is present in all the Brucella strains tested Verger, personal communication). This observation also suggest that expression of the P39 protein is not essential for bacterial survival, and that it should be possible to delete this gene (see further example A possible explanation for the failure to detect the P39 protein may be related to a difference in the specificity for the antigenic determinant on this protein. Alternatively, the protein may be physically altered through the accumulation of mutations, reducing the recognizable epitopes among strains. It is also possible that the regulation of the transcription of the P39 gene may be altered in some strains resulting in no gene expression or gene expression under specific conditions. Finally, two insertion sequences have been characterized and are known to occur more frequently in B. ovis (Halling et al.

1993; Ouahrani et al. 1993). The P39 gene or its regulation elements could be disrupted by such sequences in this species.

TABLE 2 (next page) Species and strains tested WO 98/08951 PCT/EP97/04668 46 Species and strains Immunoreactivity with Mab 5E1E8 (b iovar) and PRA39 antiserum B. abortus 544 B19 45/20 (rough)* 86/8/59 Tulya 292 B3196 870 (6) 80-236 (6) 89-43 (6) C68 (9) 75-60 (9) 76-2 99 (9) 77-9 (9) 80-133 (9) 87-46 (9) 90-64 (9) 91-28 (9) 91-135 (9) B. nielitensis Revl 16M H38 (roughn) 63/9 Ether B 11 (rough) B. suis S2(1) 1330 Thomsen 686 40(4) 513 B. canis RM6/66 B. neotomae 5K33 B. ovis Bow 63/290 Y. enterocolitica 0:9 P. nialtophilia S. urbana P. multocida E. cli 0: 157 WO 98/08951 is a variant of the smooth H38 strain PCT/EP97/04668 WO 98/08951 PCT/EP97/04668 48 Example Cloning and sequencing of the Brucella abortus 39 kDa antigen Cloning of the P39 gene. A genomic library of a B. abortus has been previously constructed in Xgtl 1 phage (Tibor et al. 1994). Immunoscreening was carried out as described in example 1. Recombinant phages were screened for expression of P39 with MAb 5E1E8.

Two positive plaques were identified; they were further amplified and their DNA purified. The sizes of the inserts, determined after restriction digest of X phages DNA with EcoRI I, were approximately of 1.6 and 3.9-kb for 1 clone gtl.2, respectively.

To determine the size of the protein recognized by the MAb 5E1E8, and whether it was produced as a P-galactosidase fusion protein, lysogens from both the Xgtl.1 and Xgtl.2 recombinant clones were produced in E. coli Y1089 and cultured in the presence of IPTG to induce the lac promoter. Lysogen production and preparation of whole cell exracts was carried out as described in Example 1. Whole-cell extracts derived from non-induced or IPTGinduced lysogens were immunoblotted with MAb 5E1E8 or anti-p-galactosidase antiserum.

Cell extracts from recombinant lysogens Agtl.1 reacted with MAb 5E1E8 and the signal intensity increased when IPTG was added; the recognized polypeptide had an apparent molecular mass greater than that of P-galactosidase and also reacted with the anti-pgalactosidase antiserum, indicating that it was a P-galactosidase fusion product. Cell extracts from recombinant lysogens Xgtl.2 strongly reacted with MAb 5E1E8 independent of IPTG induction; the expressed protein recognized has an apparent molecular mass of 39-kDa comparable to the B. melitensis B 115 native protein, indicating that the X clone gtl.2 contains the entire gene.

Localization of the P39 gene in the cloned fragment. Lambda gtl. 1 and gtl.2 inserts digested with EcoR I were sub cloned into the pTZ19R vector yielding pTZ1.1 and pTZ1.2, respectively. Restriction maps of the recombinant plasmids (Fig. 2) indicate that the pTZ1.2 insert contained the pTZ1.1 insert. As already observed with Xgtl.2 lysogen, pTZ1.2 transformed E. coli cells express the complete P39 protein; in contrast, pTZ1.1 did not express any polypeptide recognized by MAb 5E1E8. Since synthesis of P39 could be detected in pTZ1.2 independently of the insert cloning orientation in pTZ19R (data not shown), the Brucella P39 gene seems to be transcribed from its own promoter.

To localize the P39 gene, the 1.7 and 2.2-kb Xbal-EcoRl fragments of PTZ1.2 were sub cloned into the pSK vector (pSK1.2 and pSK2.2, respectively) (Fig. The plasmid WO 98/08951 PCT/EP97/04668 49 pSK2.2 did not express any gene product recognized by MAb 5E1E8 or PRA 39; on the contrary, pSK1.2 expressed a protein with the same molecular mass as the gene product from pTZl.2.

To localize more precisely the ORF encoding the P39 gene, the 1-kb SphI-EcoRI and the 0.7-kb XbaI-HindlII fragments from pTZ1.2 were sub cloned into pUC19 and pSK vectors, respectively (Fig. The plasmid pSK3.2 did not express any gene product recognized by either MAb 5E1E8 or PRA 39. On the contrary pUC3.1 expressed a protein of recognized by MAb 5E1E8 and PRA 39, independently of the induction with IPTG.

Nucleotide sequence of the full-length P39 gene. The DNA sequence of the P39 gene (Fig. 3) was determined from overlapping fragments which were subcloned into pUC 19 or pSK vectors and sequenced as described in materials and methods. A total of 1438 bases were sequenced on both insert strands. Sequence analysis revealed the presence of seven potential ATG/GTG start codons located at bases 169, 187, 241, 319, 352, 400 and 406. A stop codon TAA is located at position 1390. A putative ribosome binding site GAGG is found 10 bp upstream from the GTG at position 227. This suggests that the potential ORF codes for a protein with a molecular weight of 41.1-kDa, the sequence of which is shown in figure 3. This is in close agreement with the apparent molecular mass of the native P39. There is a GC-rich region of dyad symmetry identified 10 bases downstream from the stop codon which could function as rho-independent transcription terminator (Denoel et al; 1995; Tibor et al. 1994).

The calculated pi (4.86) is in agreement with the pi observed for the native P39 from Brucella The deduced amino acid sequence of the nucleotide sequence extending from position 909 to 929 of the P39 gene ORF is in accordance with the amino acid sequence of the native P39 protein determined by Edman degradation of partial peptide digest products. The G-C content of the 1152 bp gene sequence encoding the P39 protein is 57.1% guanidine and cytosine which is as expected for Brucella genes.

Similarities between the deduced P39 amino acid sequence and the sequences available in the databases were searched for with the FastA ans TFastA algorithms. P39 sequence shows significant similarity (optimized score 100) to the Leptothrix discophora excA excreted protein (66.1% similarity and 48.4% identity).

Discussion and conclusion. The P39 antigen gene was identified from clones derived from genomic Brucella DNA library constructed by using the expression vector Xgtl 1. A WO 98/08951 PCT/EP97/04668 subcone of a 3.9-KB EcoRI -EcoRI fragment was expressed in E. coli and the recombinant product had a similar molecular mass as the native protein. The P39 gene is transcribed from its own promoter in E. coli as it has been described for other Brucella cloned genes (Ficht et al., 1989a, 1989b; Mayfield et al. 1988; Tibor et al. 1994). In addition to the reactivity with MAb 5E1E8, the similarity between the recombinant P39 gene product and the native protein was confirmed by partial amino acid sequence of the native P39.

