EP0537342A1

EP0537342A1 - Immunogenic recombinant strains of b. anthracis and immunogenic compositions containing same

Info

Publication number: EP0537342A1
Application number: EP92917387A
Authority: EP
Inventors: Michèle Mock; Angel Cataldi; Corinne Pezard
Original assignee: Institut Pasteur de Lille
Current assignee: Institut Pasteur de Lille
Priority date: 1991-05-02
Filing date: 1992-04-30
Publication date: 1993-04-21
Also published as: FR2676068A1; FR2676068B1; WO1992019720A1

Abstract

L'invention concerne une souche recombinante de B. anthracis, caractérisée: par sa capacité à induire chez un hôte animal ou humain, la production d'anticorps protecteurs contre des souches virulentes de B. anthracis, la mutation du plasmide pX01 d'au moins un gène donné codant pour une protéine responsable d'un effet toxique chez B. anthracis, cette mutation résultant en: la délétion de tout ou partie de ce gène codant pour une protéine responsable d'un effet toxique et, l'insertion au niveau du site de délétion de ce gène dans pX01, d'une cassette d'ADN permettant la sélection de la souche ainsi modifiée et empêchant la réversion de la souche recombinante, le gène ainsi muté étant alors soit dépourvu de la capacité à produire la protéine responsable de l'effet toxique pour laquelle il code, soit capable de coder pour une protéine tronquée dépourvue de son caractère toxique. L'invention vise aussi l'utilisation d'une telle souche dans des compositions immunogènesThe invention relates to a recombinant strain of B. anthracis, characterized: by its ability to induce, in an animal or human host, the production of protective antibodies against virulent strains of B. anthracis, the mutation of the plasmid pX01 of at least a given gene encoding a protein responsible for a toxic effect in B. anthracis, this mutation resulting in: the deletion of all or part of this gene encoding a protein responsible for a toxic effect and, the insertion at the level of the deletion site of this gene in pX01, of a DNA cassette allowing the selection of the strain thus modified and preventing the reversion of the recombinant strain, the gene thus mutated then either lacking the capacity to produce the protein responsible for the toxic effect for which it codes, is capable of coding for a truncated protein devoid of its toxic character. The invention also relates to the use of such a strain in immunogenic compositions.

Description

Immunogenic recombinant strains of

B. anthracis - Immunogenic compositions containing them

The present application relates to recombinant immunogenic and non-toxicogenic strains of Bacillus anthracis and immunogenic compositions containing them.

Bacillus anthracis (B. anthracis) is responsible in animals and in humans for anthrax, which exists in cutaneous, respiratory and digestive forms. Severe forms of this disease can lead to the death of the infected person. The pathogenicity of B. anthracis is expressed in two aspects: a toxic effect manifested by the appearance of edema, and a so-called lethal toxic effect which can lead to the death of the infected subject. These effects are attributed to the presence in B. anthracis of three protein factors acting in combination by two. One of the three factors is present in both combinations and is involved in the binding of toxins from B. anthracis to the membrane of the host. The two other protein factors constitute the active elements responsible for the manifestation either of the toxic edema-like effect or of the toxic effect of a lethal nature. These two factors are respectively called edematogenic factor (English abbreviation: EF) and lethal factor (English abbreviation: LF).

The factor responsible for membrane fixation is called protective antigen (abbreviation PA) because it was initially attributed the ability to confer active protection against the disease, during immunization tests.

The three factors PA, LF and EF were purified (FISH et al 1968a J. Bacterial 95: 907-917) and the two toxins obtained by combining PA and LF on the one hand, PA and EF on the other hand have been described by LEPPLA et al (1982, PNAS - USA 79-3162-3166).

It was also known that the three genes pag, cya and lef coding respectively for the factors PA, EF and LF are distributed on a plasmid pXOl of B. anthracis described by MIKESELL P. et al (1983 Infect. Immun 39, 371-376) .

Another plasmid pX02 has been demonstrated in B. anthracis; it includes in particular the DNA sequences coding for the elements of the bacillus capsule.

The pag, cya and lef genes have been cloned and sequenced. These results have been reported respectively by WELKOS et al, 1988 in Gene, 69: 287-300, by ESCUYER et al, 1988 in Gene, 71: 293-298, and by BRAGG et al, 1989, Gene, 81: 45- 54.

In addition CATALDI et al (Molecular Microbiology 1990 4 (7), 1111-1117) described the construction and the characterization of a strain of B. anthracis devoid of the plasmid pX02 and deprived of the antigen PA by modification of the plasmid pXOl.

To carry out this construction, CATALDI et al used a mobilizable shuttle vector, transferable by conjugation of E. coli to B. anthracis. This vector included on the one hand the gene coding for the protein PA, mutated by deletion of a fragment of size less than 50 bp and on the other hand a reporter DNA cassette carrying a gene for resistance to 1 erythromycin (Erm ^r ) inserted at the site of the deletion.

The vector thus formed was introduced by conjugation into B. anthracis 7702 (Sterne strain) and the transconjugants having undergone homologous recombination between the vector introduced and the plasmid pXOL resulting in the introduction of an inactive pag gene into pXOL in place of the original pag gene, were selected.

According to the authors of the publication, the construction carried out made it possible to show that the absence of expression of PA is sufficient to completely abolish the lethal character of B. anthracis and also to confirm that PA plays a central role, allowing the penetration of EF or LF in eukaryotic cells.

According to the authors of this published article, additional research is necessary to provide information on the expression of the pathogenic character of B. anthracis and on the elements likely to intervene in immunization against this bacillus.

