Patent Publication Number: US-7906123-B1

Title: Modified Bordetella adenylate cyclase comprising or lacking CD11b/CD18 interaction domain and uses thereof

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a Continuation of U.S. application Ser. No. 11/304,590, filed Dec. 16, 2005 (now abandoned), which is a Continuation of PCT International Application No. PCT/EP2004/007811, filed on Jun. 18, 2004, which claimed the priority of European Patent Application No. 03291486.3, filed Jun. 18, 2003, the contents of each are incorporated herein by reference. 
    
    
     The invention relates to modified  Bordetella  adenylate cyclase toxins which are deficient for CD11b/CD18 binding and to their use in the preparation of pharmaceutical composition for the treatment of whooping cough and/or for the protection against  Bordetella  infection. The invention also relates to specific fragments of  Bordetella  adenylate cyclase comprising the CD11b/CD18 interaction domain and to their use, especially for targeting a molecule of interest to CD11b expressing cells. 
     The genus  Bordetella  comprises four species, i.e.,  Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica  and  Bordetella avium.    
     The bordetellae are Gram-negative coccobacilli responsible for respiratory infections.  Bordetella pertussis  and  Bordetella  parapertussis are strictly human pathogens.  Bordetella bronchiseptica  is pathogenic for various mammals, and more rarely for man, and, in distinction to  B. pertussis  and  B. parapertussis , is capable of surviving outside the host.  Bordetella avium  is pathogenic for birds. 
     The most virulent species to man is  B. pertussis , which is the etiologic agent of whooping cough, a highly contagious childhood respiratory disease, characterized by bronchopneumonia and paroxysmal coughing interrupted by inspiratory whoops. 
     The vaccination against whooping cough has hitherto been most usually carried out with the aid of inactivated whole bacteria. However, such vaccines are not always devoid of toxicity in view of the fact that the virulence factors are constituted by proteins secreted by the bacteria and not by the bacteria themselves. The proteins can thus exert serious pathological effects, even after the death of the bacteria. 
     European patent EP 0 424 518 (Institut Pasteur) recites the use of  Bordetella  adenylate cyclase as protective antigens against both  Bordetella pertussis  and  Bordetella bronchiseptica.    
     European patent EP 0 338 169 (Institut Pasteur) also describes the use of active adenylate cyclase preparations from  Bordetella  parapertussis as protective antigens against whooping cough. 
     Alternate strategies have also been developed, including the preparation of acellular vaccine using immunogenic detoxified toxins of  Bordetella.    
     An example of a vaccine based on detoxified pertussis toxin is described in U.S. Pat. No. 6,040,427 (Locht et al., 2000). 
     Among the variety of toxins produced by  B. pertussis , the adenylate cyclase (also referred hereafter by the term CyaA) is a crucial factor in the virulence strategy of the bacteria during the early phases of respiratory tract colonization (Goodwin and Weiss, 1990; Khelef et al., 1992). The toxin allows the pathogen to escape host immune surveillance, mainly, by intoxicating neutrophils and macrophages causing phagocyte impotence and inducing macrophage apoptosis (Confer and Eaton, 1982; Gueirard et al., 1998; Harvill et al., 1999; Khelef and Guiso, 1995; Khelef et al., 1993). The role of CyaA in the pathogenesis of  B. pertussis  was clearly demonstrated in mouse respiratory model. Indeed, genetically modified  B. pertussis  strains deficient for the expression of CyaA were impaired in their ability to induce pulmonary lesions and to cause lethal infection (Khelef et al., 1994; Weiss and Goodwin, 1989). On the other hand, CyaA was shown to induce protective immunity against  B. pertussis  lung colonization in a mouse model Betsou et al., “CyaC-mediated activation is important not only for toxic but also for protective activities of  Bordetella pertussis  adenylate cyclase hemolysin,”  Infect. Immure.,  (1993) 61:3583-3589; and Betsou et al., “The C-terminal domain is essential for protective activity of the  Bordetella pertussis  adenylate cyclase hemolysin,”  Infect. Immun.  (1995) 63:3309-3315, and Hormozi et al, “Adjuvant and protective properties of native and recombinant  Bordetella pertussis  adenylate cyclase toxin preparations in mice,”  FEMS Immunol Med Microbiol , (1999) 23, 273-282. 
     CyaA is a 1706 amino acid residue-long polypeptide consisting of four functional domains; the adenylate cyclase activity (AC) domain (residues 1 to 400), the hydrophobic channel-forming domain (residues 500 to 700), the calcium-binding glycin/aspartate rich repeat domain (residues 1000 to 1600), and the C-terminal domain harboring a secretion signal (residues 1600 to 1706). CyaA is able to invade eukaryotic cells and translocate its catalytic domain into the cytoplasm where, upon activation by endogenous calmodulin, it catalyzes the conversion of ATP into cAMP (Ladant and Ullmann, 1999). The accumulation of cAMP in the cell cytosol is considered to be responsible for the toxic effect of this toxin (Rogel et al., 1991). The main consequences of this intoxication are cell apoptosis and the alteration of phagocytic abilities and superoxide production (Confer and Eaton, 1982; Friedman et al., 1987; Khelef et al., 1993; Njamkepo et al., 2000; Pearson et al., 1987). 
     The whole sequence of  Bordetella pertussis  adenylate cyclase is shown in SEQ ID NO: 1. 
     The whole sequence of  Bordetella bronchiseptica  adenylate cyclase is shown in SEQ ID NO: 3. 
     CyaA requires calcium to acquire a translocation-specific conformation that allows the delivery of the catalytic domain into the cell cytosol (Rogel and Hanski, 1992; Rose of al., 1995). Primarily, CyaA is produced as an inactive protoxin, proCyaA, which after post-translational modification by an acyltransferase, the product of the cyaC gene, becomes an active toxin. This covalent post-translational fatty-acylation is required for translocation of the toxin through target cell membranes and the delivery of its catalytic AC domain as well as for the formation of hemolytic cation-selective channels. Acylation of proCyaA occurs at two different positions, Lys-983 and Lys-860, which are located within conserved RTX acylation sites (Barry et al., 1991; Hackett et al., 1994). While acylation of Lys-860 seems not to be necessary for CyaA activity, acylation of Lys-983 has been shown to be crucial (Basar of al., 2001). 
     CyaA can penetrate a wide range of cell types, including the mammalian erythrocytes lacking membrane trafficking (Bellalou et al., 1990; Gray et al., 1999; Rogel and Hanski, 1992). In contrast, CyaA toxicity effects such as the abrogation of phagocytic capacity and the induction of apoptosis were mainly elucidated on immune cells, namely neutrophils and macrophages (Confer and Eaton, 1982; Khelef et al., 1993). In addition, in a mouse respiratory infection, CyaA was shown to display specific intoxication towards alveolar macrophages (Gueirard et al., 1998). Vaccine comprising recombinant adenylate cyclase toxin produced by  B. pertussis  fixed to heterologous epitopes is also described in patent WO 93/21324 (Institut Pasteur, 1993). It has been recently demonstrated that CyaA binds specifically to target cells through the α M β 2  integrin (CD11b/CD18). This binding was saturable and completely inhibited by anti-CD11b monoclonal antibodies. CyaA displayed a selective cytotoxicity towards CD11b +  cells showing that its interaction with CD11b is required for the translocation of the catalytic domain and the subsequent cAMP increase and cell death. Moreover, sensitivity of CHO cells to CyaA cytotoxicity was dramatically increased upon expression of the CD11b/CD18 heterodimer. Furthermore, Ca 2+  ions that are required for the catalytic domain translocation into cells were also strictly necessary for CyaA interaction with CD11b (Guermonprez et al., 2001). The importance of CD11b for interaction of CyaA with cells was further demonstrated in a system where CyaA is used as a vector to deliver foreign antigens into antigen presenting cells, such as dendritic cells. Only dendritic cells of the CD11c+CD8α-CD11b high  subset were, indeed, able to display MHC class I peptide complexes corresponding to the epitope inserted in the recombinant CyaA (Guermonprez et al., 2002). 
