Patent Publication Number: US-7902349-B2

Title: Nucleic acids encoding protective epitopes of adenyl cyclase-haemolysin (AC-Hly)

Description:
This application is a continuation of application Ser. No. 09/907,951, filed Jul. 19, 2001 (now U.S. Pat. No. 6,994,854), which is a divisional of application Ser. No. 08/669,785, filed Jun. 27, 1996 (now U.S. Pat. No. 6,309,648). This application claims foreign priority to FR 95 07945, filed Jun. 30, 1995. 
    
    
     BACKGROUND OF THE INVENTION 
     The present application relates to amino acid sequences comprising epitopes of adenyl cyclase—haemolysin from  Bordetella . Adenyl cyclase-haemolysin (AC-Hly) is one of the toxins participating in the  Bordetella  infectious syndrome. AC-Hly is a bifunctional protein having an adenyl cyclase activity and a haemolytic activity. It is secreted by the bacterium. Its structural gene has been cloned and sequenced (Glaser P. et al., 1988, Molec. Microb. 2, 19-20). It is the case that this protein is part of the family of toxins termed “RTX” for “repeats in toxins” and exhibits homologies with haemolysin from  Escherichia coli  and  Actinobacillus pleuropneumoniae , and the leucotoxins from  Pasteurella haemolytica  and  Actinobacillus actinomycetemcomitans . This protein, like PTX (pertussis toxin), is capable of penetrating into eucaryotic cells such as the macrophages, of being activated by calmodulin, of synthesizing large quantities of cAMP and of disrupting cellular functions (Coote J. 1992. FEMS Microbiol. Rev. 88:137-162). 
     The inventors have identified, within this sequence, various domains having the capacity to induce the formation of protective antibodies against an infection by  Bordetella , in particular by  B. pertussis  and/or  B. parapertussis  and/or  B. bronchiseptica.    
     The subject of the invention is therefore amino acid sequences capable of entering into the composition of immunogenic compositions or of protective vaccines against  Bordetella  infections as well as antibodies directed against these amino acid sequences, capable of being used for example in immunotherapy. Immunotherapy or serotherapy is especially applicable in children, where appropriate in infants infected with  B. pertussis, B. parapertussis  or  B. bronchiseptica . The invention proposes applications in human medicine or in veterinary medicine. 
     The subject of the present invention is an amino acid sequence derived from the polypeptide sequence of adenyl cyclase-haemolysin (AC-Hly), characterized in that it is capable of inducing the formation of protective antibodies against an infection by  B. pertussis  and/or  B. parapertussis  and/or  B. bronchiseptica , and in that it is chosen from the following chains: 
     a) a sequence comprising the chain of amino acids situated approximately between position 910, preferably 913, and the last C-terminal amino acid of the polypeptide sequence of AC-Hly from  B. pertussis  (SEQ ID NO:2) or of a sequence corresponding to the preceding one in  B. parapertussis  or in  B. bronchiseptica  (SEQ ID NO: 4), the said sequence comprising a modification by addition of a fatty acid between amino acids 980 approximately and 985 approximately, preferably at the level of amino acid 983; 
     b) a sequence comprising a chain of 6 to 500 amino acids comprising amino acids 385 to 400 of the polypeptide sequence of AC-Hly from  B. pertussis  (SEQ. ID NO: 2) or of a sequence corresponding to the preceding one in  B. parapertussis  or in  B. bronchiseptica  (SEQ. ID NO: 4); 
     c) a sequence comprising the chains of amino acids defined above in a) and b), the said chains being either contiguous, or combined via the chain of amino acids naturally present between the chains defined above in a) and b) within the AC-Hly from  B. pertussis, B. parapertussis  or  B. bronchiseptica , or combined via an antigenic sequence derived from a protein different from AC-Hly, the amino acid sequence obtained having a three-dimensional conformation identical or analogous to that of the corresponding polypeptide sequence of AC-Hly from  B. pertussis, B. parapertussis  or  B. bronchiseptica.    
     The expression “amino acid sequence derived from the polypeptide sequence of AC-Hly” is understood to mean, within the framework of the invention, a sequence whose amino acids are identical, by their nature or by their linkage, to those of the polypeptide sequence of AC-Hly or are, for some of them, substituted, deleted or added, in a manner such that the immunological properties of AC-Hly are conserved. In particular, such a sequence, derived from the polypeptide sequence of AC-Hly is recognized by antibodies formed in a patient infected with  Bordetella , especially with  B. pertussis, B. parapertussis  or  B. bronchiseptica.    
     It will be considered, within the framework of the present invention, that the structure or the conformation of AC-Hly is conserved, the amino acid sequence of the invention then having a structure identical or analogous to that of AC-Hly, when the said amino acid sequence is capable, after immunization of a patient or an animal, of inducing a protective immunity against  Bordetella  infections. 
     A sequence according to the invention can be obtained by proteolysis of AC-Hly purified from  Bordetella . Preferably, this sequence is obtained by chemical synthesis or by genetic engineering techniques. 
     Thus, to produce an amino acid sequence according to the invention by genetic engineering, plasmids carrying fragments of the CyaA gene will be used. 
     By way of example, the chemical synthesis may be performed in automatic machines of the Applied Biosystem type. It is also possible to use the technique of Betsou F. et al., 1993, Infect. Immun., 61:3583-3589. 
     In this manner, it will be possible to prepare a peptide corresponding to the chain of amino acids 385 to 400, or even 385 to 500 of AC-Hly from  B. pertussis  or to the corresponding chain of AC-Hly from  B. parapertussis  or from  B. bronchiseptica . The amino acid sequence (SEQ. ID NO: 2) as well as the nucleotide sequence (SEQ. ID NO: 1) of AC-Hly from  B. pertussis  has been described in Glaser et al, 1988, Molec. Microb. 2-19-30 and is represented in  FIG. 5 . The amino acid sequence (SEQ. ID NO: 4) and the nucleotide sequence (SEQ. ID NO: 3) of AC-Hly from  B. bronchiseptica  is represented in  FIG. 6 . 
     The amino acid sequence of AC-Hly from  B. parapertussis  and the nucleotide sequence encoding AC-Hly can be obtained by conventional techniques from the DNA from a strain of  B. parapertussis , for example the strain No. 1 deposited at the CNCM on 2 Dec. 1994 under the No. I-1498. 
     When an amino acid sequence according to the invention is produced by genetic engineering from the DNA of the CyaA genes from  B. pertussis, B. parapertussis  or  B. bronchiseptica , and when it comprises the amino acid in position 983 of AC-Hly from  B. pertussis  (SEQ. ID NO: 2) or a corresponding amino acid of AC-Hly from  B. parapertussis  or  B. bronchiseptica  (SEQ. ID NO: 4), care will be taken to ensure that this sequence is produced in a cellular host also expressing the CyaC gene from the strains identified above. The expression of the CyaC gene allows the modification by addition of a fatty acid necessary to conserve in the amino acid sequence comprising residue 983. The addition of a fatty acid may, by way of example, be a palmitoylation. 
     Strains which can be used in order to have access to CyaA and CyaC genes are for example  B. pertussis  HAV deposited at the CNCM on 19 Oct. 1994 under the No. I-1485,  B. parapertussis  I-1498 and  B. bronchiseptica  973S deposited at the CNCM on 12 May 1989 under the No. I-858. 
     Moreover, the nucleotide sequences encoding AC-Hly from  B. pertussis  and from  B. bronchiseptica  are presented in  FIGS. 5 and 6  (SEQ. ID NOS: 1 and 3) respectively. 
     The nucleotide sequence of the CyaC gene which activates the CyaA gene has been described in the publication of Barry E. M. et al. (Journal of Bacteriology, January 1991, p. 720-726). 
