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WO1993015212A1 - Attenuated mutant of listeria monocytogenes; recombinant strain of listeria monocytogenes, use as heterologous vaccinal antigene vectors and use as vaccine or diagnostic composition - Google Patents

Attenuated mutant of listeria monocytogenes; recombinant strain of listeria monocytogenes, use as heterologous vaccinal antigene vectors and use as vaccine or diagnostic composition Download PDF

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WO1993015212A1
WO1993015212A1 PCT/FR1993/000105 FR9300105W WO9315212A1 WO 1993015212 A1 WO1993015212 A1 WO 1993015212A1 FR 9300105 W FR9300105 W FR 9300105W WO 9315212 A1 WO9315212 A1 WO 9315212A1
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listeria monocytogenes
gene
mutant
antigen
recombinant strain
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PCT/FR1993/000105
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French (fr)
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Pascale Cossart
Christine Kocks
Pierre Goossens
Geneviève Milon
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Institut Pasteur
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/0208—Specific bacteria not otherwise provided for
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated

Definitions

  • Attenuated mutant of Listeria monocytogenes recombinant monocytogenic strain of Listeria, use as heterologous vectors of vaccine antigen and use as vaccine or diagnostic composition
  • the present invention relates to an attenuated mutant strain of Listeria monocytogenes and its immunotherapeutic and diagnostic applications, in particular for the manufacture of a recombinant strain which can be used as a vaccine.
  • Listeria monocytogenes is an optional aerobic, non-sporulating, gram-positive bacillus widely distributed in the environment and responsible for human and animal listeriosis.
  • the disease manifests itself by opportunistic infections, either by meningitis and / or encephalitis, septicemia or by abortions, with a high mortality rate in newborns and adults whose defense mechanisms are weakened by pregnancy, therapeutically induced immunosuppression, underlying disease or old age. Listeriosis can also affect apparently healthy people.
  • Listeria monocytogenes is capable of infecting a wide variety of cell types, in vivo and in vitro, including macrophages, fibroblasts, epithelial cells and enterocytes.
  • the bacteria After entering the infected cell, the bacteria lyses the phagosome membrane with a hemolysin that it secretes. At the end of this stage, the bacteria is in the cytoplasm of the host cell.
  • Listeria monocytogenes is characterized by its ability to spread through tissues by direct cell-to-cell infection without leaving the cytoplasm (Racz et al., 1970 (9)).
  • actin F filamentous actin
  • Polymerized actin is made up of short, randomly oriented microfilaments that differ from the long actin filaments usually observed in muscle cells.
  • the bacteria are mobile and leave behind F-actin "comets" several microns in length. Some are incorporated into finger-shaped cytoplasmic protrusions, which can be internalized by neighboring cells. The two plasma membranes surrounding the bacteria are then lysed. Once in the cytoplasm of the new host cell, the bacteria can reproduce and start a new cycle of dissemination.
  • act A The gene coding for this protein, called act A, is part of an operon (Mengaud et al., 1991 (b) (8)) whose complete nucleotide sequence has recently been described (Vazquez-Boland et al., 1992 (12 )).
  • the present invention thus relates to a strain of listeria monocytogenes with attenuated virulence, characterized in that it comprises, in the act A gene or in the promoter thereof, a mutation capable of blocking or substantially modifying the expression of the protein encoded by the act A gene.
  • the mutation can be carried out according to the known techniques, in particular by insertion into the act A gene or its promoter, of a sequence of one or more bases, preferably a stable transposon, deletion of one or more bases, mutations such as mutations by site-directed mutagenesis, for example by PCR and in particular missense mutations.
  • the mutation can in particular be carried out by insertion of a transposon, such as the transposon Tn 917-lac, as described by Mengaud et al., 1991 (a) (7).
  • a transposon such as the transposon Tn 917-lac
  • the mutation is preferably carried out in the DNA fragment coding for the peptide sequence with repeating motifs comprised between amino acids 235 to 315, 350 to 360, 367 to 385 and 389 to 393 of the peptide sequence SEQ ID No. 1.
  • Another advantageous mutation site in particular for insertion, is located downstream of adenosine at position 497 of the nucleotide sequence of the act A gene.
  • This position corresponds to that between amino acids 61 and 62 of the peptide sequence SEQ ID No. 1.
  • a particularly preferred strain according to the present invention is the strain of Listeria monocytogenes LUT 12 deposited in the National Collection of Cultures of Microorganisms (C ⁇ CM) on January 30, 1992 under the number 1-1167.
  • mutants according to the invention are capable of conferring on hosts to which they have been administered protection against a subsequent infection by a pathogenic strain of Listeria monocytogenes.
  • the invention therefore also relates to a human or veterinary vaccine comprising as active component an attenuated strain of Listeria monocytogenes, as defined above.
  • This vaccine is capable of providing effective protection to humans or animals, in particular cattle and sheep against listeriosis.
  • the immune response generated by the administration of an attenuated mutant as defined results in the proliferation essentially of T lymphocytes of the CD8 subclass.
  • CD8 subclass T cells are activated by peptides linked to class antigens
  • the attenuated mutants of Listeria monocytogenes according to the invention are capable of stimulating the immune system by the route which uses MHC class I molecules.
  • the invention thus also relates to a recombinant mutant of Listeria monocytogenes characterized in that it comprises a heterologous DNA, either inserted into the genome of an attenuated mutant as defined above, or carried by a plasmid replicating in the mutant mitigated.
  • the heterologous DNA preferably consists of a heterologous gene coding for a target protective antigen of T lymphocytes of the CD8 subclass.
  • This antigen may be of bacterial origin
  • LCMV lymphocytic choriomeningitis virus
  • HAV AIDS virus
  • a recombinant mutant of Listeria monocytoge particularly interesting born contains the genes coding for the gag antigen and / or the HIV nef antigen or all or part of the gp 120 envelope of HIV 1 or gp 140 of HIV 2 or by a peptide as defined in US- 4,943,628.
  • the construction of the recombinant mutant can be carried out by transformation of an attenuated mutant as defined above, in particular of the mutant LUT 12 using an appropriate plasmid, and for example electroporation.
  • the cloning of the heterologous DNA will be carried out in E. Coli and a shuttle plasmid E. Coli - Listeria monocytogenes will be used to carry out the transformation.
  • plasmids As plasmids, mention may be made of pMKA 4 (Sullivan et al., (14)) or PHT 320 (Leredus et al., (15)).
  • the gene of interest (heterologous gene) it is advantageous to insert upstream of the gene of interest a strong Listeria promoter, such as the hly promoter.
  • the translation product of the gene of interest can be secreted, it is preferable to merge the gene of interest at the start of hly in order to use the "signal sequence" of listeriolysin O to release the protein of interest encoded by the heterologous gene in the cytoplasm of a host cell.
  • the recombinant mutants of Listeria monocytogenes as defined above are advantageously suitable for the preparation of a recombinant human or veterinary vaccine against an infection caused by a microorganism producing an antigen corresponding to the protein encoded by the heterologous DNA inserted into the recombinant Listeria monocytogenes genome.
  • the vaccines according to the invention can be administered intravenously, subcutaneously, intramuscularly or orally.
  • An appropriate dose is between 5.10 4 and 10 9 cells / kg of weight.
  • This dose varies depending on the route of administration and the sensitivity of the host.
  • the administration is preferably repeated in order to confer effective protection on the host.
  • the recombinant mutants of Listeria monocytogenes defined above are also suitable for the preparation of a diagnostic composition intended for monitoring the protective state of a human or animal host against an infection caused by a microorganism producing an antigen corresponding to the protein encoded by heterologous DNA inserted into the genome of Listeria monocytogenes recombinant or expressed in this strain when carried by a plasmid.
  • P indicates the promoter, the dotted lines the transcript and a potential transcription termination signal.
  • the potential signal sequence and the transmembrane segment are underlined.
  • the repeating pattern region is surrounded.
  • the arrow corresponds to the insertion of Tn 917-lac into the actA gene of the LUT 12 mutant.
  • the numbering begins at the NH 2 end of the mature protein.
  • the residues determined by microsequencing of the 90 kDa band are printed in bold type and marked with an asterisk.
  • the chromosomal DNA of Listeria monocytogenes was prepared as described by Mengaud et al., 1991 (b) (8).
  • the Southern blot probes were prepared by PCR, purified from agarose gels using the Geneclean kit (Bio 101, Inc, La Jolla, CA), and labeled using the Amersham Multiprime system.