Data on PA which may appear contradictory were indeed available in the prior art, establishing on the one hand that PA is the main component among the factors of toxicity required for protection against B. anthracis and on the other hand that a live vaccine based on Sterne spores is more effective than an acellular preparation of AP.

From this, the authors concluded that there is a need to clarify the role of the components of toxins or other antigens in order to find out what could be effective protection against virulent strains of B. anthracis.

According to another publication (M. MOCK Annales de l'INSTITUT PASTEUR, December 1990), analyzes of serum samples from patients infected with B. anthracis have made it possible to demonstrate antibodies directed against PA, EF and LF, this last most important trigger factor. The conclusion of the article is that LF is a potent immunogen.

The data available both on the pathogenicity of B. anthracis and on the elements involved in immunization have not so far made it possible to really determine what a protective vaccine composition against B. anthracis could be.

The results known so far made it possible to envisage that such a composition could contain strains of live B. anthracis. However, the results described in the articles cited above did not make it possible to define under what conditions these strains should be used to benefit from their supposed immunogenic components (in particular LF according to the article by MOCK) knowing that PA was recognized as playing a central role among the factors of toxicity, in order to trigger the protective immune response in animals or in humans, these strains being furthermore non-toxic and stable in the host.

The inventors of the present application have researched and developed immunogenic compositions capable of inducing protective antibodies in animals. Contrary to the case of the recombinant strain PA- of B. anthracis described in the aforementioned article by CATALDI et al, in which the mutation of the plasmid pXOl of B. anthracis related to an element not directly responsible for the toxicity, the inventors have issued the hypothesis that a B. anthracis strain devoid of at least one of its toxicity factors factor responsible for pathogenicity and generally involved in the reaction of production of protective antibodies, could enter in the constitution of an immunogenic and protective composition against B. anthracis.

Despite the finding that the lethal factor LF is a potent immunogen (MOCK, Annales de 1'INSTITUT PASTEUR), the inventors have in particular prepared and characterized a recombinant strain of B. anthracis in which the gene coding for the LF protein is mutated so that it is no longer expressed in the bacteria or so as to be expressed in the form of an inactive truncated protein. They found that the mutation of the plasmid pXOl at the lef gene level did not alter the functions other than those linked to the expression of LF in B. anthracis.

Likewise, they have shown that a mutation in the cya gene responsible for the second toxic effect, could lead to the production of recombinant B. anthracis strains of interest for the preparation of vaccines.

Consequently, a modification of the components of the toxicity of B. anthracis leading to their disappearance or their inactivation does not alter the potential of strains of B. anthracis to be immunogenic and to protect against infection by this bacillus.

The invention therefore relates to a recombinant strain of B. anthracis, characterized by:

- its ability to induce, in an animal or human host, the production of protective antibodies against virulent strains of B. anthracis,

- the mutation of at least one given gene on the plasmid pXOl coding for a protein responsible for a toxic effect in B. anthracis, this mutation resulting in:

. the deletion of all or part of this gene coding for a protein responsible for a toxic effect and, The insertion at the deletion site of this gene in pXOl, of a DNA cassette allowing the selection of the strain thus modified and preventing the reversion of the recombinant strain, the gene thus mutated then being either lacking the capacity to produce the protein responsible for the toxic effect for which it codes, is capable of coding for a truncated protein devoid of its toxic character.

The term "protective antibody" denotes, within the framework of the invention, antibodies capable of conferring protection against virulent strains of B. anthracis, when they are produced in an animal or human host. They are therefore in particular neutralizing antibodies against B. anthracis.

The protein responsible for a toxic effect of the B. anthracis strain is called a protein which enters into the composition of a toxin and is necessary for the manifestation of either edema or lethal toxicity even if this effect results from the combination of the activities of several factors. Two proteins correspond in particular to this definition: the EF protein and the LF protein which contribute to the formation of toxins, constituted in this case respectively by the combinations PA + EF and PA + LF.

The DNA cassette inserted into the deleted gene is a DNA sequence which makes it possible to verify the integration of the deleted gene into the plasmid pXOl, since this cassette is inserted at the level of the gene deletion site.

The deletion of at least part of the gene coding for the protein with toxic effect prevents the recombinant strain thus obtained from returning to its natural state of origin by reversion, and therefore can again express the protein encoded by the gene in question or produce it in an active form.

The mutation of the plasmid pXOl at the level of one of the genes coding for EF or LF is carried out in a particularly advantageous manner thanks to the implementation of an intermediate step involving a vector itself carrying the mutated cya or lef gene which by homologous recombination will make it possible to modify pXOl respectively at the level of the cya or lef gene. It is however understood that any technique making it possible to carry out the desired mutation of the cya gene or of the lef gene can be used, whether it is direct or not.

A first family of recombinant B. anthracis strains according to the invention corresponding to the preceding definition, is characterized in that, in addition to the preceding characteristics, the strain is devoid of the plasmid pX02. This plasmid pX02 is in particular necessary for the formation of the B. anthracis capsule and its absence makes it possible to attenuate the virulence of the natural strain.

Mention may be made, among the strains lacking this plasmid, of the Sterne 7702 strain described in the article by CATALDI et al (Molec. Microbiology 1990 4 (7), 1111-1117 and Sterne 1939 Onderstepoort J Vet Sci Ani Indust 13: 313-317 ).

According to a first advantageous embodiment of the invention, a recombinant strain of B. anthracis is a strain of which the lef gene of the plasmid pXOl, coding for the lethal protein LF is mutated. Such a recombinant strain expresses the toxin formed by the combination PA + EF. In this case the LF protein is no longer expressed or is expressed in the form of an inactive protein. This strain which will be designated in the following by LF strain ^" has been shown to be a stable strain in animals and capable of inducing protective antibodies against B. anthracis.