     An advantageous sequence within the framework of the present invention is the sequence corresponding to the regions termed modified region and repeat region of AC-Hly (see  FIG. 1 ). 
     When an antigenic sequence which is heterologous in relation to the polypeptide sequence of AC-Hly is present, it may be a sequence of a bacterial, viral or parasitic pathogenic organism in particular, against which the formation of protective antibodies, for example, is sought. 
     The expression “protective antibodies against an infection by  B. pertussis  and/or  B. bronchiseptica ” is understood to mean antibodies which protect against the lethal and sublethal infections induced by these bacteria, that is to say which protect against the disease and the infection. 
     Advantageously, the amino acid sequences according to the invention, which are capable of inducing the formation of protective antibodies against the infections designated above, are associated with factors involved in the virulence of  Bordetella , and are used for example in the form of polypeptide preparations or of bacterial extracts having the capacity to induce protection against persistence of the bacteria in the host. 
     An advantageous amino acid sequence within the framework of the invention is a sequence characterized in that it is in the form of a polypeptide having a three-dimensional conformation identical or analogous to that of the corresponding polypeptide sequence of AC-Hly from  B. pertussis  or from  B. parapertussis  or from  B. bronchiseptica , in that it comprises the chain of amino acids situated between positions 900 approximately, in particular 910, and the last C-terminal amino acid approximately of the polypeptide sequence of AC-Hly from  B. pertussis  (SEQ. ID NO: 2) or of a sequence corresponding to the preceding one in  B. parapertussis  or in  B. bronchiseptica  (SEQ. ID NO: 4), the said sequence comprising, in addition, a modification by addition of a fatty acid between amino acids 980 approximately and 985 approximately, in particular at the level of amino acid 983. 
     This amino acid sequence comprises the modified region and the repeat region of AC-Hly. 
     Another preferred amino acid sequence according to the invention is a sequence characterized in that it is formed by the chain of amino acids between the amino acid in position 385 approximately and approximately the last C-terminal amino acid of the polypeptide sequence of AC-Hly from  B. pertussis  (SEQ. ID NO: 2) or of a sequence corresponding to the preceding one in  B. parapertussis  or in  B. bronchiseptica  (SEQ. ID NO: 4), the said sequence comprising a modification by addition of a fatty acid between amino acids 980 approximately and 985 approximately, preferably at the level of amino acid 983. 
     A subject of the invention is also an amino acid sequence entering within the scope of the definitions given above, formed by the chain of amino acids between the amino acid in position 385 approximately and the amino acid in position 400 approximately of the polypeptide sequence of AC-Hly from  B. pertussis  (SEQ. ID NO: 2) or of a sequence corresponding to the preceding one in  B. parapertussis  or in  B. bronchiseptica  (SEQ. ID NO: 4). 
     This amino acid sequence advantageously comprises an epitope capable of inducing the formation of protective antibodies against an infection by  Bordetella  of the  B. pertussis, B. parapertussis  and  B. bronchiseptica  types. 
     According to another embodiment of the invention, the amino acid sequence is characterized in that it is formed by the chain of amino acids between the amino acid in position 385 approximately and the amino acid in position 500 approximately of the polypeptide sequence of AC-Hly from  B. pertussis  or of a sequence corresponding to the preceding one in  B. parapertussis  or in  B. bronchiseptica.    
     Advantageously, this sequence comprising amino acids 385 to 400 or 385 to 500 is presented in a conformation identical or analogous to the conformation which it possesses in the AC-Hly protein from  Bordetella.    
     The subject of the invention is also amino acid sequences obtained by deletion of polypeptide fragments from the sequence of AC-Hly from  Bordetella , whether it is the sequence purified from the bacterium, and in particular from  B. pertussis, B. parapertussis  or  B. bronchiseptica  or whether it is a sequence obtained from a recombinant protein r-AC-Hly. 
     An amino acid sequence thus obtained is advantageously the sequence called ΔCla corresponding to the AC-Hly chain modified by deletion of an ΔCla fragment represented in  FIG. 1 , corresponding to the chain of amino acids 827 to 887 of the polypeptide sequence of AC-Hly from  B. pertussis  (SEQ. ID NO: 2) or of a sequence corresponding to the preceding one in  B. parapertussis  (SEQ. ID NO: 4) or in  B. bronchiseptica , the sequence obtained comprising a modification by addition of a fatty acid between amino acids 980 approximately and 985 approximately, preferably at the level of amino acid 983. 
     Another fragment may be deleted from the AC-Hly sequence in order to form an amino acid sequence according to the invention; this is the polypeptide fragment ΔH corresponding to the chain of amino acids 385 to 827 of the polypeptide sequence of AC-Hly from  B. pertussis  (SEQ. ID. NO: 2) or of a sequence corresponding to the preceding one in  B. parapertussis  or in  B. bronchiseptica  (SEQ. ID NO: 4), the sequence obtained comprising a modification by addition of a fatty acid between amino acids 980 approximately and 985 approximately, preferably at the level of amino acid 983. 
     The subject of the invention is also the amino acid sequence forming the “repeat region” of AC-Hly from  Bordetella , between the amino acid residues 1000 and 1600 approximately. The repeat region from  B. bronchiseptica  comprises one repeat less compared to the AC-Hly from  B. pertussis . In this regard, the invention relates, for example, to the sequence comprising amino acids 1552 to 1592 of AC-Hly from  B. pertussis  (SEQ. ID NO: 2). 
     The invention also relates to the nucleotide sequences encoding the amino acid sequences described above. 
     The genes CyaA, CyaB and partially CyaD from  B. bronchiseptica  were cloned into the plasmid pFBD2 harboured by  E. coli  K12XL1 and deposited at the CNCM on 21 Jun. 1995 under the No. I-1601. 
     The plasmid pFBD2 is obtained by insertion at the BamHI site of the 8 kb fragment from  B. bronchiseptica  carrying the genes CyaA, CyaB and partially CyaD. 
     The subject of the invention is moreover polypeptide compositions comprising sequences according to the invention originating from various types of  Bordetella . For example, an advantageous polypeptide composition comprises a sequence defined above from  B. pertussis  or another sequence or several of these sequences from  B. parapertussis.    
     Another polypeptide composition of the invention comprises, in addition, one or more sequences from  B. bronchiseptica.    
     According to another embodiment of the invention, a polypeptide composition is characterized in that it comprises one or more sequences defined above from  B. parapertussis  and one or more sequences from  B. bronchiseptica.    
     The subject of the invention is also immunogenic compositions characterized in that they comprise one or more sequences defined in the preceding pages. 
     Advantageously for protection against infection by  Bordetella  and in particular for protection against persistence of the bacteria in the host, an immunogenic composition comprising the amino acid sequences of the invention may be characterized in that it comprises, in addition, a bacterial extract containing the products of expression of the vrg genes from a strain of  Bordetella  chosen from  B. pertussis, B. parapertussis  or  B. bronchiseptica  or a portion of these expression products, sufficient to induce an immune response in a host to which the extract might be administered. 