  • Hybridizations according to Southern were carried out using the rapid hybridization system (Amersham) on Nylon N membranes (Amersham) in an oven for Hybaid hybridization. II - Isolation of the LUT 12 strain and determination of the transposon insertion point
  • a library of Tn 917-lac mutants produced from the wild type strain L028 and from the plasmid p TV32 carrying the Tn 917-Lac transposon as described by Mengaud et al., 1991 a (7), was screened on plates of egg yolk agar prepared from fresh egg yolk diluted 1: 2 in 150 mM NaCl solution, and adding 12.5 ml of this mixture to 250 ml of agar with added a brain and heart infusion (BHI) at 56 ° C.
  • BHI brain and heart infusion
  • LUT 12 A lecithinase-negative mutant that did not produce egg yolk clouding even after prolonged incubation and exhibited a wild-type phenotype for all other traits examined was called LUT 12.
  • This mutant had both hemolytic activity and an in vitro growth rate identical to the wild type, but was found to be very attenuated in virulence in mice.
  • Tests were carried out on 3T3 fibroblasts (ECA CC88031146). according to the technique described by Kuhn et al., 1990 (5), except that the infections were carried out at various concentrations of inoculum: 1 to 25 ⁇ l of bacterial subcultures of 2 hours (At 600nm of 0.45) either undiluted or diluted 1/10.
  • This test reveals the ability of Listeria monocytogenes to multiply intracellularly and to disseminate on single layers of fibroblasts coated with an agar layer containing gentamicin at a lethal concentration for extracellular bacteria but not for intracellular bacteria . After several days, areas of dead cells destroyed by the bacterial infection are visible to the naked eye in the form of "patches".
  • the mutant LUT 12 bacteria were unable to form plaques on single layers of 3T3 fibroblasts.
  • Suspensions containing macrophages have. were prepared from bone marrow of a C57BL / 6 female mouse aged 7 weeks, and cultured in RPMI medium containing 10% fetal calf serum in the presence of supernatant L. 4.10 5 macrophages derived from bone marrow obtained on the day 6 were sown in round glass strips (diameter 12 mm) the day before use. The macrophages were infected with an MI (multiply of infection) of 0.04 (one bacterium for 25 macrophages, resulting in approximately 1% of infected cells), so as to be able to observe individual points of infection, generated by the offspring of a single bacterium. The infection was carried out as described for a macrophage J774.
  • MI multiply of infection
  • This test was carried out on single layers of J774 in 25 cm 3 plastic tissue culture flasks.
  • the cells were infected with an MDI of 10 bacteria per cell.
  • the number of intracellular bacteria was calculated after 2, 6 and 10 hours of growth on a medium containing gentamicin (5 ⁇ g / ml) by lysis of the cell monolayers, washing with cold distilled water and spreading of appropriate dilutions. on plates containing BHI medium.
  • the intracellular behavior of the LUT 12 mutant was observed under the electron microscope. Macro J774 phages were infected with wild-type bacteria or the mutant strain for 30 minutes, followed by incubation for 60 to 210 minutes in medium containing gentamicin. For the mutant and the wild type, free bacteria could be observed in the cytoplasm at 1.5 hours of infection. At that time, the wild type and the mutant were surrounded by a thin layer of creped granular material, but only the wild type had filamentary material assembled on its surface, consisting of actin filaments. At 4 h after infection, the wild-type bacteria were surrounded by thick layers of actin F filaments. On the contrary, the LUT 12 mutant bacteria were almost naked. Even the thin creped coating observed at the early time of infection was gone.
  • This mutant was analyzed by Southern blot to determine the number of transposons inserted in its chromosome.
  • the chromosomal DNA was digested with Bam HI, Eco RO, Hind III, Kpn I and Pst I.
  • Two different probes corresponding to Tn 917-lac were used (Shaw and Clewell, 1985 (11)): a probe covering 515 base pairs of the erythromycin resistance gene, obtained by PCR with the oligonucleotides 5'-TTG GAA CAG GTA AAG GGC ATT TAA-3 '(position 821 to 844) and 5'-AGT AAA CAG TTG ACG ATA TTC TCG-3' (position 1313 to 1336), and a probe covering the internal Hind III fragment of the transposon obtained by PCR with the oligonucleotides 5'-ACA ATT AAT GTC TCC CAT ATT-3 '(position 3082 to 3102) and 5' (ACT GAT AAT TAA CCA AAA CAG-3 '(position 4295-4315).
  • the transposon-chromosome junction was cloned from a chromosomal DNA library obtained by restriction with Eco RI / Kpn I in pUC 18.
  • a clone comprising an insertion segment, corresponding to the chromosome-transposon junction was isolated and sequenced directly from the plasmid by using an oligonucleotide hybridizing with the right end of the transposon (5'-CTA AAC ACT TAA GAG AAT TG-3 ', position 5244 to 5263).
  • the transposon was inserted after adenine
  • the LUT 12 mutant lecithinase-negative phenotype is probably due to a polar effect of the insertion mutation in act A, since the 3rd gene of the operon plcB codes for lecithinase.
  • Mutants were produced by homologous recombination between the chromosome of Listeria monocytogenes and fragments corresponding to parts of the plcB gene and open reading frames ORFX / Y and ORFZ (fig. (A)) located downstream of the act A gene by insertion of plasmids at various sites.
  • a transformation of the mutant LUT 12 carried out with act A also shows that the wild type phenotype is restored, which excludes the possibility of a spontaneous mutation at another site of the chromosome.
  • the nucleotide sequence of the act A gene suggests that the gene codes for a protein of 639 amino acids with a signal sequence and a transmembrane region (Vazquez Boland et al., 1992 12 ().
  • the bacterial isolates were cultured in 200 ml of brain-heart infusion broth (BHI, DIFCO Laboratories, Detroit, Michigan) supplemented with erythomycin at 5 ⁇ g / ml for LUT 12, with stirring at 160 rpm on a Gyrotory G10 agitator. (New Brunswick Scientific) at 37 ° C for 18 h.
  • the bacteria were collected by centrifugation (5000 g for 20 minutes) and washed three times in a saline solution of phosphate buffer (PBS).
  • PBS phosphate buffer
  • the pellet obtained was resuspended in 4 ml of PBS and SDS was added to a final concentration of 1%. At this concentration of SDS, the cells of L. monocytogenes do not lyse. The absence of bacterial lysis was checked under the microscope. After 5 minutes of stirring at room temperature, the bacteria were centrifuged (50,000 g for 10 minutes) and the supernatant concentrated by ultrafiltration on microconcentrators (Centricom 30, Araicon) and stored at -20 ° C.
  • the protein concentration was determined using the bicinchoninic acid method (Pierce). The protein concentration was adjusted to 300 ⁇ g / ml for electrophoresis. 10 ⁇ l of extract were mixed with 10 ⁇ l of buffer (2% SDS, 10% glycerol, 5% mercaptoethanol, 0.002% bromophenol blue and 0.02 M Tris HCl), boiled for 3 minutes at 100 ° C. The electrophoresis was carried out at 60 mA for 120 minutes through discontinuous polyacrylamide gels
  • the bacteria were then treated with sulfosuccinimido biotin (sulfo-NHS-biotin; Pierce) at a final concentration of 0.5 mg / ml for 2 minutes with moderate agitation.
  • sulfosuccinimido biotin sulfo-NHS-biotin; Pierce
  • the cells were washed three times with PBS at pH 7.4 and extracted by extraction with SDS.
  • the extracts corresponding to 7 ⁇ g of protein per lane were deposited on SDS gels and transferred as described by De Rycke et al., 1989 (2) on nitrocellulose (BA 85, Schleicher and Sch ⁇ ll.
  • the nitrocellulose filters were saturated for overnight in PBS 0.5% gelatin and incubated for 1.5 hours with streptavidin conjugated to peroxidase (Jackson) in PBS containing 0.5% gelatin and 0.1 M Tween 20. After different washes in the same buffer, the reaction bands were revealed with 0.5 mg / ml of 4-chloro-1-naphthol (Biorad) and 0.03% v / v of H 2 O 2 in water. Analyzes of the electrophoresis gels show a 90 kDa band for the wild type which is absent in the LUT 12 strain. This band is also found in the plcB mutants and the LUT 12 mutants transformed by act A mentioned above.
  • the 90 kDA band was isolated and the sequence of the 6 amino acids of the NH 2 end was determined and compared with the amino acid sequence deduced from the nucleotide sequence of the act A gene.
  • the extracts on SDS corresponding to 100 ⁇ g of proteins per channel were boiled in an SDS sampling buffer containing 7% (w / v) of urea before performing electrophoresis on 7.5% SDS gels .
  • the separated proteins were transferred to a Problott membrane (Applied Biosystems) in 50 mM Tris - 50 mM borate for 17 hours at 4 to 5 volts / cm.