The inventors have noticed that a deletion at the level of the lef gene under the conditions set out above does not modify the properties of the bacterium apart from the absence of toxicity resulting from the modification at the level of the LF protein and does not is also not lethal to the bacteria.

A recombinant strain of B. anthracis which is particularly preferred in the context of the implementation of the invention is therefore characterized in that it is capable of expressing the proteins PA and EF and in that it does not express the LF protein.

In this regard, the invention relates in particular to the strain LF ^" (RP10) deposited on May 2, 1991 at the CNCM (National Collection of Culture and Microorganisms) under the number 1-1095.

According to another advantageous embodiment of the invention, the recombinant strains of B. anthracis are characterized in that the mutated pXOl gene is the cya gene coding for the edematogenic protein EF.

In this case the plasmid pXOl either does not express the EF protein, or expresses it in an inactive truncated form as regards its toxic nature.

A preferred strain EF ^" is the strain RP9 deposited on May 2, 1991 at the CNCM under the number 1-1094.

In particular, strains producing only the toxin PA + LF are obtained. Once the lethality of the LF protein can be controlled, these strains can be used for the production of immunogenic compositions.

According to another embodiment of the invention, the recombinant strain of B. anthracis entering one of the groups defined above, is characterized in that the plasmid pXOl is further mutated at the level of the gene coding for the protein PA, in such a way that this protein is either not expressed in the recombinant strain, or is not functional in the recombinant strain, as regards its role in the toxic activity of the strain by attachment to the cell membranes of the host.

According to this embodiment, the strain formed produces only the EF protein or only the LF protein or one of these proteins accompanied by an inactive PA protein with regard to toxicity. Consequently, the induction of the toxic effect resulting from the penetration of B. anthracis into the cells via the PA protein cannot take place and the toxic effect due to LF or EF cannot be manifested.

A strain thus defined deficient in PA, at least in active form is the strain RP8 deposited at the CNCM under the number 1-1093, on May 2, 1991.

Other strains of interest in the context of the invention include a mutation in several of the protein factors involved in the pathogenicity of B. anthracis. In particular, these strains can be "doubly mutant" and in this regard, we will mention the strains RP31 (EF ⁺ , LF ^" , PA ^" ), RP4 (LF ⁺ , EF ^" , PA ^" ) and RP42 (PA \ EF ^" , LF ^" ).

Such strains can therefore also be of interest for the production of vaccines.

The DNA cassette integrated into the recombinant B. anthracis strain at the level of pXO1 carries, according to a first embodiment of the invention, a gene for resistance to an antibiotic and in particular a gene for resistance to anamycin. Other resistances o can be implemented, for example through resistance to erythro-ycine.

According to another embodiment of the invention, this cassette is provided with a metabolic marker and its presence in the recombinant strain is verified by a reaction of the enzyme-substrate type.

Whatever its nature, the cassette allows the selection of strains of B. anthracis which have undergone a mutation at pXOl level.

The deleted fragment of the gene involved in the toxic activity at pXOl level may consist of all or part of the cya gene or of the lef gene in particular. The size of the deleted fragment is advantageously greater than 0.1 kb if the absence of production of the protein naturally encoded by the gene is sought, without taking into account the part of the gene essential for the expression of this protein.

Advantageously, the deleted fragment of the cya gene or of the lef gene is a fragment of at least 0.5 kb, preferably greater than 1 kb. The deletion can relate to the promoter of the cya or lef genes but it also includes the deletion of a part of the coding sequence of the protein in order to avoid in particular any reversion of the deleted gene.

It is understood that recombinant strains of B. anthracis can also be characterized by a mutation of the plasmid pXOl by mutation of the two genes lef or cya, according to what is described above for each of the genes. These strains can be used in the context of the invention for the production of protective antibodies.

In another particular embodiment of the invention, the recombinant strain of B. anthracis is further characterized in that it further comprises a heterologous nucleic acid, if necessary mutated, coding for a determined immunogenic factor, under the control of regulatory elements allowing its expression in B. anthracis.

This heterologous gene codes for a so-called immunogenic factor, capable of driving the production of antibodies in a host to which the recombinant B. anthracis is administered. This immunogenic factor can benefit, if appropriate, from the environment provided by B. anthracis in which it is expressed, to lead to the production of neutralizing antibodies.

In other words, the heterologous factor is immunogenic as such or is constituted by a hapten rendered immunogenic by virtue of its expression within B. anthracis and / or by virtue of its association with a carrier molecule. In this case, the recombinant B. anthracis strain behaves as a vector of immunogenicity or as a carrier strain.

Interestingly, it is therefore possible to associate in B. anthracis the mutated nucleotide sequences of the plasmid pXOl with a heterologous nucleic acid coding for any amino acid sequence capable of being of interest in veterinary or human medicine from the point of view of vaccination.

The heterologous sequence can be transferred into B. anthracis using a plasmid, in particular a replicable and mobilizable conjugation plasmid.

Following its integration into B. anthracis, this sequence can be maintained on this exogenous plasmid or, on the contrary, integrated, for example, into the plasmid DNA of pXOl under conditions which do not affect the characteristics of the recombinant strain modified according to the above. in terms of its toxicity. Advantageously, the recombinant strains of B. anthracis can be stored and used in the form of spores. Spores represent an interesting mode of conservation because of their transient latent nature. Once administered to the animal or human host these spores germinate to give the recombinant bacteria.