The deduced sequence of the P39 protein is 66% similar to the Leptothrix discophora excA deduced protein sequence (Corstjens, 1993). The excA gene encodes an excreted protein in this species. However, we have no indication that the P39 gene product could be excreted and no functional role can be attributed to this protein based on its amino acid sequence. The construction of a P39 gene Brucella deletant strain will give us evidence to propose a role for this protein and allow us to further study its cellular immunogenicity (see example 8).

Example4 Cloning and sequencing of the Brucella abortus 15 kDa antigen The construction of a genomic library of B. abortus strain 544 in the phage vector lambda gtl1 has been described (Tibor, Infect. Immun. 1994). An aliquot of this library was plated and 30.000 plaques were screened for reactivity with the Br25 monoclonal (Silenus, Australia) essentially as described (Tibor et al. Infect. Immun, 1994). Two positive plaques were identified and the phages were purified until all of the plaques reacted with the monoclonal Purified phages were amplified and DNA was extracted for further analysis. Digestion with the restriction enzyme EcoR1 yielded three fragments of 3.7, 1.7 and 0.83 kbp respectively, apart from the lambda right and left arms. The EcoR1 fragments were cloned into the EcoR1 site of pBluescript and transformants were induced with IPTG. Induced cells were boiled in SDS sample buffer and analysed on Western blot for reactivity with the monoclonal. One recombinant clone displayed a reaction profile showing three signals at kDa, 20 kDa and 29 kDa respectively. The corresponding recombinant plasmid was termed WO 98/08951 PCT/EP97/04668 51 pBBr25-1 and was shown to contain the 0.83 kbp DNA insert.

Sequence analysis of the insert DNA revealed two open reading frames (ORF) in the 842 bp fragment, one on each strand. The largest ORF is capable of encoding a protein of 174 amino acids whereas the smaller ORF encodes 104 amino acids.

To identify the ORF encoding the protein recognized by the Br25 monoclonal, constructs were made to express both ORF in E. coli in the vector pIGALMPH. For the largest ORF, the fusion protein starts 28 amino acids before the first methionine encoded by the ORF. This should result in a protein with a calculated mass of 29.3 kDa. For the smaller ORF, the expressed information starts at the fifth codon relative to the first methionine codon in that ORF. The calculated mass of the latter fusion protein is 17,5 kDa.

Both constructs were induced in E. coli [SG4044/C1 85 7 and the cell extracts were analysed by western blot using the anti crolac tag monoclonal as well as the Br25 monoclonal.

For the construct expressing the largest ORF, a protein of about 29 kDa was revealed with the anti crolac tag monoclonal. However, no reaction with the Br25 monoclonal could be demonstrated. A similar experiment with the smaller ORF revealed a protein of about 15 kDa which strongly reacted with the BR25 monoclonal.

This experiment indicates that the smaller ORF encodes the protein (called 15 kDa antigen from now on) that reacts with the BR25 monoclonal. The nucleic acid sequence and corresponding amino acid sequence of this 15 kDa antigen and corresponding gene from B.

abortus is shown on figure 4.

Example Bacterial expression of the B. abortus genes encoding the 39 kDa and 15 kDa antigens 1.1. Expression in E. coli of the 15 kDa antigen of B. abortus.

The features of the vectors used for the expression of the Brucella antigens have been described before (Van Gelder et al., 1993: Gilot et al., 1993) and are summarized in figure Briefly, a construct to fuse the 104 codon long ORF encoding the 15 kDa protein to WO 98/08951 PCT/EP97/04668 52 the crolac leader peptide in the vector plGALMPH was designed. The vector was opened with restriction enzyme BamH1, sticky ends filled and then treated with Ncol. The Brucella gene fragment was treated with Fokl, sticky ends filled and then cut with Ncol and the resulting 130 bp fragment was cloned. The Brucella (15 kDa antigen encoding)-gene was then completed by treating the former construct with Ncol and Stul and by insertion of the Brucella Ncol/Ava2 fragment, of which the Ava2 site had been filled. The resulting plasmid was designated The Brucella gene information expressed in this construct is starting at the fifth codon of the ORF, relative to the first methionine codon. The calculated MW of the resulting fusion protein is 17479 d. Analysis of the protein produced in the bacteria containing this plasmid by SDS/PAGE revealed an induced protein migrating at the 15 kDa position (figure 6).

Upon lysis of the bacteria by French press action,the fusion protein was found to be mainly membrane associated. The protein was solubilized in 6M guanidinium chloride and purified by IMAC (Immobilized Metal Ion Affinity Chromatography) to yield a preparation of higher than 95% purity (figure An overall yield of 7 mg purified protein per liter fermentation broth was obtained.

This purified recombinant 15 kDa antigen was then used in indirect ELISA to evaluate the reactivity with sera from different animals (see example 6).

1.2. Expression of 39 kDa antigen of B. abortus.

This antigen has been expressed as a fusion protein in the pIGFHIO and the pIGALMPH vectors (figure 5c and 5a respectively). The (39 kDa antigen encoding)-gene was cloned as a Bgl2/Xbalfragment in the vectors opened with BamH1 and Xbal. In both cases a good expression level was obtained but the proteins are trapped in inclusion bodies. The pIGALMPH construct (IMPHBru39) was used for large scale preparation and purification. To allow purification, the inclusion bodies are separated from the rest of the cell debris by centrifugation, extensively washed with Tris buffer containing 1.05% Triton X100 pH 6.8 and subsequently solubilized in 6M Guanidinium chloride to allow further purification of the 39 kDa protein on IMAC. From a fermentation of 15L, about 550 mg of purified 39 kDa fusion protein was obtained upon IMAC chromatography (figure This purified recombinant P39-antigen is subsequently evaluated serologically (see example 6).

WO 98/08951 PrT/P9a7/0m6 53 1.3. Construction of new expression vectors to improve solubility.

Since both proteins (15 kDa and 39 kDa) produced as crolac-fusions were highly insoluble and could not be kept in solution without denaturing agents, an attempt to express them in another configuration was made.

To allow fast purification of the expressed protein, the gene was cloned into an expression vector to fuse the N-terminus of the protein to a small leader peptide with the sequence: vector pIGRIHisG: leader peptide M G H H H H H or vector plGRIHisA: leader peptide M AH H H H H H These vectors should allow to obtain a more soluble protein.

The constructions were made in both vectors and for both proteins (15 kDa and 39 kDa). The expression vectors pIGRIHisA or plGRIHisG (see figure 5b) were opened with Nsil, blunted and treated with Xbal. The (39 kDa antigen encoding) gene was inserted as a Bgl2 (filled)/Xbal fragment. In this way only about 45 bp (encoding 15 aa) of the ORF beyond the putative start codon GTG at position 241are missing.