  • the proteins were stained for 5 seconds using 0.1% amido black in a solution of 1% acetic acid and 40% methanol, and rinsed thoroughly with water.
  • a 90 kDa strip was cut in several lanes.
  • the membrane proteins were sequenced by degradation according to Edman in a 740 A sequencer from Applied Biosystems, with an on-line HPLC PTH 120 A analyzer programmed by the manufacturer for the Problott membrane.
  • the amino acid sequences were analyzed on a Data General MV 10000 computer at the Scientific Computing Unit of the Institut Pasteur.
  • the Ala-Thr-Asp-Ser-Glu-Asp sequence of the isolated protein corresponds exactly to the amino acids of the cleavage site of the predicted signal sequence according to the peptide-predicted sequence from the act A gene (fig. B).
  • A is a protein of 610 amino acids with a calculated molecular weight of 67 kDA. It has an apparent molecular weight of 90 kDA and is expressed on the surface of the bacteria.
  • LUT 12 has been studied, after intravenous injection, in the spleen and liver of mice, which are the main target organs where L. monocytogenes of wild type express their pathogenicity.
  • the clinical trials used were as follows: the livers and spleens of infected mice were harvested at various times after infection, and homogenized to allow the release of bacteria, and live bacteria were counted in vitro.
  • the LD 50 of the actA LUT 12 mutant, after intravenous injection in C3H pure line mice was a factor 3 log 10 higher than that of wild-type Listeria monocytogenes (2.5 ⁇ 10 7 versus 2.5 ⁇ 10 4 ).
  • the growth kinetics of the actA mutant and the virulent wild-type strain in the liver and spleen were compared. After intravenous injection of a maximum sublethal dose of the actA mutant (1.5 ⁇ 10 7 organisms) or two different doses of virulent L. monocytogenes (7 ⁇ 10 3 or 6 ⁇ 10 4 ), the number of bacteria in the liver and in the spleen of infected mice was determined at varying durations during infection.
  • the persistence of the actA mutant at a stable level for 4 days in the liver may reflect either a balance between bacterial multiplication and bacterial death, or survival of the bacteria without multiplication.
  • the bacterial growth curves in the liver and spleen of mice treated with ampicillin one were compared to those of control mice.
  • Ampicillin inhibits the synthesis of peptidoglycan and is bactericidal on bacteria in the active multiplication phase.
  • the infected mice were treated twice with 15 mg of ampicillin intraperitoneally from day 1, 2 or 3 of the infection; the liver and spleen were removed a day later, and the number of remaining bacteria was determined and compared with that obtained from mice not treated with the antibiotic.
  • actA mutants are deficient in cell-to-cell dissemination in vitro, the persistence of actA in the liver is likely due to infection of neighboring cells after lysis of the first infected host cells; consequently, Listeria monocytogenes can be exposed to bactericidal effectors present in an extracellular medium and its capacity for local dissemination can be reduced.
  • extra-cellular Listeria monocytogenes are phagocytosed by macrophages activated by interferon ⁇ , they may be unable to reach the cytosol and to continue their intracellular cycle.
  • actA mutants were eliminated from the spleen and liver sooner than the wild-type strain, suggesting that the protective effectors of the host are rapidly induced in mice infected with the actA mutant.
  • the inventors determined when the non-specific resistance stopped expressing.
  • mice injected intravenously an unrelated intracellular pathogen, Yersinia enterocolitica Ye8081 0: 8 (16,17) either in naive mice or in mice infected with the mutant actA, 4, 6.5, and 8, 5 weeks before the injection. They compared the number of bacteria in the spleen and in the liver in these two groups of mice. No difference was observed between the two groups at any time during the test. They then performed the following experiments,
  • the LD 50 of wild-type L. monocytogenes was determined in mice infected 6 weeks previously with the actA mutant, and in control mice: a difference of around 100 was observed between the two groups (2 , 2 ⁇ 10 6 and 2.5 ⁇ 10 4 respectively).
  • the growth curves of wild-type L. monocytogenes were compared in the liver and spleen of naive mice and of mice immunized for 6 weeks, during the first 3 days of infection. A significant slowdown in bacterial growth was observed from day 1 in the spleen, and from days 2 to 3 in the liver.
  • this specific inhibition of the growth of wild-type L. monocytogenes was still effective 8.5 weeks after infection with the actA mutant (decrease of 4.01 log 10 in the bacterial count of the spleen 48 hours after an inoculum 5 ⁇ 10 4 ).
  • a protection transfer was carried out in naive syngeneic "receptors" using spleen cells harvested 7 days after an intravenous injection of 1.5 ⁇ 10 7 actA mutant bacteria.
  • the "receptors” were exposed to intravenous infection with a lethal dose of wild-type L. monocytogenes for 1 hour, and the number of bacteria was determined in the liver and in the spleen of the "receptors" two days after infection.
  • the immune splenic cell population namely Thy-1 + lymphocytes, either CD4% or CD8 ⁇ before passive transfer (Table 1 below).
  • the transfer of untreated splenocytes resulted in a 3-4 log 10 reduction in the number of bacteria in the spleen.
  • This protection was transferred by T lymphocytes because a depletion of Thy-1 + lymphocytes abolished the reduction in the burden of bacteria in the spleen.
  • the high level of protection conferred by immune splenocytes after 7 days was only slightly affected by the depletion of the CD4 subpopulation.
  • the majority of the protective effect conferred by the immune splenocytes after 7 days was sensitive to depletion of the CD8 subpopulation, but could not be attributed solely to the CD8 + lymphocyte subpopulation.
  • the actA mutant of L. monocytogenes is thus capable of inducing the generation of specific CD8 + lymphocytes, protecting against Listeria.
  • the actA mutant has a functional listeriolysin-O gene which allows it to escape from the phagosome and enter the cytosol; it is likely that the actA mutant is capable of stimulating the production of CD8 + T lymphocytes which protect against Listeria, recognized arising from natural peptides of L. monocytogenes.
  • the ability of the actA mutant to transiently multiply in the organs of infected mice and to secrete a sufficient amount of bacterial proteins is probably critical to allow efficient production of protective CD8 + lymphocytes.
  • the transposon Tn917-lac is inserted into actA, the second gene of the operon lecithinase, and has a polar effect on the expression of the plcB gene coding for a lecithinase.
  • the results obtained in the present invention indicate that lecithinase does not play an essential role in the induction of protective immunity against L. monocytogenes.
  • the results of the present invention show that actA attenuated mutants are capable of inducing protective CD8 + T lymphocytes against Listeria, and that a state of protective immunity against wild-type L. monocytogenes can be established by a single infection.
  • actA mutants enter the cytosol of infected cells and multiply in this compartment, we can consider their use as a living vector to deliver heterologous proteins in the cytosol and promote the production of CD8 + T lymphocytes; such living vectors capable of transiently multiplying are supposed to deliver a sufficient charge of heterologous proteins in the cytosol, for a short period of time.
  • these Listeria of attenuated virulence can be useful for the screening and characterization of bacterial or parasitic peptides specific for alleles of the locus encoding the molecules of CMH class 1.
  • Some bacteria and parasites reside in the vacuo- laires; in addition, the growth kinetics may be low.
  • this tool could be very useful for defining the specificity of CD8 + lymphocytes which are produced in response to infection.
  • Spleen cells were isolated from C3H mice on day 7 after intravenous injection of 1.5 ⁇ 10 7 actA mutants. Thy-1 + , CD4 + or CD8 + cells were removed in vitro before adoptive transfer.
  • the monoclonal antibodies used here were Anti-Thy-1, 2 J1j (ATCC TIB 184), anti-CD4 RL1724 (Ceredig, R. Lowenthal, JW Nabholz M. and MacDonald, HR, 1985, Nature 314 98), and anti- CD8 31M (Sarmiento M. Glasebrook. AL and Fitch FW, 1980, J. Immunol. 125-2665).

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Abstract

The invention discloses an attenuated mutant of Listeria monocytogenes incorporating in the act A gene or in its promotor a mutation capable of blocking or modifying the expression of the protein coded by the act A gene. This mutant can be used as a livin g vector for the expression of an heterologous ADN, particularly a gene coding for a viral, bacterial or parasitic protector antigene which is the target of T cells of subclass CD8. The recombinant mutant strains thus obtained may be used as a vaccine or diagnostic composition for checking the protection state of a host.

