# Patent

## Title
Formulation processes for pharmaceutical compositions of recombinant .beta.

## Abstract

The adenyl cyclase derivatives are atoxic derivatives in which one or several amino acids are different from those present in the toxic protein, the antibodies formed against these derivatives recognize, however, the toxic protein. These derivatives can be used as vaccines.

## Background
The subject of the invention is adenyl cyclase derivatives, their preparation by mutagenesis and their biological uses, in particular as vaccines.

Vaccinations against acute infections caused in vertebrates by pathogenic bacteria of the genus Bordetella or Bacillus anthracis generally require the utilization of virulent or attenuated whole bacteria.

However, such vaccines are not necessarily devoid of toxicity due to the nature of the diseases caused by these bacteria.

In fact, it is often proteins produced by bacteria, and not the bacteria themselves, which are responsible for the virulence. Even after bacterial death, these proteins may be responsible for many pathological effects.

In the pathology induced by Bordetella pertussis a filamentous hemagglutinin (FHA) as well as two toxins lead to an unregulated increase in the production of cyclic AMP in the host cells. These toxins are the pertussis toxin (Ptx or LPF) which activates the cyclase of the host, and the adenyl cyclase, activated by calmodulin (AC).

Finally, a hemolysin is associated with the adenyl cyclase.

The adenyl cyclase of B. pertussis was called cyclolysin to point out this fact.

In the light of these observations, the inventors have investigated vaccination agents in which the bacteria of the virulent or attenuated strains are not longer involved, but rather the proteins produced, are modified in an appropriate manner in order to destroy their original toxicity. In particular, adenyl cyclase has been investigated in this respect.

The aim of the invention is to provide novel derivatives of adenyl cyclase, these derivatives being devoid of toxic activity but being capable of forming antibodies which also recognize the toxic proteins.

It also aims to provide a procedure for preparing these derivatives by in vitro mutagenesis of the genes coding for the toxic adenyl cyclase.

Another aim of the invention consists in the provision of vaccines which ensure an effective protective action against the infections produced by pathogenic agents.

The adenyl cyclase derivatives of the invention are characterized in that they contain one or more amino acids different from those of the toxic protein these changes conferring on them an atoxic character. The amino acids that have been changed are implicated in the catalytic mechanism and/or in the mode of activation by calmodulin and/or the transfer of information from the activator site to the catalytic site of the adenyl cyclase and do not appreciably affect the threedimensional configuration of the toxic protein, so that the antibodies formed against the atoxic derivatives also recognize the toxic protein.

Another characteristic of the invention relates to the whole protein, where the amino acids responsible for the hemolytic activity have been altered or deleted.

FIGS. 1a to 1f represent the nucleotide sequence of the active part of the gene coding for the adenyl cyclase of B. pertussis and the corresponding amino acid sequence of the adenyl cyclase expressed in E. coli.

FIG. 2 represents the complete amino acid sequence of the polypeptide of the adenyl cyclase of B. anthracis (upper line) and the N-terminal polypeptide sequence of the adenyl cyclase of B. pertussis (lower line).

FIGS. 3a to 3j represent the nucleotide sequence of the active part of the gene coding for the adenyl cyclase of B. anthracis and the corresponding amino acid sequence of the adenyl cyclase expressed in E. coli.

The amino acid(s) of replacement are selected in the light of their physico-chemical properties, so as to destroy the toxicity without affecting the structure of the protein appreciably, at least in respect to its recognition by the immune system.

In possessing the same geometry as the toxic protein, these derivatives advantageously play the role of decoy for the immune system without, however, exerting toxic catalytic activity.

Among the properties making it possible to operate on the original toxic character of the protein and to destroy it, mention may especially be made of the size of the amino acids, their hydrophobic or hydrophilic character, their charge, their propensity to form or destroy the secondary structures of the protein their action on the structure of water, their helix-forming or -breaking character, their interaction with all or part of the ATP involved as substrate or with calmodulin.

According to a preferred embodiment of the invention, the adenyl cyclase derivatives contain one or advantageously at least two amino acids replacing wild type amino acids in the catalytic site, i.e. in the domain responsible for the production of cyclic AMP (cAMP). This arrangement makes it possible to suppress the production of cAMP or to make it minimal while conserving the immunogenic properties of the protein.

The different amino acid(s) in the catalytic site are selected more especially to modify the structure of water, or the interaction of the protein with water or with the phosphate, the nucleic base and/or the sugar of the ATP, so that the catalytic activity or the control of this activity of the site is destroyed.