For the 39 kDa antigen fusion proteins (designated HisGBru39 resp. HisABru39) a good expression level was obtained. Analysis of the soluble and insoluble fractions after French press lysis of induced cells showed that about 20 to 30% of the expressed protein was in soluble form whereas the remaining part was present in inclusion bodies. WB analysis of the expressed protein using the 5E1E8 MAb directed to the 39 kDa protein confirmed the identity of the protein produced.

To express the (15 kDa antigen encoding)-gene in the same configuration, the vector was again opened with Nsil and blunted, followed by Xbal digest. The insert fragment was recovered from the former construct plGALMPHBr25 as a BamHl (filled)/Xbal fragment (supra). Both plasmids (plGRIHisABr25 and plGRIHisGBr25) were introduced in E. coli strain SG4044 and the expression was analysed. Upon coomassie staining of the gel. no induced protein was apparent.

These results indicate that the expression level of the (15 kDa antigen encoding)-gene in the latter constructs is considerably lower than with the plGALMPHBr25 construct.

Therefore, only the latter will be used for production of the 15 kDa fusion protein.

WO 98/08951 PCT/EP97/04668 54 Example6: Serological evaluation of the recombinant 39 kDa and 15 kDa B. abortus antigens The purified recombinant proteins (described in Example 5 were used in an indirect ELISA setup to study the reactivity with sera from Brucella infected animals. The antigens were diluted from the stock solution containing 6M guanidinium chloride or 8M urea into a 0.1M carbonate buffer at pH 9.5, to obtain a final concentration of 5gg/ml of the recombinant protein. From this solution, 0.1 ml was applied into each well of the microplate and incubated for 1 hour at 37°C. The wells were subsequently saturated with 0.1% casein solution (1 hour at 37°C) and washed. The sera were diluted 1/100 in a buffer containing 0.01 casein and 0.1 ml of this dilution was incubated into each well (37°C for 1 hour). All sera were tested in duplo in adjacent wells. Bound antibodies were revealed with a species specific (goat. sheep or cattle) second antibody conjugated to horse radisch peroxidase (HRPO). Finally. colour development was obtained with hydrogen peroxide/tetra-methyl-benzidin as the substrate/chromogen couple. Colour development was usually stopped after 30 min by adding 0.1 ml of 2N sulfuric acid.

When both recombinant antigens described in Example 5 were coated simultaneously, they were each diluted to a final concentration of 5 tg/ml in carbonate buffer pH 9.5 as described above.

For the interpretation of the ELISA results, the cut off value was defined as the mean of the negative control sera plus three times the standard deviation When signal to noise ratio is used, the noise is defined as the mean of the negative sera and the cut off is placed at a S/N of 2.

Sera from goats, sheep and cattle were analysed in the ELISA described above. For each sample, the analysis was done with both antigens separately as well as in combination.

The cut off was determined with a set of sera from certified brucellosis free animals from each species. Results obtained with some individual goat sera are shown in figure 8.

As far as ovine sera are concerned, the distribution found when a set of 9S field sera was investigated is shown in table 3. This table indicates that the combination of both antigens allows the detection of over 90% of ovine sera, positive in classical serology. Moreover, the combi ELISA detects 25 sera of animals which are negative in classical serology but originating from an infected herd. This results in a level of 48% positive sera. whereas WO 98/08951 PCT/EP97/04668 classical serology detects only 24 In addition, the combi ELISA has a higher sensitivity than the ELISA using both antigens separately, indicating complementarity between both antigens in the combi ELISA.

Table 3 Results obtained with ovine sera reacting in ELISA with the 39 kDa and 15 kDa antigens of B. abortus, either alone or in combination.

Antigen Sensitivity kDa 87.5 39 kDa 33 combi (15 kDa 39 kDa) 92 Total number of sera tested: 98; sensitivity is calculated as the number of sera reacting in ELISA divided by the number of sera positive in classical serology (CFT and Rose Bengale test) In another set of 49 ovine sera, all positive in classical serology, the detection level (sensitivity) for the combi ELISA was 96%.

For cattle sera from field infected animals, the 39 kDa antigen detected 17% of animals positive in classical serology, whereas the combination ELISA showed a sensitivity of 22%.

In these cases, signals were generally rather low, resulting in a S/N ratio under Exampli 7: Evaluation of the 39 kDa antigen as a stimulatory antigen for the cellular immune system 20 Delayed type hypersensitivity test. Fifteen Brucella naturally infected cows were tested for DTH reaction with brucellergen, F4 and peak 2 recovered from phenyl Superose (purified 39 kDa antigen) (Table Among these 15 cows, one animal (cow 1) reacted neither to brucellergen, F4 nor purified 39 kDa antigen. The cows (cow 2, 3 and 4) presenting a WO 98/08951 PCT/EP97/04668 56 positive reaction with brucellergen did not react to F4 or purified 39 kDa antigen. The eleven remaining cows reacted to the three allergens but displayed a slightly weaker reaction to F4 (2.15 mm mean skin thickness increase) than with brucellergen (2.76 mm). The reactivity of the purified 39 kDa antigen was similar to F4.

To evaluate the specificity of a DTH test with the 39 kDa antigen and brucellergen, 11 bovines were experimentally infected with bacteria that are known or presumed to induce immunological cross-reactions with Brucella (see Example These animals were intradermally injected with the three allergens preparations. Results are shown in table Except for one of the two S. urbana-infected cows which slightly reacted to brucellergen and the 39 kDa antigen, none of the other infected animals reacted positively to either allergen.

The reaction observed with one S. urbana-infected cow was however limited to the injection point and did not correspond to the typical aspect of the DTH reaction on Brucella-infected cows since a positive reaction was already observed after 24 h and progressively decreased after 48 h.

These results suggests that Brucella specific epitopes on the 39 kDa antigen are probably recognized by DTH specific effective T cells in most Brucella- infected cows presenting a clear DTH reaction to brucellergen.

Lymphocyte blastogenic assay. PBMC from a Brucella-infected cow that elicited the highest DTH response to brucellergen (batch 96G091) (cow 3) and from an non-infected cow were used to test the 5 fractions of brucellergen (batch 8A083). PBMC from the non-infected cow did not proliferate to any of the allergen preparations at either time whereas PBMC from the infected cow proliferated strongly to brucellergen, fraction F4 and F5 after 9 days of culture with a SI (stimulation index) of 21,2, 22.7 and 20 respectively (Fig. 9).

The reactivity of the purified 39 kDa antigen in LBT assay at day 9 was then tested on PBMC from 4 other Brucella-infected cows and compared with two brucellergen preparations (batches 96G091 and 8A083) (Table The 39 kDa antigen induced a particularly high proliferation of PBMC from all 4 infected cows (5.9 SI 35.5) when compared with both brucellergen preparations (10 SI 23 and 4.1 SI 7).

Specificity of the 39 kDa antigen in LBT test was assessed on PBMC from cows infected with Y. enterocolitica 0:9, E. coli 0:157 and S. urbana. None of these PBMC responded to brucellergen or 39 kDa antigen (Table 6).