     The CD11b protein is a member of the large family of β 2  integrins, the leukocyte adhesion molecules, which comprizes LFA1 (CD11a), MAC-1 (CD11b) and p150,95 (CD11c). The members of this family differ by their α-chain which is expressed as an obligate heterodimer with a β chain (CD18) (Amaout, 1990). CD11b, also known as complement type 3 receptor (CR3), is expressed on macrophages, neutrophils, dendritic cells, NK cells, peritoneal B-1 cells and a subset of CD8+ T cells (Arnaout, 1990; Bell et al., 1999). It plays a key role in leukocyte adhesive functions and triggers phagocytosis of complement coated particles (Diamond and Springer, 1993). CD11b binds various ligands, such as the intracellular adhesion molecule ICAM-1, fibrinogen, coagulant factor X and inactivated complement component C3b (iC3b) (Altieri and Edgington, 1988; Beller et al., 1982; Diamond et al., 1990; Wright et al., 1988). 
     Based on the binding properties of CyaA to CD11b/CD18, European patent application EP1188446 (Institut Pasteur) describes proteinaceous vectors comprising recombinant  Bordetella  species adenylate cyclase for targeting a molecule of interest, and especially an antigen to dendritic cells. 
     The present invention is now based on the discovery that one or several regions of the  Bordetella pertussis  adenylate cyclase comprised within the amino acid sequence extending from amino acid 1166 to amino acid 1281 (SEQ ID NO:2) are critical for the interaction of the toxin with CD11b/CD18. This region, necessary to provide binding capacity of CyaA to CD11b/CD18 can further be combined with other regions of CyaA acting as accessory regions. 
     This discovery affords the opportunity to prepare an efficient and versatile molecule delivery vector capable of targeting a molecule of interest to dendritic cells. Alternatively, the deletion of the identified CD11b/CD18 interaction domain of adenylate cyclase can be used advantageously to design a safe a cellular vaccine for the protection against  Bordetella  infection, and especially,  Bordetella pertussis  infection. 
     The invention also provides the use of the identified CD11b/CD18 interaction domain to generate neutralizing antibodies, capable of blocking the interaction of native adenylate cyclase produced by infectious bacteria with cell receptors. 
     It is thus an object of this invention to provide a protein consisting of a  Bordetella  adenylate cyclase which is modified in the CD11b/CD18 interaction domain by one or more amino acid deletion, substitution, or insertion, wherein said protein is deficient for CD11b/CD18 binding but is specifically reactive with antisera recognizing a wild-type  Bordetella  adenylate cyclase. 
     The protein of the invention can be used, as the active principle, in a vaccine against whooping cough. The mutation(s) within the CD11b/CD18 interaction domain thus preserves immune cells from potentially negative effects, such as signalling upon the integrin engagement by the toxoid and/or some functional interference due to competition for binding to CD11 b with the CyaA toxoid, which also serves as the complement receptor CR3. 
     As used herein, the term “polypeptide” refers to a single chain of amino acids linked by peptide bonds, comprising at least 6 amino acids, preferably at least 10 amino acids, and more preferably at least 50 amino acids. 
     The term “protein” refers to a macromolecule which essentially consists of one or more polypeptides. 
     The term “ Bordetella  adenylate cyclase” encompasses, within the present invention, the calmodulin-dependent adenylate cyclase which is naturally synthesized in  Bordetella  species, and which is a major virulence factor mandatory for the initial phases of bacterial colonization in the lung. 
     In one preferred embodiment, the protein of the invention is obtained by modification of the  Bordetella pertussis  adenylate cyclase, the agent of whooping cough in human. 
     In  Bordetella pertussis , the adenylate cyclase is synthesized and secreted in the form of a polypeptide of 1706 amino acids (SEQ ID NO:1): The calmodulin-dependent catalytic activity is localized in the first 400 amino acids, this domain being hereafter referred to as “the N-terminal catalytic domain”. As previously reported, in order to be active, said adenylate cyclase toxin is rendered invasive and hemolytic when post-translationally modified by the coexpression of the cyaC gene product. 
     According to the present invention, the expression “CD11b/CD18 interaction domain” refers either to
     a. the CD11b/CD18 interaction domain of  Bordetella pertussis  extending from amino acid 1166 to amino acid 1281 of  Bordetella pertussis  adenylate cyclase (SEQ ID NO:2), or   b. the domain of the adenylate cyclase of a  Bordetella  species corresponding to the CD11b/CD18 interaction domain of  Bordertella pertussis , as identified by aligning the sequence of the adenylate cyclase of said  Bordetella  species with the sequence of adenylate cyclase of  Bordetella pertussis  using an algorithm for searching best local alignment.   

     An example of an algorithm for searching best local alignment is the BLAST algorithm (Altschul et al., 1990). 
     The CD11b/CD18 interaction domain of  Bordetella bronchiseptica  is represented by SEQ ID NO: 4. 
     As used herein, the expression “deficient for CD11b/CD18 binding” means that the protein of the invention does not compete with the wild-type  Bordetella  adenylate cyclase for binding to CD11b/CD18 α m β 2  expressing cells. The “CD11b/CD18 α m β 2 ” or “CD11b/CD18” refers to the cellular receptor of the  Bordetella  adenylate cyclase (Guermonprez et al., 2001). Examples of binding assays to evaluate specific binding of a recombinant toxin to CD11b/CD18 α m β 2  expressing cells are described in the following experimental part. The protein of the invention preferably has less than 50% of binding affinity to CD11b/CD18 α m β 2  as compared to wild-type  Bordetella  adenylate cyclase. Most preferably, the protein of the invention has less than 10% and more preferably less than 5% of the assayed binding affinity. 
     As used hereafter, the term “CD11b expressing cells” relates to the cells that express the CD11b/CD18 α m β 2  on their surface. In particular, these cells are granulocytes/neutrophils, macrophages, NK cells, subsets of T CD8+ and B cells and myeloid dendritic cells. 
     To provide the protein of the invention, the CD11b/CD18 interaction domain of a  Bordetella  adenylate cyclase is modified by insertion, deletion or substitution of one or more amino acid, the resulting protein being deficient for CD11b/CD18 binding. 
     In one embodiment of the invention, the CD11b/CD18 interaction domain is modified by insertion of a peptide therein. For example, a sequence consisting of between 6 to 12 residues is inserted in the CD11b/CD18 interaction domain. 
     Specific embodiments include  Bordetella pertussis  adenylate cyclase modified by insertion between residues 1166 and 1167 or between residues 1281 and 1282 (the number indicates the position of the amino acids in the wild type  Bordetella pertussis  adenylate cyclase), of a peptide containing between 6 to 12 amino acids. Examples of epitope insertions of the FLAG sequence at these positions are described in the following Experimental Part, hereafter referred to as CyaA1166/FLAG and CyaA1281/FLAG. 
     Alternatively, the residues which are shown to be involved in the binding to CD11b/CD18 can be deleted or replaced by non-functional residues. 
     In one specific embodiment, the  Bordetella  adenylate cyclase is modified by insertion, deletion or substitution of one or more amino acid in the region extending from residue 1208 to 1243 in  Bordetella pertussis  adenylate cyclase or in corresponding regions of other  Bordetella  adenylate cyclases. 
     Preferred embodiments of the protein of the invention include a  Bordetella pertussis  adenylate cyclase containing deletions of one or more of the amino acids or their replacement by non-functional amino acids. 
     In one preferred embodiment, the  Bordetella  adenylate cyclase is modified by the complete deletion of the CD11b/CD18 interaction domain. 
     According to another specific embodiment of the invention, the  Bordetella pertussis  adenylate cyclase is modified by deletion of the amino acids extending from position 1245 to position 1273, these amino acids being optionally replaced by non functional amino acids, for example an octapeptide as exemplified in the Experimental Part, hereafter referred to as the CyaAΔ1245-1273. 
     Additionally, to ensure complete safety of the administration in living organism of the protein of the invention, the  Bordetella  adenylate cyclase is modified such that the catalytic activity is ablated. According to one embodiment of the invention, the  Bordetella  adenylate cyclase is further modified by insertion, deletion or substitution of one or more amino acids in the N-terminal catalytic domain, wherein said modified  Bordetella  adenylate cyclase has a catalytic activity which is decreased as compared to the wild-type  Bordetella  adenylate cyclase catalytic activity. Preferably, the catalytic activity represents less than 10% of the catalytic activity of the wild-type  Bordetella  adenylate cyclase and is more preferably non significant. 
     Examples of mutants in the N-terminal catalytic domain are described in the Art (for example in WO 93/21324, Institut Pasteur). 
     Embodiments of the protein of the invention include modified  Bordetella  species adenylate cyclase lacking at least the amino acids 1 to 300 of the N-terminal catalytic domain and preferably lacking amino acids 1 to 373. 
