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WO1998008951A1 - Nouveaux antigenes de la brucella, polypeptides recombines, acides nucleiques codant ceux-ci et leur utilisation dans des procedes et materiels diagnostiques et prophylactiques - Google Patents

Nouveaux antigenes de la brucella, polypeptides recombines, acides nucleiques codant ceux-ci et leur utilisation dans des procedes et materiels diagnostiques et prophylactiques Download PDF

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Publication number
WO1998008951A1
WO1998008951A1 PCT/EP1997/004668 EP9704668W WO9808951A1 WO 1998008951 A1 WO1998008951 A1 WO 1998008951A1 EP 9704668 W EP9704668 W EP 9704668W WO 9808951 A1 WO9808951 A1 WO 9808951A1
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Prior art keywords
brucella
polypeptide
species
recombinant
kit
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PCT/EP1997/004668
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English (en)
Inventor
Eric Saman
Gérard DUBRAY
Jean-Michel Verger
Jean-Jacques Letesson
Anne Tibor
Original Assignee
Innogenetics N.V.
Institut National De La Recherche Agronomique
Facultes Universitaires Notre-Dame De La Paix
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Application filed by Innogenetics N.V., Institut National De La Recherche Agronomique, Facultes Universitaires Notre-Dame De La Paix filed Critical Innogenetics N.V.
Priority to CA002263791A priority Critical patent/CA2263791A1/fr
Priority to EP97941971A priority patent/EP0922103A1/fr
Priority to AU43818/97A priority patent/AU724849B2/en
Publication of WO1998008951A1 publication Critical patent/WO1998008951A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/23—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Brucella (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • 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 ant ⁇ gen(s).
  • Brucellosis is an infection due to a small lntracellular 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 Withm the genus Brucella, six closely related species have been described (Fekete et al.. 1992, Verger et al., 1985; Verstraete &
  • bovine brucellosis is mainly based on serological tests to detect the presence of anti-lipopolysaccharide (LPS) antibodies (Abs)
  • LPS anti-lipopolysaccharide
  • these tests by themselves do not allow the detection of all Brucella-mitcvz ⁇ animals (Biasco et al 1994; Dohoo et al 1986, Diaz-Apa ⁇ cio 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 animals infected with virulent strains (Alton, 1978; Nicoletti, 1980).
  • serological cross-reactions between B. abortus and many other bacterial species were described (Corbel, 1985; Garin-Bastuji et al. 1992; Weynants et al. 1995)).
  • Brucellergen ® (Rh ⁇ ne-Merieux), consisting of a mixture of 20 to 30 cytoplasmic proteins and prepared from a rough (R) strain of B. melitensis Bl 15 (Jones et al. 1973) has proved to be valuable in detecting Brucella-m ' fected bovines (Fensterbank, 1977) and is a useful diagnostic method complementary to serology (Fensterbank, 1982).
  • 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.
  • 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 oi 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 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 15kDa 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.
  • 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 th ⁇ ir 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 comprising a recombinant Brucella strain in which the gene encoding the Brucella 39kDa antigen and/or tne gene encoding the Brucella 15 kDa antigen has been deleted or inactivated.
  • the present invention relates to an isolated Brucella antigen characterized by an ammo acid sequence showing at least 60% , preferably at least 70% , more preferably at least 80% homology to any of the amino acid sequences as shown in SEQ ID NO 2 or 4, or fragments of said antigen, consisting of at least 9 contiguous amino acids selected from said amino acid sequences.
  • 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.
  • isolated thus implies that the antigen is in a different state and environment than the naturally occurring antigen.
  • 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.
  • 39kDa antigen and 15kDa antigen refer to antigens 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.
  • homologous and homoology 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.
  • 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.
  • 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 2), 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.
  • 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 4), 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 d e 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:
  • 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 differentia] recognition by sera from field-infected individuals as opposed to sera from vaccinated individuals.
  • (Brucella) field-infected individuals refers to individuals which have been infected by the Brucella pathogen, and in which the infection leads to brucellosis disease.
  • (Brucella) vaccinated individuals refers 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.
  • (Brucella) contacted individuals refers to individuals which have been in contact with the Brucella pathogen, said contact possibly leading to subsequent disease, or to subsequent protection.
  • cutoff value mean optical density of the control sera + 2 (or 3) standard deviations.
  • 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).
  • 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 T- cell 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.
  • the present invention also relates to antigens characterized by an ammo acid sequence showing a homology of at least 60% , preferably at least 70% , and even more preferably at least 80% or 90% to the ammo 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.