     In addition to the presence of various adhesins and toxins, Bordetellae are characterized by regulation of the expression of the factors involved in their virulence. In other words, Bordetellae undergo phase variations and modulations. 
     Bordetellae, depending on their environment, can become “avirulent”, that is to say incapable of inducing lethality, an inflammatory reaction and pulmonary lesions in the murine model of respiratory infection. They undergo either a phase modulation or a phase variation. The phase variation is observed at a frequency ranging from 10 −3  to 10 −6  and is practically reversible. It results in a stoppage of the expression of the toxins and adhesins described above and in the expression of other factors still not well characterized (change of the Phase I “virulent” bacteria to Phase IV “avirulent” bacteria). The phase I and phase IV bacteria have been described by Lacey B. 1960, J. Hyg, 58:57-93. The phase modulation, phenotypically similar to the phase variation, is completely reversible and results in a stoppage of the synthesis of the adhesins and toxins during environmental changes (composition of the culture medium, temperature and the like). 
     The phase variation and the phase modulation observed in  Bordetella  are under the control of two regulatory genes, bvg A and bvg S (Arico B et al., 1989, Proc. Natl. Acad. Sci. USA, 86:6671-6675). 
     The bvg S gene encodes a protein sensitive to the external conditions. This protein modulates, by phosphorylation, the activity of the protein encoded by the bvg A gene, which is, on the one hand, a positive activator of the transcription of the genes encoding the factors for virulence (vag genes for “vir activated genes”) mentioned above (Uhl M. A. and Miller J, 1994, Proc. Natl. Acad. Sci. USA 91:1163-1167), and on the other hand a repressor of the transcription of certain genes (Beattie D. T. et al., J. of Bacteriology, Jan. 93, p. 159-527). The genes whose expression is repressed are called vrg genes for “vir repressed genes” and are yet poorly characterized. It has however been shown that the vrg 6 gene from  B. pertussis  encodes a protein having a role in the persistence of the bacterium in the host (Beatties D. et al., 1992, Infect. Imm. 60:571-577). In  B. bronchiseptica , two proteins encoded by the vrg genes have been characterized: they are proteins of the flagella type ( B. bronchiseptica  phase I is an immotile bacterium which does not synthesize flagella but which synthesizes adhesins and toxins, and  B. bronchiseptica  phase IV is a motile bacterium which synthesizes flagella). 
     The presence, in the immunogenic composition, of a bacterial extract comprising the products of expression of the vrg genes would make it possible to enhance the humoral and/or cellular immune response obtained after infection in vaccinated subjects and would also contribute to the protection against the persistence of the bacterium. 
     The bacterial extract termed “vrg bacterial extract” which is in question above contains all the constituents of the external membrane of a phase IV bacterium, that is to say of a bacterium not expressing the vag genes, including the LPS endotoxin. This endotoxin may however be eliminated or detoxified. 
     This extract may be present in the form of a suspension. 
     The “vrg” bacterial extracts used to carry out the invention are preferably extracts termed “urea extracts”. 
     A “urea extract” is composed of a mixture of proteins expressed at the surface of the bacterium and which are separated from the bacterium after incubation of the latter with 5 M urea. The “vrg” urea extract contains several proteins as yet not characterized, the flagella and the LPS. 
     The use of urea extracts allows the production of a vaccine which is cheaper compared with a vaccine which would be obtained from the proteins contained in the extracts, in purified form. 
     In addition, the inventors have observed that the urea extracts used may induce a T type cell immuno response (lymphoproliferation), thus behaving like the cellular vaccine used up until now. 
     On the contrary, exclusively acellular compositions are thought not to induce a T response, a reaction which nevertheless occurs in the event of infection. 
     The vrg urea extracts are respectively prepared from phase IV bacteria. Where appropriate, the phase IV bacteria are replaced with bacteria whose bvg S gene is mutated such that the bacteria express only the proteins encoded by the vrg genes. 
     The preparation of these extracts is described in detail in the experimental part. 
     Thus, the invention preferably relates to an immunogenic composition comprising both a vag urea extract from  B. pertussis  and a vrg urea extract from  B. pertussis.    
     An appropriate  B. pertussis  strain for the preparation of these extracts is the HAV strain entering within the framework of the invention and deposited at the CNCM (Collection Nationale de Cultures de Microorganismes in Paris), on 19 Oct. 1994 under the No. I-1485. To prepare the vag urea extract, the HAV strain can be used directly since it is a phase I strain. 
     On the other hand, the vrg urea extract is obtained from a phase IV strain derived from the phase I strain for example by mutation of the bvg S gene from the bacterium or by culture of the said phase I strain in a medium containing only magnesium sulphate, so as to obtain the expression of the  B. pertussis  vrg genes alone. 
     In the same manner and where appropriate to improve the immune response in the host to which the immunogenic composition might be administered, the latter comprises, in addition, one or more adhesins or toxins from  B. pertussis, B. parapertussis  or  B. bronchiseptica , chosen from FHA, AGGs or PRN and PTX or a portion of these proteins, sufficient to induce an immune response in a host to which the extract might be administered. 
     Advantageously, the amino acid sequences derived from the AC-Hly toxin and, where appropriate, the proteins expressed by the vrg genes are obtained from the HAV strain deposited at the CNCM under the No. I-1485. 
     Likewise, the amino acid sequences from  B. parapertussis  used within the framework of the invention are obtained from strain No. 1 deposited at the CNCM under the No. I-1498. 
     Moreover, the amino acid sequences from the  bronchiseptica  strain which are used within the framework of the invention may be obtained from the strain 9735 deposited at the CNCM under the No. I-858. 
     The references given above for the various  Bordetella  strains are indicated either to give access to the sequence of the proteins and where appropriate to reproduce this sequence by chemical synthesis, or to obtain the amino acid sequences of the invention by proteolysis of the proteins from  Bordetella , or to give access to the DNA of the strains and thus to allow the production of the amino acid sequences of the invention by genetic engineering techniques. 
     The subject of the invention is also a vaccine composition comprising, as active ingredient, an immunogenic composition defined above, in combination with a pharmaceutically acceptable vehicle and, where appropriate, with an adjuvant. 
     The invention also relates to a process for the preparation of monoclonal antibodies recognizing the AC-Hly from  B. pertussis , the AC-Hly from  B. parapertussis  and the AC-Hly from  B. bronchiseptica , comprising the steps of:
         immunizing an animal, for example a Balb/c mouse with a peptide comprising the sequence of amino acids 385 to 400 of AC-Hly from  B. pertussis  (SEQ. ID NO: 2), the immunization being, where appropriate, carried out by means of repeated administrations of the peptide;   fusing the spleen cells of the immunized animal with myeloma cells to form a hybridoma;   culturing the hybridoma under conditions allowing the production of antibodies;   recovering the antibodies directed against the sequence of amino acids 385 to 400 of the AC-Hly from  B. pertussis.          