For example, hydrophobic amino acids may be charged with hydrophilic amino acids of the same size or vice versa or amino acids having differing electrical charge may be used, or alternatively amino acids of different size which prevent the introduction of the substrate ATP, or the cyclization reaction to cAMP.

As examples, mention should be made of the replacement of one or at least two lysines, which interact with the ATP, with amino acids of the same volume, hydrophilic but devoid of charge such as glutamine (Q).

Within the catalytic peptide sequence, the GLNVHAKS core may be modified, and G and A each replaced by one of the following amino acids M, L, I, V, or F. As indicated K may be replaced by Q but also by M, L, I, V.

The meanings of the letters commonly used to designate the above amino acids and those mentioned hereafter are given at the end of the description.

The domain responsible for the hemolytic activity may advantageously be deleted in the whole protein, for example by cutting the gene at the Bgl II (AGATCT) sites and by deleting the corresponding fragment. That amounts to removing the portion of the protein situated between EI xxx and EI xxx.

As an additional arrangement, it is possible to take advantage of the fact that the protein does not contain a cysteine residue in order to introduce this amino acid by localized mutagenesis. The latter then may be subject to an appropriate chemical treatment (oxidation for example or reaction with SH reagents of the metallic type, organomercurials or iodoacetamide), the result of which will be the inactivation of the protein.

The different amino acids replacing the wild type amino acids may also be selected from among amino acid analogues which can be incorporated by the translation machine, if necessary, chemically modified. As examples, mention should be made of flourophenylalanine, bromotryptophan, or nor-leucine.

In another embodiment of the invention, which may be implemented, if appropriate, with the above preferred embodiment, the amino acid(s) of replacement are incorporated in the calmodulin activator site, and are selected as a function of the same parameters, in order to make possible a change in the binding to the activating molecule of the host.

Thus, a negatively charged and helix-breaking, hydrophilic aspartic acid residue will advantageously replace a helix-forming and hydrophobic tryptophan residue of larger size in the region of binding to the calmodulin.

In yet another embodiment, implemented alone, or in combination with at least one of those which precede the amino acid(s) of replacement, such as those considered above, are present in the region responsible for the transfer of information from the activator site to the catalytic site.

Preferably, the derivatives of the invention are such as those obtained by expression in a host organism of a gene coding for a protein with adenyl cyclase activity, this gene having been modified in vitro, after cloning, by directed mutagenesis.

In addition to the elements of mutagenesis described below, it is also possible to operate by deletion of known regions of the genome, or by insertion of synthetic oligonucleotides so as to destroy a site endowed with activity.

The derivatives of the invention are more particularly mutants of adenyl cyclase of Bordetella responsible for respiratory diseases of vertebrates such as B. pertussis and B. parapertussis in man and B. bronchiseptica and B. avium in animals.

The active part of the toxic adenyl cyclase of B. pertussis expressed by the cloned gene has the sequence (I) given in the FIGS. 1a to 1f. This sequence is reported in the application EP 88/401.935.7 of Jul. 25, 1988.

The protein corresponding to the sequence (I), which contains 1706 amino acids, comprises two domains exhibiting two principal activities.

The 400 to 500 amino acids of the N-terminal part are responsible for the adenyl cyclase activity and the 1300 to 1200 of the carboxy-terminal part are responsible for the hemolytic activity and the secstory properties of the protein.

In the catalytically active part responsible for the synthesis of cAMP, the adenyl cyclase appears to be organized in two domains: the N-terminal domain of 25 kDa which contains the catalytic site and the C-terminal domain of 12 kDa more particularly the sequence corresponding to the residues 235-254 which contains the principal binding site to calmodulin.

In another embodiment of the invention, the adenyl cyclase derivatives are mutants of an adenyl cyclase of the type expressed by B. anthracis. The nucleotide sequence of the gene coding for this adenyl cyclase and the sequence of this latter are reported in the French application filed today in the names of the applicants, entitled "SEQUENCES OF NUCLEOTIDES EXPRESSING THE ADENYL CYCLASE OF B. ANTHRACIS, PROTEINS HAVING THE ACTIVITY OF THIS ADENYL CYCLASE AND BIOLOGICAL USES".

The amino acid sequence of the adenyl cyclase of B. anthracis (upper line) shows several regions in common with that secreted by B. pertussis (lower line) (see FIG. 2).