  • 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
  • Said “analogues” may be the result of strain to strain variations of the Brucella
  • (species) antigens of the invention may be the result of modifications introduced in the original polypeptide sequences, said modifications bringing about a desirable side effect to the polypeptide molecule (e.g. better physicochemical properties, more efficient purification, more efficient coating characteristics, more stable etc.).
  • the 39kDa antigen of Brucella abortus represented by SEQ ID NO 2
  • has homologous counterparts in other Brucella species like e.g. Brucella me lit ens i , Brucella ovis, Brucella suis, etc.
  • 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”
  • the 15kDa antigen of Brucella abortus, represented by SEQ ID NO 4 has homologous counterparts in other Brucella species, like e.g. Brucella melitensis, Brucella ovis, Brucella suis, etc.
  • the family of proteins homologous to the sequence represented in SEQ ID NO 4 are called throughout the current specification "the I5k ⁇ a Brucella antigens”.
  • 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.
  • proteins modified by additional substituents attached to the amino acids side chains such as glycosyl units, lip ids, or inorganic ions such as phosphates, as well as modifications relating to chemical conversions of the chains, such as oxidation of sulfhydryl groups.
  • 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.
  • polypeptides of the invention can be prepared by classical chemical synthesis.
  • the synthesis can be carried out in homogeneous solution or on solid phase.
  • polypeptides of the invention can also be prepared in solid phase according to the methods described by Atherton and Shepard in their book entided "Solid phase peptide synthesis” (IRL Press, Oxford, 1989).
  • polypeptides according to this invention can also be prepared by means of recombinant DNA techniques as described by Maniatis et al. (1982).
  • the invention relates to a polynucleic acid comprising a sequence of at least 10 contiguous nucleotides selected from: (a) the polynucleic acid sequences which code for any of the polypeptides described above, or (b) the polynucleic acid sequences which are degenerate as a result of the genetic code to the polynucleic acid sequences as defined in (a), and which still encode a polypeptide as described above, or (c) the polynucleic acid sequences which hybridize to any of the polynucleic acids as defined in (a) or (b).
  • the present invention relates to a polynucleic acid sequence, in an isolated form, comprising a contiguous sequence of at least 10 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.
  • 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.
  • hybridizes to refers to preferably stringent hybridization conditions (Maniatis et al. , 1982), allowing hybridisation between sequences showing at least 70% , 80% , 90% , 95 % or more homology with each other.
  • in isolated form refers to the fact that said polynucleic acid is 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 (e.g. more than 85%, more than 90%, more than 95 % homologous) to any of the nucleic acid sequences represented by SEQ ID NO 1 or 3.
  • homologous and homologous 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.
  • the invention relates to a polynucleic acid as described above comprising a sequence of at least 10 contiguous nucleotides selected from:
  • the invention relates to a polynucleic acid as described above comprising a sequence of at least 10 contiguous nucleotides selected from:
  • the present invention relates to any nucleotide sequence which, upon expression, gives rise to a polypeptide sequence as represented in SEQ
  • 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 process comprising essentially the following steps:
  • 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.
  • 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 mto 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.
  • 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.
  • probe refers to single stranded sequence-specific ohgonucleotides which have a sequence which is sufficiently complementary to hybridize to the target sequence to be detected.
  • 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 (B. abortus, B. melitensis, B. ovis, B. suis). 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.
  • these probes should be stringently hybridized at their appropriate temperature in order to attain sufficient specificity.
  • these probes or variants thereof can be caused to hybridize specifically at the same hybridization conditions (i.e. the same temperature and the same hybridization solution).
  • 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 TMAC1 solutions (Jacobs et al., 1988).
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • primer refers to a smgle 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.
  • 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 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,
  • nucleic acid sequence-based amplification NASBA, Guatelli et al , 1990 Compton, 1991
  • TAS transcription-based amplification system
  • SDA strand displacement amplification
  • SDA strand displacement amplification
  • the amplified products can be conveniently labelled either using labelled primers or by incorporating labelled nucleotides. Labels may be isotopic ( 2 P, 35 S, etc.) or non-isotopic (biotin, digoxigenin, etc.).
  • the amplification reaction is repeated between 20 and 80 times, advantageously between 30 and 50 times.
  • 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.
  • operably linked refers to a juxtaposition wherein the components are configured so as to perform their usual function.
  • control sequences operably linked to a coding sequence are capable of effecting the expression of the coding gene.
  • 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.
  • 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 me polypeptides of the invention.
  • 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 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”.
  • the current invention also provides for a recombinant vector allowing expression of the polypeptides of the invention as fusion proteins, i.e. whereby the ammo acid chain of the polypeptide of the invention is linked to a heterologous ammo acid sequence at the amino-terminal or carboxy-terminal end.