     Alternatively, dipeptide insertions can be done into the ATP-binding site between residues 188 and 190 of adenylate cyclase of  Bordetella pertussis , or the corresponding residues in adenylate cyclase from other  Bordetella  species. 
     It is also shown in the present invention that acylation of the  Bordetella  adenylate cyclase is involved in CD11b/CD18 binding and subsequent translocation of the toxin into the cell. Accordingly, in one preferred embodiment of the protein of the invention, the protein is not acylated. Especially, the  Bordetella  adenylate cyclase is further modified in the amino acids which are acylated post-translationally. These amino acids correspond to Lys-983 and Lys-860 of the  Bordetella pertussis  adenylate cyclase. 
     In this particular embodiment, the protein is not acylated in position 983 and/or 860 of the adenylate cyclase sequence. 
     In another embodiment, the protein of the invention is acylated. 
     The protein of the invention is preferably immunogenic, yet substantially non toxic protein, i.e. a protein that is at least deficient for cell receptor binding, and optionally in adenylate cyclase activity, but which is still specifically recognized by anti-adenylate cyclase toxin antibodies. 
     The invention also relates to the pharmaceutical composition comprising the protein defined above, in combination with a pharmaceutically acceptable vehicle. 
     According to one embodiment, said composition is a vaccine suitable for administration in a human or an animal. The vaccine is preferably capable of inducing immunity against whooping cough. Such vaccine comprises an immunoprotective and non-toxic amount of the protein of the invention. Said composition may further comprise one or several suitable priming adjuvants accordingly. Other antigens which are known to be desirably administered in conjugation with the protein of the invention may also be included in the vaccine of the invention. Such additional components include other known protective antigens of  Bordetella , tetanus toxoid and/or diphteria toxoid. 
     Naturally, the invention further relates to a method for immunizing a human or an animal against  Bordetella  infection and/or symptoms associated to disease caused by  Bordetella  infection, which comprises administering the vaccine of the subject invention to such human or animal. 
     The route of administration of the vaccine of the invention may be any suitable route which delivers an immunoprotective amount of the protein of the invention to the host. However, the vaccine is preferably administered parenterally via the intramuscular or subcutaneous routes. Other routes of administration may also be employed, where desired, such as oral administration or via other parenteral routes, i.e., intradermally, intranasally or intravenously. 
     Another aspect of the present invention relates to the use of the protein of the invention, in the preparation of a medicament for the treatment, in human or in an animal, of disease symptoms associated with whooping cough and/or for protecting a human or an animal against the disease symptoms associated with  Bordetella  infection. 
     Naturally, the invention further relates to a method for treating a human or an animal against  Bordetella  infection and/or symptoms associated to disease caused by  Bordetella  infection, which comprises administering the medicament of the subject invention to such human or animal. 
     Another aspect of the invention is a polypeptide capable of binding to CD11b/CD18 integrin, said polypeptide being either
     a. a fragment of a  Bordetella  adenylate cyclase having between 30 to 500 amino acids, preferably between 50 to 300, and more preferably between 50 to 150 amino acids, said fragment comprising the CD11b/CD18 interaction domain of said  Bordetella  adenylate cyclase, or comprising a fragment of said wild type CD11b/CD18 interaction domain sufficient to retain the capacity to bind to CD11b/CD18, or,   b. a variant of said fragment having at least 70% identity preferably at least 80% identity and more preferably at least 90% identity with said fragment, wherein said variant retains the capability to bind to CD11b/CD18.   

     The  Bordetella  adenylate cyclase is preferably selected among  Bordetella pertussis, Bordetella parapertussis  and  Bordetella bronchiseptica , and more preferably  Bordetella pertussis.    
     The polypeptides of the invention will be selected among those which adopt an appropriate conformation to bind to the CD11b/CD18. 
     In specific embodiments, the polypeptides of the invention may comprise other accessory regions of the  Bordetella  adenylate cyclase, which are involved in optimal binding to CD11b/CD18. The regions include more specifically, amino acid sequences comprised in the region extending from 1416 to 1648. 
     In one preferred embodiment, the polypeptide of the invention is a variant as defined above in b., consisting of one or more fragments from 10 to 50 amino acids of the CD11b/CD18 interaction domain. For example, in one preferred embodiment, said polypeptide comprises at least fragments from 10 to 50 amino acids of the region of  B. pertussis  adenylate extending from amino acid 1208 to amino acid 1243 of  B. pertussis  adenylate cyclase. 
     Percentage identity corresponds to the percentage of amino acids of the variant sequence which are identical to the wild-type sequence when both sequences are aligned using the BLAST algorithm. The expression “retains the capacity to bind to CD11b/CD18” means that the variant retains at least 80% of the binding affinity to CD11b/CD18 as compared to the wild-type corresponding fragment from which it can be aligned, and preferably, at least 90% of the binding affinity to CD11b/CD18. 
     According to one preferred embodiment, said polypeptide is specifically reactive with antisera recognizing  Bordetella  wild-type adenylate cyclase, preferably  Bordetella pertussis  adenylate cyclase. More preferably, said polypeptide is capable, when administered to a mammal, of raising antibodies recognizing specifically  Bordetella  adenylate cyclase. 
     In one specific embodiment, said polypeptide is a fragment of the  Bordetella pertussis  adenylate cyclase. In another specific embodiment, is said polypeptide essentially consists of the CD11b/CD18 interaction domain, and more specifically to CD11b/CD18 interaction domain of  B. pertussis , extending from amino acid 1166 to amino acid 1281 of  B. pertussis  adenylate cyclase (SEQ ID NO:2). 
     In other specific embodiments, said polypeptide further comprises an acylation domain of the  Bordetella  adenylate cyclase and/or the hydrophobic domain. Said acylation domains are included in the corresponding regions extending from residue 700 to residue 1000 of SEQ ID NO: 1, as described in WO 93/21324 and comprise Lys 983 and/or Lys 860. The hydrophobic domain corresponds to the region extending from residue 500 to residue 700 of SEQ ID NO: 1. 
     Preferably, said polypeptide is not toxic when administered in vivo to a mammal. 
     The polypeptides of the invention compete for the binding of the CD11b/CD18 integrin with wild-type adenylate cyclase. 
     The invention thus relates to the use of the polypeptide as defined above, in the preparation of a vaccine or a medicament for the prevention or treatment, in human or in an animal, of disease symptoms associated with whooping cough and/or for protecting a human or an animal against the disease associated with  Bordetella  infection. 
     More specifically, the invention concerns the use of said polypeptide of the invention to generate protective antibodies against  Bordetella  infection. 
     It has already been reported that adenylate cyclase is an efficient molecule delivery vector capable of targeting different antigens to dendritic cells leading especially to the generation of potent CD4+ as well as CD8+ T cell responses (EP1188446, Institut Pasteur). 
     The present invention now relates to the use of the polypeptides of the invention, in the preparation of a vector for targeting a molecule of interest, specifically to CD11b expressing cells. 
     The term “specifically” means within the context of the present invention that the polypeptide when used as a vector for a molecule of interest, is directed preferentially to CD11b expressing cells according to the high binding affinity of the CD11b/CD18 interaction domain with the CD11b/CD18, thereby offering means to target the molecule of interest at the surface of said cells or within said cells in a selective way with respect to other cells. 
     In particular, in one embodiment, the targeting of said molecule or peptide is effective in vivo. In other embodiments, the targeting of said molecule is effective in vitro or ex vivo. By “in vitro”, it is meant that the target cells are cells, which are cultured in vitro. By “ex vivo”, it is meant that the target cells are cells, which have been extracted from a living organism, are cultured in vitro and are intended to be readministered in a living organism. 
     The invention thereby provides means appropriate for the design of compositions suitable for administration to animal or human hosts requiring targeting of certain leukocytes and in particular myeloid dendritic cells, neutrophils or macrophages. 
     The invention more specifically relates to a vector for targeting a molecule of interest to CD11b expressing cells, characterized in that said vector comprises the polypeptide capable of binding to CD11b/CD18, as defined above, coupled to said molecule of interest. 
     The invention also relates to a method for in vitro targeting a molecule of interest to CD11b expressing cells, said method comprising:
     a. providing CD11b expressing cells extracted from a living organism,   b. culturing said CD11b expressing cells with the vector of the invention under appropriate conditions for targeting said vector to said CD11b expressing cells.   

     The invention also provides CD11b-expressing cells comprising a molecule of interest as obtainable by the above-defined method. 