WO 98/08951 PCT/EP97/04668 57 Discussion and conclusion. The value of the DTH test with brucellergen as a complementary tool to serology .in ELISA anti-LPS antibodies for brucellosis diagnosis is clearly demonstrated in our study and confirms earlier observations (Fensterbank, 1977).

Interestingly, purified 39 kDa antigen elicited a DTH reaction in most cows presenting a DTH reaction with brucellergen. The relatively moderate DTH reaction of 39 kDa antigen in some animals does not imply a weak immunogenicity of the 39 kDa antigen, but the superior potency of brucellergen can in some cases be attributed to its complexity which enables the stimulation of T cells of multiple specificities. This observation also suggests that an allergen composed of only one protein would not allow the detection of all infected animals.

In view of these results, we examined the in vitro cellular immune response to the various fractions of brucellergen. With freshly isolated lymphocytes, the maximal brucellergen-specific cellular repertoire is available for recognition of particular antigens.

Experiments using different protein fractions to stimulate lymphocyte proliferation suggest that, besides the enriched 39 kDa antigen fraction, no additional brucellergen fraction (overlapping the 35-60-kDa molecular mass range) from the 8A083 batch is able to induce lymphocyte proliferation to the same extend as 39 kDa antigen enriched fractions. One may speculate that the increased immunogenicity of 39 kDa antigen over other fractions of brucellergen may be attributed to the preferential expansion of lymphocyte clones which are sensitive to Brucella 39 kDa antigen in infected animals. Further analysis of the proliferative response with additional Brucella-infected animals confirmed that 39 kDa antigen protein constitutes a preferential target antigen for T lymphocytes of Brucella-infected cows. Although substantial variations in lymphocyte responsiveness among animals were observed, this variability may be explained by various factors including molecular histocompatibility complex polymorphism, infectious dose, age of animals or resistance to bacterial infections.

Additionally, it is conceivable that some animals harbour more T cells specific for other Brucella antigens than to the cytoplasmic 39 kDa antigen. The variability of the proliferative responses to the different brucellergen preparations tested may correlate with qualitative and quantitative variations in protein compositions observed in SDS-PAGE protein profiles (data not shown).

The specificity of the cellular immune response not only to brucellergen but also to 39 kDa antigen was ascertained by the fact that T lymphocytes from non-infected cows anc WO 98/08951 PCTIEP97/04668 animals infected with bacteria cross-reacting with Brucella were not stimulated by any of the antigens. This could not have been presupposed as it has been shown that a Brucella porin induces a high response of PBMC from one heifer infected with the E. coli 0116:H31 strain, suggesting a possible cross-reaction with the porin of E. coli (Baldwin et al. 1985). The fact that the DTH test and the lymphoblastogenic test with 39 kDa antigen seems to be not only species-specific but also sensitive suggests that this antigen could be very useful for the diagnosis of brucellosis in cattle.

TABLE 4 Brucella Skin thickness increase (mm) ELISA antinaturally LPS antibodie infected-cows Brucellergen F4 P39 titer (/ml) ident. N' 96G091 96G091 1 0.3 0.5 0.5 2 2.5 0.5 0.5 24 3 4.5 0.4 0.4 8 4 1.7 0.2 0.2 4.3 1.5 1.5 6 2.5 1.9 1.9 7 1.2 1.3 1.3 23 8 2.2 1.2 1.2 9 1.3 1.2 1.2 7 3.0 1.5 1.5 11 4.6 3.3 3.3 4 12 4.0 4.4 4.5 4 13 1.4 4.0 3.9 4 14 3.0 1.3 1.3 4 3.0 2.0 2.0 WO 98/08951 WO 9808951PCT/EP97/04668 TABLE r 1 Number of cows Bacteria strain Dose Skin thickness increase (mm)

ELISA

anti-LPS Ab titer Brucellergen F4 P39 96091 (U/mi) r t E. coli 0: 15 7 P. maltophilia P. multocida S. urbana Y. enterocolitica 0:9 KNG 1024 1.8 1012 1.05 1012 0.24 1012 0.72 1012 1012 <1 <1 <1 <1 1.3 <1 <1 <1 1 1.3 6 <1I <1I 7.7,.4.9.

L I TABLE 6 Bacterial strain Number Brucellergen Brucellergen P39 of cows 966091 8A083 B. abortus 4 1.0 4.1 6.2 23 6.8 25.2 12 5.6 5.9 7.0 35.5 Y. enterocolitica 4 1.5 SI 1 1.5 SI 1 1.5 SI 0:9 S. urbana 2 1.5 SI 1 1.5 SI 1 1.5 SI I E. ccli 0: 157 2 1.5 SI 1 1.5 SI 1 1.5 SI WO 98/08951 PCT/EP97/04668 Example 8: Construction and characterization of Brucella strains deleted for the genes encoding the 39 kDa and/or 15 kDa antigens.

Principle In the following example, known vaccine strains B. abortus B19 and B. melitensis Rev.1) are modified by deletion of one (or more) gene(s) known to encode an immunodominant protein which induces the humoral immune response in the infected individual, assuming that this protein is essential neither for bacterial survival nor for conferring protection. This should lead to strains that fail to elicit antibodies to the deleted protein, hence allowing distinction between animals vaccinated with one of these strains and animals with field infection.

Description of the deletion strategy.

The strategy used implies a double homologous recombination event leading to the replacement of the resident wild type gene by a genetic marker (Halling et al., 1991; Tatum et al. 1994). A suicid vector containing the Brucella DNA insert where the gene of interest has been replaced by the gene encoding kanamycin resistance, but leaving sufficient flanking sequence to allow recombination, is introduced into a recipient Brucella strain by conjugation with an E. coli donor. Two types of transconjugants resistant to kanamycin can be obtained.

"Integrants" resulting from a single recombination event between the chromosomal copy and the residual flanking regions from the incoming gene. These clones have integrated the entire plasmid and are therefore ampicillin resistant. "Deletants" on the contrary will be kanamycin resistant ampicillin sensitive and will be isolated on condition that the deleted gene of interest is not essential for Brucella survival. This strategy allows selection of deletants after one step.

After characterization of the deletants by Southern- and immunoblotting, the effect of deletion on virulence and/or on protection can be studied in the mouse model.

In an attempt to develop a positive selection system for deletant strains in a background of integrants, a new selection system based on the sacBR genes has been applied to Brucella.

The sacB gene of Bacillus subtilis, regulated in cis by the sequence sacR. encodes a levan sucrase that catalyses hydrolysis of sucrose as well as synthesis of levans (Gay et al, 1983) The expression of the sacB gene in the presence of sucrose is lethal in many gram WO 98/08951 PCT/EP97/04668 61 negative bacteria and this gene is therefore useful as a positive selection marker in these bacteria (Simon et al, 1991; Kon6 et al, 1991). When the gene transfer suicid plasmid is carrying the SacB gene, integrants will be sensitive to sucrose whereas deletion strains will be resistant since they do not carry the SacB gene. The following tabkle summarizes the different phenotypes to be expected.