  • 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 recombmant 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 thereof, has been inserted under the control of the appropiate regulatory elements,
  • the invention further relates to an antibody recognizing specifically any of the polypeptides 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 antibody, more particularly a monoclonal antibody, characterized in that it is specifically raised against an antigenic determinant of an isolated 39kDa Brucella polypeptide, more particularly against an antigenic determinant of the 39kDa Brucella abortus polypeptide as represented in SEQ ID NO 2.
  • the invention relates to the monoclonal antibody 5E1E8 recognizing the 39kDa Brucella antigen of the invention, prepared as described further in the examples section.
  • A435E1E8 has been deposited with the ECACC on July 31 , 1996 under the (provisional) access ion number 96073115.
  • the invention also relates to the 39 kDa Brucella polypeptides, or fragments thereof, recognized by the above-mentioned antibodies, more specifically recognized by the monoclonal antibody 5E1E8.
  • a further embodiment of the present invention relates to an antibody, more particularly a monoclonal antibody, characterized in that it is specifically raised against an antigenic determinant of an isolated 15kDa Brucella polypeptide, more particularly against an antigenic determinant of the 15kDa Brucella abortus polypeptide as represented in SEQ ID NO 4.
  • the antibodies of the invention are different from the Br25 monoclonal antibody (Silenius).
  • the invention also relates to the 15 kDa Brucella polypeptides, or fragments thereof, recognized by the above-mentioned antibodies.
  • antigenic determinant refers 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.
  • 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.
  • 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.
  • fragments derived from these monoclonal antibodies such as Fab, F(ab)' ⁇ and 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).
  • an antibody, or an antigen-binding fragment F(ab') 2 , 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
  • 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-B rucella 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.
  • anti-idiotype antibodies refers to monoclonal antibodies raised against the antigenic determinants of tlie variable region of monoclonal antibodies themselves raised against the Brucella polypeptides of tlie invention These antigenic determinants of immunoglobulins are known as ldiotypes (sets of ldiotopes) and can therefore be considered to be the "fingerprint" of an antibody (for review see de Preval, 1978; Fleishmann and
  • Monoclonal anti-idiotypic antibodies have the property of forming an immunological complex with the idiotype of the monoclonal antibody against which they were raised.
  • the monoclonal antibody is often referred to as Abl
  • 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
  • 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 anu-Brucella (species) antibodies, Brucella
  • 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.
  • sample may refer to any biological sample (tissue or fluid) possibly containing Brucella nucleic acid sequences, antibodies or polypeptides.
  • the detection method is the detection of antibodies against Brucella (species), and die preferred sample in that case is serum or plasma.
  • the invention thus preferably relates to a method for detecting antibodies to Brucella
  • kits 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.
  • 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 .
  • the polypeptides used in the above-described method for detection of an -Brucella species antibodies can be replaced by anti-idiotype antibodies as described above, acting as their equivalents.
  • 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.
  • 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.
  • 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
  • polypeptides of the invention in a sample, whereby the polypeptides of the invention are immobilized on a solid support, eventually on a membrane strip.
  • Different polypeptides of the invention may be immobilized together or next to each other (e.g. in the form of parallel lines which is the case in the Line Immuno Assay (LI A)).
  • 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:
  • 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
  • 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 presence 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 Mania ⁇ s et al. (1982).
  • 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 o ⁇ discriminated).
  • Amplification of nucleic acids present in a sample prior to detection in vitro ma ⁇ accomplished by preferably first extracting the nucleic acids present in the sample according 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.
  • 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.
  • 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.
  • 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.
  • 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 detecting and/or quantifying the cellular immune response raised against said polypeptides.
  • 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:
  • 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 zn -Brucella (species) antibodies as defined above present in a biological sample
  • a kit for determining the presence of zn -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 me buffer enabling a binding reaction to occur between these polypeptides and the an -Brucella antibodies possibly present in the biological sample, means for detecting the immune complexes formed in the preceding binding reaction, possibly also including an automated scanning and interpretation device for inferring the presence of m ⁇ -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.
  • the kit according to this aspect of the invention comprises both
  • kits 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
  • OMP outer membrane protein
  • Brucella antigen International application WO96/17065 published on 06.06.96
  • other bacterial antigenic proteins or peptides in general.
  • 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
  • kits present in a biological sample, said kit comprising an antibody as described above, with said antibody being preferably bound to a solid support.
  • the invention relates to a diagnostic kit for die 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.
  • 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 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 tlie 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.
  • the probes are immobilized in a Line Probe Assay (LiPA) format.