The invention also relates to an immunogenic composition allowing the production of neutralizing and protective antibodies against B. anthracis in the host to which it is administered, characterized in that it comprises, as active principle, a recombinant strain of B. anthracis according to the invention, in mixture with an acceptable pharmaceutical vehicle.

As an example of an immunogenic composition, mention may be made of one which would include, as active principle, the strain RP42 or the strain RP9.

The invention also relates to a mixed vaccine composition characterized in that it comprises, as active principle, a recombinant strain of B. anthracis of the invention under conditions such that its administration to a determined host allows the production of protective antibodies both against infection with B. anthracis and against the organism from which the heterologous amino acid sequence originates.

Such compositions can be administered by injection or by any mode of administration usually used for vaccination.

Advantageously, they can also comprise an adjuvant making it possible to increase the level of the reaction.

The doses which can be administered in particular to animals, of these compositions, will be determined according to the animal which it is sought to protect. We considers that the administration of doses ranging from 10 to 100 times, or even more, the dose of Sterne strain used (10 ⁶ strains in mice), can be considered.

Also within the scope of the invention is a recombinant vector, in particular chosen from vectors of the conjugative type capable of infecting both Gram-negative and Gram-positive bacteria, in particular E. coli and B. anthracis, stable and not integrative in B. anthracis and comprising at a site which is not essential for its replication, on the one hand a mutated sequence of at least one gene coding for a protein responsible for the toxic effect of the toxins of B. anthracis, such so that it does not allow the production of this protein or that this protein is produced in an inactive form with regard to the toxic effect, and on the other hand a DNA cassette capable of behaving like a reporter, by example by conferring resistance to an antibiotic for example kanamycin or by allowing a metabolic reaction on a given substrate, when this vector is introduced into B. anthracis.

A preferred recombinant vector is the vector in which the mutated pXO1 gene is the cya gene. According to another advantageous embodiment of the invention, this recombinant vector comprises the lef gene mutated at the plasmid pXOl level.

Other usable recombinant vectors are characterized in that the plasmid pXOl is mutated at the level of two genes, for example at the level of the LF and PA genes, or EF and PA or EF and LF.

Vectors which are particularly advantageous for carrying out the invention are the plasmid PMMA110 and the plasmid pCPLUO. The recombinant vector according to the invention can also contain, in addition to the sequences described above, a heterologous DNA coding for a determined immunogenic protein.

The invention further relates to a process for the preparation of a recombinant strain of B. anthracis corresponding to the above definitions, comprising the following steps:

the modification of specific strains of B. anthracis by a recombinant vector as described above under conditions allowing the integration of the mutated sequence of the cya gene or of the mutated sequence of the lef gene as well as of the reporter cassette at the level the plasmid pXOl and, where appropriate, the heterologous nucleic acid, such that the mutated cya sequence or the mutated lef sequence is inserted by homologous recombination into pXOl respectively at the level of the cya gene or of the original lef gene,

the recovery of the recombinant B. anthracis strains, after elimination of the recombinant vector not integrated in pXOl.

The DNA sequences coding for a protein responsible for the toxic effect of B. anthracis also fall within the scope of the invention, characterized in that they are mutated by deletion under conditions such as the protein for which they code. naturally is not produced or is produced in an inactive truncated form with regard to toxicity, in particular what it is the coding sequences of the lef gene or of the cya gene.

The invention also relates to the plasmid pXOl mutated according to what has been defined above at the level of the lef gene or the cya gene. Other advantages and properties of the invention also appear in the figures and examples which follow.

Figure 1: diagram of the mutagenesis of the cya gene (A) and the lef gene (B)

Figure 2: proteins contained in different supernatants of B. anthracis cultures analyzed by immunoblot and verification with anti EF (A /), anti LF (B /) or anti PA (C /) immunosera Samples 20 μg of:

1/7702 strain of B. anthracis 2 / EF ^" strain of B. anthracis 3 / PA ^" strain of B. anthracis 4 / LF ^" strain of B. anthracis

E X E M P L E S

I - CONSTRUCTION OF MUTANT STRAINS

MATERIALS AND METHODS

Bacterial strains, plasmids and culture media

The bacterial strains and the plasmids used are listed in Table I. The shuttle conjugation vector between Escherichia coli and Bacillus anthracis is pAT18, used in these experiments to transform B. anthracis. It contains an erythromicin resistance gene capable of being expressed in both Gram negative and Gram positive bacteria.

L broth or L agar media were used to cultivate E. coli (Miller 1972, Experiments in molecular genetics, Cold Spring Harbor Laboratory). B anthracis was cultivated routinely in a BHI medium (Difco laboratories) or in a NBY medium for the preparation of spores. An R medium (Ristroph and Ivins 1983, Inf. Imm. 39: 483-486) was used for the production of toxins. Antibiotics were used at the following concentration: ampicillin at 100 μg / ml in an E. coli culture; kanamycin at 50 μg / ml and 20 μg / ml respectively in cultures of E. coli and B. anthracis; erythromycin at 180 μg / ml and 10 μg / ml for cultures of E. coli and B. anthracis respectively. DNA techniques

pXOL was prepared according to the method of Green et al 1985 (Inf. Imm. 49: 291-297). The preparation of plasmid DNA from E. coli and B. anthracis was carried out according to the method of Birnboim and Doly (Methods for recombinant DNA technology Maniatis et al 1982). The DNA cassette conferring resistance to kanamycin was prepared using the Geneclean kit (Bio 101, La Jolla, California) after electrophoresis on agarose gel.