Table 7 Phenotype of the possible recombinant strains resulting from recombination.

Strain Nal/Kan Nal/Kan/Amp Nal/Kan/Suc5 parental (NalR)* integrant deletant Parental strain is resistant to Nalidixic acid Experimental part Brucella strains resistant to nalidixic acid (NalR) have been selected and isolated, and are used as recipient strains. A mobilizable suicide vector based on ColE1 has been constructed. For each gene of interest, the KanR gene has been inserted into this vector flanked by the 5' and 3' regions of the Brucella gene of interest.

For the use of the sacBR marker, NalR Brucella strains have first been shown to grow in the presence of 5 sucrose. To demonstrate that the sacBR marker was toxic for Brucella in the presence of 5 sucrose, this marker was cloned into a broad host range plasmid and was then introduced into Brucella by conjugation. The sensitivity of the Brucella transconjugants (containing plasmids carrying the sacBR marker) seemed to depend on the medium composition. However, the sacBR marker has proved to be toxic for Brucella after integration of this marker into the Brucella chromosome. Hence, the sacBR marker provided an effective counter-selectable marker in B. abortus and was used to improve the efficiency of isolation of deletants. The sacBR marker was inserted into the plasmids constructed to delete the genes encoding the 15 kDa (=br25 gene) and 39 kDa (=P39 gene) antigens of the invention. The WO 98/08951 PCT/EP97/04668 62 construction of the deletion plasmids used to carry out the deletion of the P39 gene (P39 deletion plasmid) and the br25 gene (P15 deletion plasmid) is shown in figure 10a and respectively, and is specified below.

Construction of the P39 deletion plasmid The EcoRI-XbaI Brucella DNA fragment of 1650 bp, encoding the 39 kDa antigen was inserted into the corresponding sites of the MCS (multiple cloning site) of the plasmid pBluescript. This plasmid was then digested with BsmI, blunted and BamHI linkers were added. The plasmid was subsequently digested with BamHI and BglII and the larger fragment was purified and ligated to the KanR gene isolated from pUC4K (Pharmacia, Uppsala) by BamHI digest.

The RK2 oriT (transfer origin) was isolated from the pTJS82 plasmid (Cornelis, 1989.) as a 760 bp EcoRI fragment and inserted into the unique EcoRI site of the plasmid described above. The layout of the final construct is shown in figure Construction of the P15 (br25) deletion plasmid.

The pBluescript plasmid was modified by deletion of the Sspl fragment, containing the fl origin, and replacement of this fragment by the RK2 oriT, isolated from pTJS82. This construct was designated pSK-oriT. To introduce the br25 gene fragments, the following steps were taken.

Step 1: The 400 bp BglI-PstI fragment containing the sequences downstream from the br25 ORF was made blunt ended and inserted into the SmaI site of pSK-oriT.

Step2: A 240 bp fragment upstream of the br25 ORF was isolated with NcoI and HindIII. The Ncol site was blunted before the HindlIl digest and the resulting frament was cloned into the plasmid obtained in step 1, opened with HindIII and EcoRV.

Step3: The KanR gene was isolated from pUC4K as a Pstl fragment and cloned into the PstI site of the plasmid obtained from step 2 to obtain the plasmid pDBr25.3.

Step4: To introduce the sacBR marker, the plasmid pDBr25.3 was digested with BamHI, blunted and treated with Scal. The fragment containing the oriT and the kanR gene is isolated (fragmentl). The sacBR marker was available as a 2.6 kBp BamHI-Pstl fragment in the pBluescript vector. This plasmid was opened with Pstl, blunted and treated with Scal.

WO 98/08951 PCT/EP97/04668 63 The fragment containing the colEl and sacBR genes was purified and ligated to fragment 1 to obtain vector pDSBr25. This vector was used for the deletion experiments. The structure of the deletion casette is shown in figure Results of the deletion experiments.

In Table 8 the different Brucella strains for which deletion mutants have been isolated, are listed together with the genes which have been deleted. In Table 9 the number of strains which have been isolated with a deletion in a particular gene has been listed for each of the parental strains studied.

All the deletant strains have been analysed by Southern blot and hybridization with a kanamycin gene probe and a probe derived from the gene to be deleted. The Kan probe was prepared from a 1.28 kb EcoR1 fragment isolated from pUC4K (Pharmacia). The probe to detect the P39 gene was prepared from a 1208 bp BglII/Bsml fragment. This fragment is localised inside the P39 ORF and is absent on the plasmid used to delete the P39 gene. The gene probe was prepared from a 840 bp EcoRI/EcoRI fragment containing the br25 gene.

All probes were labeled with biotin by random priming with the Neblot phototope kit (Biolabs).

Whole genomic DNA from the Brucella strains to be analysed was digested with HindIII, gel separated and blotted onto a nylon membrane (immobilon-S) with a Hybaid vaccuum blotter (Biozym) and hybridized successively with 2 different probes. Hybridization and chemiluminescent detection were performed according to the Phototope detection protocol (Biolabs).

For the strains deleted in the P39 gene, two bands (1650 and 850 bp) were detected in the parental wt (wild type) strain using the P39 gene probe (figure 1 la). In the P39 deletant strains, these bands were absent. DNA from the wt strain did not hybridize to the kanamycin probe, but in the deletant strains, two bands (1800 and 950 bp) were revealed as expected since the kanamycin gene contains a HindIII site (figure lb).

For the br25 gene probe, a doublet at 3500 bp was revealed in the parental wt strain.

In the deletant strains, a doublet at about 4000 bp was detected (figure 12a). This is different from the P39 situation, since the br25 gene probe still contains the flanking fragments used in WO 98/08951 PCT/EP97/04668 64 the deletion plasmid. When the kanamycin probe was used, no signal was obtained on the wt parental strain and a doublet at about 4000 bp was detected in the deleted strains (figure 1-1b).

Table 8: Brucella strains with confirmed deletions and the genes affected.

Strain Gene deleted Corresponding antigen B. abortus B 19 P39(bru39) 39 kDa B. mnelitensis 16M br25, P39(bru39) 25 kDa, 39 kDa B. melitensis H38 P39(bru39) 39 kDa B. melitensis Rev. 1 P39(bru39), br25 39 kDa, 25 kDa B. suis 1330 P39(bru39) 39 kDa Table 9: Numer of deletion strains isolated for each parental strain and per gene studied.

Strain P39(bru39) B. abortus 544 0 0 B. abortusBl19 0 B. melitensis 16M 1 2 B. melitensis B 11 10

ND

B. melitensis H38 2 0 B. mielitensis Rev. 1 IM(623 B. suis 1330 1 0 deposited at NCTC on July 31, 1996 under No. 12944 2 deposited at NCTC on July 31, 1996 under No. 12942 one of the six strains isolated was deposited at NCTC on July 31, 1996 under No. 12943 WO 98/08951 PCT/EP97/04668 Discussion Frequentc of plasmid transfer We have clearly observed a positive relationship between the number of generations of the Brucella recipient strain (cell growth stage) and the number of Brucella transconjugants obtained after conjugation. This confirms in part what has been reported elsewhere for the efficiency of transposition after electroporation (Gerhardt et al, 1990). Because both mutation processes make use of the same bacterial recombination system, they could be influenced by the same factors.