The most important similarity corresponds to a peptide of 24 amino acids (from position 342 to position 365 in the adenyl cyclase of B. anthracis: GVATKGLNVHGKSSDWGPVAGYIP). This sequence contains five gly residues and the core sequence G--GKS (AKS in B. pertussis) which is often found in proteins having an affinity for nucleotides.

Two other regions show a weaker similarity. They correspond in B. anthracis to the domains extending from the positions 487 to 501  (PLTADYDLFALAPSL), on the one hand, and from 573 to 594 (DVVNHGTEQDNEEFPEKDNEIF), on the other.

As a variant, the derivatives of the invention are such as those obtained by chemical treatment of the modified sequence of adenyl cyclase.

The invention also relates to polyclonal antibodies directed against these modified adenyl cyclases as well as the monoclonal antibodies capable of recognizing them specifically.

In conformity with the invention, the adenyl cyclase derivatives defined above are obtained by in vitro mutagenesis of the cloned gene expressing the toxic protein, followed by expression of the gene in appropriate hosts, for example E. coli, B. avium, or Alcaligenes eutrophus with a subsequent chemical treatment if necessary (particular case of the proteins with cysteine, for example). This expression is carried out in high yields in particular in E. coli. The gene can be reintroduced into the pathogenic bacterium in order for it to express the atoxic protein. The bacterium thus modified also is part of the invention.

The procedure for the preparation of the atoxic adenyl cyclase derivatives according to the invention comprises the in vitro mutagenesis of the cloned gene expressing the toxic adenyl cyclase. This mutagenesis is carried out in the region coding for the catalytic site, and/or the calmodulin activator site and/or the site responsible transfer of information from the calmodulin activator site to the catalytic site and/or the region responsible for the hemolytic activity. The mutagenesis introduces one or more codons coding for amino acids different from these present in the toxic protein, or deletes codons present in the original gene to confer on the mutant an atoxic character without appreciably affecting its three dimensional configuration shown by the toxic protein, to permit antibodies formed against the atoxic derivatives to also recognize the toxic protein.

The mutagenesis advantageously affects the codons coding for the amino acids defined above.

According to a preferred embodiment of the invention, the system of in vitro mutagenesis implemented is that of Amersham or any other company offering systems of the same type comprising the utilization of a mutant oligonucleotide to direct the mutagenesis. This oligonucleotide is constructed to include the codon encoding the amino acid which is desired to be introduced. The desired final codon may be introduced in a single step, or through an introduction of an intermediary codon, then in a later step, to the introduction of the desired final codon.

As an alternative, it is also possible to use a mutant oligonucleotide containing an ambiguity and thus capable of coding for two different amino acids. Similarly, it is possible to use an oligonucleotide which lacks one or more codons, to produce a deletion.

The mutant oligonucleotide advantageously contains at least fifteen, and in particular from twenty to twenty-five, nucleotides. In conformity with the standard method of in vitro mutagenesis, utilizing the Amersham kit, the following steps are performed:

hybridization of the mutant oligonucleotide to a DNA sequence of a single-stranded DNA matrix appropriately constructed from a vector by in vitro mutagenesis. A phage, in particular a derivative of M 13, is advantageously utilized as DNA single-stranded vector;

synthesis in the presence of a thio-dCTP and ligation to the mutant DNA strand with a polymerase such as the Klenow fragment of the DNA polymerase I of E. coli.

removal of the non-mutant single-stranded DNA, for example by filtration;

cleavage of the non-mutant DNA strand with the aid of a restriction enzyme, such as Ncil;

digestion of the non-mutant strand with the aid of an exonuclease, in particular exonuclease III;

repolymerization and ligation of the partially defective DNA;

transformation of competent cell hosts with the DNA.

This procedure has the advantage of giving rise to the atoxic derivative in high yield. It is clear, however, that other embodiments of the mutagenesis for example utilizing restriction sites in order to create insertions, deletions, or replacements, can be implemented so that modifications can be made to the method based on the utilization of the kit sold by Amersham in order to introduce suitable codons and to obtain the atoxic adenyl cyclase derivatives of the invention.

The gene coding for the adenyl cyclase expressed by one of the microorganisms mentioned above is advantageously cloned according to the procedure described in the application Fr 87/10614 of Jul. 24, 1987 in the name of the applicants.