     The process described above advantageously allows, by using for the immunization an antigen specific for the AC-Hly from  B. pertussis , monoclonal antibodies to be obtained which recognize both the AC-Hly from  B. pertussis  and those from  B. parapertussis  and from  B. bronchiseptica . In addition, such monoclonal antibodies advantageously have protective capacities in relation to the infection of a human or animal host by  Bordetella  of the  B. pertussis, B. parapertussis  and/or  B. bronchiseptica  type. Thus, the monoclonal antibodies obtained by using the process described above can be used for the treatment of patients or animals infected with one or more strains of  Bordetella  chosen from  B. pertussis, B. parapertussis  or  B. bronchiseptica.    
     In general, the subject of the invention is monoclonal antibodies characterized in that they recognize the sequence of amino acids 385 to 400 of the AC-Hly from  B. pertussis.    
     The invention relates, in this regard, to monoclonal antibodies which recognize an epitope comprising the sequence of amino acids 385-400 of the AC-Hly from  B. pertussis , produced by the hybridoma B5-4 deposited at the CNCM on 19 Jun. 1996 under the No. I-1734. 
     Other advantageous monoclonal antibodies are produced against the sequence of the last 217 amino acids of the AC-Hly from  B. pertussis  and are produced by the hybridoma E17-21 deposited at the CNCM on 19 Jun. 1996 under the No. I-1733. 
     The subject of the invention is also monoclonal antibodies directed against any of the amino acid sequences described above. Monoclonal antibodies directed specifically against the C-terminal sequence of the AC-Hly are among the advantageous antibodies of the invention. Special antibodies are for example directed against a sequence derived from the corresponding chain comprising the last 217 amino acids of the AC-Hly from  B. pertussis , especially the amino acids 1488 to 1705 of the AC-Hly from  B. pertussis  or the amino acids 1489 to 1706 of the AC-Hly from  B. bronchiseptica . These monoclonal antibodies may be prepared by a process analogous to that which is described above for the monoclonal antibodies obtained against the epitope contained in the sequence of amino acids 385 to 400 approximately of the AC-Hly from  B. pertussis.    
     The antibodies of the invention may be humanized, for example, by replacing the hypervariable part of a human immunoglobulin, having no antibody function, with a hypervariable region of a monoclonal immunoglobulin obtained by means of the technique described above. 
     Techniques which make it possible to humanize antibodies have for example been described by Waldmann T., June 1991, Science, vol. 252, p. 1657-1662; Winter G. et al., 1993, Immunology Today, vol. 14, No. 6, p. 243-246; Carter et al., May 1992, Proc. Natl. Acad. Sci. USA, vol. 89, p. 4285-4289; Singer et al., 1 Apr. 1993, Journal of Immunology, vol. 150, No. 7, p. 2844-2857. 
     The subject of the invention is also polyclonal sera as obtained by immunization of an animal with an amino acid sequence corresponding to the definitions given above and recovering the antibodies formed which are capable of recognizing the sequences used for the immunization. 
     Where appropriate, the immunization comprises the administration, with the amino acid sequences, of an adjuvant. 
     According to a specific embodiment of the invention, the immunization is carried out with one or more amino acid sequences and with an immunogenic composition comprising various antigens of AC-Hly. 
     The invention also relates to a pharmaceutical composition comprising, as active ingredient, monoclonal antibodies according to the invention. 
     These antibodies, in particular the antibodies directed against the sequence comprising the amino acids 385 to 400 and/or the antibodies directed against the C-terminal part of the AC-Hly protein, can be used as medicinal product in immuotherapy in a host infected with  B. pertussis  and/or  B. parapertussis  and/or  B. bronchiseptica.    
     Use will be made for example of the monoclonal antibodies produced by the hydridoma B5-4 deposited at the CNCM under the No. I-1734 or by the hybridoma E17-21 deposited at the CNCM under the No. I-1733. 
     The antibodies according to the invention may also be used as means for analyzing the  Bordetella  strains collected. The monoclonal antibodies directed against the amino acid sequence 358 to 400 or against the C-terminal sequence of the AC-Hly protein are thus appropriate for the analysis of the  B. pertussis  strains. 
     Also entering within the framework of the invention is a process for the in vitro detection of an infection by a  Bordetella  especially by  B. pertussis, B. parapertussis  or  B. bronchiseptica , characterized in that a sample of biological liquid from a patient or from an animal capable of being infected by  Bordetella  is brought into contact with monoclonal antibodies defined above or with a polyclonal serum defined above and in that an immunological reaction is detected between the said monoclonal or polyclonal antibodies and bacteria of the genus  Bordetella  when they are present in the sample. 
     The detection of infection with a bacterium of the  Bordetella  genus, especially by  B. pertussis, B. parapertussis  and  B. bronchiseptica , may also be carried out on a sample of a biological fluid from a patient or from an animal by bringing the sample tested into contact with an amino acid sequence corresponding to the definitions above, followed by the detection of an immunological reaction between the said amino acid sequences and the antibodies present in the sample tested. 
     The invention also relates to a pharmaceutical composition comprising, as active ingredient, nucleotide sequences described above. 
     The use of the nucleotide sequences in pharmaceutical compositions may be implemented with reference to the technique described by Donnelly et al. (Nature Medecine, 1995, vol. 1. No. 6, p. 583-587). 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1 : Schematic representation of the truncated proteins of the AC-Hly used: the construction of the plasmids and the production of the corresponding truncated proteins has been described in previous publications from Sebo P. et al., 1991, Gene, 104:19-24 and Sebo P. et al., 1993, Mol. Microb. 9:999 1009. The numbers placed in the column “Mr” indicate the relative molecular masses of the toxins, calculated from their deduced amino acid sequences. The numbers which follow the symbol Δ in the name of the plasmid are the numbers for the first and the last amino acid of the parts deleted from the reading frame of cyaA. The numbers which follow the symbol ΔC represent the missing C-terminal residues. The cyaA alleles are coexpressed with the cyaC gene under the control of signals for initiation of transcription and of translation of the lacZ gene identical to that present in the plasmid pCACT3 (Betsou F. et al., 1993, Infect. Immun. 61:3583-3589). ΔC1307 was produced from PDIA 5240 (pACTΔC1307) in the presence of the cyaC gene expressed in trans from a compatible plasmid pPS4C (Betsou F. et al., 1993, Infect. Immun. 61:3583-3589 and Sebo P. et al., 1991, Gene, 104:19-24). 
         FIG. 2 : Characterization of  B. pertussis  and of the r-AC-Hly protein: 200 ng of purified  B. pertussis  and of r-AC-Hly were subjected to electrophoresis on an 8-25% SDS-PAGE gel and the proteins were stained with Coomassie blue (A) or transferred to a HYBOND C-Super membrane and incubated with mixed anti-r-AC-Hly sera from 20 mice, 22 days after the vaccination (B) or with monoclonal antibodies specific for the AC-Hly from  B. pertussis  (C) or a mixture of sera from infected children (infections confirmed by culture) (D) or a serum from mice infected with  B. pertussis  18323 collected two weeks after the infection (E) or a serum from mice infected with  B. pertussis  collected two months after the infection (F). The immunodetection was performed with peroxidase-labelled rabbit anti-mouse antibodies. Line 1:  B. pertussis  AC-Hly; line 2: r-AC-Hly; line 3: ΔHR2; line 4: ΔH; line 5: ΔCla; line 6: ΔC217; line 7: ΔC1307; the numbers indicate the molecular weight markers. 
         FIG. 3 : Inhibition of the adenylate cyclase (A), haemolytic (B) and cytotoxic (C) activities of the AC-Hly from  B. pertussis  by antisera directed against r-AC-Hly fragments obtained in various mice and by sera from mice and children infected with  B. pertussis : the AC-Hly was incubated with various sera as described in the Materials and Methods part. The bars indicate the standard deviation (n=4). 
         FIG. 4 : Protective activities of r-AC-Hly and of its truncated proteins. 3- to 4-week-old mice were immunized twice, at two week intervals, with 15 μg of r-AC-Hly (Δ-Δ), ΔCla (▴-▴), ΔC1307 (●-●), or ΔHR2 ([ ]-[ ]), ΔC217 (▪-▪), ΔH (□-□) or  B. pertussis  AC-Hly (◯-◯) adsorbed onto aluminium hydroxide, or with a buffer containing aluminium hydroxide alone as control (□-[ ]). They are infected via the intranasal route two weeks later with 10 5  CFU of  B. pertussis  18323. The curves show the standard geometric deviation (bars) for six mice per point. 
         FIG. 5 : Nucelotide sequence of the gene encoding the AC-Hly from  B. pertussis  (SEQ. ID NO: 2) and the corresponding amino acid sequence. 
         FIG. 6 : Nucelotide sequence of the gene encoding the AC-Hly from  B. bronchiseptica  (SEQ. ID NO: 3) and the corresponding amino acid sequence (SEQ. ID NO: 4). 
     