Conjugation procedure

The recombinant shuttle plasmids were transferred by conjugation into B. anthracis 7702 (Sterne strain). The filter conjugation system of Trieu Cuot et al (1987 FEMS Microbiology Letters 48, 289-294) was used according to the description of Cataldi et al. E. coli and B. anthracis were grown in L and BHI media. 5 10 ⁸ E. coli cells and 10 ⁸ B. anthracis cells were mixed, washed to remove the antibiotics and loaded onto a 0.45 μ Millipore filter placed on BHI agar. After 15 hours of incubation at 37 ° C, the cells were resuspended and spread on a selective medium containing the appropriate antibiotics. The resistance of B. anthracis to colicins E3 and D was used to select by donor donors sensitive to E. coli. Plasmid DNA was purified from the transconjugants and used to transform E. coli. Plasmids were tested for absence of rearrangements by analysis with restriction enzymes. Isolation of mutant strains of B. anthracis

The mutant strains of B. anthracis were obtained by culture for several generations in the absence of erythromycin but in the presence of kanamycin pcr select the recombination events between the mutated gene containing the cassette and pXOl and to select the strains having lost the recombinant plasmid. Among all the clones tested resistant to kanamycin, all were sensitive to erythromycin and devoid of plasmids. The characterization of the recombinants was completed by preparing the DNA of pXOl and by subcloning the genes inactivated in pUC18 in E. coli. The plasmid DNA was purified from the Kan ^r -Amp ^r transformants (kanamycin-resistant to ampicillin-resistant) and tested for the absence of rearrangements, by analysis with restriction enzymes.

Adenylate cyclase test

Adenylate cyclase was tested in culture supernatants of B. anthracis in R media as described by Ladant 1988 (J Biol Chem 263: 2612-2618). The enzymatic activity is expressed in units / ml corresponding to 1 nM / min / ml.

Protein analysis

An electrophoresis on 8% polyacrylamide gel in the presence of SDS was carried out according to the description by Laemmli 1970. The gels were either stained with coomassie blue or subjected to an immunoblot analysis (Towbin et al 1979, Proc. Natl. Acad. Sci. USA 76: 4350-4354). Western blots were made with a serum obtained from rabbits immunized either with the EF62 protein or with the LF protein of B. anthracis purified from the gel. Immunodetected proteins were visualized using protein A labeled with 1251 and by autoradiography with X-ray films.

Spore preparation

For the preparation of B. anthracis spores, a colony is streaked on an NBY medium in inclined agar and incubated for 7 days at 30 ° C. Spore formation was checked by microscopic examination. 5ml of sterile distilled water was added to each tube. The spore suspension was transferred to a sterile tube incubated in water at 65 ° C for 30 minutes to kill any bacilli that may exist in this culture. The culture containing at least 90% of spores and 10 ⁸ CFU / ml was collected by centrifugation and suspended in water (1/20 v / v of the original culture). The dilutions of all the spores were spread out for counting viable spores on a BHI agar medium. The set of spores was divided into aliquots in 5 ml and stored at 4 ° C.

Before each infection experiment, the aliquots were diluted in physiological water and the dilutions were spread on a BHI agar dish for determination by counting the viability.

The spores of B. subtilis were prepared in the same way. Mice infection

Uncontaminated female Swiss mice aged 3 to 6 weeks were supplied by the Saint Aubin Les Elboeuf laboratory, France. The animals were fed with sterilized water supplemented with vitamins (pH 3). Mortality was followed by inoculating subcutaneously (se) groups of 10 mice with appropriate dilutions of spore suspension (in a volume of 0.2 ml) obtained from the parental strain or mutants. Virulence was estimated by determining the 50% lethal dose (LD 50) in groups of mice 3 to 10 weeks old by the statistical method. The survival of the bacteria was followed in the tissues of the host by infecting the mice at the level of the right plantar pad (in a volume of 0.1 ml) with large doses of spore suspension coming from the tested strains. Groups of 5 mice were sacrificed at given intervals and their feet were sectioned, washed in sodium hypochloride solutions (1 min) to kill skin contaminants and with sterile PBS pH 7.2 (1 min ). Then 0.1 ml volumes of the serial dilutions (10 times) were spread on an agar mixture containing trypticase. The colonies were counted after 16 hours of incubation at 37 ° C and the results were expressed as the log 10 of bacterial counts per foot. At the same time the inflammation reaction induced in the plantar pad of infected mice was measured at given intervals using a caliper (Schelltaster, Hessen). RESULTS

Construction of mutant strains of B. anthracis deficient in EF toxin or LF toxin

The genes coding for the LF protein (lef gene) or for the EF protein (cya gene) were inactivated according to the Cataldi technique both by deletion and by insertion of a cassette resistant to kanamycin. The constuction initially carried out in E. coli was transferred by conjugation into the Sterne 7702 strain of B. anthracis. The cya clone and sequence gene (Mock and all988 Gene 64: 277-284) was subcloned into E. coli in the plasmid pUC8 (pMM861).

The recombinant plasmid pMM861 (Labruyère et al 1990 Bioche istry 29: 4922-4928) coding for the cya locus was subjected to a partial enzymatic digestion at the Hind III site so as to create a Hind III deletion fragment of 1 Kb to inside the cya gene. The plasmid was then cut at its unique BglII site in the gene and the 1 Kb DNA cassette conferring resistance to kanamycin was ligated to the plasmid. The resulting recombinant plasmid pMMA862 was characterized from transformants E. coli Ampr, Kanr. The profiles of the plasmid preparation pMMA862 obtained with restriction enzymes showed that the cya gene was mutated in line with what we wanted ^• 1. Then a 4.2 Kb EcoRI-BamHI fragment of the plasmid pMMA862 was inserted into the mobilizable shuttle plasmid pATlδ (Ermr) (pMMAHO).