     According to the present invention, the expression “molecule of interest” refers to any molecule, preferably a molecule which is not a fragment of a  Bordetella  species adenylate cyclase. 
     The molecules of interest can also be selected among the nucleic acids, such as DNA, RNA, oligonucleotides, antisense DNA, plasmids and cosmids. They can also be selected among the peptides or polypeptides, and especially, the enzymes, co-enzymes, receptor ligands, haptens, antigens, antibodies and fragments thereof. Naturally, the person skilled in the Art will select the appropriate molecule depending upon the desired use. 
     Molecules of interest can be selected among the active principle of the medicament, the immunotoxins, the antioxidants, the antibiotics, the growth factors, the intracellular hormones, the cytokines, the toxins, the neuromediators, the antimicrobial agents, especially, antiviral, antibacterial, antiparasital or antitumoral and more generally, any therapeutical or prophylactic agent of interest. 
     According to one specific embodiment, a molecule of interest is selected among the group consisting of: peptides, glycopeptides, lipopeptides, polysaccharides, oligosaccharides, nucleic acids, lipids and chemicals. 
     In specific embodiments, a molecule of interest is a heterologous antigen or epitope, the term “heterologous” referring to an antigen or epitope other than the adenylate cyclase antigenic determinant comprised in the vector itself. 
     The molecule of interest is coupled to the polypeptide of the invention to provide the vector of the invention. 
     As used herein, the term “coupled” means any interaction allowing physical association of the molecule of interest and the polypeptide. Preferably, the coupling is covalent. It can be direct covalent coupling or indirect coupling by the use of a linkage agent to form a conjugate. Chemical linkage methods are well known in the Art. Chemical linkage can be selected for example among maleimide, peptidic, disulfide or thioether linkage. For example, disulfide linkage using N-pyridyl sulfonyl-activated sulfhydryl can be used. 
     One specific method consists in adding a linker to the polypeptide, said linker consisting of at least one cysteine which can be easily used for disulfide linkage. Another approach consists of coupling chemically a biotinyl moiety, which enables the coupling of other molecules associated to streptavidin. 
     Multiple molecules can be chemically coupled to the polypeptide of the invention by means of a disulfide bond to different cysteine residues, provided that the coupling does not prevent interaction with the CD11b/CD18. 
     The functional properties of the CD11b expressing cells define furthermore a use of said polypeptides of the invention in the manufacturing of a proteinaceous vector for drug targeting to these specific cells. In this context, in one specific embodiment of the invention, the so-called molecule of interest is an active principle of a medicament. Said active principle may be chemically or genetically coupled to the polypeptide of the invention. Advantageously, a molecule of interest is an anti-inflammatory drug which is, when coupled to the adenylate cyclase toxin, specifically targeted to the surface of the cells involved of the inflammatory response, such as neutrophils. 
     Since CD11b expressing cells and more specifically the myeloid dendritic cells, the neutrophils and the macrophages are involved in essential functions of the immune and innate defence system, in particular in inflammatory and specific immune responses, in a preferred embodiment of the invention, the vector of the invention is more specifically designed to prime CD4+ and CD8+ cells response, said response following the targeting of the molecule of interest to CD11b expressing cells, in particular myeloid dendritic cells. 
     In this context, the molecule of interest is or comprises preferably an epitope or an antigen. More specifically, the molecule of interest can be especially an antigen selected from the group consisting of: a poliovirus antigen, an HIV virus antigen, an influenza virus antigen, a lymphocytic choromeningitidis virus, eptitope, a human papillomavirus (HPV) antigen, a bacterial antigen, a  mycobacterium tuberculosis  antigen for instance. 
     The invention thus provides means to prime CD4+ and CD8+ cells response in a patient, either by in vivo targeting antigen or epitope to CD11b expressing cells or by ex vivo targeting antigen or epitope to extracted CD11b expressing cells and re-administering the resulting cells to said patient. 
     Accordingly, the invention relates to a method for in vitro targeting an antigen or an epitope to CD11b expressing cells, said method comprising
     a. providing CD11b expressing cells extracted from a living organism, and,   b. culturing said CD11b expressing cells with the vector of the invention carrying an antigen or an epitope as a molecule of interest under appropriate conditions for targeting the vector to said CD11b expressing cells.   

     Preferably, CD11b-expressing cells extracted from a living organism are myeloid dendritic cells. 
     The invention also provides CD11b-expressing cells comprising a heterologous antigen or epitope obtainable by the above-defined method. 
     The invention thus relates to a cell therapy product for immunizing a human or an animal against an antigen, characterized in that it comprises an efficient amount of CD11b expressing cells comprising a heterologous antigen or epitope obtainable by the above-defined method, in combination with a pharmaceutically acceptable vehicle. 
     The invention further relates to a use of CD11b-expressing cells comprising said antigen or epitope as obtainable by the above-defined method, in the preparation of a cell therapy product for immunizing a human or an animal against an antigen. 
     More specifically, the invention provides a method for immunizing a patient against an antigen, said method comprising:
     a. extracting CD11b expressing cells from said patient,   b. in vitro culturing said CD11b expressing cells with a vector of the invention carrying an antigen or an epitope as a molecule of interest, under conditions appropriate for targeting said vector to said cells,   c. re-administering an efficient amount of said cells comprising said vector to said patient to prime a CD4+ and/or CD8+ response,
 
thereby immunizing said patient to said antigen.
   

     According to a preferred embodiment of the invention, said CD11b-expressing cells are myeloid dendritic cells. 
     The invention thus also relates to the pharmaceutical composition comprising the vector of the invention carrying an epitope or an antigen as the molecule of interest, in combination with a pharmaceutically acceptable vehicle. 
     According to one embodiment, said composition is a vaccine suitable for administration in a human or an animal. Preferably, the vaccine is capable of inducing immunity against poliovirus, HIV or a lymphocytic choromeningitidis virus. Of course, the type of immunity induced will depend upon the selected antigen which is carried by the vector. In another embodiment, the vaccine is capable of inducing immunity against whooping cough. 
     Such vaccines comprise an immunoprotective and non-toxic amount of the vector of the invention. Said composition may further comprise suitable priming adjuvants accordingly. 
     The invention further relates to a method for immunizing a human or an animal against a pathogen infection, which comprises administering the vaccine comprising an immunoprotective and non toxic amount of the vector of the subject invention to such human or animal. 
     The invention is also directed to the means for preparing the polypeptides, proteins or the vector of the invention. Especially, those means comprise a nucleic acid encoding one of the following polypeptides:
     a. the protein of the invention which is deficient for CD11b/CD18 binding;   b. the polypeptide of the invention which is capable of binding to the CD11b/CD18 integrin; or   c. the vector for targeting a molecule of interest to CD11b expressing cells.   

     Especially, the nucleic acid of the invention can be derived from the DNA encoding wild-type adenylate cyclase of any  Bordetella  strain using known techniques, e.g., isolating the gene from a gene bank, making complementary or cDNAs from mRNA templates or via the polymerase chain reaction or from isolates of clinical species. Alternatively, the DNA encoding wild-type adenylate cyclase may be synthesized by standard DNA synthesis technique. Various  Bordetella  strains are publicly available from commercial depositories. 
     Modifications of the wild-type DNA encoding  Bordetella  adenylate cyclase can be obtained by genetic engineering of the wild-type DNA using conventional molecular biology technologies. 
     Another object of the invention concerns a recombinant nucleic acid constituted by the nucleic acid encoding the polypeptide, the protein or the vector of the invention, cloned into an expression vector appropriate for the expression of the encoded polypeptide or protein in a host cell. Optionally, the recombinant DNA molecule comprise additional coding sequence of a carrier polypeptide which has immunostimulating properties, such as an adjuvant, or which is useful in expressing, purifying and/or formulating the polypeptides of the invention. This coding sequence can be placed in frame with the coding sequence of the polypeptide, protein or vector for targeting molecule of the invention. 
     The selection of the expression vector will, of course, depend upon the host cell employed. 
     Preferably, said expression vector is a plasmid, a cosmid, a phagemid or a viral DNA. 
     The invention is also directed to a method for preparing the protein of the invention deficient for CD11b/CD18 binding; the polypeptide capable of binding CD11b/CD18 as defined above; or the vector for targeting a molecule of interest to CD11b expressing cells, said method comprising the steps of incorporating the recombinant nucleic acid as defined above in an appropriate host cell for the expression of the corresponding polypeptide, protein or vector of interest; culturing the transformed recombinant cells and recovering the synthesized recombinant polypeptide, protein or vector of the invention. 