    
    
     EXAMPLES 
     Materials and Methods 
     Bacteria Strains, Plasmids and Growth Conditions: 
     The virulent strain  B. pertussis  18323 (ATCC 9797) was cultured on a Bordet Gengou agar medium supplemented with 15% defibrinated sheep blood (BG) at 36 EC for 72 hours and again for 24 h. Subcultures in a liquid medium were performed in a Stainer-Scholte medium (Stainer D. W. and Scholte M. J., 1971, J. Gen. Microb., 63:211-220) for 20 h at 36 EC until an optical density at 650 nm of 1.0 is obtained (OD 620 =1.0). 
     The plasmids used for the production of the recombinant protein r-AC-Hly and its truncated derivatives in  E. coli  have been described in the following publications: (Betsou F., P. Sebo and N. Guiso, 1993, CyaC-mediated activation is important not only for toxic but also for protective activities of  Bordetella pertussis  adenylate cyclase-haemolysin, Infect. Immun. 61:3583-3589; Iwaki M., Ullmann A. and P. Sebo, 1995, “Oligomerization of the adenylate cyclase toxin of  Bordetella pertussis : Evidence from in vitro complementation of individually inactive mutants”. Submitted; and Sebo P., P. Glaser, H. Sakamoto and A. Ullmann, 1991, High level synthesis of adenylate cyclase toxin of  Bordetella pertussis  in a reconstructed  Escherichia coli  system, Gene 104: 19-24. 
     The plasmids allow the production of the various proteins ( FIG. 1 ) in the presence of the activating protein CyaC.  E. coli  XL-1 Blue strains (Stratagene) comprising the respective plasmids were cultured at 37 EC on a 2xYT medium containing 100 μg/l ampicillin at an optical density OD 600  of 0.5 to 0.7, and induced in order to obtain the production of AC-Hly by IPTG (1 mM) for four additional hours (Betsou F., P. Sebo, and N. Guiso, 1993, Infect. Immun. 61:3583-3589). 
     Tests of the Adenylate Cyclase, Haemolytic and Cytotoxic Activities 
     The adenylate cyclase activity was measured according to the procedure described by Ladant D., C. Brezin, I. Crenon, J. M. Alonso, and N. Guiso, (1987,  Bordetella pertussis  adenylate cyclase: purification, characterization and radioimmunoassay, J. Biol. Chem. 261:16264-16269). One unit (U) of adenylate cyclase activity corresponds to 1 nmol of cAMP formed per minute at 30 EC, at pH 8.0. The haemolytic and cytotoxic activities of AC-Hly were determined at 37 EC using washed sheep erythrocytes (109/ml) according to the description of Bellalou J., H. Sakamoto, D. Ladant, C. Geoffroy and A. Ullmann, (1990, Deletions affecting haemolytic and toxin activities of  Bordetella pertussis  adenylate cyclase, infect. Immun. 58: 3242-3247). The protein concentrations were determined by the method of Bradford (A rapid and sensitive method for the quantification of micrograms of protein, utilizing the principle of protein-dye binding. Anal. Biochem. 72:248-257). 
     Tests of Inhibition of Adenylate Cyclase 
     The purified AC-Hly from  B. pertussis  was incubated at 100 U/ml in 50 mM Tris-HCl at pH 7.6, with 0.2 mM CaCl 2  and 0.1 NP-40 with various sera diluted 100-fold for 18 to 20 h at 4 EC; the post-incubation adenylate cyclase activity (IA) of the samples was measured. The AC (adenylate cyclase) activity after incubation with the serum from mice immunized with aluminium hydroxide alone (CA) was taken as reference for 100% activity (in other words 0% inhibition). 
     The percentage inhibition of the AC activity was calculated as follows:
 
% inhibition=100%−(100%× IA/CA ).
 