In this procedure, use is made of the adenyl cyclase-calmodulin interaction which is expressed by the production of cAMP. The cloning of the gene is carried out in a receptor strain deficient in adenyl cyclase, carrying a plasmid expressing high levels of calmodulin. It should be noted that the genes which cooperate in this cloning procedure, namely the one which codes for the adenyl cyclase and the one which codes for calmodulin, are of different origins.

The cloned DNA fragment of the active part of the gene coding for the adenyl cyclase of B. pertussis, which can be used in the mutagenesis procedure of the invention, corresponds to the sequence of the active part of the genes coding for the adenyl cyclase of B. Pertussis and B. anthracis respectively as shown in the FIG. 1a to 1f or also 3a to 3j.

The catalytic part for the synthesis of cAMP of the DNA fragment includes the sequence of nucleotides localized between the positions 0 and 2050, with the position 0 corresponding to the BamHI restriction site.

Any sequence of nucleotides which hybridizes with the sequence of nucleotides of FIGS. 1a to 1f or 3a to 3j and codes for a protein with adenyl cyclase activity, whether this sequence is obtained by reverse enzymatic transcription of the corresponding RNA or by chemical synthesis, can be used in the procedure according to the invention for the purposes of mutagenesis.

The invention also relates to nucleotides sequences comprising or constituted by the gene coding for at least the active part of an adenyl cyclase, but mutated so as to code for an adenyl cyclase derivative such as that defined above as well as the mutant oligonucleotides.

Thus, in relation to the DNA fragment of the FIGS. 1a to 1f, a nucleotide sequence according to the invention contains a CAA codon in the place of the AAA codon at position 58, or also a CAG codon in the place of the AAG codon at position 65. Preferably, these oligonucleotides are unable to make possible a nonspecific hybridization with the vector M13.

The mutant oligonucleotides contain for example a CAA codon in the place of a AAA codon at position 58 and contain all or part of the sequence GTG GCC ACC CAA GGA TTG G.

It may also involve oligonucleotides with a CAG codon in the place of a AAG codon at position 65 and containing all or part of the sequence GTGCACGCCCAGTCGTCCG.

Other oligonucleotides contain instead of a TGG condon at position 242 a GAC codon and correspond to all or part of the sequence CTTGTTGGACAAAATCGC, or a GAC codon in the place of the GAC codon of the preceding oligonucleotide.

A mixture of oligonucleotides can make it possible to obtain several mutants in a single experiment.

As an alternative, the atoxic derivatives of the invention may be obtained by subjecting the protein sequence of an adenyl cyclase, or at least its active part, to a chemical treatment. For example, the inactivation treatments used in vaccination, such as in the case of tetanus vaccinations.

The toxicological assays carried out on the protein derivatives of the invention have demonstrated the absence of toxicity.

The strongly immunogenic properties of the derivatives of the invention are thus advantageously taken advantage of for the development of vaccines.

The vaccines of the invention are characterized in that they are molecular vaccines containing at least one atoxic derivative of adenyl cyclase such as that defined above, in combination with a pharmaceutical vehicle.

They may also contain the bacterium carrying the mutated genes and thus rendered atoxic.

Vaccination is carried out according to the usual dosages and with the usual forms of administration.

In an advantageous manner, the adenyl cyclase derivatives of the invention give rise to an immunological cross-reaction with antibodies directed against the adenyl cyclase of B. pertussis. Advantageous compositions of vaccines contain the adenyl cyclase derivative of the invention in combination with a preparation of killed B. pertussis bacteria used as adjuvant.

In order to illustrate the invention, examples are reported below of the preparation of adenyl cyclase mutants of B. pertussis. In these examples, reference is made to the FIG. 1a to 1f and 3a to 3j, which present the nucleotide sequences of the active part of the gene coding for the adenyl cyclase of B. pertussis and B. anthracis, respectively, cloned in E. coli and the corresponding amino acid sequence expressed, and to FIG. 2 which shows the complete sequence of the polypeptide of the adenyl cyclase of B. anthracis (upper line) and the N-terminal polypeptide sequence of the adenyl cyclase of B. pertussis (lower line).

1. Cloning of the gene coding for the adenyl cyclase of B. pertussis.

cya.sup.-, restriction.sup.- strains of E. coli are transformed by a plasmid carrier of a gene specifying the synthesis of calmodulin such as the plasmid pVUC-1, or any other plasmid expressing the calmodulin of the incompatibility group ColE1 and carrier of the gene for resistance to ampicillin.