     Another aspect of the invention is a host cell transformed with the recombinant nucleic acid of this invention and thus comprising the nucleic acid or the recombinant nucleic acid as defined above. In one embodiment, the recombinant nucleic acid can be integrated into the host cell&#39;s genome by conventional techniques, including homologous recombination. 
     Preferred host cells of the invention include those belonging to the species  E. coli  and the genus  Bordetella . Other host cells which may be suitable include, but are not limited to, mammalian cells, insect cells, yeast and other bacterial cells. 
     The invention also encompasses the polyclonal serum obtainable by the immunization of an animal or a human with the polypeptide, the protein, the vector or with the composition of the invention. 
     In one preferred embodiment, the polyclonal serum is obtainable by the immunization of an animal or a human with the polypeptide consisting of the CD11b/CD18 interaction domain of  Bordetella  adenylate cyclase, preferably the CD11b/CD18 interaction domain of  Bordetella pertussis  adenylate cyclase, extending from amino acid 1166 to amino acid 1281. 
     The invention also relates to monoclonal antibody directed specifically against the polypeptides of the invention comprising the CD11b/CD18 interaction domain. 
     In one preferred embodiment, the monoclonal antibody is directed against an epitope located in the CD11b/CD18 interaction domain, preferably against an epitope located in the CD11b/CD18 interaction domain of  Bordetella pertussis  adenylate cyclase, extending from amino acid 1166 to amino acid 1281. 
     Preferably, said polyclonal serum, or monoclonal antibody is capable of blocking the binding of wild-type adenylate cyclase to CD11b/CD18. The blocking can be assayed by evaluating the capacity of a mixture of said polyclonal serum or monoclonal antibody with a wild-type adenylate cyclase to bind to CD11b/CD18 as compared to the capacity of wild-type adenylate cyclase alone. 
     In one specific embodiment, said medicament provides passive immunization against  Bordetella  infection. 
     For use in human organism, the antibodies of the invention can be humanized for instance by the replacement of the hypervariable part of a human immunoglobulin, which has no antibody function, by a hypervariable region of a monoclonal immunoglobulin obtained from the technique described above. 
     For example, techniques for humanizing antibodies were 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 invention also concerns a pharmaceutical composition, comprising the polyclonal serum or the monoclonal serum, in combination with a pharmaceutically acceptable vehicle. 
     The invention also relates to the use of a polyclonal serum or a monoclonal to antibody of the invention, in the preparation of a medicament for the treatment, in human or in animal, of disease symptoms associated with whooping cough and/or for protecting a human or an animal against the disease symptoms associated with  Bordetella  infection. 
     The following experimental part shows the results identifying (i) the role of post-translational acylation in CyaA interaction with CD11b and (ii) the CD11b interaction domain in  Bordetella pertussis  adenylate cyclase. 
    
    
     
       LEGENDS TO THE FIGURE 
         FIG. 1 . CyaA Binds Specifically to CD11b Cells and Inhibits both CyaA-Biotin and Anti-CD11b Monoclonal Antibody Binding to these Cells 
       (A) CHO cells or CHO-CD11b cells were incubated with the indicated concentrations of CyaA. Surface-bound CyaA was detected by FACS, with anti-CyaA Mab (5G12). Results are expressed as ΔMFI=(MFI value of cells incubated with CyaA)—(MFI value of cells incubated without CyaA) and are representative of at least 2 independent experiments. 
       (B) CHO-CD11b cells were preincubated with the indicated concentrations of CyaA. Then, CyaA-biotin (30 nM) or anti-CD11b Mab (2 μg/ml) was added separately in the continuous presence of the toxin and their binding was measured by FACS. 
       (C) After preincubation with the indicated concentrations of CyaA, CHO-CD11b cells or CHO-CD11c cells were incubated with either anti-CD11b or anti-CD11c monoclonal antibody, respectively, in the continuous presence of the toxin. Then, antibody binding was determined by FACS. 
       For (B) and (C), results are expressed as percentage of binding=(sample binding)/(maximum binding)×100 and are representative of at least 2 independent experiments. 
         FIG. 2 . Direct Binding of CyaA or ProCyaA to CHO Tranfectants. 
       CHO-CD11b cells (A) or CHO cells (B) were incubated with the indicated concentrations of CyaA or proCyaA. Surface-bound CyaA was detected with anti-CyaA Mab (5G12). Results are expressed as ΔMFI=(MFI value of cells incubated with CyaA)—(MFI value of cells incubated without CyaA) and are representative of 2 independent experiments. 
         FIG. 3 . CyaA Acylation is Required for Stable Association with CHO-CD11b Cells 
       CHO-CD11b cells were preincubated with the indicated concentrations of CyaA or proCyaA. CyaA-biotin (A) or anti-CD11b Mab (B) was then added, in the continuous presence of CyaA or proCyaA. Surface bound CyaA-biotin or anti-CD11b Mab was measured by FACS. Results are expressed as percentage of binding=(sample binding)/(maximum binding)×100 and are representative of at least 2 independent experiments. 
         FIG. 4 . CyaA Acylation is Required for CyaA Induced-cAMP Accumulation and Cytotoxicity 
       CHO-CD11b cells were incubated with either CyaA or proCyaA at the indicated concentrations for 20 min at 37° C. Then, cells were lysed and cAMP was measured (A). In parallel, toxicity was determined by measuring the amount of lactate dehydrogenase released in the medium after incubation of CHO-CD11b cells for 4 hours at 37° C. in the presence of the indicated concentrations of either CyaA or proCyaA (B). Results are representative of at least 2 independent experiments. 
         FIG. 5 . The Catalytic Domain is not Required for CyaA Interaction with CD11b Cells 
       CHO-CD11b cells were preincubated with the indicated concentrations of CyaA, CyaA 1-384 or CyaA 373-1706. Cells were then incubated with either CyaA-biotin (A) or anti-CD11b Mab (B). Binding of CyaA-biotin and anti-CD11b Mab was measured by FACS. Results are expressed as percentage of binding=(sample binding)/(maximum binding)×100 and are representative of at least 2 independent experiments. 
         FIG. 6 . Direct Binding of CyaA Fragments to CD11b Cells 
       CHO-CD11b cells (A, C) or CHO cells (B, D) were incubated with the indicated concentrations of CyaA, CyaA 1-384 and CyaA 373-1706. Then, surface—bound CyaA was detected with anti-CyaA 5G12 Mab that recognizes the catalytic domain (A, B) or with anti-CyaA 6D7 Mab that recognizes the repeat domain (C, D). Results are expressed as ΔMFI=(MFI value of cells incubated with CyaA or CyaA fragments)−(MFI value of cells incubated without CyaA or CyaA fragments) and are representative of at least 2 independent experiments. 
         FIG. 7 . CyaA-Biotin Binding to CHO-CD11b Cells in the Presence of CyaA-FLAG Mutants 
       CHO-CD11b cells were preincubated with CyaA or CyaA FLAG mutants (30 nM). Then, CyaA-biotin was added, in the continuous presence of CyaA or CyaA-FLAG molecules. Surface bound CyaA-biotin was detected by FACS with streptavidin-PE. Results are expressed as percentage of binding=(sample binding)/(maximum binding)×100 and are representative of at least 2 independent experiments. 
         FIG. 8 . SDS-Page Analysis of the Purified CyaA Preparations and Their Invasive Activity on Erythrocytes 
       (A) CyaA/FLAG molecules together with the wild type CyaA were purified from urea extracts by DEAE- and Phenyl sepharose chromatographies as previously described (Karimova et al., 1998). About 3 μg of each protein was analyzed on a 7.5% acrylamide gel stained with Coomassie blue. (B) Invasive activity of CyaA/FLAG molecules on sheep erythrocytes. Two micrograms of various CyaA proteins were incubated with 5×10 8  washed sheep erythrocytes for 30 minutes and the amount of AC activity translocated into the cells was determined as previously described (Osicka et al., 2000). The values represent the average from three experiments performed in duplicates (n=6). 
         FIG. 9 . CyaA Binding to CHO-CD11b Cells in the Presence of Selected CyaA-FLAG Mutants 
       CHO-CD11b cells were preincubated with CyaA or CyaA FLAG mutants at various concentrations ranging from 7.5 nM to 240 nM. CyaA-biotin was added, in the continuous presence of CyaA molecules and surface bound CyaA-biotin was revealed. Results are expressed as percentage of binding=(sample binding)/(maximum binding)×100 and are representative of at least 2 independent experiments. 