  • LiPA Line Probe Assay
  • a LiPA support may contain on its surface different oligonucleotide probes derived from the polynucleic acid sequences according to die invention which hybridize specifically with certain strains of Brucella (such as B. abortus, B. melitensis, B. ovis, B.
  • 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 WO94/12670 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 advantages make the LiPA format liable for the use of Brucella detection in a routine setting
  • 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.
  • 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 mvention 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.
  • 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.
  • the vaccine composition as described above may comprise as an active principle one of the anti-idiotype antibodies as described above.
  • 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 general.
  • Brucella immunogenic components such as outer membrane proteins ((OMP) Cloeckaert et al. 1991) or other bacterial or immunogenic components general.
  • 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.
  • nucleic acids are injected into the individual and that they express in situ the immunogenic protein / peptide which they are encoding, dius conferring in vivo protection to the vaccinated host (e.g. Ulmer et al. , 1993).
  • the active ingredients of such a vaccine composition may be administered orally, subcutaneously, conjunctivally, intramuscularly, mtranasally, 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 m the art, etc.
  • the invention also provides for recombinant Brucella (species) strains m 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.
  • the current invention provides for a recombinant Brucella (species) strain 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. l 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 B19 ⁇ P39 and B. melitensis Rev.l ⁇ P39 have been deposited at the NCTC on July 31 , 1996 under accession numbers NCTC 12944 and NCTC 12942 respectively.
  • 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. l strains deleted in the gene encoding me 15kDa antigen of the invention (Br25 gene).
  • An example of such a deleted vaccine strain i.e. B. melitensis Rev. l ⁇ Br25 has been deposited at the NCTC on July 31, 1996 under accession number NCTC 12943.
  • 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 invention also relates to a vaccine composition which provides protective immunity against Brucella (species) infection in a mammal (human, ruminants) and which still allows die 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 die Brucella 39kDa antigen of the invention and/or the gene encoding the Brucella 15 kDa antigen of the invention has been deleted or inactivated.
  • the Brucella strain in which the gene(s) of the invention is (are) deleted is die 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 agamst brucellosis, yet permitting differentiation between Brucella field infected and vaccinated individuals, because of their distinctive immunological signature.
  • a diagnostic screening test e.g. a serological assay
  • an antigen encoded by one of the deleted genes l e more particularely the 15 kDa antigen and/or 39 kDa antigen
  • the current invention also relates to a combined method of vaccination against and detection of brucellosis, said method comprising
  • 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
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • the invention relates to the combined use of
  • FIGURE LEGEND 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.
  • FIGURE LEGEND 39 kDa Brucella antigen (poly)peptides and/or the 15 kDa Brucella antigen (poly)peptides
  • Fig. 1 Purification of the 39 kDa antigen of B. abortus starting from brucellergen.
  • F ⁇ gure5a Prokaryotic expression vector pIGALMPH. 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 crolacI-H ⁇ s6 tag at their N-termmal end.
  • FIG. 5b Prokaryotic expression vector p ⁇ ORHTSA. Heterologous expression is under control of the early lambda PI 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 ⁇ bosomal R ⁇ A terminator is also indicated (rrnBTlT2) Some unique restriction sites are also indicated.
  • Figure 5c Prokaryotic expression vector pIGFHIO. Heterologous expression is under control of the early lambda promoter PI. The mT ⁇ F-hexahistidine gene fragment encodmg the fusion partner is indicated upstream from the BamHI site. All fusion proteins expressed in this vector contain the mT ⁇ F-H ⁇ s6 tag at their N-terminal end. The ⁇ bosomal RNA terminator is also indicated, as well as some unique restriction sites.
  • Figure & Induction of the 15 kDa Brucella protein in E.coli. Lysate from umnduced 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 , umnduced 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.
  • Figure 6h Analysis on SDS PAGE of purified 15 kDa fusion protein and detected by coomassie blue staining. Lane 1 , 10 ⁇ protein; lane 2, 3 ⁇ g protein. Lane 3, marker proteins indicated in kDa.
  • Figure 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 7b Purified 39 kDa fusion protein analysed on SDS PAGE and detected by coomassie blue staining. Lane 1, 10 ⁇ g protein; lane 2, 3 ⁇ g 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 die 15 kDa antigen was 0.130, for the 39 kDa antigen it was 0.073 and for the combined ELISA it was 0.156.
  • Fig. 9 Proliferative response of PBMC from _5rt.ce//fl-infected cows to fractions of brucellergen (batch 8A083).