Mutagenesis of the lef gene was carried out in a similar manner. The lef gene is contained in a 3.5 Kb SacI-ClaI fragment of the plasmid pXOl from B. anthracis. This fragment was first cloned into pUClδ and has allowed to express the lef gene in E. coli cells carrying the corresponding plasmid pCPL11. The 3.5 Kb fragment was then inserted into pAT18 (pCPLlOO). The recombinant plasmid pCPLlOO was subjected to a partial enzymatic digestion with EcoRI so as to create an EcoRI deletion fragment of 0.8 Kb inside the gene. The resulting plasmid was cut at its unique Xhol site inside lef and the Kan ^r cassette was ligated to the plasmid. The corresponding recombinant plasmid pCPLUO was characterized from the transformants E. coli, Erm ^r , Kan ^{1 *} . The restriction enzyme profile of pCPLUO showed that the lef gene was mutated as desired.

The inactivated genes were then transferred into B. anthracis 7702: PMMA110 and pCPLUO carrying respectively the mutated cya and lef genes made it possible to transform E. coli HB101 carrying pRK212.1 for mobilization. The strategy of transformation by the vector developed by Trieu Cuot was used between E. coli and the B. anthracis 7702 strain of Sterne so as to transfer the DNA constructs made in E. coli to B. anthracis by conjugation.

Although the transfer frequencies were low (10 ^"8 ) of the B. anthracis Kan ^r , Erm ^r transconjugants were obtained. The homologous recombination events between PMMA110 or pCPLUO and pXOl were then selected to verify the introduction of the gene Inactive cya or inactive lef gene in pXOl. For this, the transconjugants were cultivated over several generations without erythromycin (Erm) (antibiotic selection for the shuttle plasmid) but in the presence of kanamycin (Kan) (selective for the cassette). All the clones tested according to this procedure were sensitive to Erm and resistant to Kan, showing loss or PMMAllO pCPLUO and 1 ^"integration cassette Kan ^r in pXOl. The B. anthracis strains presumed to be recombinant EF ^" or LF ^" carrying respectively pXOl-cya ^" or pXOl-lef ^" were first characterized according to the technique described in the Materials and Methods section by analysis with restriction enzymes then by the test of 1 ^• adenylate cyclase and immunoblot experiments.

Production of PA, LF and EF toxins in culture supernatants of mutant strains of B. anthracis

The adenylate cyclase activity of the EF component was determined in different strains of B. anthracis (Table 2). No activity was detected in the culture supernatants of the mutant strain EF ^", thus confirming ^1" cya gene inactivation on pXOl. In the culture supernatant of the mutant strain LF ^"adenylate cyclase activity was the same (200 u / ml) than the activity usually found in the culture supernatants of strains 7702.

The mutant B. anthracis strains were then characterized by immunoblot analysis using a serum directed against PA, EF or LF. The sera were obtained according to the procedure described in Materials and Methods. As shown in FIG. 2, the three proteins making up the two toxins of B. anthracis, PA, EF and LF are produced in B. anthracis 7702 when the bacteria are cultivated on an R medium under defined conditions. No LF-related components were produced by the LF ^* mutant strains when PA and EF were produced. Furthermore, no EF-related component was detected in the supernatants of the mutant strains. EF ^" , confirming the absence of adenylate cyclase activity in the culture supernatants of the EF mutants ^" (Table 2). In the culture supernatants of the PA mutants ^", any components related to PA was detected while LF and EF were still produced.

Virulence of different strains of B. anthracis in mice

Groups of 10 Swiss mice were inoculated with increasing doses of different strains of B. anthracis, and mortality was followed for 15 days. The strain 7700 corresponding to the Sterne 7702 strain devoid of the plasmid pXOl coding for the toxins was completely avirulent (LD 50 10 ⁹ ), as are the mutant strains PA ^" obtained by Cataldi. The inactivation of EF significantly influenced the LD 50 per 1.0 log unit (LD 50 10 ⁷ spores per mouse against 10 ⁶ spores per mouse for the strain 7702 Tern). This indicates that the expression of virulence was partially affected by the absence of adenylate. On the contrary, lethality was completely abolished in the absence of the lethal factor since the strains of B. anthracis mutants LF ^" were avirulent (LD 50 10 ⁹ ). The mutant strain LF ^" completely avirulent but nevertheless producing the toxin of edema (PA + EF) could induce edema of the skin after subcutaneous injections, whereas the mutant strains PA ^" or EF ^" did not induce It was important to verify this property in vivo and for this, mice were inoculated with different strains of B. anthracis (10 ⁸ , 10 ⁷ or 10 ⁶ spores per mouse) in the right plantar pad. edema in the foot pad was measured with a caliper. results obtained are reported in FIG. 2. The mutant strain LF ^" was capable of inducing edema of the skin like the strain Sterne 7702. On the contrary, the mutant strains of B. anthracis 7700 PA ^" or EF ^" did not induce edema of the skin. For all strains of B. anthracis, the inflammation reaction increased 2 days after the injection (the mice died in this experiment, the LD 50 in the leg pad was stronger 10 ^8). When the mice were inoculated with 10 ⁸ spores of strain 7702 Sterne edema was observed for 6 days, with the mutant strain LF ^'edema was visible for 2 days only. with strain B. subtilis MSY no inflammation reaction (10 ⁶ , 10 ⁷ or 10 ⁸ spores in suspension) was observed Finally, to verify whether the induction of edema was dependent on bacterial survival in the mouse paw pad infected, a bacterial culture has been This was followed when the mice were inoculated with 10 ⁸ spores of all the strains of B. anthracis tested.