     
    
    
     EXPERIMENTAL PART 
     A. Material and Methods 
     A.1 Production, Purification and Modification of the CyaA-Derived Proteins 
     DNA manipulations were performed according to standard protocols (Sambrook et al., 1989) in the  Escherichia coli  strain XL1-Blue (Stratagene, Amsterdam, Netherlands) as host cells. The plasmids coding for a non-acylated wild type proCyaA (pACT7), acylated wild type CyaA (pT7CACT1) and a recombinant detoxified CyaA-E5-CysOVA harbouring a unique cysteine residue and the OVA epitope in its catalytic domain (pCACT-E5-CysOva) were already described (Gmira et al., 2001; Guermonprez et al., 2001; Osicka et al., 2000; Sebo et al., 1991). The plasmid encoding CyaA 373-1706 (pTRCyaAΔ1-373) is a derivative of pTRCAG (Gmira et al., 2001) in which the DNA sequence coding for the catalytic domain of the toxin (comprised between the NdeI and BstBI sites) was deleted and replaced by an appropriate synthetic double stranded oligonucleotide encoding the amino acid sequence: Met-Gly-Cys-Gly-Asn. 
     Protocol for CyaA production has already been described elsewhere (Karimova et al., 1998). All proteins were expressed in  E. coli  BLR strain (Novagen, Merck KG, Darmstadt, Germany), and were purified to more than 95% homogeneity (as judged by SDS-gel analysis) from inclusion bodies by a two-step procedure including DEAE-Sepharose and Phenyl-sepharose chromatographies as described in Guermonprez et al., 2000. Purified CyaA-E5-CysOVA protein was labeled on its unique cysteine residue with the sulfhydryl reagent N-(6-(Biotinamido)hexyl)-3′-(2′-pyridyldithio) propiamide (Biotin-HPDP) (Pierce, Bezons, France) according to the manufacturer&#39;s instructions. The biotinylated-CyaA was re-purified on DEAE-Sepharose in order to remove the unreacted Biotin-HPDP reagent. CyaA-1-384 was expressed and purified as described in Ladant et al., 1992. 
     Toxin concentrations were determined spectrophotometrically from the adsorption at 278 nm using a molecular extinction coefficient of 141 mM −1  cm −1  for the full length CyaA toxins, 113 mM 1  cm −1  for the CyaA 373-1706 and 28 mM −1  cm −1  for CyaA 1-384. 
     The CyaA-FLAG molecules were constructed using the previously defined permissive insertion sites along the CyaA molecule (Osicka et al., 2000). We generated a set of 17 CyaA constructs, which carried at the individual permissive positions a synthetic octapeptide insert Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys for the FLAG epitope (Sigma, Saint Quentin Fallavier, France). To achieve this, three double stranded synthetic oligonucleotide pairs (5′-GTACTGATTATAAAGATGACGATGACAAATCAC+5′-GTACGTGATTTGTCAT CGTCATCTTTATAATCA, 5′-GTACTTATCGATTATAAAGATGACGATGACAAA 5′-GTACTTTGTCATCGTCATCTTTATAATCGATAA and 5′-GTACGTGGATTATAAA GATGACGATGACAAAGC 5′-GTACGCTTTGTCATCGTCATCTTTATAATCCAC, respectively) (SEQ ID NOs: 5 to 10), encoding the FLAG epitope in the required reading frames, were inserted into the unique BsrG I sites previously introduced within the cyaA (Osicka et al., 2000). Correct insertions were checked by DNA sequencing, the recombinant CyaA molecules were expressed in  E. coli  and purified. The invasive capacity of selected CyaA/FLAG molecules were characterized, using sheep erythrocytes as target cells as previously described (Osicka et al., 2000). 
     A.2 Production of Anti-CyaA Monoclonal Antibodies 
     BALB/c mice were initially immunized intraperitoneally with CyaA toxin (20 μg in alum). At approximately two weeks interval, mice were boosted with 10 μg CyaA in alum for 3 times. Throughout the immunization protocol, mice were bled and their sera tested for the presence of anti-CyaA antibodies by ELISA. When significant sera titers were detected, a last boost was given to these mice and their splenocytes were fused with P3X63 myeloma cells (ATCC, Manassas, USA) 3 days later. The generated hybridomas were screened for the production of CyaA specific monoclonal antibodies by ELISA. Highly productive hybridomas were then selected and cloned by single-cell limiting dilutions and subsequently used to make ascites in BALB/c nude mice to generate large amounts of anti-CyaA monoclonal antibodies. The monoclonal antibodies were purified from ascites using T-GelTM purification kit (Pierce, Bezons, France) according to manufacturer instructions. The antibody concentration was measured with Bio-Rad protein assay (Bio-Rad, Marnes la Coquette, France). Two of these monoclonal antibodies were used in this study: antibody 5G12 that reacts with an epitope localized within amino acid 1 to 190, and antibody 6D7 that reacts with an epitope localized within amino acids 1006 to 1706. 
     A.3 Cells and Culture 
     Chinese Hamster Ovary cells transfected with human CD11b/CD18 (CHO-CD11b cells), human CD11c/CD18 (CHO-CD11c cells) or transfected with the vector alone (CHO cells) were a kind gift of D. Golenbock (Boston University School of Medicine, Boston, Mass.) and were cultured in the presence of neomycin as described previously (Ingalls et al., 1998). 
     A.4 Antibodies 
     Monoclonal antibodies specific for human CD11b (ICRF44, mouse IgG1, κ) and human CD11c (B-Ly6, mouse IgG1, κ) were purchased from BD Pharmingen (Le Pont de Claix, France). 
     A.5 Binding Assays 
     The assays were performed as described in Guermonprez et al., 2001. Briefly, 2×10 5  cells were incubated with the indicated concentrations of CyaA molecules in DMEM medium containing 4.5 mg/ml glucose (Life Technologies, Cergy Pontoise, France), without serum, in 96-well culture plates for 30 min on ice. After washing, anti-CyaA catalytic domain Mab (5G12) or anti-CyaA repeat domain Mab (6D7) was added at 25 μg/ml. In some experiments, cells were preincubated with the indicated concentrations of CyaA molecules for 30 min on ice. Then, CyaA-biotin (30 nM), anti-CD11b Mab (2 μg/ml) or anti-CD11c Mab (2 μg/ml) (BD Pharmingen) were added separately in the continuous presence of the toxins. 
     After washing and removing supernatant, cells were stained with goat anti-mouse IgG-PE (Caltag, Le Perray en Yvelines, France) or with streptavidin-PE (BD Pharmingen) at 1:300 dilution. After the last wash, cells were analyzed by flow cytometry on a FACStar™ (Becton Dickinson, Le Pont de Claix, France) in the presence of 5 μg/ml propidium iodide. Aggregated and dead cells were substracted by gatings based on propidium idiode exclusion. The binding data were deduced from the mean fluorescence intensity (MFI) and expressed as ΔMFI=(MFI value of cells incubated with CyaA)−(MFI value of cells incubated without CyaA) or as percentage of binding=(sample binding)/(maximum binding)×100. The maximum binding corresponds to (MFI value of cells incubated with CyaA or anti-CD11b in the absence of competitor)−(MFI value of cells incubated with medium alone). The sample binding corresponds to (MFI value of cells incubated with CyaA or anti-CD11b in the presence of competitor)−(MFI value of cells incubated with medium alone). 
     A.6 cAMP Assay 
     Cyclic AMP accumulated in cells exposed to the CyaA toxin was performed essentially as described in Guermonprez et al., 2001. Briefly, 5×10 5  cells were incubated with the indicated concentrations of CyaA in DMEM+glucose for 20 min at 37° C. After washing, cAMP accumulated in cell cytosol was released by lysis with 0.1 N HCl and boiling for 5 min at 120° C. After neutralization with 0.1 N NaOH, the samples were then added to microtiter plates previously coated with a cAMP-BSA conjugate and, then incubated with an appropriate dilution of anti-cAMP rabbit antiserum. After washing, anti-cAMP antibodies were revealed with anti-rabbit antibodies coupled to alkaline phosphatase. The cAMP content of each sample was determined from comparison with a standard curve obtained by adding known cAMP concentration. 
     A.7 CyaA Invasive Activity 
     Invasive activity of CyaA molecules was determined as described previously in Osicka et al., 2000. Briefly, sheep erythrocytes were incubated with toxin for 30 min and the invasive activity was measured as the AC activity translocated into erythrocytes and protected against digestion by extracellularly added trypsin. 