Into the cya.sup.- strains are then introduced vectors containing the DNA of B. pertussis completely fragmented in various ways (partial cleavages by the restriction enzymes EcoRI and SauIIIa, sonication followed by addition of EcoRI "linkers", for example).

In view of the problems posed by the incompatibility of other plasmids having the ColEI replicon, the cloning of fragments of the total DNA of B. pertussis partially cleaved by the enzyme SauIIIa was carried out in the compatible vector pACYC184, at the unique BamH1 site. The receptor strains were transformed and spread on McConkey Maltose plates. Several clones fermenting maltose were isolated. It was then demonstrated that these clones produce cyclic AMP. Moreover, the plasmids extracted from the producer strains were integrated by transformation into strains which do not contain the plasmid pVUC-1 or do contain it. In the former case, the clones are white on the McConkey maltose media, red in the latter, confirming that it is indeed the presence of the plasmids giving rise to the synthesis of calmodulin which has made the cloning possible.

2. In vitro mutagenesis of the cloned gene coding for the adenyl cyclase.

By operating as described by Carter et al. in Nucl. Acids Res. 13, 4431-43, 1985, a AAA codon (coding for K) is replaced by a CAA codon (coding for Q) at position 58 by using the oligonucleotide GTGGCCACCCAAGGATTGG. Similarly, a AAG codon (coding for K) at position 65 is replaced by a CAG codon (coding for Q) by utilizing the oligonucleotide GTGCACGCCCAGTCGTCCG.

The two modifications are carried out on the DNA fragment BamHI-EcoRV cloned in the phage M13tg130.

The TGG codon at position 242 (coding for tryptophan) is first modified to a GAC codon (coding for aspartic acid) by using the oligonucleotide CTTGTTGGACAAAATCGC. The GAC codon is then modified to a GAG codon (coding for glutamic acid) by using the oligonucleotide CTTGTTGGAGAAAATCGC. It should be noted that in this case the mutagenesis affects a codon which is not found in a zone of similarity with the nucleotide sequences of other genes coding for an adenyl cyclase, more particularly that of B. anthracis.

The modifications at position 242 were carried out on the DNA fragment EcoRV-EcoRI cloned in the phage M13tg130.

For each mutagenesis, a control is performed on the mutated fragment for the absence of any other mutation by the method of sequencing with a dideoxynucleotide.

3. Preparation of the atoxic derivatives of adenyl cyclase.

By expression of the mutated gene in an E. coli strain according to conventional techniques the atoxic adenyl cyclase is obtained which is then purified.

It will be noted that the replacement of tryptophan by aspartate reduces by a factor of 1,000 the activatability of the enzyme by calmodulin, while its basal activity is maintained.

The replacement of lysine at position 65 diminishes the intrinsic basal activity of the enzyme by a factor of at least 100.

The single letters used to designate the amino acids have the following meanings: Aspartic acid D Glutamic acid E Alanine  A Arginine R Asparagine N Cysteine C Glutamine Q Glycine  G Histidine H Isoleucine I Leucine  L Lysine   K Methionine M Phenylalanine F Proline  P Serine   S Threonine T Tryptophan W Tyrosine Y Valine   V ______________________________________

## Claims

1. An adenyl cyclase derivative wherein: amino acid sequence of a Bacillus anthracis adenyl cyclase comprising an amino acid substitution in at least one site of said derivative, wherein said sites are selected from the group consisting of amino acids 342-365 of the Bacillus anthracis sequence of FIG. 2 and the calmodulin binding site, and said amino acid substitution is selected from the group consisting of: group consisting of glutamine, asparagine, aspartic acid, glutamic acid, methionine, leucine, isoleucine, and valine; the group consisting of aspartic acid, glutamic acid, asparagine, and glutamine; and of a tryptophan residue as indicated in ii); and

2. An adenyl cyclase derivative, wherein: amino acid sequence of a Bordetella pertussis adenyl cyclase comprising an amino acid substitution in at least one site of said derivative, wherein said sites are selected from the group consisting of amino acids 54-77 of the Bordetella pertussis sequence of FIG. 2 and the calmodulin binding site, and said amino acid substitution is selected from the group consisting of: group consisting of glutamine, asparagine, aspartic acid, glutamic acid, methionine, leucine, isoleucine, and valine; the group consisting of aspartic acid, glutamic acid, asparagine, and glutamine; and of a tryptophan residue as indicated in ii).

3. The adenyl cyclase derivative of claim 1 or 2, wherein said amino acid substitution comprises a replacement of a lysine residue of said adenyl cyclase with an amino acid selected from the group consisting of glutamine, asparagine, aspartate, and glutamate.