For all B. anthracis strains, the log 10 of bacterial count per leg decreased rapidly 24 hours after injection by more than 1.0 log unit and remained stable for 10 days. This indicated that the induction of edema was not correlated with bacterial survival at the point of inoculation. When the mice were inoculated with a suspension of 10 ⁸ spores of B. subtilis, the log 10 of the bacterial count decreased significantly. The spores were destroyed 5 days after infection. These data demonstrated that the B. anthracis strain (even strain 7700) survived longer than the B. subtilis strain in host tissue.

REPLACEMENT SHEET TABLE 1:

Bacterial strains and plasmids used

Bacterial strains Plasmid strains Characteristics

B. nthracis 7702 pXOl PA ⁺ EF ⁺ LF ⁺

B.anthracis 7700 Nal ^r

B. anthracis RP8 PA ^" pXOl-pag 322 PA ^" EF ⁺ LF * Erm ^r

B. anthracis RP9 EF ^" pXOl-cya 303 PA ⁺ EF ⁺ LF ^" Kan ^r

B.anthracis RP10 pXOl-lef 238 PA ⁺ EF ⁺ LF ^" Kan ^r

LF ^" E.COli HB101 ppRRKK221122..11 Amp ^r

E.COli JM105 B. Subtilis SMY Plasmids pAT18: shuttle plasmid pXOl, Erm ^{1 *} (Trieu Cuot et al, 1987) pCPLlOO: recombinant plasmid pATlδ carrying the 3.5 kb Clal-Sacl DE fragment of PXOl encoding LF. Erm ^r pCPLUO: derivative of pCPLlOO carrying a Kan ^r cassette of 1.5 kb inserted into the structural gene

LF deleted from a 0.8 kb EcoRI Kan ^r fragment.

Erm ^r pMMA861: recombinant plasmid pUC8 carrying the 3.17 kb EcoRI-BamHI fragment of pXOl coding for EF (Mock 1987) Amp ^r pMMA862: derivative of pMMA861 carrying the Kan ^r cassette of 1.5 kb inserted into the structural gene

EF deleted from the HindIII fragment Amp ^r fragment

Kan ^r pMMUO: insert of 3.57 kb of pMMA861 DNA cloned into pATlδ Erm ^r , Kan ^r B. anthracis 7702 Sterne strain 1939 J Vet Sci Ani

Indust 13: 313-317

B. anthracis 7700 strain derived from 7702 devoid of pXOl

B. anthracis RP8 strain derived from 7702 E.coli HB101 Trieu Cuot et al, 19δ7 FEMS

Microbiol Letters 4δ: 289-294

E.coli JM105 Yannish-Perron et al, 19δ5 Gene

33: 103-109

TABLE 2:

Adenylate cyclase activity in the various culture supernatants of B. anthracis

Strains Adenylate cyclase activity nmol / min / ml supernatant

Mutant EF ^" 0

Mutant PA ^" 300

Mutant LF ^" 200

Tern strain 7702 200

Construction of doubly mutant strains of B. anthracis

These strains were constructed according to the techniques described in the previous pages. They produce only one of the PA, EF or LF components. Methods

The names of the strains and their characteristics are as follows:

- RP31: EF ⁺ LF ^" PA ^"

The lef gene is inactivated by insertion of the kanamycin resistance cassette (Kan ^R ).

The pag gene is inactivated by insertion of the erythromycin resistance cassette (Erm).

- RPA4: LF ⁺ EF ^" PA ^"

The pag gene is inactivated by insertion of the Erm ^R resistance cassette.

The cya gene is inactivated by insertion of the Kan resistance cassette.

- RP42: PA ⁺ EF ^" LF ^*

The cya gene is inactivated by insertion of the Erm ^R resistance cassette.

The lef gene is inactivated by insertion of the Kan ^R resistance cassette.

These three strains were characterized biochemically by analysis of the proteins contained in their culture supernatants (according to the Western Blot method).

II - IMMUNOPROTECTION EXPERIENCES

Experiments have been undertaken in mice, in order to study the immunoprotective power of the mutant strains. The animals received, subcutaneously, spores of RP42 (PA ⁺ , EF ^" , LF ^" ) and RP9 (PA ⁺ , EF ⁺ , LF ^" ) a control batch of animals receiving nothing.

After 40 days, the challenge was achieved by subcutaneous injection of a lethal dose (1.5 × 10 ° spores) of the Sterne strain.

The percentage of surviving animals was determined:

90% and 85% of the animals having received RP42 and RP9 respectively survived the injection of the lethal dose of the Sterne strain, while no protection was observed in the control group.

These first results are significant and indicate that the strains constructed are capable of inducing active immunoprotection in mice.

Claims

1. Recombinant strain of B. anthracis, characterized by:

- the mutation on the plasmid pXOl of at least one given gene coding for a protein responsible for a toxic effect in B. anthracis, this mutation resulting in:

. deletion of all or part of this gene coding for a protein responsible for a toxic effect and, insertion at the deletion site of this gene in pXOl, of a DNA cassette allowing the selection of the strain thus modified and preventing the reversion of the recombinant strain, the gene thus mutated then being either devoid of the capacity to produce the protein responsible for the toxic effect for which it codes, or capable of coding for a truncated protein devoid of its toxic character.

2. Recombinant strain of B. anthracis according to claim 1, characterized in that it is devoid of the plasmid pX02, in particular in that it is the strain STERNE 7702.

3. Recombinant strain of B. anthracis according to any one of claims 1 or 2, characterized in that the mutated gene of pXOl is the lef gene coding for the lethal protein LF.