Description

Mutant atténué de Listeria monocytogénes; souche recombinante de Listeria monocytogènes, utilisation comme vecteurs hètèrologues d'antigène vaccinal et utilisation comme vaccin ou composition diagnostique Attenuated mutant of Listeria monocytogenes; recombinant monocytogenic strain of Listeria, use as heterologous vectors of vaccine antigen and use as vaccine or diagnostic composition
La présente invention concerne une souche mutante atténuée de Listeria monocytogenes et ses applications immuno-thérapeutiques et diagnostiques, notamment pour la fabrication d'une souche recombinante utilisable en tant que vaccin.  The present invention relates to an attenuated mutant strain of Listeria monocytogenes and its immunotherapeutic and diagnostic applications, in particular for the manufacture of a recombinant strain which can be used as a vaccine.
Listeria monocytogenes est un bacille aérobie facultatif, non sporulant, à gram-positif très répandu dans l'environnement et responsable de la listériose humaine et animale. La maladie se manifeste par des infections opportunistes, soit par une méningite et/ou encephalite, des septicémies ou par des avortements, avec un taux de mortalité élevé chez les nouveau-nés et les adultes dont les mécanismes de défense sont affaiblis par la grossesse, une immunosuppression thérapeutiquement induite, une maladie sous-jacente ou la vieillesse. La listériose peut aussi atteindre des sujets apparemment sains.  Listeria monocytogenes is an optional aerobic, non-sporulating, gram-positive bacillus widely distributed in the environment and responsible for human and animal listeriosis. The disease manifests itself by opportunistic infections, either by meningitis and / or encephalitis, septicemia or by abortions, with a high mortality rate in newborns and adults whose defense mechanisms are weakened by pregnancy, therapeutically induced immunosuppression, underlying disease or old age. Listeriosis can also affect apparently healthy people.
Listeria monocytogenes est capable in vivo comme in vitro d'infecter une grande variété de types cellulaires, notamment les macrophages, les fibroblastes, les cellules épithéliales et les entérocytes.  Listeria monocytogenes is capable of infecting a wide variety of cell types, in vivo and in vitro, including macrophages, fibroblasts, epithelial cells and enterocytes.
Après sa pénétration dans la cellule infectée, la bactérie lyse la membrane du phagosome grâce à une hémolysine qu'elle secrète. Au terme de cette étape, la bactérie est dans le cytoplasme de la cellule hôte.  After entering the infected cell, the bacteria lyses the phagosome membrane with a hemolysin that it secretes. At the end of this stage, the bacteria is in the cytoplasm of the host cell.
En outre, Listeria monocytogenes se caractérise par son aptitude à se propager dans les tissus par infection directe de cellule à cellule sans quitter le cytoplasme (Racz et al., 1970 (9)).  In addition, Listeria monocytogenes is characterized by its ability to spread through tissues by direct cell-to-cell infection without leaving the cytoplasm (Racz et al., 1970 (9)).
Peu de temps après son entrée dans la cellule hôte, la bactérie s'entoure d'actine filamenteuse (actine F) qui est ultérieurement se réarrange en "comète" derrière la bactérie dans la direction opposée au mouve ment (Tilney et al., 1989, (13); Mounier et al., (1990) (1). L'actine polymérisée est constituée de microfilaments courts, orientés au hasard qui diffèrent des filaments d' actine longs habituellement observés dans les cellules musculaires. Shortly after entering the host cell, the bacteria surround themselves with filamentous actin (actin F) which is later rearranged into a "comet" behind the bacteria in the opposite direction to the movement. ment (Tilney et al., 1989, (13); Mounier et al., (1990) (1). Polymerized actin is made up of short, randomly oriented microfilaments that differ from the long actin filaments usually observed in muscle cells.
Les bactéries sont mobiles et laissent derrière elles des "comètes" d'actine F de plusieurs μm de longueur. Certaines sont incorporées dans des protubérances cytoplasmiques en forme de doigt, qui peuvent être internalisées par les cellules voisines. Les deux membranes plasmiques entourant la bactérie sont alors lysées. Une fois dans le cytoplasme de la nouvelle cellule hôte, la bactérie peut se reproduire et commencer un nouveau cycle de dissémination.  The bacteria are mobile and leave behind F-actin "comets" several microns in length. Some are incorporated into finger-shaped cytoplasmic protrusions, which can be internalized by neighboring cells. The two plasma membranes surrounding the bacteria are then lysed. Once in the cytoplasm of the new host cell, the bacteria can reproduce and start a new cycle of dissemination.
Pendant ce processus de dissémination, les cellules de Listeria monocytogenes sont protégées du système immunitaire de l'hôte, et la dissémination de cellule à cellule représente par conséquent un facteur clé de virulence.  During this spreading process, Listeria monocytogenes cells are protected from the host's immune system, and cell-to-cell spread is therefore a key factor in virulence.
En isolant et analysant un mutant Tn 917-Lac inapte à se disséminer de cellule à cellule, les inventeurs ont pu identifier une protéine de Listeria monocytogenes impliquée dans l'assemblage de l'actine induit par la bactérie.  By isolating and analyzing a Tn 917-Lac mutant incapable of spreading from cell to cell, the inventors were able to identify a protein of Listeria monocytogenes involved in the assembly of actin induced by the bacterium.
Le gène codant pour cette protéine dénommée act A, fait partie d'un opéron (Mengaud et al., 1991 (b) (8)) dont la séquence nucléotidique complète a été récemment décrite (Vazquez-Boland et al., 1992 (12)).  The gene coding for this protein, called act A, is part of an operon (Mengaud et al., 1991 (b) (8)) whose complete nucleotide sequence has recently been described (Vazquez-Boland et al., 1992 (12 )).
La présente invention a ainsi pour objet une souche de listeria monocytogenes à virulence atténuée, caractérisée en ce qu'elle comporte, dans le gène act A ou dans le promoteur de celui-ci, une mutation capable de bloquer ou de modifier sensiblement l'expression de la protéine codée par le gène act A.  The present invention thus relates to a strain of listeria monocytogenes with attenuated virulence, characterized in that it comprises, in the act A gene or in the promoter thereof, a mutation capable of blocking or substantially modifying the expression of the protein encoded by the act A gene.
La mutation peut être réalisée selon les techniques connues, notamment par insertion dans le gène act A ou son promoteur, d'une séquence d'une ou plusieurs bases, de préférence un transposon stable, délétion d'une ou plusieurs bases, mutations telles que mutations par mutagénèse dirigée, par exemple par PCR et notamment mutations faux-sens. The mutation can be carried out according to the known techniques, in particular by insertion into the act A gene or its promoter, of a sequence of one or more bases, preferably a stable transposon, deletion of one or more bases, mutations such as mutations by site-directed mutagenesis, for example by PCR and in particular missense mutations.
La mutation peut notamment être réalisée par insertion d'un transposon, tel que le transposon Tn 917-lac, comme décrit par Mengaud et al., 1991 (a) (7).  The mutation can in particular be carried out by insertion of a transposon, such as the transposon Tn 917-lac, as described by Mengaud et al., 1991 (a) (7).
La mutation est effectuée de préférence dans le fragment d'ADN codant pour la séquence peptidique à motifs répétés comprise entre les aminoacides 235 à 315, 350 à 360, 367 à 385 et 389 à 393 de la séquence peptidique SEQ ID N° 1.  The mutation is preferably carried out in the DNA fragment coding for the peptide sequence with repeating motifs comprised between amino acids 235 to 315, 350 to 360, 367 to 385 and 389 to 393 of the peptide sequence SEQ ID No. 1.
Un autre site de mutation avantageux, notamment d'insertion est situé en aval de l'adénosine en position 497 de la séquence nucléotidique du gène act A.  Another advantageous mutation site, in particular for insertion, is located downstream of adenosine at position 497 of the nucleotide sequence of the act A gene.
Cette position correspond à celle entre les aminoacides 61 et 62 de la séquence peptidique SEQ ID N° 1.  This position corresponds to that between amino acids 61 and 62 of the peptide sequence SEQ ID No. 1.
Une souche particulièrement préférée selon la présente invention est la souche de la Listeria monocytogenes LUT 12 déposée à la Collection Nationale de Cultures de Micro-organismes (CΝCM) le 30 janvier 1992 sous le numéro 1-1167.  A particularly preferred strain according to the present invention is the strain of Listeria monocytogenes LUT 12 deposited in the National Collection of Cultures of Microorganisms (CΝCM) on January 30, 1992 under the number 1-1167.
Les mutants selon l'invention sont aptes à conférer à des hôtes auxquels ils ont été administrés une protection contre une infection ultérieure par une souche pathogène de Listeria monocytogenes.  The mutants according to the invention are capable of conferring on hosts to which they have been administered protection against a subsequent infection by a pathogenic strain of Listeria monocytogenes.
L'invention a donc également pour objet un vaccin humain ou vétérinaire comprenant en tant que composant actif une souche atténuée de Listeria monocytogenes, telle que définie précédemment.  The invention therefore also relates to a human or veterinary vaccine comprising as active component an attenuated strain of Listeria monocytogenes, as defined above.