Tests of Inhibition of the Haemolytic Activity
 
     One unit (U) of toxin and 5 μl of the various sera were mixed in 1 ml of 10 mM Tris-HCl at pH 8, with 2 mM CaCl 2 , 150 mM NaCl and 1 μM bovine brain calmodulin and preincubated for 20 min. at 4 EC. Washed sheep erythrocytes (10 9 ) were added and the remaining haemolytic activity was determined after incubation for 3 h at room temperature. The unlysed erythrocytes were recovered in the form of a pellet after centrifugation at 2000 rpm and the optical density of the haemoglobin released (RH) into the supernatants was measured at 541 nm. The haemolytic activity of the toxin incubated with the serum of mice immunized with only aluminium hydroxide (CH) was taken as reference for 100% activity (0% inhibition). The sheep erythrocytes incubated without the toxin were used as control for nonspecific lysis. The percentage inhibition of the haemolytic activity was calculated as follows: % inhibition=100%−(100%×RH/CH). 
     Tests of Inhibition of the Cytotoxic Activity 
     One unit (U) of toxin and 5 μl of various sera were preincubated at 4 EC for 20 minutes in a total volume of 1 ml of 10 mM Tris-HCl at pH 8, with 2 mM CaCl 2 , 150 mM NaCl, 5 mM glucose, 1 mg/ml BSA and 1 μM bovine brain calmodulin. Then 10 9  washed sheep erythrocytes were added and the incubation was continued at 37 EC for 30 minutes. In order to stop the activity of the toxin, 50 μl of erythrocyte suspension were injected into 1 ml of 50 mM boiling sodium acetate at pH 5.2 and heated at 100 EC for 5 min. The quantity of cAMP formed in the lysed erythrocytes (IT) was determined by a standard ELISA method (Pradelles P. Grassi J. Chabardes D., Guiso N. 1989, Analytical Chemistry, 61:447-450). The toxin incubated at 4 EC was used as negative control of cytotoxicity. The activity of the toxin incubated with the serum of mice immunized with aluminium hydroxide alone (CT) was considered as being the 100% activity (0% inhibition). The percentage inhibition of the cytotoxic activity was calculated as being: % inhibition=100%−(100%×IT/CT). 
     Electrophoresis and Immunoblotting Methods 
     An SDS-PAGE electrophoresis was performed on an 8-25% ready-for-use polyacrylamide gel for the PhastSystem (Pharmacia) and the separated proteins were electrotransferred from the polyacrylamide gels to Hybond C-Super membranes (Amersham). After blocking, the membranes were incubated at a dilution of 10 −3  with polyclonal sera at 4 EC overnight. The immunochemical detection was performed using horseradish peroxidase-labelled sheep anti-mouse immunoglobulins and an enhanced chemiluminescence system (ECL-Amersham). 
     Production and Purification of AC-Hly. 
     The AC-Hly from  B. pertussis , the r-AC-Hly (recombinant AC-Hly) produced from  E. coli  and the various recombinant truncated proteins were extracted from bacteria with urea and purified on calmodulin affinity columns according to the method described by Guiso N., M. Szatanik and M. Rocandourt (1991, Protective activity of  Bordetella  adenylate cyclase against bacterial colonization. Microb. Pathog. 11:423-431). The enzymatic preparations were preserved in 8 M urea with 50 mM Tris-HCl at pH 8, with 0.2 mM CaCl 2 , at −20 EC. All the preparations were analysed in order to determine their degree of purity by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). 
     Active Immunization 
     The purified preparations of r-AC-Hly and of the truncated proteins were adsorbed at 60 μg/ml on aluminium hydroxide (250 μg/ml). To carry out an active immunization, 3- to 4-week-old female Balb/c mice (CERJ, St Berthevin, France) were treated by subcutaneous injection of 15 μg of protein antigens twice, at two weeks interval. The controls received only buffer with aluminium hydroxide. The mice were then bled one week following the last injection, so as to allow access to the circulating antibodies present. Sublethal respiratory infection was achieved two weeks after the second immunization. 
     Intranasal Infection of the Mice 
     The  B. pertussis  strain was cultured on a Bordet Gengou BG medium for 48 h according to the preceding description and the bacteria were resuspended in 1% casamino acids. Sublethal quantities were administered by intranasal injections of 50 μl of the bacterial suspensions. The infected mice were sacrificed by cervical dislocation 1 hour after the exposure (at a time designated by “day 0”) and on different days after day 0 (6 mice during each stage). The lungs were collected aseptically and homogenized in salt with a tissue grinder. Dilutions of the homogeneous lung preparations were sampled on a BG medium and colony forming units (cfu) were counted after 3 days of incubation at 36 EC. All the experiments were performed at least twice and gave coherent results. 
     Preparation of the Immune Sera 
     To obtain the sera from the infected mice, 10 female Balb/c mice (4 weeks old) were infected intranasally with 2×10 5  virulent  B. pertussis  bacteria according to the technique described by Guiso N. Rocancourt, M. Szatanik and J. M. Alonso, (1989 , Bordetella  adenylate cyclase is a virulence associated factor and a protective antigen, Molec. Pathog. 7:373-380). The mice were bled 14 days after the infection or on different designated days following these 14 days. 
     The mouse polyclonal sera directed against the truncated derivatives of AC-Hly were collected one week after the second injection given to the mice with each of the antigens considered. 
     The serum of infected patients was prepared by mixing polyclonal immune sera from 10 selected nonvaccinated children infected with  B. pertussis . These children were more than 8 months old, so as to exclude the presence of maternal antibodies in them, and less than 2 years old so as to be certain of their clinical history. 
     Results 
     Immunological Properties of the r-AC-Hly Protein and of Its Truncated Variants 
     It has been shown in the past that the modification of AC-Hly occurring through the CyaC protein from  B. pertussis  is essential for its protective activity (Betsou et al., CyaC-mediated activation is important not only for toxic but also for protective activities of  Bordetella  pertussis adenylate cyclase-haemolysin, Infect. Immun., 61:3583-3589). It was therefore important to determine if this modification itself contributed to the formation of the protective epitope in its linear form or if this modification induced a conformational modification in the toxin, a modification required for the presentation of the protective epitopes. Consequently, the immunological and protective properties of a number of truncated proteins were examined; the truncated proteins in question are schematically represented in  FIG. 1 . 
     These proteins were produced in  E. coli  in the presence of the CyaC protein using the plasmids pCACT or pDIA so as to allow acylation by CyaC by means of fatty acid chains, of the constructs containing the site of modification. As shown in  FIG. 2A , the preparations purified from all the proteins contained a major polypeptide corresponding to the expected molecular weight. These proteins were checked by a Western Blot test in order to verify their recognition by various sera. As shown in  FIG. 2B , the serum produced against the r-AC-Hly protein recognized the r-AC-Hly protein and also recognized the AC-Hly protein purified from  B. pertussis  as well as all the truncated forms derived from r-AC-Hly. In accordance with  FIG. 2A , the purified polypeptide preparations comprising the total length of the protein, in other words the preparations of AC-Hly, r-AC-Hly and the truncated polypeptides ΔCla and ΔC217, also contained several fragments recognized by a polyclonal serum obtained against the purified protein r-AC-Hly ( FIG. 2B ). Monoclonal antibodies ( FIG. 2 ) prepared specifically against the adenylate cyclase domain of AC-Hly also recognized these fragments of AC-Hly, ΔCla and ΔC217, showing that these fragments were proteolytic fragments truncated in the C-terminal part and containing the AC domain which copurified with AC-Hly on calmoduline-agarose. However, the monoclonal antibody did not recognize the proteins ΔH or ΔHR2 which lacked residues 385 to 828 and 1489 to 1706 respectively, but this antibody recognized the protein ΔC1307. Using these observations in particular, the inventors established that the region of the molecule located between amino acids 385 and 400 participated in the formation of an epitope. 
     Quite remarkably and contrary to what could happen with the anti-r-AC-Hly serum, the sera obtained from infected children and used in the form of a mixture did not recognize the AC domain of AC-Hly (ΔC1307; line 7), and neither did they recognize the protein lacking the 217 terminal residues of AC-Hly (ΔC217; line 6) nor the protein containing the last 217 residues but lacking hydrophobic regions, which is modifed, and the major part of the repeat region of AC-Hly (ΔHR2, line 3). However, these human sera recognized the AC-Hly from  B. pertussis  and r-AC-Hly ( FIG. 2D , lines 1 and 2) and the two truncated proteins possessing modified and repeat regions (last 900 residues) of AC-Hly ( FIG. 2D , lines 4 and 5). An identical recognition pattern ( FIG. 2F ) was obtained with mouse serum infected with  B. pertussis  18323 (reference strain) and collected rapidly after the infection (14 days). The sera collected long after the infection (35 days) recognized the C-terminal part of AC-Hly present in the protein ΔHR2 ( FIG. 2F , line 3), whereas they continued not to recognize the AC domain ( FIG. 2F , line 7) and the protein ΔC217 lacking only the last 217 residues ( FIG. 2F , line 6). These results strongly suggest that the anti-AC-Hly antibodies synthesized after infection with  B. pertussis  are predominantly directed against the C-terminal region comprising the modification region and the repeat region of AC-Hly (last 900 residues). Furthermore, neither the protein ΔC217 lacking the last 217 residues of AC-Hly, nor the protein ΔHR2 which contained the last 217 residues were recognized by the immune and murine sera, suggesting that these polyclonal sera recognize a specific structure of the repeat region of AC-Hly, which structure is abolished by any of the two nonoverlapping deletions. 
     It is important to note that the sera from infected patients or mice recognized only the polypeptides stretching over the entire length of the protein (AC-Hly, r-AC-Hly, ΔCla and ΔH) but not their proteolytic fragments. The absence of recognition, by these sera, of the proteolysis products, which contain the AC domain and are cleaved in their C-terminal part (see above  FIG. 2C ) indicates, in addition, that the region of modification and the repeat region of AC-Hly (last 900 residues) must be intact to be recognized by these sera. 
     Inhibition of the Adenylate Cyclase, Haemolytic and Cytotoxic Activities by Sera Directed Against Truncated Derivatives of AC-Hly 