4. The adenyl cyclase derivative of claim 1 or 2, wherein the amino acid sequence of said derivative further comprises an amino acid analogue selected from the group consisting of fluorophenylalanine, bromotryptophan, and norleucine in said sites.

5. The adenyl cyclase derivative of claim 2, wherein said amino acid substitution comprises a replacement of a tryptophan residue with an amino acid selected from the group consisting of aspartate, glutamate, asparagine, and glutamine, wherein said replacement is in the calmodulin binding domain.

6. An adenyl cyclase derivative of Bordetella pertussis wherein the amino acid sequence of said derivative consists essentially of an amino acid sequence of Bordetella pertussis adenyl cyclase comprising a replacement of lysine at position 58 according to the Bordetella pertussis sequence of FIG. 2 with glutamine.

7. An adenyl cyclase derivative of Bordetella pertussis, wherein the amino acid sequence of said derivative consists essentially of an amino acid sequence of Bordetella pertussis adenyl cyclase comprising a replacement of lysine at position 65 of the Bordetella pertussis sequence of FIG. 2 with glutamine.

8. An adenyl cyclase derivative of Bordetella pertussis, wherein the amino acid sequence of said derivative consists essentially of an amino acid sequence of Bordetella pertussis adenyl cyclase comprising a replacement of tryptophan at position 242 of the Bordetella pertussis sequence of FIG. 2 with an amino acid selected from the group consisting of aspartic acid and glutamic acid.

9. An adenyl cyclase derivative of Bacillus anthracis, wherein the amino acid sequence of said derivative consists essentially of an amino acid sequence of Bacillus anthracis adenyl cyclase comprising a replacement of lysine at position 346 of the Bacillus anthracis sequence of FIG. 2 with glutamine.

10. An adenyl cyclase derivative of Bacillus anthracis, wherein the amino acid sequence of said derivative consists essentially of an amino acid sequence of Bacillus anthracis adenyl cyclase comprising a replacement of lysine at position 353 of the Bacillus anthracis sequence of FIG. 2 with glutamine.

11. An adenyl cyclase derivative of Bordetella pertussis, wherein the amino acid sequence of said derivative comprises a substitution of the amino acid sequence GLNVHAKS of the catalytic site of Bordetella pertussis with the amino acid sequence: wherein V.

12. A nucleotide sequence encoding the adenyl cyclase derivative of claim 1.

13. A process for obtaining an atoxic adenyl cyclase derivative, wherein said process comprises: sequence of a mutant gene of an adenyl cyclase, wherein: anthracis adenyl cyclase comprising an amino acid substitution in at least one site of said derivative, wherein said sites are selected from the group consisting of amino acids 342-365 of the Bacillus anthracis sequence of FIG. 2 and the calmodulin binding site, and said amino acid substitution is selected from the group consisting of: group consisting of glutamine, asparagine, aspartic acid, glutamic acid, methionine, leucine, isoleucine, and valine; the group consisting of aspartic acid, glutamic acid, asparagine, and glutamine; and of a tryptophan residue as indicated in 2); and

14. The process of claim 13, wherein said host cell is Escherichia coli.

15. A process for obtaining an atoxic adenyl cyclase derivative, wherein said process comprises: sequence of a mutant gene of an adenyl cyclase, wherein: pertussis adenyl cyclase, comprising an amino acid substitution in at least one site of said derivative wherein said sites are selected from the group consisting of amino acids 54-77 of the Bordetella pertussis sequence of FIG. 2 and the calmodulin binding site, and said amino acids substitution is selected from the group consisting of: group consisting of glutamine, asparagine, aspartic acid, glutamic acid, methionine, leucine, isoleucine, and valine; the group consisting of aspartic acid, glutamic acid, asparagine, and glutamine; and of a tryptophan residue as indicated in 2); and

16. The process of claim 15, wherein said amino acid substitution comprises a replacement of lysine at position 58 of the Bordetella pertussis sequence of FIG. 2 with glutamine.

17. The process of claim 15, wherein said amino acid substitution comprises a replacement of lysine at position 65 of the Bordetella pertussis sequence of FIG. 2 with glutamine.

18. The process of claim 15, wherein said amino acid substitution comprises a replacement of tryptophan at position 242 of the Bordetella pertussis sequence of FIG. 2 with an amino acid selected from the group consisting of aspartic acid and glutamic acid.