Ce vaccin est apte à conférer une protection efficace à l'homme ou à l'animal notamment aux bovins et ovins contre la listériose. This vaccine is capable of providing effective protection to humans or animals, in particular cattle and sheep against listeriosis.
La réponse immunitaire générée par l'administration d'un mutant atténué tel que défini se traduit par la prolifération essentiellement de lymphocytes T de la sous-classe CD8.  The immune response generated by the administration of an attenuated mutant as defined results in the proliferation essentially of T lymphocytes of the CD8 subclass.
Les lymphocytes T de la sous-classe CD8 sont activés par des peptides liés à des antigènes de classe CD8 subclass T cells are activated by peptides linked to class antigens
I du CMH (complexe majeur d'histocompatibilité) générés par la protéolyse de protéines synthétisées ou libérées dans le cytoplasme d'une cellule présentant l'antigène. I of the MHC (major histocompatibility complex) generated by the proteolysis of proteins synthesized or released in the cytoplasm of a cell presenting the antigen.
Ainsi, les mutants atténués de Listeria monocytogenes selon l'invention, sont aptes à stimuler le système immunitaire par la voie qui utilise les molécules de classe I du CMH.  Thus, the attenuated mutants of Listeria monocytogenes according to the invention are capable of stimulating the immune system by the route which uses MHC class I molecules.
II est par conséquent possible en transformant Listeria monocytogenes à l'aide d'un plasmide approprié d'introduire un gène hétérologue provenant de n'importe quel organisme et d'utiliser les souches recombinantes obtenues comme système d'expression d'ADN hétérologue.  It is therefore possible by transforming Listeria monocytogenes using an appropriate plasmid to introduce a heterologous gene from any organism and to use the recombinant strains obtained as a heterologous DNA expression system.
L'invention a ainsi également pour objet un mutant recombinant de Listeria monocytogenes caractérisé en ce qu'il comporte un ADN hétérologue, soit inséré dans le génome d'un mutant atténué tel que défini précédemment, soit porté par un plasmide se répliquant dans le mutant atténué.  The invention thus also relates to a recombinant mutant of Listeria monocytogenes characterized in that it comprises a heterologous DNA, either inserted into the genome of an attenuated mutant as defined above, or carried by a plasmid replicating in the mutant mitigated.
l'ADN hétérologue consiste de préférence en un gène hétérologue codant pour un antigène protecteur cible de lymphocytes T de la sous-classe CD8.  the heterologous DNA preferably consists of a heterologous gene coding for a target protective antigen of T lymphocytes of the CD8 subclass.
Cet antigène peut être d'origine bactérienne This antigen may be of bacterial origin
(par exemple de mycobactéries), parasitaire, (par exemple de Leishmania, Tripanosoma ou Toxoplasma) ou viral (virus de la grippe, virus de la chorioméningite lymphocytaire (LCMV) ou virus du Sida (VIH)). (e.g. mycobacteria), parasitic (e.g. Leishmania, Tripanosoma or Toxoplasma) or viral (flu virus, lymphocytic choriomeningitis virus (LCMV) or AIDS virus (HIV)).
Un mutant recombinant de Listeria monocytoge nés particulièrement intéressant comporte les gènes codant pour l'antigène gag et/ou l'antigène nef du VIH ou tout ou partie de l'enveloppe gp 120 du VIH 1 ou gp 140 du VIH 2 ou par un peptide tel que défini dans US-4 943 628. A recombinant mutant of Listeria monocytoge particularly interesting born contains the genes coding for the gag antigen and / or the HIV nef antigen or all or part of the gp 120 envelope of HIV 1 or gp 140 of HIV 2 or by a peptide as defined in US- 4,943,628.
La construction du mutant recombinant peut être réalisée par transformation d'un mutant atténué tel que défini ci-dessus, notamment du mutant LUT 12 à l'aide d'un plasmide approprié, et par exemple électroporation.  The construction of the recombinant mutant can be carried out by transformation of an attenuated mutant as defined above, in particular of the mutant LUT 12 using an appropriate plasmid, and for example electroporation.
Avantageusement, le clonage de l'ADN hétérologue sera réalisé chez E. Coli et on utilisera un plasmide navette E. Coli - Listeria monocytogenes pour réaliser la transformation.  Advantageously, the cloning of the heterologous DNA will be carried out in E. Coli and a shuttle plasmid E. Coli - Listeria monocytogenes will be used to carry out the transformation.
Comme plasmides, on peut citer pMKA 4 (Sullivan et al., (14)) ou PHT 320 ( Leredus et al., (15)).  As plasmids, mention may be made of pMKA 4 (Sullivan et al., (14)) or PHT 320 (Leredus et al., (15)).
Pour permettre l'expression du gène d'intérêt (gène hétérologue), il est avantageux d'insérer en amont du gène d'intérêt un promoteur fort de Listeria, tel que le promoteur hly.  To allow the expression of the gene of interest (heterologous gene), it is advantageous to insert upstream of the gene of interest a strong Listeria promoter, such as the hly promoter.
Pour que le produit de traduction du gène d'intérêt puisse être sécrété, il est préférable de fusionner le gène d'intérêt au début de hly afin d'utiliser "la séquence signal" de la listériolysine O pour libérer la protéine d'intérêt codée par le gène hétérologue dans le cytoplasme d'une cellule d'hôte.  So that the translation product of the gene of interest can be secreted, it is preferable to merge the gene of interest at the start of hly in order to use the "signal sequence" of listeriolysin O to release the protein of interest encoded by the heterologous gene in the cytoplasm of a host cell.
Les mutants recombinants de Listeria monocytogenes tels que définis ci-dessus conviennent de manière avantageuse pour la préparation d'un vaccin recombinant humain ou vétérinaire, contre une infection provoquée par un microorganisme produisant un antigène correspondant à la protéine codée par l'ADN hétérologue inséré dans le génome de Listeria monocytogenes recombinant.  The recombinant mutants of Listeria monocytogenes as defined above are advantageously suitable for the preparation of a recombinant human or veterinary vaccine against an infection caused by a microorganism producing an antigen corresponding to the protein encoded by the heterologous DNA inserted into the recombinant Listeria monocytogenes genome.
Les vaccins selon l'invention peuvent être administrés par voie intra-veineuse, sous-cutanée, intramusculaire ou orale. Une dose appropriée se situe entre 5.104 et 109 cellules/kg de poids. The vaccines according to the invention can be administered intravenously, subcutaneously, intramuscularly or orally. An appropriate dose is between 5.10 4 and 10 9 cells / kg of weight.
Cette dose varie en fonction de la voie d ' administration ainsi que de la sensibilité de l'hôte.  This dose varies depending on the route of administration and the sensitivity of the host.
L'administration est de préférence répétée afin de conférer une protection efficace à l'hôte.  The administration is preferably repeated in order to confer effective protection on the host.
Les mutants recombinants de Listeria monocytogenes définis ci-dessus sont également appropriés à la préparation d'une composition de diagnostic destinée au contrôle de l'état de protection d'un hôte humain ou animal contre une infection provoquée par un microorganisme produisant un antigène correspondant à la protéine codée par l'ADN hétérologue inséré dans le génome de Listeria monocytogenes recombinant ou exprimé dans cette souche lorsque porté par un plasmide.  The recombinant mutants of Listeria monocytogenes defined above are also suitable for the preparation of a diagnostic composition intended for monitoring the protective state of a human or animal host against an infection caused by a microorganism producing an antigen corresponding to the protein encoded by heterologous DNA inserted into the genome of Listeria monocytogenes recombinant or expressed in this strain when carried by a plasmid.
Il suffira d'injecter localement la composition de diagnostic selon l'invention par voie souscutanée par exemple et d'observer après un certain temps de latence si une réaction inflammatoire a lieu ou non, à la manière du test à la tuberculine utilisé pour contrôler l'état de protection d'un hôte contre le bacille de la tuberculose.  It will suffice to locally inject the diagnostic composition according to the invention by subcutaneous route for example and to observe after a certain lag time whether an inflammatory reaction takes place or not, in the manner of the tuberculin test used to control the protection status of a host against the tuberculosis bacillus.
On décrira ci-après l'obtention de la souche mutante LUT 12 de Listeria monocytogenes ainsi que ses propriétés en se référant à la fig. annexée représentant:  The production of the mutant strain LUT 12 of Listeria monocytogenes and its properties will be described below, with reference to FIG. attached representing:
A : l'opéron lécithinase de Listeria monocytogenes (Vasquez - Boland et al., 1992 (12) avec la position du transposon dans le mutant LUT 12.  A: the lecithinase operon of Listeria monocytogenes (Vasquez - Boland et al., 1992 (12) with the position of the transposon in the mutant LUT 12.