  • PBMC (2 x lOVwell) 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 me deletion experiments as described in example
  • Fig. 11 Southern blot analysis of vaccinal deletants and their parental strains. DNA is cut with Hindlll. Genes P39 (fig. 11A) and kan (fig. 1 IB) were used as probes. Lane 1 : DNA molecular weight marker, fragment sizes were 23130, 9416, 6557, 4361 , 2322, 2027, 1353,
  • lane 2 B. melitensis Rev. 1 DNA
  • lane 3 B. melitensis Revl ⁇ P39 DNA
  • lane 4 B. abortus B 19 DNA
  • lane_£ B. abortus B19 ⁇ P39 DNA.
  • Fig. 12 Southern blot analysis of DNA's cut with Hindlll 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; lan£_i: B. melitensis 16 ⁇ br25 (clone 2) DNA; lane. 4: B. melitensis 16M ⁇ br25 (clone 19) DNA.
  • PBMC Proliferative response of PBMC from 4 B rucella-m ected cows to 2 batches of brucellergen (96G091, 8A083), and 39 kDa antigen.
  • PBMC (2 lOVwell) were cultured during 8 days with 1 ⁇ g 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).
  • Gly Gly, Ala, Thr, Pro, Ser He (I) He, Met, Leu, Phe, Val, Tyr
  • Lys (K) Lys, Arg, Glu, Gin, His
  • Example 1 Material and methods
  • Bacterial strains and vectors 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 Veterinaire (I.N.R.V., Geneva).
  • 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 (I.N.R.A., Nouzilly, France).
  • Bacteriophage ⁇ gtl 1 , E. coli Y1089 and Y1090 were provided by Amersham E. coli DH5 ⁇ F', 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 [Cl-857] as described by Gottesman et al. 1981.
  • the expression vector pIGALMPH drives the transcription of the heterologous gene by the rightward promoter of phage lambda. Transcription can thus be contolled by the lambda Cl repressor, which is provided from a compatible plasmid present in me host cell. When die Cl ⁇ allel is used, a temperature induction of the transcription can be achieved.
  • the vector also encodes a 50 ammo 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 m 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 die latter vector, used in the current invention, are described in Fig. 5b (pIGRHISA/G) and Fig. 5c (pIGFHIO).
  • Hybridomas were produced as described elsewhere (Cioeckaert et al. 1990). Briefly, BALB/c mice between 8 and 12 weeks of age. were infected by mtrape ⁇ toneal (l.p.) injection of 10" B. abortus B3 smooth cells. After 4 months, mice were boosted l.p. with 1 mg of Brucellin. 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.
  • the Br25 monoclonal antibody was obtained from Silenus (Victoria, Australia) as AMD-Br-25 (cat. No 12VET03).
  • the filters were removed and washed m TBST (20 mM T ⁇ s-HCi pH 8.0, 0.5 M NaCl, 0.05% Tween 20) and immunoscreening was done as described previously (Tibor et al. 1994) with ant ⁇ -39kDa antigen monoclonal antibody 5E1E8 or ant ⁇ -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.
  • Lysogen production and preparation of whole-cell extracts To determine whether the putative recombinant phages produced ⁇ -galactosidase fusion proteins, lysogens of each of the phages were generated in E coli Y1089. Crude extracts were prepared as described by Hyunh et al. (1985), boiled for 5 mm m SDS-PAGE sample buffer (Laemmh 1970) resolved on a 12% SDS polyacryiamide gel and immunoblotted with Mab 5E1E8 or BR25 and the anti- ⁇ -galactosidase polyserum by using the procedure described above Controls, consisting of lysates of E. coli Y1089, infected by a non recombinant lambda gtl 1 were included in each experiment.
  • 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.,
  • DNA sequencing was performed by the primer extension dideoxy chain termination sequencing method of Sanger (Sanger et al.. 1977)) with Sequenase Version 2.0 (U.S. 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 me 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.
  • PBMC 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 peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • NUNC Rockslide, DK.
  • Brucellergen and purified P39 (1 ⁇ g/well) were added at the initiation of the cultures; PHA (1 g/well) was used as a control of proliferation.
  • the cultures were pulsed with 0.8 ⁇ ci [ 3 H] thymidine/well for 18 h and harvested onto glass fibber filters with a cell harvester (Titertek 550, Flow Laboratories).
  • 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.
  • Negative control sera were obtained from a Brucella free sheep flock (INRA, Tours) or from goats roaming in a brucellosis free area (CNEYA. 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 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.
  • 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 4 and displayed a single band of 39-kDa (Fig.
  • 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 2).
  • MAb 5E1E8 and PRA 39 gave no reaction with whole-cell lysates prepared from Y. enterocolitica 0:9, P. maltophilia, 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.