     B. Results 
     B.1 CyaA Specifically Binds to CD11b +  Cells and Inhibits CyaA-biotin and Anti-CD11b Binding to CD11b +  Cells 
     In order to investigate the role of biological and structural properties of CyaA in its interaction with CD11b, two complementary assays were developed; a binding assay and a competition assay. The binding assay consisted in incubation of CyaA molecules with transfected CHO cells expressing human CD11b/CD18 (CHO-CD11b cells), or with mock transfected CHO cells and subsequent detection of the cell-associated toxin with an anti-CyaA monoclonal antibody (5G12) specific for the catalytic domain. As shown in  FIG. 1A , using this assay, CyaA binding was specifically detected on CD11b +  cells. In the competition assay, different CyaA molecules (mutants or fragments) can be tested for their ability to compete with CyaA-biotin, or anti-CD11b monoclonal antibody (Mab) binding to CD11b +  cells. Here, the CHO-CD11b cells were incubated with CyaA at different concentrations for 30 minutes on ice. Then, in the continuous presence of CyaA, CyaA-biotin (30 nM) or anti-CD11b Mab (2 μg/ml) were added and their binding to the cells was evaluated by FACS. As shown in  FIG. 1B , CyaA efficiently inhibited both CyaA-biotin and anti-CD11b binding to CHO-CD11b cells in a dose dependent manner. This inhibitory effect was specific for CD11b since CyaA was completely unable to compete with another ligand (anti-CD11c Mab) for its specific receptor (CD11c) expressed by CHO cells ( FIG. 1C ). 
     B.2 Lack of CyaA Acylation Affects its Binding to CD11b + Cells 
     Since it is well established that CyaA needs a postranslational palmitoylation to perform its invasive activity and to form hemolytic membrane channels, we tested whether the lack of acylation affects CyaA interaction with CD11b +  cells. In a binding assay, CHO cells or CHO-CD11b cells were incubated with either CyaA or non acylated proCyaA. The binding was evaluated using anti-CyaA catalytic domain Mab (5G12). As shown in  FIG. 2A , at low concentrations, binding of both CyaA and proCyaA molecules to CD11b +  cells was rather comparable, with a slightly more efficient binding of the acylated CyaA. This could be due to its enhanced interaction with cell membrane, a better adapted conformation of CyaA for binding and/or higher affinity of CyaA for the CD11b receptor. Indeed, the proCyaA binding reached saturation at substantially higher protoxin concentrations, as compared to CyaA binding. The simplest explanation of this observation could be that proCyaA binds CD11b +  cells with lower affinity than CyaA. At high protoxin concentration, aggregates and/or oligomers of proCyaA would bind to the cells and, consequently, higher amounts of proCyaA are found to be bound by the antibody detection system. In contrast, very low binding of CyaA or proCyaA to control CHO cells was detected ( FIG. 2B ). 
     B.3 Acylation Stabilizes Interaction of CyaA with CD11b +  Cells 
     To further analyze the role of CyaA acylation in interaction of the toxin with CD11b +  cells, we tested the ability of non-acylated proCyaA to compete with CyaA for binding to CHO-CD11b cells. As shown in  FIG. 3A , when compared to the acylated CyaA, the non-acylated proCyaA exhibited a significantly reduced capacity to compete with biotinylated CyaA for binding to CD11b +  cells. To determine if the lack of inhibition was due to an inefficient interaction with CD11b, we evaluated the capacity of proCyaA to block anti-CD11b binding to CHO-CD11b cells. Indeed, compared to CyaA, proCyaA was unable to inhibit anti-CD11b binding to CHO-CD11b cells ( FIG. 3B ). 
     Since supraphysiological production of cAMP and cell intoxication are the consequences of CyaA interaction with CD11b +  cells, we then analyzed, using CHO-CD11b cells, if these toxin functions are dependent on CyaA acylation. As expected, in contrast to the acylated toxin, proCyaA did not induce any cAMP increase in CHO-CD11b cells ( FIG. 4A ) and had no significant cytotoxic effect on these cells ( FIG. 4B ). Taken together, these results clearly demonstrate that acylation of CyaA is necessary for a functional interaction of the toxin with CD11b +  cells and that the binding of proCyaA to CD11b is insufficient to trigger cytotoxic effects on CD11b-expressing cells. 
     B.4 The Catalytic Domain is not Required for cyaA Interaction with CD11b 
     Functionally, CyaA is composed of two main domains harboring independent activities. The N-terminal domain harbors the adenylate cyclase activity (aminoacids 1-400), whereas the carboxy-terminal hemolysin moiety (aminoacids 400-1706) is responsible for the delivery of the AC domain into target cells and the hemolytic activity of  B. pertussis . To examine the role of these two functional domains of CyaA in binding to CD11b +  cells, we tested the ability of the catalytic domain encoded by residues 1 to 384, CyaA 1-384, and of the hemolytic moiety, encoded by residues 373-1706, CyaA 373-1706, to compete for binding to CHO-CD11b cells with CyaA-biotin. As shown in  FIG. 5A , the catalytic domain was unable to inhibit CyaA-biotin binding to CHO-CD11b cells whereas CyaA 373-1706 exhibited the same binding inhibition as the full-length CyaA. Similarly, the catalytic domain was also unable to inhibit binding of the anti-CD11b Mab to CHO-CD11b cells ( FIG. 5B ). Moreover, direct binding assays with an anti-CyaA Mab (5G12) specific for the catalytic domain, could not reveal any significant association of CyaA 1-384 to the surface of CHO-CD11b cells, while binding of CyaA was readily detected ( FIG. 6A ). Direct binding of CyaA 373-1706 to CHO-CD11b cells could not be detected with the 5G12 Mab which recognizes an epitope located within the first 200 amino acids of CyaA, but was clearly demonstrated by using another anti-CyaA Mab (6D7), specific for the repeat domain ( FIG. 6C ). Again, only very weak binding of CyaA or CyaA 373-1706 was detected with the 6D7 Mab on CHO cells lacking CD11b ( FIGS. 6B  and D). Altogether, these results clearly demonstrate that catalytic domain is not necessary for CyaA interaction with CD11b and that the CyaA/CD11b interaction domain is located in the CyaA 373-1706 fragment. 
     B.5 CyaA Domain Interacting with CD11b is Located within the CyaA Repeat Region 
     To identify the region of CyaA that interacts with CD11b, we expressed and purified different sub-fragments of the C-terminal region CyaA 373-1706 (encompassing residues 373-1490, or 700-1706, or 700-1490, or 1006-1706) that were tested in the competition assay. However, none of these polypeptides were able to compete in a significant manner with the binding of CyaA-biotin to CHO-CD11b cells. This might be due to the fact that these isolated fragments adopt an altered conformation. Therefore, we used a mutational approach to locate the CD11b binding domain of CyaA. Seventeen different modified CyaA molecules were constructed by insertion of the FLAG epitope (of amino acid sequence: DYKDDDDK) at various defined positions throughout the toxin polypeptide as detailed in Material and Methods. We hypothesized that insertion of a heterologous and highly charged peptide at certain positions of the CD11b-binding domain might disrupt its capacity to interact with CD11b. The 17 FLAG-tagged CyaA molecules were expressed and purified to homogeneity and tested for the capacity to inhibit binding of CyaA-biotin to CHO-CD11b cells (note that in two cases, CyaAΔ510-515/FLAG and CyaAΔ1245-1273/FLAG, the amino acids 510 to 515 or 1245 to 1273 of CyaA, respectively were deleted and replaced by the inserted FLAG epitope). As shown in  FIG. 7 , insertion of the FLAG epitope at 3 different sites located between residues 1166-1281 totally abrogated the interaction with CD11b. The corresponding modified CyaA were essentially unable to compete with CyaA-biotin for CD11b binding, when tested at 30 nM concentrations. In contrast, all other FLAG-tagged recombinant CyaAs were able to compete with CyaA-biotin for binding to CD11b +  cells, although with variable efficiency. Noticeably, the three recombinant CyaA constructs with the FLAG epitope inserted close to the carboxy-terminal end of the protein (i.e. at position, 1416, 1623 and 1648) were also partially impaired in their capacity to compete for CD11b binding with CyaA-biotin. 