B : la séquence d'aminoacides de la protéine codée par le gène act A. Les traits noirs renforcés représentent des gènes dont les produits ont été caractérisés (mpl : metalloprotéase, Domann et al., 1991, (3) act A :B: the amino acid sequence of the protein encoded by the act A gene. The reinforced black lines represent genes whose products have been characterized (mpl: metalloprotease, Domann et al., 1991, (3) act A:
(présente invention), pic B : lécithinase) ORFX, ORFY et ORFZ sont des cadres de lecture ouverte. (present invention), pic B: lecithinase) ORFX, ORFY and ORFZ are open reading frames.
P : indique le promoteur, les lignes en pointillées le produit de transcription et un signal de terminaison de transcription potentiel.  P: indicates the promoter, the dotted lines the transcript and a potential transcription termination signal.
La séquence signal potentielle et le segment transmembranaire sont soulignés. La région de motifs répétés est entourée. La flèche correspond à l'insertion de Tn 917-lac dans le gène actA du mutant LUT 12.  The potential signal sequence and the transmembrane segment are underlined. The repeating pattern region is surrounded. The arrow corresponds to the insertion of Tn 917-lac into the actA gene of the LUT 12 mutant.
La numérotation débute à l'extrémité NH2 de la protéine mature. Les résidus déterminés par microséquençage de la bande de 90 kDa sont imprimés en caractères gras et marqués par un astérisque. The numbering begins at the NH 2 end of the mature protein. The residues determined by microsequencing of the 90 kDa band are printed in bold type and marked with an asterisk.
I - Techniques générales de clonage et d'analyse d'ADN Toutes les techniques de clonage et d'analyse ont été effectuées conformément aux protocoles standards (Sambrook et al. 1989 (10)) ou suivant les instructions du fabricant. I - General DNA cloning and analysis techniques All the cloning and analysis techniques were carried out in accordance with standard protocols (Sambrook et al. 1989 (10)) or according to the manufacturer's instructions.
l'ADN chromosomique de Listeria monocytogenes a été préparé comme décrit par Mengaud et al., 1991 (b) (8). Les sondes pour Southern blot ont été préparées par PCR, purifiées à partir des gels d'agarose en utilisant le kit Geneclean (Bio 101, Inc, La Jolla, CA), et marquées en utilisant le système Multiprime d'Amersham.  the chromosomal DNA of Listeria monocytogenes was prepared as described by Mengaud et al., 1991 (b) (8). The Southern blot probes were prepared by PCR, purified from agarose gels using the Geneclean kit (Bio 101, Inc, La Jolla, CA), and labeled using the Amersham Multiprime system.
Les hybridations selon Southern ont été réalisées à l'aide du système d'hybridation rapide (Amersham) sur des membranes de Nylon N (Amersham) dans un four pour hybridation Hybaid. II - Isolement de la souche LUT 12 et détermination du point d'insertion du transposon Hybridizations according to Southern were carried out using the rapid hybridization system (Amersham) on Nylon N membranes (Amersham) in an oven for Hybaid hybridization. II - Isolation of the LUT 12 strain and determination of the transposon insertion point
Une banque de mutants Tn 917-lac, produits à partir de la souche de type sauvage L028 et du plasmide p TV32 portant le transposon Tn 917-Lac comme décrit par Mengaud et al., 1991 a (7), a été criblée sur des plaques de gélose au jaune d'oeuf préparée à partir de jaune d'oeuf frais dilué au 1:2 dans une solution de NaCl 150 mM, et d'addition de 12,5 ml de ce mélange à 250 ml de gélose additionnée d'une infusion de cerveau et de coeur (BHI) à 56°C. A library of Tn 917-lac mutants, produced from the wild type strain L028 and from the plasmid p TV32 carrying the Tn 917-Lac transposon as described by Mengaud et al., 1991 a (7), was screened on plates of egg yolk agar prepared from fresh egg yolk diluted 1: 2 in 150 mM NaCl solution, and adding 12.5 ml of this mixture to 250 ml of agar with added a brain and heart infusion (BHI) at 56 ° C.
Un mutant lécithinase-négatif ne produisant pas d' opacification du jaune d'oeuf même après une incubation prolongée et montrant un phénotype de type sauvage pour tous les autres caractères examinés a été appelée LUT 12.  A lecithinase-negative mutant that did not produce egg yolk clouding even after prolonged incubation and exhibited a wild-type phenotype for all other traits examined was called LUT 12.
A - Caractéristigues biologigues de ce mutant A - Biological characteristics of this mutant
Ce mutant avait à la fois une activité hémolytique et un taux de croissance in vitro identiques au type sauvage, mais se révélait de virulence très atténuée chez la souris.  This mutant had both hemolytic activity and an in vitro growth rate identical to the wild type, but was found to be very attenuated in virulence in mice.
TOXICITE  TOXICITY
La DL50 était plus élevée d'un facteur 4 logLD 50 was 4-fold higher
10 que la DL50 des bactéries du type sauvage (108,55 bactéries au lieu de 104,25). 10 as the LD 50 of wild type bacteria (10 8.55 bacteria instead of 10 4.25 ).
TESTS DE FORMATION DE PLAGES SUR CULTURES DE FIBROBLASTES  BEACH TRAINING TESTS ON FIBROBLAST CULTURES
Des essais ont été effectués sur des fibroblastes 3T3 (ECA CC88031146). selon la technique décrite par Kuhn et al., 1990 (5), sauf que les infections étaient réalisées à des concentrations variées d'inoculum : 1 à 25 μl de sous-cultures bactériennes de 2 heures (A600nm de 0,45) soit non diluées soit diluées au 1/10. Cet essai révèle l'aptitude de Listeria monocytogenes à se multiplier de manière intracellulaire et à se disséminer sur des couches simples de fibroblastes revêtues d'une couche de gélose contenant de la gentamicine à une concentration létale pour les bactéries extracellulaires mais non pour les bactéries intracellulaires. Après plusieurs jours, des zones de cellules mortes détruites par l'infection bactérienne sont visibles à l'oeil nu sous forme de "plages". Tests were carried out on 3T3 fibroblasts (ECA CC88031146). according to the technique described by Kuhn et al., 1990 (5), except that the infections were carried out at various concentrations of inoculum: 1 to 25 μl of bacterial subcultures of 2 hours (At 600nm of 0.45) either undiluted or diluted 1/10. This test reveals the ability of Listeria monocytogenes to multiply intracellularly and to disseminate on single layers of fibroblasts coated with an agar layer containing gentamicin at a lethal concentration for extracellular bacteria but not for intracellular bacteria . After several days, areas of dead cells destroyed by the bacterial infection are visible to the naked eye in the form of "patches".
Les bactéries LUT 12 mutantes étaient inaptes à former des plages sur des couches simples de fibroblastes 3T3.  The mutant LUT 12 bacteria were unable to form plaques on single layers of 3T3 fibroblasts.
ESSAI DE DISSEMINATION SUR DES MACROPHAGES DE MOELLE OSSEUSE  DISSEMINATION TEST ON BONE MARROW MACROPHAGES
Une observation au microscope optique de la dissémination de Listeria monocytogenes sur des couches simples de macrophages primaires de moelle osseuse a en outre été réalisée comme suit.  In addition, observation of the spread of Listeria monocytogenes on single layers of primary bone marrow macrophages was carried out under the light microscope as follows.
Des suspensions contenant des macrophages ont. été préparées à partir de moelle osseuse d'une souris femelle C57BL/6 âgée de 7 semaines, et cultivées dans un milieu RPMI contenant 10% de sérum foetal de veau en présence de surnageant L. 4.105 macrophages dérivés de moelle osseuse obtenus au jour 6 ont été ensemencés dans les lamelles en verre rondes (diamètre 12 mm) la veille de l'utilisation. Les macrophages ont été infectés avec une MI (multiplicte d'infection ) de 0,04 (une bactérie pour 25 macrophages, résultant en à peu près 1% de cellules infectées), de manière à pouvoir observer des points individuels d'infection, générés par la progéniture d'une seule bactérie. L'infection a été réalisée comme décrit pour un macrophage J774. Suspensions containing macrophages have. were prepared from bone marrow of a C57BL / 6 female mouse aged 7 weeks, and cultured in RPMI medium containing 10% fetal calf serum in the presence of supernatant L. 4.10 5 macrophages derived from bone marrow obtained on the day 6 were sown in round glass strips (diameter 12 mm) the day before use. The macrophages were infected with an MI (multiply of infection) of 0.04 (one bacterium for 25 macrophages, resulting in approximately 1% of infected cells), so as to be able to observe individual points of infection, generated by the offspring of a single bacterium. The infection was carried out as described for a macrophage J774.