  • 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 (Hailing et al. 1993: Ouahrani et al. 1993). The P39 gene or its regulation elements could be disrupted by such sequences in this species.
  • E. coli 0: 157 - ** is a variant of the smooth H38 strain
  • lysogens from both the ⁇ gtl . l and ⁇ gtl .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 IPTG- induced lysogens were immunoblotted with MAb 5E1E8 or anti- ⁇ -galactosidase antiserum.
  • the 1-kb Sphl-EcoRl and the 0.7-kb Xbal-HimHU fragments from pTZl .2 were sub cloned into pUC19 and pSK vectors, respectively (Fig. 2).
  • the plasmid pSK3.2 did not express any gene product recognized by either MAb 5E1E8 or PRA 39.
  • pUC3.1 expressed a protein of 20-kDa, recognized by MAb 5E1E8 and PRA 39, independently of the induction with IPTG.
  • Nucleotide sequence of the full-length P39 gene 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.
  • the deduced ammo acid sequence of the nucleotide sequence extending from position 909 to 929 of the P39 gene ORF is in accordance with the ammo acid sequence of me 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
  • P39 sequence shows significant similarity (optimized score > 100) to the Leptoihrix 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 ⁇ gtl l . A 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.
  • 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.
  • 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).
  • Example 4 Cloning and sequencing of the Brucella abortus 15 kDa antigen
  • One recombinant clone displayed a reaction profile showing three signals at 15 kDa, 20 kDa and 29 kDa respectively.
  • the corresponding recombinant plasmid was termed pBBr25-l and was shown to contain the 0.83 kbp DNA insert.
  • Rxamplp ft Bacterial expression of the B. abortus genes encoding the 39 kDa and 15 kDa antigens
  • pIGALMPHBr25 Ncol I Aval fragment, of which the Aval site had been filled.
  • pIGALMPHBr25 Ncol I Aval fragment, of which the Aval site had been filled.
  • 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.
  • the fusion protein 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 6). An overall yield of 7 mg purified protein per liter fermentation broth was obtained.
  • IMAC Immobilized Metal Ion Affinity Chromatography
  • 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 7). This purified recombinant P39-antigen is subsequently evaluated serologically (see example 6). 1.3. Construction of new expression vectors to improve solubility.
  • the vector was again opened with Nsil and blunted, followed by Xbal digest.
  • the insert fragment was recovered from the former construct pIGALMPHBr25 as a BamHl(f ⁇ ed)/Xbal fragment (supra).
  • Both plasmids (pIGRIHisABr25 and pIGRIHisGBr25) were introduced in E. coli strain SG4044 and the expression was analysed. Upon coomassie staining of the g ⁇ l. no induced protein was apparent.
  • the 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
  • the cut off value was defined as the mean of the negative control sera plus three times the standard deviation (SD).
  • SD standard deviation
  • the noise is defined as the mean of the negative sera and the cut off is placed at a S/N of 2.
  • 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.
  • ** 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)
  • Example 7 Evaluation of the 39 kDa antigen as a stimulatory antigen for the cellular immune system 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 4). 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 positive reaction widi brucellergen did not react to F4 or purified 39 kDa antigen.
  • 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).
  • 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 and 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.
  • E. coli 0 157 2 1.5 ⁇ SI ⁇ 1 1.5 ⁇ SI ⁇ 1 1.5 ⁇ SI ⁇ 1
  • Example 8 Construction and characterization of Brucella strains deleted for the genes encoding the 39 kDa and/or 15 kDa antigens.
  • known vaccine strains e.g. B. abortus B19 and B. melitensis Rev. l
  • B. abortus B19 and B. melitensis Rev. l 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 mat this protein is essential neither for bacterial survival nor for conferring protection.
  • the strategy used implies a double homologous recombination event leading to me replacement of the resident wild type gene by a genetic marker (Hailing 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 trans conjugants resistant to kanamycin can be obtained. "Integrants" resulting from a single recombination event between the chromosomal copy and me residual flanking regions from the incoming gene.
  • 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,
  • sacB gene in the presence of sucrose is lethal in man ⁇ ' gram negative bacteria and this gene is therefore useful as a positive selection marker in these bacteria (Simon et al, 1991 ; Kone et al, 1991).
  • integrants 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.
  • Table 7 summarizes the different phenotypes to be expected.
  • Table 7 Phenotype of the possible recombinant strains resulting from recombination.
  • KanR nalidixic acid
  • NalR Brucella strains have first been shown to grow 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 trans conjugants seemed to depend on tlie medium composition.
  • the sacBR marker has proved to be toxic for Brucella after integration of this marker into the Brucella chromosome.