Depending on the gene and on the recipient strain, the percentage of deletants varies between 0% and 16% of the screened recombinants with the first selection method.

Conjugation being an efficient transfer method, the introduction of the suicide plasmid in Brucella does not seem to be the limiting step. However, it has to be noticed that although a high number of Brucella transconjugants have been obtained in the majority of conjugation attempts, for some strains or plasmids used, conjugation resulted in a very low number of recombinants. If conjugation is the limiting step in these cases, plasmid introduction by electroporation might be helpful.

Frequency of deletion versus integration A double homologous recombination event is necessary for deletion to occur and the frequency of double recombination is very low in Brucella spp. in our hands. On the one hand, the recipient Brucella strains used are all resistant to nalidixic acid caused by a mutation in host DNA gyrase. This enzyme is required for homologous recombination. The effect of the mutation on the enzyme activity during recombination has, as far as we are aware, not been described. However, Nal R strains are currently proposed as recipient strains in deletion protocols. On the other hand, DNA entering the cell by conjugation is single stranded and is then copied and circularised. DNA introduced in bacteria by electroporation is a double stranded circular molecule. This difference in DNA structure may account for the higher recombination frequency reported after electroporation by research groups deleting other Brucella genes (Halling et al. 1991; Tatum et al. 1994).

The recombination frequency seems to be positively correlated with the size of the WO 98/08951 PCT/EP97/04668 66 flanking regions inserted in the suicid vector. Longer flanking regions have been cloned and used to delete the Ompl9 gene resulting in several potential deletants for strain 544 (unpublished). Longer segments have also been cloned for the br25 gene. However even with these longer sequences the recombination frequency remains low.

Positive selection procedure With this in mind the selection procedure using the SacBR system for positive selection was introduced. During our experiments it was found that susceptibility to ampicillin for the screening of deletants versus integrants is not entirely reliable and southern blot analysis is strictly needed to confirm the nature of a phenotypic deletant candidate. Some AmpS clones were identified as integrants whereas some clones resistant to 10 mg/ml of ampicillin could be deletants.

By using SacBR as positive selection marker the percentage of deletants can reach 100% of the clones that grow in presence of 5 sucrose. Despite this improvement of the selection efficiency, some deletants have not been isolated a P39 deletant of strain 544).

Biological characterisation of the deletant strains isolated.

The effect(s) of the deletions on Brucella survival and virulence in vivo as well as the influence on the protection conferred by the deleted vaccine strains has been evaluated in the mouse model. Results concerning the residual virulence of the P39 deleted vaccine strains Rev. 1 and B19 are discussed below.

Brucella spp. are described as facultative intracellular pathogens able to survive and multiply inside macrophages. Study of the survival of the deletant strains in vitro in macrophages could also give information about the potential role of the studied gene in bacterial virulence.

The deletion strains have been studied for alterations in their bacterial characteristics and metabolic behaviour. None of the deletion strains studied shows a detectable effect on lysis by phages, metabolic profiles on selected amino acids and sugar substrates, CO, requirement, production of HS, dye sensitivity, urease activity, and agglutination with specific anti-sera.

However, B19AP39 and Rev. 1AP39 deletants seem to grow faster than their parental strains on Trypcase soy agar. Their growth rate in liquid medium still has to be measured.

WO 98/08951 PCT/EP97/04668 67 It has to be noticed that no P39 deletant has been isolated for the B. abortus 544 virulent strain. Such a deletant would allow the evaluation of the role of the 39 kDa antigen, if any, in the virulence of B. abortus. P39 deletants are available for B. melitensis strain B 115.

Brucellin, the allergen used in skin tests, is prepared from the cytoplasmic fraction of this strain and the 39 kDa antigen has been identified as the major antigen present in this allergen preparation (unpublished). Brucellin from the P39 deleted B115 strain has been prepared and has been tested in skin tests on Brucella infected cattle or guinea pigs. It is expected that this experiment can contribute to the evaluation of the effective contribution of the 39kDa antigen in the DTH response to brucellin injection. Other brucellin components might also be identified as major T antigens in this same way.

Study) of the residual virulence of mutant strains.

The residual virulence of the P39 deleted vaccinal strains B. melitensis Rev. and B.

abortus B19 was compared with the respective parental strains in the mouse model. The experimental setup is briefly outlined below.

Female CD-1 mice, 6 weeks of age, were injected subcutaneously with 1.2 x 108 CFU in 0.2 ml from either B. melitensis Rev.1, Rev.lAP39, B. abortus B19 or B19AP39. Mice were killed at 1, 3, 6, 9, 12, 15 weeks after infection and their spleens aseptically removed, weighed and kept frozen (-20 Spleens were then homogenised in saline, serially diluted and plated on Tryptic soy agar (TSA-Ye). Practical detection limit was 1 CFU per organ and the number of CFU per spleen was expressed as the log CFU to normalize the distribution of individual counts, required for variance analysis. Mean and standard deviation of transformed values per group were then computed.

The results from these experiments can be summarized as follows. Brucella spleen counts from mice injected with strain RevlAP39 decreased as regularly as those infected with strain Rev. 1 from week 3 to week 12. Although the number of Brucella infected spleens was similar at weeks 1, 3, 6 and 9 for both strains, mice infected with strain Rev. 1AP39 had spleen counts which were higher than those infected with the parental strain strain Rev.1. Three mice were still infected with Rev. 1 at week 15 whereas all mice injected with strain RevlAP39 were Brucella free at that time.

Spleen counts from mice injected with strain B19 decreased rapidly from week 1 to WO 98/08951 PCT/EP97/04668 68 week 6. Only one mouse was still found to be infected at weeks 6 and 9 and no B19 bacteria were recovered from spleen at weeks 12 and 15 post infection. Although the number of B 19AP39 Brucella infected spleens decreased less rapidly than with the parental strain B19, all mice were Brucella free from week 12 onwards. As described for Rev. 1, spleen counts were higher in mice injected with strain B19AP39 than in mice injected with strain B19.

Example 9. Vaccination and challenge experiments in the mouse model.

Vaccine strains. All Brucella strains were grown on Tryptic soy agar (TSA-Ye) slants for 24h. They were harvested in sterile buffered saline solution (BSS), adjusted spectrophotometrically at 600nm to 1 x 109 colony forming units and diluted to 1 x 10' c.f.u./0.2ml. Viable counts per 0.2 ml retrospectively determined by dilution and enumeration on TSA-Ye plates were 1.05 x 10 5 for B. abortus B19, 1.08 x 105 for B. abortus B19AP39, 0.80 x 10' for B. melitensis Rev.1 and 0.81 x 10 s for B. melitensis Rev. 1AP39.