     To further characterize the CyaA domain that interacts with CD11b, we focused on the three CyaA/FLAG molecules that failed to inhibit CyaA-biotin binding to CD11b +  cells, in addition to four other CyaA/FLAG molecules shown to bind CD11b +  cells as efficiently as intact CyaA. These CyaA molecules were again expressed and purified close to homogeneity ( FIG. 8A ) and their cell-invasive activity was examined by analyzing their capacity to penetrate sheep erythrocyte membranes (RBC) and to deliver the catalytic domain into a compartment inaccessible to externally added trypsin. As shown in  FIG. 8B , except for CyaA1387/FLAG, the invasive activity of all other tested CyaA/FLAG molecules was affected to some extent by insertion of the FLAG peptide. The invasive activity of CyaA524/FLAG, which reflects the capacity of CyaA to translocate the catalytic domain into erythrocytes, was completely ablated by the insertion of the FLAG peptide at residue 524. The capacities of the other proteins, CyaA424/FLAG, CyaA722/FLAG and CyaA1166/FLAG and, to a lesser extent, of CyaAΔ1245-1273/FLAG and CyaAΔ1281/FLAG proteins to penetrate into RBC were, however, comparable. 
     The ability of these molecules to compete with CyaA-biotin for binding to CHO-CD11b cells was tested in a dose dependent manner, as shown in  FIG. 9 . As expected, the CyaA1166/FLAG, CyaAΔ1245-1273/FLAG, and CyaAΔ1281/FLAG proteins were unable to inhibit CyaA-biotin binding to CD11b +  cells, even at concentrations as high as 240 nM. In contrast, all other CyaA/FLAG constructs inhibited the CyaA-biotin binding in a dose dependent manner, similarly to intact CyaA. The lack of inhibition of binding by CyaA1166/FLAG, CyaAΔ1245-1273/FLAG and CyaA1281/FLAG could, hence, not be attributed to a generalized conformational disruption of the toxin caused by FLAG insertion, because the invasive activity of these constructs on RBC was comparable to the activity of CyaA424/FLAG protein, which interacted very efficiently with CD11b +  cells. 
     In conclusion, these results provide a compelling evidence that the portion of the CyaA RTX repeat domain delimited by residues 1166 and 1281 and comprising a predicted loop (residues 1208-1243) located between two conserved RTX repeat blocks (Osicka et al., 2000), is crucial for interaction of CyaA with CD11b +  cells and it most likely represents the main integrin-binding domain of CyaA. 
     C. Discussion 
     The biological activity of the adenylate cyclase toxin (ACT or CyaA) is entirely dependent on a covalent post-translational fatty-acylation. In the absence of acylation of the conserved Lys-983 residue, CyaA cannot deliver its catalytic domain into erythrocyte cytosol and is unable to form hemolytic channels (Barry et al., 1991; Basar at al., 2001; Hackett et al., 1994). CyaA was shown to penetrate with detectable efficiency a large variety of eukaryotic cells. It was, however, demonstrated that its primary target cells are myeloid cells such as neutrophils and lung macrophages that are particularly sensitive to CyaA and are paralyzed and committed to apoptosis upon exposure to CyaA (Confer and Eaton, 1982; Khelef and Guiso, 1995; Khelef et al., 1993). We have, indeed, recently shown that the toxin has a specific cellular receptor, an α M β 2  integrin (CD11b/CD18), which is exclusively expressed on immune cells such as neutrophiles, macrophages or dendritic cells and that expression of CD11b most likely accounts for the high sensitivity of these cells to CyaA (Guermonprez at al., 2001). In the present study, the inventors showed that CyaA acylation plays a major role in its interaction with CD11b +  cells. Indeed, albeit non-acylated proCyaA was able to bind CD11b +  cells as efficiently as CyaA, it was inefficient in competing with acylated CyaA for binding to CHO-CD11b +  cells and was completely unable to block anti-CD11b Mab binding to these cells. This suggests that while still interacting with CD11b, the nature of interaction and in particular the affinity and/or stability of the proCyaA-CD11b complex differs significantly from that involved in CD11b interaction of the mature CyaA. Moreover, although proCyaA is still able to bind the CD11b receptor, this interaction does not allow membrane penetration of the protoxin. Hence, the acylation may be needed to confer a translocation-competent conformation of CyaA that is required for the delivery of the catalytic domain to the cell cytosol where it can catalyze the conversion of ATP to cAMP. 
     Functionally, CyaA can be divided in two main domains; one endowed with adenylate cyclase activity domain located between residues 1 to 400, and one responsible for hemolytic activity located within residues 400 to 1706 (Ladant and Ullmann, 1999). After toxin interaction with target cells, the catalytic domain can be directly translocated across the plasma membrane of erythrocytes. The present data show that albeit the catalytic domain plays a key role in the cytotoxic activity of CyaA by catalyzing conversion of ATP to cAMP, this domain is not required for binding of CyaA to its receptor. These results further show that the CyaA/CD11b interaction domain is located in the hemolysin moiety and more precisely in a portion of the glycine- and aspartate-rich RTX repeat region comprising residues 1166 to 1281, as delineated by the insertion sites of the FLAG epitope in constructs with strongly affected binding to CD11b +  cells. In particular, a predicted loop structure interposed between two RTX repeat blocks and comprising the residues 1208 to 1243 (Osicka et al., 2000), could play a crucial role in interaction of CyaA with CD11b +  cells. The loss of interaction with CD11b of the CyaA1166/FLAG, CyaAΔ1245-1273 and CyaA1281/FLAG constructs, respectively, could be due to structural alterations selectively affecting a functionally essential segment involved specifically in the interaction of the CyaA protein with CD11b. This appears much plausible, since all three constructs that failed to bind CD11b still exhibited a substantial cell-invasive activity (20% to 50% of that of intact CyaA) in the surrogate assay system on erythrocytes, where toxin activity does not depend on interaction with CD11b. This indicates that FLAG insertions at positions 1166, 1245 and 1281, did not impair the overall structure of CyaA but rather selectively ablated the capacity of those constructs to interact with the CD11b +  cells. Altogether these results suggest that residues 1166 to 1281 of CyaA delineate an essential portion of the integrin binding domain involved in toxin interaction with the α M β 2  integrin (CD11b/CD18). 
     This conclusion is supported by results showing that all CyaA variants with FLAG peptide inserted within the first 800 residues of CyaA fully competed for binding to CD11b with biotinylated intact CyaA. In contrast, the CD11b-binding capacity was somewhat reduced also for proteins CyaA1416/FLAG, CyaA/FLAG1623 and CyaA/FLAG1648, suggesting that an accessory CD11b-interacting domain of CyaA might be located towards the carboxy-terminal end of the RTX repeat portion of the toxin. 
     The present results that identify the region 1166-1287 as a major CD11b binding motif of CyaA offer an attractive explanation for the previous observation that binding of CyaA to CD11b was strictly calcium-dependent (Guermonprez et al., 2001). As the RTX domain is involved in calcium-binding and undergoes major structural rearrangement upon calcium binding (Rose et al., 1995), one can speculate that the CD11b binding motif located in the region 1166-1287 might be exposed only in the calcium-bound conformation of RTX domain. The CD11b binding motif identified here within the amino-acid region 1166-1287 of CyaA, is precisely localized between the second and 3 rd  block of RTX repeats. One can hypothesize that the α-helical structuration of this segment is involved in the formation of a docking site for CD11b. 
     CyaA has been used in several passive and active protection protocols in mouse models of pertussis. Immunization with anti-CyaA specific antibodies or with purified CyaA reduced the time course of the respiratory tract colonization by  B. pertussis  and protected the mice against a lethal intranasal infection (Guiso et al., 1989; Guiso et al., 1991). Moreover, antibodies specific for CyaA were detected in the sera of human infants infected with  B. pertussis  (Arciniega et al., 1991; Guiso et al., 1993). The present results suggest that a CyaA molecule lacking CyaA/CD11b interaction domain can be designed for the production as a safe acellular vaccine for protection against  B. pertussis  infection. The catalytic activity of such a molecule can be easily inactivated by dipeptide insertions within the ATP-binding site, located between residues 188 and 189 of CyaA (Fayolle et al., 1996), while the deletion within the CD11b interaction domain could preserve immune cells from potentially negative effects, such as signaling upon the integrin engagement by the toxoid and/or some functional interference due to competition for binding to CD11b with the CyaA toxoid, which also serves as the complement receptor CR3. 
     In conclusion, the present data provide important new insights into the role of acylation and of different domains of the adenylate cyclase of  B. pertussis  in its interaction with CD11b +  cells as well as in the subsequent biological activities triggered by this interaction. 
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