  • the sacBR marker provided an effective counter-selectable marker in B. abortus and was used to improve the efficiency of isolation of deletants.
  • the construction of the deletion plasmids used to carry out the deletion of the P39 gene (P39 deletion plasmid) and the br25 gene (PI 5 deletion plasmid) is shown in figure 10a and 10b respectively, and is specified below.
  • the RK2 oriT (transfer origin) was isolated from the pTJS82 plasmid (Cornells, 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 10a.
  • 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.
  • Stepl The 400 bp BgH-Pst fragment containing the sequences downstream from the br25 ORF was made blunt ended and inserted into the Smal site of pSK-oriT.
  • Step2 A 240 bp fragment upstream of the br25 ORF was isolated widi Ncol and H dm. The Ncol site was blunted before the H dIII digest and the resultmg frament was cloned into the plasmid obtained in step 1 , opened with H.fldlll and EcoRV.
  • Step3 The KanR gene was isolated from pUC4K as a Pstl fragment and cloned into the Pstl 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 Seal. The fragment containing die oriT and the kanR gene is isolated (fragment 1). The sacBR marker was available as a 2.6 kBp BamH -Pst fragment in the pBluescript vector. This plasmid was opened with Pstl, blunted and treated with S al 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 10b.
  • 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 EcoRI fragment isolated from pUC4K (Pharmacia).
  • the probe to detect the P39 gene was prepared from a 1208 bp BgRllBsml fragment. This fragment is localised inside the P39 ORF and is absent on the plasmid used to delete me P39 gene.
  • the br25 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
  • Table 8 Brucella strains with confirmed deletions and the genes affected.
  • 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 me 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.
  • 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.
  • 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.
  • 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 (Hailing et al. 1991 ; Tatum et al. 1994).
  • the recombination frequency seems to be positively correlated with the size of the 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.
  • SacBR SacBR
  • 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 (e.g. a P39 deletant of strain 544).
  • deletion strains have been studied for alterations in their bacterial characteristics and metabolic behaviour None of the deletion strai ⁇ s studied shows a detectable effect on lysis by phages metabolic profiles on selected amino acids and sugar substrates, CO 2 requirement, production of H 2 S, dye sensitivity, urease activity, and agglutination with specific anti-sera.
  • B19 ⁇ P39 and Re ⁇ 1 ⁇ P39 deletants seem to grow faster than their parental strains on Trypcase soy agar Their growth rate in liquid medium still has to be measured 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 die 39 kDa antigen, if any, in the virulence o B. abortus.
  • P39 deletants are available for B. melitensis strain Bl 15.
  • Brucellin the allergen used in skin tests, is prepared from die 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.
  • mice Female CD-I mice, 6 weeks of age, were injected subcutaneously with 1.2 x 10 8 CFU in 0.2 ml from either B. melitensis Rev. l , Rev.l ⁇ P39, B. abortus B19 or B19 ⁇ P39. Mice were killed at 1 , 3, 6, 9, 12, 15 weeks after infection and their spleens aseptically removed, weighed and kept frozen (-20°C).
  • 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.
  • mice injected with strain Revl ⁇ P39 decreased as regularly as those infected wid 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. l ⁇ P39 had spleen counts which were higher than those infected with the parental strain strain Rev.1. Three mice were still infected widi Rev. l at week 15 whereas all mice injected with strain Revl ⁇ P39 were Brucella free at that time. Spleen counts from mice injected with strain B19 decreased rapidly from week 1 to week 6.
  • 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 IO 9 colony forming units (c.f.u. )/ml and diluted to 1 x IO 5 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 s for B. abortus B19, 1.08 x IO 5 for B. abortus B19 ⁇ P39, 0.80 x 10 s for B. melitensis Rev. l and 0.81 x 10 5 for B. melitensis Rev. l ⁇ P39. These suspensions were used for vaccinations.
  • 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 IO 5 c.f.u. per 0.2 ml for B. abortus 544 and to 1 x IO 4 c.f.u. per 0.2 ml for B. melitensis H38. Viable counts retrospectively determined were 2.07 x 10 5 for B. abortus 544 and 0.82 x 10 for B. melitensis H38. These suspensions were used for challenge experiments.
  • mice Female CD-I mice, 6 weeks old and obtained from Charles River (Elbeuf,
  • Deleted vaccina! 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 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 intrape ⁇ toneal route (0.2 ml of the bacterial suspension described above) .
  • mice immunized with B. melitensis Revl ⁇ P39 strain and B. melitensis Revl parental strain were significantly protected against the B. melitensis H38 virulent challenge as compared to control mice and again no significant difference in protection was found m protective capacity between the parental Revl strain and the Revl strain deleted m P39.