     It was examined if sera from mice infected with  B. pertussis  or sera from human patients infected with  B. pertussis  or alternatively polyclonal sera directed against the various truncated forms of r-AC-Hly, obtained by immunization with the purified truncated proteins, could specifically inhibit one of the activities of the toxin. Since a control was necessary prior to these experiments, the ELISA titres of the various sera were determined using the complete r-AC-Hly as coating antigen for these tests. These titres were similar, with the exception of the titre obtained with the anti-ΔC1307 serum which did not recognize AC-Hly in ELISA although it recognized it in a Western Blot test like the other sera. As shown in  FIG. 3A , the adenylate cyclase activity was inhibited by all of the sera obtained after immunization of mice with purified r-AC-Hly and with truncated derived proteins. However, none of the sera from the infected mice or from the infected human patients inhibited the AC activity of AC-Hly under identical conditions. This is in agreement with the result obtained by the Western Blot analysis (see above) showing that these sera contain antibodies predominantly directed against the C-terminal part of AC-Hly (last 900 residues) and not against the AC domain. Indeed, the haemolytic activity of the protein AC-Hly was inhibited by the sera from infected children and was less strongly inhibited by the sera from infected mice. However, a strong inhibition of the haemolytic activity was observed with the antisera obtained by immunization with the proteins AC-Hly, r-AC-Hly, ΔH, ΔCla and ΔHR2 from  B. pertussis  ( FIG. 3B ). This inhibition was probably due to the presence of specific antibodies against the C-terminal part of AC-Hly. Indeed, the anti-ΔC1307 serum directed against the AC domain of AC-Hly which lacked such antibodies did not inhibit the haemolytic activity of AC-Hly. In a completely advantageous manner, the serum directed against the protein ΔC217 lacking only the last 217 residues did not inhibit the haemolytic activity of AC-Hly at a measurable level either. This indicates that the last 217 residues are either the target of the neutralizing antibodies or are involved in the formation of a structure which favours the synthesis of neutralizing antibodies directed against other parts of AC-Hly. Together, these results indicate, in addition, that the antibodies synthesized after infection are mainly directed against the C-terminal haemolysin part of AC-Hly and are capable of neutralizing the haemolytic activity of this protein but not the enzymatic activity of its N-terminal adenylate cyclase domain. As shown in  FIG. 3C , a significant inhibition of the cytotoxic activity of AC-Hly was observed with sera obtained against intact AC-Hly and against the truncated proteins ΔH, ΔCla and ΔHR2 which all contain both the AC domain and the last 217 residues. The antisera also neutralized the AC and haemolytic activities. On the contrary, the 2 sera which inhibited either the haemolytic activity alone (for example the sera from infected mice or patients) or the AC activity alone (the anti-ΔC1306 and anti-ΔC217 sera) did not inhibit the cytotoxic activity of AC-Hly. Thus, it appeared that the presence of antibodies against the AC domain and against the last 217 residues of AC-Hly may be required in order to neutralize its cytotoxic activity. 
     Protective Activity of the Various Recombinant AC-Hly Constructs 
     In order to locate the epitopes of AC-Hly required to obtain a protective activity, the protective activity of various purified truncated proteins using the murine respiratory model was tested. Groups of mice were immunized twice with aluminium hydroxide alone (control), or with purified truncated proteins adsorbed on aluminium hydroxide and then these mice were brought into contact, via the intranasal route, with sublethal doses of virulent  B. pertussis  18323 strains. This model reflects the capacity of the bacteria to adhere, colonize, survive and multiply in the respiratory system of mice. As shown in  FIG. 4 , the bacteria multiply rapidly in the lungs of the control mice for 6 days after infection, and then begin to be eliminated from the lungs. No multiplication of the bacteria was observed in the lungs of mice immunized with the proteins AC-Hly or r-AC-Hly from  B. pertussis  and after 3 days the number of bacteria began to increase ( FIG. 4 ). In agreement with previous observations (Betsou F., Sebo and N. Guiso, 1993, CyaC-mediated activation is important not only for toxic but also for protective activities of  Bordetella pertussis  adenylate cyclase-haemolysin, Infect. Immun. 61:3583-3589), the protective efficacy of  B. pertussis  AC-Hly was higher than that of r-AC-Hly. A protection similar to that induced by r-AC-Hly was also obtained with the protein ΔCla, suggesting that the part deleted in the protein ΔCla between residues 827 and 887 was not essential for the induction of protective immunity. The protein ΔH, missing from residues 385 to 828, exhibited a weaker protective activity than ΔCla. No protection was induced by the protein ΔC1307 lacking the entire haemolysin part of AC-Hly. In a more advantageous manner, the protein ΔC217 lacking the last 217 residues and the protein ΔHR2 containing the last 217 residues of AC-Hly did not make it possible to induce protection. The protective activity is therefore correlated with the pattern of recognition of the individual constructs by the sera from infected patients or from infected mice. 
     Discussion 
     The antibodies directed against AC-Hly are usually present in the sera from infected children (Arciniaga J. L., E. L. Hewlett, F. D. Johnson, A. Deforest, S. G. F. Wassilak, I. M. Onorato, C. R. Manclark and D. L. Burns, 1991, J. Infect. Dis. 163:135-142; and Guiso N., E. Grimprel, I. Anjak and P. Bégué, 1993, Eur. J. Clin. Microbiol. and Infect. Dis. In press) and it has in the past been demonstrated that immunization with AC-Hly protects mice against bacterial colonization by  Bordetella  (Khelef, N., H. Sakamoto and N. Guiso, 1992, Microb. Pathog. 12:227-235). The production of a set of truncated forms of recombinant AC-Hly has made it possible to carry out a study of the importance of various domains of the protein in the induction of protective immunity by vaccination with AC-Hly. The results presented above show that the anti-AC-Hly antibodies, synthesized after infection by  B. pertussis , are predominantly directed against the modified domain and the repeat domain of AC-Hly (about 800 residues). Indeed, only the truncated forms of AC-Hly possessing these in tact regions were recognized by sera from infected mice and patients in a Western-Blot test and these proteins were the only ones to exhibit the capacity of inducing protection in mice. This indicates that at least in the case of mice, a relatively limited set of identical or overlapping epitopes may be advantageous both for inducing the synthesis of anti-AC-Hly antibodies by the infected subjects and for inducing the synthesis of protective antibodies obtained after vaccination with AC-Hly. The experiments reported previously have shown that the elimination of the last 217 residues of AC-Hly which are in the distal position relative to the sites of modification of the protein by the product of expression of the CyaC gene, at the level of the lysine residue 983, abolished the recognition of this protein (ΔC217) by sera from infected mice and patients and also abolished its protective activity. However, this was not due to a defect in modification of the protein ΔC217 since this protein was acylated at the level of the fatty acid chains when it was produced in the presence of the protein CyaC. Furthermore, the 217 residues per se were not recognized by the sera and did not show any protective activity when they were presented in the protein ΔHR2 which, for its part, lacked the modification site (lysine 983) and a large proportion of the repeat region. 
     This led to the conclusion that both the last 217 residues and the modification region were important for the protective activity of AC-Hly. Thus, interaction between the modified region and the last 217 residues of AC-Hly would be required for the formation or the activity of the protective epitopes of AC-Hly. Surprisingly, neither the protein ΔC217 missing from the last 217 residues of AC-Hly, nor the protein ΔHR2 lacking from the modified region and from a large portion of the repeats but containing the last 217 residues were recognized by the polyclonal sera from infected patients and/or from mice (fresh serum). The antisera could have a narrow specificity against a single epitope, or against a small group of epitopes located at the breakpoint of the repeat regions present in the proteins ΔC217 and ΔHR2 (proline 1489). However, this possibility does not appear highly probable with mixed polyclonal antisera. In addition, the sera from infected human patients and mice recognized most of the full length polypeptides of the various proteins but did not recognize the cleaved C-terminal proteolytic fragments present in these preparations, which fragments were recognized by an anti-AC monoclonal antibody. Taken together, these results suggest that the formation of protective epitopes on AC-Hly and the recognition of AC-Hly by the sera from infected subjects require the presence of a specific structure formed only when the modified and repeat regions of AC-Hly are present. It is conceivable that the formation of this structure could require the modification, by acylation, of the fatty acid chains after the translation of AC-Hly at the level of the lysine residue 983 and could be abolished by the elimination of the C-terminal part of AC-Hly. In this regard, it is important that both the haemolytic and cytotoxic activities of AC-Hly are lost when the last 75 residues of AC-Hly, which contain the unmodified secretory signal, are eliminated, and are not directly involved in the activity of formation of pores (4 and 29). These observations support, in addition, the hypothesis that the extreme part of the C-terminal region of AC-Hly plays an essential role in the overall structure of AC-Hly. 
     It has already been suggested that major protective epitopes of AC-Hly could be located in the AC portion (Guiso, N., M. Rocancourt, M. Szatanik and J. M. Alonso, 1989, Molec. Pathog. 7:373-380 and Guiso, N. M. Szatanik and M. Rocancourt, 1991, Microb. Pathog. 11:423-431). The results presented here show that contamination by AC-Hly fragments extending over the modified and repeat regions of AC-Hly might enter into the production of a protective activity associated with the fragments of the AC domain present in the preparations described in the prior state of the art. Nevertheless, the capacity of these contaminants in minor quantities to produce a protective activity may be doubtful. Another interpretation might be that, under certain conditions, the region between amino acids 385 and 450 or 500 could also play a role in the protective activity of AC-Hly. The structure of this region could be modified in AC-Hly in the absence of the last 217 amino acid residues or in the absence of acylation. The structure of the N-terminal part of 40-50 kDa and the presentation to the immune system could induce protection when this structure is cleaved in relation to the rest of the AC-Hly molecule in  B. pertussis  culture supernatants. One explanation for this hypothesis could be that a monoclonal antibody directed against the part of the molecule located between amino acids 385 and 400 is a protective antibody. 
     It was also examined whether a correlation exists between the protective activity of a truncated protein obtained from r-AC-Hly in vivo and its activity in inducing the synthesis of antibodies which could neutralize the toxic activity in vitro. Such a correlation was not established. While all the proteins having a protective activity induced the synthesis of neutralizing antibodies, the protein ΔHR2, which was not at all protective, induced a strongly neutralizing antibody response. This suggests that the presence of neutralizing antibodies in relation to the toxic activity of AC-Hly is not a reliable measure of the induction of protection against infection by  B. pertussis.    
     It is moreover important to note that the protein ΔCla, lacking a portion of the hydrophobic domain, exhibited a protective activity. This recombinant protein is a good candidate for inclusion in acellular vaccines against pertussis since it does not exert any residual cytotoxic activity. 
     Preparation of a vrg Urea Extract 
     The vrg urea extracts are prepared from the same 3 phase IV bacterial species, that is to say from bacteria no longer expressing any vag gene but expressing the vrg genes. The products of these genes are not yet well characterized, with the exception of the flagella of  B. bronchiseptica . The vrg urea extracts are prepared like the vag urea extracts. Description of the culture media Dehydrated Bordet Gengou medium 
     Composition: 
     