Après 30 minutes et après 8 heures, ces monocouches cellulaires ont été fixées et colorées avec une solution de Giemsa. Après 8 heures, la progéniture des bactéries de type sauvage s'était disséminée à de nombreuses cellules hôtes nouvelles, et des bactéries portant des protubérances à leur extrémité pouvaient être observées. Au contraire, la progéniture du mutant LUT 12 est restée enfermée à l'intérieur d'une seule cellule infectée. Les bactéries mutantes ont, soit formé des microcolonies ou étaient disséminées dans le cytoplasme des cellules hôtes, mais aucune protubérance contenant des bactéries ne pouvait être détectée. After 30 minutes and after 8 hours, these cell monolayers were fixed and stained with a Giemsa solution. After 8 hours, the offspring of wild-type bacteria had spread to many new host cells, and bacteria with protuberances at their ends could be observed. On the contrary, the LUT 12 mutant's offspring remained trapped inside a single infected cell. The mutant bacteria either formed microcolonies or were disseminated in the cytoplasm of the host cells, but no protuberance containing bacteria could be detected.
Ce résultat indique que la bactérie mutante se multiplie à l'intérieur des cellules infectées, mais est incapable d'infecter des cellules adjacentes par dissémination de cellule à cellule.  This result indicates that the mutant bacteria multiplies inside infected cells, but is unable to infect adjacent cells by cell-to-cell dissemination.
TEST DE CROISSANCE SUR DES MACROPHAGES J774 GROWTH TEST ON J774 MACROPHAGES
Un essai démontrant que la bactérie mutante LUT 12 était apte à se multiplier de manière intracellulaire a été réalisé au moyen d'un test de croissance sur des macrophages J774. An assay demonstrating that the mutant LUT 12 bacterium was capable of multiplying intracellularly was carried out by means of a growth test on J774 macrophages.
Cet essai a été réalisé sur des couches simples de J774 dans des flacons de culture de tissus en matière plastique de 25 cm3. Les cellules étaient infectées avec une MDI de 10 bactéries par cellule. Le nombre de bactéries intracellulaires était calculé après 2, 6 et 10 heures de croissance sur un milieu contenant de la gentamicine (5 μg/ml) par lyse des mono-couches cellulaires, lavage avec de l'eau distillée froide et étalement de dilutions appropriées sur des plaques contenant un milieu BHI. This test was carried out on single layers of J774 in 25 cm 3 plastic tissue culture flasks. The cells were infected with an MDI of 10 bacteria per cell. The number of intracellular bacteria was calculated after 2, 6 and 10 hours of growth on a medium containing gentamicin (5 μg / ml) by lysis of the cell monolayers, washing with cold distilled water and spreading of appropriate dilutions. on plates containing BHI medium.
Après une durée de 10 heures, les courbes de croissance des bactéries de type sauvage et LUT 12 étaient identiques.  After 10 hours, the growth curves of wild type bacteria and LUT 12 were identical.
OBSERVATIONS AU MICROSCOPE ELECTRONIQUE  COMMENTS TO THE ELECTRONIC MICROSCOPE
Le comportement intracellulaire du mutant LUT 12 a été observé au microscope électronique. Des macro phages J774 étaient infectés avec des bactéries de type sauvage ou la souche mutante pendant 30 minutes, suivie par une incubation de 60 à 210 minutes dans un milieu contenant la gentamicine. Pour le mutant et le type sauvage, on pouvait observer des bactéries libres dans le cytoplasme à 1 h 1/2 d'infection. A ce moment, le type sauvage et le mutant étaient entourés par une couche mince de matériel granulaire crêpelé, mais seul le type sauvage comportait du matériel filamentaire assemblé à sa surface, constitué de filaments d'actine. A 4 h après l'infection, les bactéries de type sauvage étaient entourées par d'épaisses couches de filaments d'actine F. Au contraire, les bactéries mutantes LUT 12 étaient presque nues. Même le revêtement fin crêpelé observé au moment précoce de l'infection avait disparu. The intracellular behavior of the LUT 12 mutant was observed under the electron microscope. Macro J774 phages were infected with wild-type bacteria or the mutant strain for 30 minutes, followed by incubation for 60 to 210 minutes in medium containing gentamicin. For the mutant and the wild type, free bacteria could be observed in the cytoplasm at 1.5 hours of infection. At that time, the wild type and the mutant were surrounded by a thin layer of creped granular material, but only the wild type had filamentary material assembled on its surface, consisting of actin filaments. At 4 h after infection, the wild-type bacteria were surrounded by thick layers of actin F filaments. On the contrary, the LUT 12 mutant bacteria were almost naked. Even the thin creped coating observed at the early time of infection was gone.
Pour visualiser l'association d'actine F bactérienne de manière spécifique, les auteurs ont réalisé des colorations en double fluorescence à l'aide de FITC-phalloïdine, une toxine fongique se liant à l'actine F, et avec un sérum anti-L.monocytogenes. suivi par un second anti-corps couplé à la rhodamine, pour détecter les bactéries. Les macrophages J774 étaient infectés depuis 4h avec les bactéries de type sauvage ou mutant. Tandis que les bactéries de type sauvage se coloraient positivement avec la FITC-phalloidine, les bactéries mutantes LUT 12, bien que détectables avec le sérum anti-L.monocytogenes restaient invisibles avec la coloration de l'actine.  To visualize the association of bacterial actin F in a specific way, the authors carried out double fluorescence stains using FITC-phalloidin, a fungal toxin binding to actin F, and with an anti-L serum. .monocytogenes. followed by a second antibody coupled to rhodamine, to detect bacteria. The J774 macrophages were infected for 4 hours with wild type or mutant bacteria. While wild-type bacteria stained positively with FITC-phalloidin, LUT 12 mutant bacteria, although detectable with anti-L.monocytogenes serum, remained invisible with actin staining.
Ces résultats démontrent que les bactéries LUT 12 s'échappent des phagosomes aussi efficacement que les bactéries de type sauvage et se multiplient dans le cytoplasme. Cependant, les bactéries mutantes ne sont jamais associées avec de l'actine F, sont incapables de se déplacer à l'intérieur de la cellule, et ne peuvent infecter des cellules voisines par dissémination directe. Ces observations suggèrent que le mutant LUT 12 est déficient en un composant nécessaire pour le procédé de formation de filaments d'actine induit par Listeria monocytogenes. These results demonstrate that LUT 12 bacteria escape phagosomes as efficiently as wild type bacteria and multiply in the cytoplasm. However, the mutant bacteria are never associated with actin F, are unable to move inside the cell, and cannot infect neighboring cells by direct spread. These observations suggest that the LUT 12 mutant is deficient in a component necessary for the actin filament formation process induced by Listeria monocytogenes.
B - DETERMINATION DU POINT D'INSERTION DU TRANSPOSON B - DETERMINATION OF THE TRANSPOSON INSERTION POINT
Ce mutant a été analysé par Southern blot pour déterminer le nombre de transposons insérés dans son chromosome.  This mutant was analyzed by Southern blot to determine the number of transposons inserted in its chromosome.
L'ADN chromosomique a été digéré par Bam HI, Eco RO, Hind III, Kpn I et Pst I.  The chromosomal DNA was digested with Bam HI, Eco RO, Hind III, Kpn I and Pst I.
On a utilisé deux sondes différentes correspondant à Tn 917-lac (Shaw et Clewell, 1985 (11)): une sonde recouvrant 515 paires de bases du gène de résistance à l'érythromycine, obtenue par PCR avec les Oligonucléotides 5'-TTG GAA CAG GTA AAG GGC ATT TAA-3' (position 821 à 844) et 5'-AGT AAA CAG TTG ACG ATA TTC TCG-3' (position 1313 à 1336), et une sonde recouvrant le fragment interne Hind III du transposon obtenue par PCR avec les oligonucléotides 5'-ACA ATT AAT GTC TCC CAT ATT-3' (position 3082 à 3102) et 5' (ACT GAT AAT TAA CCA AAA CAG-3' (position 4295-4315).  Two different probes corresponding to Tn 917-lac were used (Shaw and Clewell, 1985 (11)): a probe covering 515 base pairs of the erythromycin resistance gene, obtained by PCR with the oligonucleotides 5'-TTG GAA CAG GTA AAG GGC ATT TAA-3 '(position 821 to 844) and 5'-AGT AAA CAG TTG ACG ATA TTC TCG-3' (position 1313 to 1336), and a probe covering the internal Hind III fragment of the transposon obtained by PCR with the oligonucleotides 5'-ACA ATT AAT GTC TCC CAT ATT-3 '(position 3082 to 3102) and 5' (ACT GAT AAT TAA CCA AAA CAG-3 '(position 4295-4315).