  • 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 br25 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.
  • Brucella ribosomal protein L7/L12 is a major component in the antigenicity of brucellin INRA for delayed-type hypersensitivity in Brucella-sensitized guinea pigs. Infect. Immun. 62:5361- 5366.
  • Diaz-Aparicio E. , C. Mann, B. Alonso-Urmeneta, V. Aragon, S. Perez-Ortiz, M. Pardo, J.M. Blasco, R. Diaz and I. Moriyon. 1994. Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats. J. Clin. Microbiol. 32: 1159-1165.
  • BLV bovine leukemia virus
  • HCV hepatitis C virus
  • Tatum FM Cheville NF and Morfitt D (1994). Cloning, characterization and construction of htrA and htrA-like mutants of Brucella abortus and heir survival in BALB/c mice.
  • Vanfleteren J.R. , J.G. Raymaekers, S.M. Van Bun and L.A. Mehus. 1992. Peptide mapping and microsequencing of proteins separated by SDS-PAGE after limited in situ acid hydrolysis. Biotechniques 12:550.

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Abstract

La présente invention concerne des antigènes purs et isolés de la Brucella, des acides nucléiques codant lesdits antigènes, ainsi que des procédés et trousses diagnostiques faisant appel à ces antigènes et acides nucléiques pour détecter l'infection par Brucella chez l'homme et le bétail. L'invention se rapporte également à des polypeptides recombinés, à leur procédé de préparation et à leur utilisation dans des procédés et des matériels de diagnostic de l'infection par Brucella. On décrit également l'utilisation de ces antigènes isolés, ou de ces polypeptides recombinés, ou de ces acides nucléiques comme principe actif d'une composition de vaccin contre l'infection par Brucella. On décrit également une composition de vaccin comprenant une souche recombinée de Brucella, dans laquelle le(s) gène(s) codant lesdits antigènes a/ont été spécifiquement supprimé(s).
PCT/EP1997/004668 1996-08-28 1997-08-27 Nouveaux antigenes de la brucella, polypeptides recombines, acides nucleiques codant ceux-ci et leur utilisation dans des procedes et materiels diagnostiques et prophylactiques WO1998008951A1 (fr)

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EP97941971A EP0922103A1 (fr) 1996-08-28 1997-08-27 NOUVEAUX ANTIGENES DE LA $i(BRUCELLA), POLYPEPTIDES RECOMBINES, ACIDES NUCLEIQUES CODANT CEUX-CI ET LEUR UTILISATION DANS DES PROCEDES ET MATERIELS DIAGNOSTIQUES ET PROPHYLACTIQUES
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WO2005055873A2 (fr) 2003-12-05 2005-06-23 Innfocus, Llc Dispositif ameliore a implant de traitement du glaucome
US7364745B2 (en) * 2004-02-06 2008-04-29 Virginia Tech Intellectual Properties, Inc. Development of a live, attenuated, recombinant vaccine for Brucellosis
CN100410389C (zh) * 2005-02-04 2008-08-13 中国农业科学院兰州兽医研究所 检测布鲁氏菌的方法和用于该方法的引物
US10191063B2 (en) 2013-03-21 2019-01-29 Caprion Proteomics Inc. Brucellosis, Q-fever, and lyme disease biomarkers and uses thereof
RU2593712C2 (ru) * 2014-09-30 2016-08-10 Федеральное Казенное Предприятие "Щелковский Биокомбинат" СПОСОБ ПОЛУЧЕНИЯ КОНЦЕНТРАТА КУЛЬТУРЫ КЛЕТОК БРУЦЕЛЛ ИЗ ШТАММА Brucella abortus 19 ДЛЯ ПРИГОТОВЛЕНИЯ БРУЦЕЛЛЕЗНЫХ АНТИГЕНОВ, БРУЦЕЛЛЕЗНЫЕ АНТИГЕНЫ (ТРИ ВАРИАНТА), СПОСОБ ИЗГОТОВЛЕНИЯ БРУЦЕЛЛЕЗНОЙ ДИАГНОСТИЧЕСКОЙ СЫВОРОТКИ И ТЕСТ-СИСТЕМЫ ДЛЯ ДИАГНОСТИКИ БРУЦЕЛЛЕЗА ЖИВОТНЫХ (ТРИ ВАРИАНТА)
CN111796091A (zh) * 2020-07-20 2020-10-20 天康生物股份有限公司 用于区分动物感染布氏杆菌或布氏杆菌菌影疫苗的试剂盒

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AU724849B2 (en) 2000-10-05

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