       
         
           
               
               
               
             
               
                   
               
               
                   
                 1 liter 
                 5 liters 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
            
               
                   
                 Bordet Gengou medium 
                 30 
                 g 
                 150 
                 g 
               
               
                   
                 Glycerol 
                 10 
                 ml 
                 50 
                 ml 
               
               
                   
                 Pyrolysed water 
                 1 
                 liter 
                 5 
                 liters 
               
               
                   
                 Adjust the pH to 7.4 
               
               
                   
               
            
           
         
       
     
     Heat 
     Autoclave for 15 minutes at 120 E 
     Store at 4 EC 
     At the time of use: 
     Bordet Gengou is enriched with 15 to 20% sheep or horse blood. Melt the tubes and keep melted at 54 EC. Add 2.5 ml of sheep blood to each tube, in a sterile manner. Pour the contents of the tube into a sterile Petri dish. 
     Remark: 
     For the identification of  Bordetella pertussis  from a nasopharyngeal sample, use fresh dishes (maximum 7 days at 4 EC) 
     CSM Agar Medium 
     To prepare 2 liters of a 10-fold concentrated solution: 
     
       
         
           
               
               
               
               
             
               
                   
               
             
            
               
                   
                 Sodium hydrogen glutamate 
                 107 
                 g 
               
               
                   
                 (Ref. Prolabo No. 27872.298) 
                   
                   
               
               
                   
                 L-Proline 
                 2.4 
                 g 
               
               
                   
                 (Ref. Merck No. 7434) 
                   
                   
               
               
                   
                 NaCl (Ref. Prolabo 
                 25 
                 g 
               
               
                   
                 No. 27810.295) 
                   
                   
               
               
                   
                 H 2 PO 4  (Ref. 
                 5 
                 g 
               
               
                   
                 Prolabo No. 26926.298) 
                   
                   
               
               
                   
                 KCl (Ref. Prolabo 
                 2 
                 g 
               
               
                   
                 No. 26759.291) 
                   
                   
               
               
                   
                 MgCl 2  (Ref. Prolabo 
                 1 
                 g 
               
               
                   
                 No. 25108.295) 
                   
                   
               
               
                   
                 Tris-base (Ref. Merck 
                 15.2 
                 g 
               
               
                   
                 No. 8382.2500) 
                   
                   
               
               
                   
                 Casamino acids (Ref. Difco 
                 5 
                 g 
               
               
                   
                 No. 0288-01-2) 
                   
                   
               
               
                   
                 1% CaCl 2  solution in 
                 20 
                 ml 
               
               
                   
                 pyrolysed water 
                   
                   
               
               
                   
                 (Ref. Prolabo No. 22317.297 
                   
                   
               
               
                   
                 Pyrolysed water qs 
                 1 
                 liter 
               
               
                   
               
            
           
         
       
     
     Dissolve the various constituents in a portion of the final volume of water. Adjust the pH to 7.4 using hydrochloric acid. Fill to the final volume and store at −20 EC. 
     At the time of use, mix: 
     100 ml of the 10-fold concentrated solution 
     900 ml of pyrolysed water 
     1 g of (2,6-O-dimethyl)cyclodextrin, reference Aldrich No. 51166-71-3 
     15 g of Bacto agar, reference Difco No. 0140-01 Distribute, in fractions of 20 ml, into glass tubes. 
     Sterilize and add the sterile complement. 
     Solution of complement: 
     Mix: 
     1 ml of solution of complement concentrated 10-fold 
     100 mg of glutathion, reference Merck No. 4090 
     9 ml of pyrolysed water 
     Filter this solution on a 0.22 μm Millex filter. 
     Add 200 μl of this solution to 1 tube of 20 ml of medium. 
     Stainer Culture Medium 
     A. Basal Medium 
     To prepare 2 liters of a 10-fold concentrated solution: 
     
       
         
           
               
               
               
               
             
               
                   
               
             
            
               
                   
                 Sodium hydrogen glutamate 
                 214.0 
                 g 
               
               
                   
                 (Ref. Prolabo No. 27872.298) 
                   
                   
               
               
                   
                 L-Proline (Ref. Merck No. 7434) 
                 4.8 
                 g 
               
               
                   
                 NaCl (Ref. Prolabo No. 27810.295) 
                 50.0 
                 g 
               
               
                   
                 H 2 PO 4  (Ref. 
                 10.0 
                 g 
               
               
                   
                 Prolabo No. 26926.298) 
                   
                   
               
               
                   
                 KCl (Ref. Prolabo No. 26759.291) 
                 4.0 
                 g 
               
               
                   
                 MgCl 2  (Ref. Prolabo 
                 2.0 
                 g 
               
               
                   
                 No. 25108.295) 
                   
                   
               
               
                   
                 Tris-base (Ref. Merck 
                 30.5 
                 g 
               
               
                   
                 No. 8382.2500) 
                   
                   
               
               
                   
                 1% CaCl 2  solution in pyrolysed 
                 40 
                 ml 
               
               
                   
                 water (Ref. Prolabo No. 22317.297 
                   
                   
               
               
                   
                 Pyrolysed water qs 
                 2 
                 liters 
               
               
                   
               
            
           
         
       
     
     Dissolve the various constituents in a portion of the final volume of water. Adjust the pH to 7.6 using hydrochloric acid. Fill to the final volume and distribute this concentrated solution which may be stored at −20 EC for several weeks. 
     At the time of use, dilute the medium, sterilize it at 120 EC for 15 minutes and then add the complement sterilized by filtration. 
     B. Solution of Complement 
     To prepare 200 ml of a 10-fold concentrated solution: 
                                                L-cystine (Ref. Prolabo No.23260.184)   8   g           Concentrated HCl   20   ml                    
Dissolve. Over this preparation, pour the following mixture dissolved beforehand:
 
     
       
         
           
               
               
               
               
             
               
                   
               
             
            
               
                   
                 FeSO 4 .7H 2 O 
                 2 
                 g 
               
               
                   
                 (Ref. Prolabo No. 24244.232) 
                   
                   
               
               
                   
                 L (+) ascorbic acid 
                 4 
                 g 
               
               
                   
                 (Ref. Prolabo No. 20155.237) 
                   
                   
               
               
                   
                 Nicotinic acid 
                 0.8 
                 g 
               
               
                   
                 (Ref. Merck No. 6817) 
                   
                   
               
               
                   
                 Pyrolysed water 
                 120 
                 ml 
               
               
                   
               
            
           
         
       
     
     Bring to 200 ml with pyrolysed water, distribute the solution in fractions of 1, 2, 3 or 4 ml and freeze at −20 EC. 
     At the time of use, dilute the solution 10-fold in pyrolysed water and add: glutathion (Ref. Merck No. 4090) . . . 100 mg/10 ml of diluted complement, sterilize this solution by filtration (0.22 μm Millex filter for single use) and add 1 ml of sterile solution to 100 ml of sterile basal medium. 
     2.1.2. Preparation of the vrg Urea Extract 
     Centrifuge the bacterial suspension for 30 minutes at 5000 g, at 4 EC. 
     Resuspend the bacterial pellet in 5M urea prepared in PBS buffer (described further on) in an amount of a volume equal to 5 times the wet weight of the bacterial pellet. 
     Leave stirring for 1 hour at 4 EC and then centrifuge for 40 minutes for 40,000 g, at 4 EC, 
     Store the supernatant at −80 EC until use. 
     2.1.3. Inactivation of the vrg Urea Extract 
     After removal of the urea by passage over a G25 column, the vrg urea extract is diluted in PBS so as to obtain a protein concentration of 300 μg/ml. 
     Add dropwise a volume of glutaraldehyde at 2.5% so as to obtain a final concentration of 0.05%. 
     Leave for 2 hours at room temperature while mixing regularly. 
     Stop the reaction by addition of lysine (final concentration 0.02M). 
     After 2 hours at room temperature, the urea extract is adsorbed on aluminium hydroxide prepared in PBS (1 mg/ml), overnight at 4 EC, with gentle stirring. The vaccine is then ready for immunization of the animal in an amount of 10 to 20 μg/injection (described further on).