4. Recombinant strain of B. anthracis according to any one of claims 1 or 2, characterized in that the mutated gene of pXOl is the cya gene coding for the edematogenic protein EF.

5. Recombinant strain of B. anthracis according to any one of claims 1 to 3, characterized in that that it is capable of expressing the PA and EF proteins and that it does not express the LF protein.

6. Recombinant strain of B. anthracis according to any one of claims 1 to 4, characterized in that the plasmid pXOl is further mutated at the level of the gene coding for the PA protein, so that the PA protein is n is not expressed in the recombinant strain, or is not functional in the recombinant strain.

7. Recombinant strain of B. anthracis according to one of claims 1 or 2, characterized in that it is doubly mutated at the level of the plasmid pXOl.

8. Recombinant strain of B. anthracis according to claim 7, characterized in that the lef and pag genes are inactivated, and in particular in that it is the strain RP31.

9. Recombinant strain of B. anthracis according to claim 7, characterized in that the pag and cya genes are inactivated, and in particular in that it is the RP4 strain.

10. Recombinant strain of B. anthracis according to claim 7, characterized in that the cya and lef genes are inactivated, and in particular in that it is the RP42 strain.

11. A recombinant strain of B. anthracis according to any one of claims 1 to 10, characterized in that the DNA cassette carries a gene for resistance to an antibiotic, for example kanamycin or a metabolic marker.

12. Recombinant strain of B. anthracis according to any one of claims 1 to 11, characterized in that the deleted fragment of the pXOl gene is a fragment of at least 0.1 kb, preferably 1 kb, enough to prevent the production of the protein it naturally codes for.

13. A recombinant strain of B. anthracis according to any one of claims 1 to 12, characterized in that it also comprises a heterologous nucleic acid, if necessary mutated, coding for a determined factor which is immunogenic or capable of being so, under the control of regulatory elements allowing its expression in B. anthracis.

14. Recombinant strain of B. anthracis according to claim 13, characterized in that the heterologous nucleic acid is carried by the plasmid pXOl.

15. A recombinant strain of B. anthracis according to any one of claims 1 to 14, characterized in that it is in the form of spores.

16. Recombinant strain according to claim 2 or 4, characterized in that it is the strain RP9 deposited on May 2, 1991 at the CNCM under the number 1-1094.

17. Recombinant strain according to claim 2 or 3, characterized in that it is the strain RP10 deposited on May 2, 1991 at the CNCM under the number 1-1095.

18. Recombinant strain according to claim 1 or claim 2, characterized in that it is the strain RP8 deposited on May 2, 1991 at the CNCM under the number 1-1093.

19. Immunogenic composition, allowing the production of neutralizing and protective antibodies against B. anthracis in the host to which it is administered, characterized in that it comprises, as active principle, a recombinant strain of B. anthracis according to any one of claims 1 to 12, 16 to 18 in admixture with an acceptable pharmaceutical carrier.

20. Mixed vaccine composition, characterized in that it comprises, as active principle, a strain recombinant B. anthracis according to any one of claims 13 to 18, under conditions such that its administration to a determined host allows the production of protective antibodies both against infection by B. anthracis and against the organism of which the heterologous amino acid sequence is derived.

21. Immunogenic composition according to any one of claims 19 or 20, characterized in that it comprises, as active principle, RP42 or RP9 strains.

22. Immunogenic composition according to any one of claims 19 to 21, characterized in that it comprises an adjuvant.

23. Recombinant vector, especially chosen from conjugative type vectors capable of infecting both Gram-negative and Gram-positive bacteria, in particular E. coli and B. anthracis, stable and non-integrative in B. anthracis and comprising at a site which is not essential for its replication, on the one hand a mutated sequence of at least one gene coding for a protein responsible for the toxic effect of the toxins of B. anthracis, in such a way that it does not allow the production of this protein or that this protein is produced in an inactive form as regards the toxic effect and on the other hand a DNA cassette capable of behaving as a reporter, for example by conferring resistance to an antibiotic by for example kanamycin or by allowing a metabolic reaction on a given substrate, when this vector is introduced into B. anthracis.

24. Recombinant vector according to claim 23, characterized in that the mutated pXOl gene is the lef gene.

25. Recombinant vector according to claim 23, characterized in that the mutated pXO1 gene is the çya gene.

26. Recombinant vector according to claim 23, characterized in that the plasmid pXOl is mutated at the level of two genes, and in particular at the level of the LF and PA or, EF and PA or, EF and LF genes.

27. Recombinant vector according to any one of claims 23 to 26, characterized in that it also contains a heterologous DNA coding for a specific immunogenic protein.

28. DNA sequence coding for a protein responsible for the toxic effect of B. anthracis, characterized in that it is mutated by deletion under conditions such that the protein is not produced or is produced in a truncated form inactive, the mutation of the sequence being in particular located at the level of the lef gene or of the cya gene.

29. Process for the preparation of a recombinant strain of B. anthracis according to any one of claims 1 to 6 or 16 to 18 comprising the following steps:

modification of specific strains of B. anthracis by a recombinant vector according to any one of claims 23 to 25 or 27 under conditions allowing the integration of the mutated sequence of the cya gene or of the mutated sequence of the lef gene as well as of the reporter cassette and, where appropriate, of the heterologous nucleic acid at the level of the plasmid pXOl in such a way that the mutated cya sequence or the mutated lef sequence is inserted by homologous recombination into pXOl respectively at the level of the cya gene or of the lef d gene 'origin, recovery of the recombinant B. anthracis strains, after elimination of the fraction of the recombinant vector not integrated into pXOl.