These suspensions were used for vaccinations.

Challenge strains. Brucella abortus 544 and Brucella melitensis H38 challenge strains were grown on TSA-Ye slants for 24h. Both strains were harvested in BSS, adjusted spectrophotometrically as described for vaccine strains, and diluted to 2 x 105 c.f.u. per 0.2 ml for B. abortus 544 and to 1 x 104 c.f.u. per 0.2 ml for B. melitensis H38. Viable counts retrospectively determined were 2.07 x 10' for B. abortus 544 and 0.82 x 104 for B. melitensis H38. These suspensions were used for challenge experiments.

Mice. Female CD-1 mice, 6 weeks old and obtained from Charles River (Elbeuf, France) were held for 1 week before being randomly allocated to experimental lots 5 weeks before immunization.

Protection experiments. Deleted vaccinal strains as well as the parental strains were tested in the mouse model for their ability to protect against a virulent challenge. Deleted vaccinal strains, parental vaccine strains as positive control, and BSS as negative control were WO 98/08951 PCT/EP97/04668 69 injected subcutaneously into 12 mice per group. In each case 0.2 ml of the vaccine strain suspension described above was used. Thirty days later, the challenge strain was administered by the intraperitoneal route (0.2 ml of the bacterial suspension described above) Six mice from each group were randomly killed by cervical dislocation to isolate spleen, 15 or 56 days post-challenge. Spleens were weighed and kept frozen at -20 0 C. Each spleen was homogenized, diluted and spread on TSA-Ye. Colonies of Brucella were enumerated andin absence of any growth, the spleen was considered to be infected by 5 bacteria or less (Bosseray et al., 1984 Dev. Biol. Stand. 257-270). The number of c.f.u. per spleen was then transformed to y log(x/logx). This last transformation normalizes the distribution of individual counts, required for variance analysis (Bosseray and Plommet, 1976, J. Biol. Stand.

341-351).

B. abortus B19AP39 strain induced a significant protection (measured by the number of c.f.u. found in the spleen) against the B. abortus 544 challenge when compared to the control (=non-vaccinated) group, 2 weeks post-challenge (p <0.001) as well as 8 weeks postchallenge (p <0.05) (figure 14). Mice immunized with the B19 parental strain were protected as expected (2 weeks post-challenge: p <0.05; 8 weeks post-challenge p No significant difference was found between the two B19 strains; the spleen counts were equivalent in mice vaccinated with the B19 parental strain to those measured in mice vaccinated with the B19AP39 strain.

Similar results were obtained in mice immunized with B. melitensis RevlAP39 strain and B. melitensis Revl parental strain (figure 13). Mice were significantly protected against the B. melitensis H38 virulent challenge as compared to control mice and again no significant difference in protection was found in protective capacity between the parental Rev strain and the Revl strain deleted in P39.

These results indicate that the deletion of the P39 gene does not significantly change the capacity of the strains to induce protective immunity in the mouse model.

Conclusion. Although slight changes were observed in the spleen infection kinetics between P39 deleted strains and the parental strains, no significant difference in residual virulence could be shown. The protective activity of the deleted vaccinal strains was compared to their parental strains in the mouse model and no significant difference was shown. Deletion of the P39 gene had no effect on the ability of the strain to protect against a virulent challenge.

These deleted vaccinal strains are therefore potential candidates for differentiating vaccinated from field-infected animals.

Using br25 and/or P39 deleted vaccinal strains for vaccination would subsequently allow to distinguish vaccinated animals from field infected animals due to a different immunological fingerprint of the vaccine strains on the one hand, and strains responsible for field infection on the other hand. More specifically, animals vaccinated with a P39 and/or deleted vaccine strain would show no immunological recognition of the 39 kDa and/or 15 kDa antigens of the invention, while field infected animals would.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

S

S

*ft WO 98/08951 PCT/EP97/04668 71

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Claims (27)

1. An isolated Brucella antigen or fragment thereof, wherein said antigen or said fragment is characterized by an amino acid sequence showing at least 60%, preferably at leat 70%, more preferably at least 80% homology to the amino acid sequence set forth in SEQ ID NO: 2 or 4, wherein said fragment comprises at least 9 contiguous amino acids of SEQ ID NO: 2 or 4 wherein said antigen or said fragment has at least one of the following immunological properties: being specifically recognized by sera from Brucella field-infected individuals, 0 and/or being specifically recognized by the cellular immune response from Brucella contacted individuals, and/or being able to elicit a Brucella specific immune response when used for vaccination of individuals prone to brucellosis disease.

2. An isolated Brucella abortus antigen according to claim 1 characterized by any of the amino acid sequences represented by SEQ ID NO: 2 or 4, or a fragment thereof, said fragment consisting of at least 9 contiguous amino acids selected from said amino acid sequences. :0

3. A polynucleic acid comprising a sequence of at least 10 contiguous nucleotides selected from: the polynucleic acid sequences which code for any of the polypeptides according to claims 1 to 2, or any of the polynucleic acid sequences as represented by SEQ ID NO: 1 or 3, or the polynucleic acid sequences which are degenerate as a result of the genetic code to the polynucleic acid sequences as defined in and and which still encode a polypeptide according to claims 1 to 2, or ;0 the polynucleic acid sequences which hybridize to any of the polynucleic acids as defined in to Q.'pcVp.\2142105 spo-.199.o-17 July, 2000 -84-

4. An oligonucleotide probe comprising a fragment of a polynucleic acid according to claim 3, with said probe being able to act as a specific hybridization probe for detecting the presence of Brucella polynucleic acids in a sample.

5. An oligonucleotide primer comprising a fragment of a polynucleic acid according to claim 3, with said primer being able to initiate specific amplification of Brucella polynucleic acids in a sample.

6. An isolated antibody, more particularly a monoclonal antibody, characterized in that it specifically recognizes a Brucella polypeptide represented by SEQ ID NO: 2 or a fragment of said polypeptide, said fragment comprising at least 9 contiguous amino acids of SEQ ID NO: 2, and more particularly the monoclonal antibody secreted by the hybridoma A435E1E8 deposited at the ECACC on July 31, 1996 under No. 96073115. S 15

7. A method for in vitro diagnosis of Brucella (species) infection in an individual .o comprising the steps of contacting a sample taken from said individual, said sample possibly containing anti-Brucella (species) antibodies, Brucella (species) antigens and/or Brucella (species) nucleic acids, with a polypeptide or peptide according to any of claims 1 to 2, under conditions allowing the formation of an immunological complex, and 20 subsequently detecting the complexes formed.

8. A method for in vitro diagnosis of Brucella (species) infection in an individual comprising the steps of contacting a sample taken from said individual, said sample possibly containing anti-Brucella (species) antibodies, Brucella (species) antigens and/or Brucella (species) nucleic acids, with an oligonucleotide probe according to claim 4, under conditions allowing the formation of a hybridization complex, with said nucleic acids of said sample being possibly amplified prior to hybridization, and subsequently detecting the complexes formed.

9. A method for in vitro diagnosis of Brucella (species) infection in an individual comprising the steps of contacting a sample taken from said individual, said sample Q:OprWVpa\2142105 firstresponse.99.doc-18 July. 2000 possibly containing anti-Brucella (species) antibodies, Brucella (species) antigens and/or Brucella (species) nucleic acids, with an antibody according to claim 6, under conditions allowing the formation of an immunological complex, and subsequently detecting the complexes formed.

A method for detecting and/or quantifying the cellular immune response of an individual against a Brucella polypeptide with said method comprising: measuring a delayed type hypersensitivity reaction upon subcutaneous injection of a polypeptide according to any of claims 1 to 2 in said individual, or measuring the stimulation of periferal blood lymphocytes isolated from the individual to be tested, upon addition of a polypeptide according to any of claims 1 to 2 to said lymphocytes under conditions allowing the immune recognition of said polypeptide. 4 S•

11. A method for detecting individuals having been in contact with Brucella (species) comprising: contacting a polypeptide according to any of claims 1 and 2 with the cellular immune system of the individual, either in vitro or in vivo, and 20 detecting and/or quantifying the cellular immune response raised against said polypeptides, with a method according to claim

12. A recombinant Brucella strain in which the gene(s) encoding at least one of the polypeptides according to any of claims 1 and 2 has (have) been deleted or inactivated.

13. A recombinant Brucella according to claim 12, with said strain Brucella abortus B19AP39, as deposited with the NCTC on July 31, 1996 under accession number NCTC 12944, or Brucella melitensis rev.1 AP39, as deposited with the NCTC on July 31, 1996 under accession number NCTC 12942, or B. melitensis rev.lABr25, as deposited with the NCTC on July 31, 1996 under accession number NCTC 12943. QAOpr\Vpa2l142105 firstresponsl99.doc-17 July, 2000 -86-

14. A vaccine composition comprising as an active principle at least one of the polypeptides according to any of claims 1 and 2, or at least one of the polynucleic acids according to claim 3, or recombinant Brucella strain according to claim 12, said active principle being combined with a pharmaceutically acceptable carrier.

A combined method for vaccination against and detection of brucellosis, said method comprising: vaccinating an individual liable to become infected by brucellosis with a recombinant Brucella strain according to claim 12, and detecting in an individual liable to be infected by brucellosis an immune response against a polypeptide as defined in any of claims 1 and 2, with a method according to any of claims 7, 10 and 11, and said combined method further characterized by the fact that it enables the differentiation between vaccinated and field-infected individuals.

16. A recombinant vector for cloning and/or expression purposes, into which a polynucleic acid according to claim 3, or part thereof, has been inserted and wherein, in case of an expression vector, the coding sequence of said polynucleic acid is operably linked to a control sequence enabling the expression of the coding sequence by a specific 20 host.

17. A host cell transformed by any recombinant vector according to claim 16, with said host cell being preferably a prokaryotic organism, and more preferably E. coli, a S Salmonella species or a lactic acid bacterium, or with said host cell being a lower eukaryotic cell (like yeast) or a higher eukaryotic cell.

18. A recombinant polypeptide encoded by a polynucleic acid according to claim 3, or part thereof, said recombinant polypeptide produced by: transforming a recombinant vector according to claim 16 into a suitable host cell, culturing said transformed cellular host under conditions which allow the Q:Opl\Vpa\2142105 first respomc. 199.do- 17 July. 2000 -87- expression and possibly secretion of the encoded polypeptide, and recovering the expressed polypeptide from the culture.

19. A recombinant polypeptide produced by: transforming a recombinant vector according to claim 16 into a suitable host cell, culturing said transformed cellular host under conditions which allow the expression and possibly secretion of the encoded polypeptide, and recovering the expressed polypeptide from the culture whereby said recombinant vector according to claim 16 comprises a sequence encoding a heterologous polypeptide and this sequence is fused in frame to a polynucleic acid according to claim 3, or part thereof.

20. A kit for the in vitro diagnosis of Brucella (species) infection in an individual, said S 15 kit being a kit for determining the presence of anti-Brucella (species) antibodies present in a biological sample, and said kit comprising at least one polypeptide or peptide according to any of claims 1 and 2, or a recombinant peptide according to any of claims 18 and 19, preferentially in combination with other polypeptides or peptides from Brucella, with said (poly)peptides being preferably immobilized on a solid substrate.

21. A kit for the in vitro diagnosis of Brucella (species) infection in an individual, said kit being a kit for the detection of antigens of Brucella (species) present in a biological sample, and said kit comprising an antibody according to claim 6, with said antibody being S: **preferably bound to a solid support.

22. A kit for the in vitro diagnosis of Brucella (species) infection in an individual, said kit being a kit for determining the presence of Brucella polynucleic acids present in a biological sample liable to contain them and said kit comprising: possibly at least one primer or a set of primers as defined on claim at least one oligonucleotide probe as defined in claim 4, with said probe(s) R being preferentially immobilized on a solid substrate, and more preferentially Q:AOp\Vpa214215 frstrespwoe.199.6-1S/ 7 /00 -88- on a membrane strip.

23. A kit for vaccination against and detection of brucellosis, with said kit comprising at least the following components: a vaccine composition comprising as an active principle a recombinant Brucella strain according to claim 12, and a polypeptide or peptide according to any of claims 1 and 2, or a recombinant polypeptide according to any of claims 18 and 19, said kit further characterized by the fact that its components enable the differentiation between vaccinated and field-infected individuals.

24. The combined use of: a recombinant Brucella vaccine strain according to claim 12 for use in vaccination, and S 15 at least one polypeptide or peptide according to any of claims 1 and 2, or a o recombinant polypeptide according to any of claims 18 and 19, for use in a diagnostic method.

25. Use of any of the (poly)peptides according to any of claims 1, 2, 18 and 19, or any 20 of the antibodies according to claim 6, or any of the polynucleic acids according to any of claims 3, 4 and 5, in the manufacture of a medicament.

26. Use of any of the (poly)peptides according to any of claims 1, 2, 18 and 19, or any of the antibodies according to claim 6, or any of the polynucleic acids according to any of claims 3, 4 and 5, in the manufacture of a medicament, more particularly in the preparation of a vaccine or in the preparation of a diagnostic composition.

27. An isolated Brucella antigen according to claim 1 or claim 2, or a polynucleic acid according to claim 3, or an oligonucleotide probe according to claim 4, or an oligonucleotide primer according to claim 5, or an antibody according to claim 6, or a S method according to any one of claims 7-11, or a recombinant Brucella strain according to Q:AOpeVpa\2121105 fir rsponse.199.doc-18/07/00 -89- claim 12 or claim 13, or a vaccine composition according to claim 14, or a combined method according to claim 15, or a recombinant vector according to claim 16, or a host cell according to claim 17, or a recombinant polypeptide according to claim 18 or claim 19, or a kit according to any one of claims 20-23, or the combined use according to claim 24, or the use according to claim 25 or claim 26, substantially as herein before described with reference to the figures and/or examples. o9