La jonction transposon-chromosome a été clonée à partir d'une banque d'ADN chromosomique obtenue par restriction avec Eco RI/Kpn I dans pUC 18. Un clone comportant un segment d'insertion, correspondant à la jonction chromosome-transposon a été isolé et séquence directement à partir du plasmide par utilisation d'un oligonucléotide s'hybridant avec l'extrémité droite du transposon ( 5'-CTA AAC ACT TAA GAG AAT TG-3', position 5244 à 5263).  The transposon-chromosome junction was cloned from a chromosomal DNA library obtained by restriction with Eco RI / Kpn I in pUC 18. A clone comprising an insertion segment, corresponding to the chromosome-transposon junction was isolated and sequenced directly from the plasmid by using an oligonucleotide hybridizing with the right end of the transposon (5'-CTA AAC ACT TAA GAG AAT TG-3 ', position 5244 to 5263).
Le transposon était inséré après l'adénine The transposon was inserted after adenine
497 dans la séquence nucléotidique du fragment Hind III - Eco RI du gène act A de l'opéron identifié par Mengaud et al., 1991 (b) (8), dont la séquence nucléotidique a été décrite par Vazquez-Boland et al., 1992 (12). Le point d'insertion du transposon Tn 917-lac est représenté sur le shcéma de la fig. (A). 497 in the nucleotide sequence of the Hind III - Eco RI fragment of the act A gene of the operon identified by Mengaud et al., 1991 (b) (8), whose nucleotide sequence has been described by Vazquez-Boland et al., 1992 (12). The insertion point of the Tn 917-lac transposon is shown in the diagram of FIG. (AT).
Le phénotype lecithinase-négatif du mutant LUT 12 est vraisemblablement dû à un effet polaire de la mutation par insertion dans act A, dans la mesure où le 3ème gène de l'opéron plcB code pour la lécithinase.  The LUT 12 mutant lecithinase-negative phenotype is probably due to a polar effect of the insertion mutation in act A, since the 3rd gene of the operon plcB codes for lecithinase.
Des études complémentaires ont été réalisées qui ont démontré que la perte de l'activité de polymérisation de l'actine était bien due à une perte d'exprèssion du gène Act A.  Additional studies have been carried out which have shown that the loss of actin polymerization activity was due to a loss of expression of the Act A gene.
Des mutants ont été réalisés par recombinaison homologue entre le chromosome de Listeria monocytogenes et des fragments correspondant à des parties du gène plcB et des cadres de lecture ouverte ORFX/Y et ORFZ (fig. (A)) situés en aval du gène act A par insertion de plasmides en divers sites.  Mutants were produced by homologous recombination between the chromosome of Listeria monocytogenes and fragments corresponding to parts of the plcB gene and open reading frames ORFX / Y and ORFZ (fig. (A)) located downstream of the act A gene by insertion of plasmids at various sites.
Des études d'immunofluorescence par utilisation de FITC-phalloïdine et marquage à la rhodamine de bactéries dans des macrophages J 774 infectés ont montré que les mutants plcB, ORFX/Y et ORFZ étaient associés à des filaments d'actine F tout comme les bactéries de type sauvage. Ces études ont été complétées par des études de microscopie électronique qui ont montré que ces mutants étaient aptes à stimuler l'assemblage de l'actine de la même manière que le type sauvage.  Immunofluorescence studies using FITC-phalloidin and rhodamine labeling of bacteria in infected J 774 macrophages have shown that the mutants plcB, ORFX / Y and ORFZ were associated with actin F filaments as did bacteria of wild type. These studies were supplemented by electron microscopy studies which showed that these mutants were able to stimulate actin assembly in the same way as the wild type.
Ces analyses montrent par conséquent que des mutations en aval de act A n'affectent pas l'assemblage de l'actine A et suggèrent que l'incapacité du mutant LUT These analyzes therefore show that mutations downstream of act A do not affect the assembly of actin A and suggest that the inability of the mutant LUT
12 à polymériser l' actine cellulaire est due à l'absence d'expression du gène act A. 12 to polymerize cellular actin is due to the lack of expression of the act A gene.
Une transformation du mutant LUT 12 réalisée avec act A montre par ailleurs que le phénotype de type sauvage est restauré, ce qui exclut la possibilité d'une mutation spontanée en un autre site du chromosome.  A transformation of the mutant LUT 12 carried out with act A also shows that the wild type phenotype is restored, which excludes the possibility of a spontaneous mutation at another site of the chromosome.
Ces résultats démontrent ainsi que le produit du gène act A est nécessaire pour l' assemblage de l'actine de Listeria monocytogenes et par conséquent pour son pouvoir pathogène. These results demonstrate that the product of the act A gene is necessary for the assembly of Listeria monocytogenes actin and therefore for its pathogenic power.
Le produit du gène act A a été déterminé comme décrit ci-après :  The act A gene product was determined as described below:
III - Analyse du produit du gène act A III - Analysis of the act A gene product
La séquence nucléotidique du gène act A laisse supposer que celui-ci code pour une protéine de 639 aminoacides avec une séquence signal et une région transmembranaire (Vazquez Boland et al., 1992 12(). The nucleotide sequence of the act A gene suggests that the gene codes for a protein of 639 amino acids with a signal sequence and a transmembrane region (Vazquez Boland et al., 1992 12 ().
Des études complémentaires ont été réalisées d'une part par analyse comparée des protéines de surface de Listeria monocytogenes de type sauvage et de la souche LUT 12.  Additional studies were carried out on the one hand by comparative analysis of the surface proteins of wild type Listeria monocytogenes and of the strain LUT 12.
Les isolats bactériens ont été cultivés dans 200 ml de bouillon d'infusion cerveau-coeur (BHI, Laboratoires DIFCO, Détroit, Michigan) additionné d'érythomycine à 5 μg/ml pour LUT 12, sous agitation à 160 tpm sur un agitateur Gyrotory G10 (New Brunswick Scientific) à 37° C pendant 18 h.  The bacterial isolates were cultured in 200 ml of brain-heart infusion broth (BHI, DIFCO Laboratories, Detroit, Michigan) supplemented with erythomycin at 5 μg / ml for LUT 12, with stirring at 160 rpm on a Gyrotory G10 agitator. (New Brunswick Scientific) at 37 ° C for 18 h.
Les bactéries ont été récoltées par centrifugation (5000 g pendant 20 minutes) et lavées à trois reprises dans une solution saline de tampon phosphate (PBS).  The bacteria were collected by centrifugation (5000 g for 20 minutes) and washed three times in a saline solution of phosphate buffer (PBS).
Le culot obtenu a été remis en suspension dans 4 ml de PBS et du SDS a été ajouté à une concentration finale de 1 %. A cette concentration de SDS, les cellules de L. monocytogenes ne se lysent pas. L'absence de lyse bactérienne a été vérifiée au microscope. Après 5 minutes d'agitation à température ambiante, les bactéries ont été centrifugées (50.000 g pendant 10 minutes) et le surnageant concentré par ultrafiltration sur des microconcentrateurs (Centricom 30, Araicon) et conservé à -20 ° C . The pellet obtained was resuspended in 4 ml of PBS and SDS was added to a final concentration of 1%. At this concentration of SDS, the cells of L. monocytogenes do not lyse. The absence of bacterial lysis was checked under the microscope. After 5 minutes of stirring at room temperature, the bacteria were centrifuged (50,000 g for 10 minutes) and the supernatant concentrated by ultrafiltration on microconcentrators (Centricom 30, Araicon) and stored at -20 ° C.
La concentration en protéines a été déterminée à l' aide de la méthode à l'acide bicinchoninique (Pierce). La concentration en protéines a été ajustée à 300 μg/ml pour l'électrophorèse. 10 μl d'extrait ont été mélangés avec 10 μl de tampon (SDS à 2 %, glycérol à 10 %, mercaptoéthanol à 5 %, bleu de bromophénol à 0,002 % et Tris HCl 0,02 M), bouillis pendant 3 minutes à 100° C. L'électrophorèse a été effectuée à 60 mA pendant 120 minutes à travers des gels discontinus de polyacrylamide The protein concentration was determined using the bicinchoninic acid method (Pierce). The protein concentration was adjusted to 300 μg / ml for electrophoresis. 10 μl of extract were mixed with 10 μl of buffer (2% SDS, 10% glycerol, 5% mercaptoethanol, 0.002% bromophenol blue and 0.02 M Tris HCl), boiled for 3 minutes at 100 ° C. The electrophoresis was carried out at 60 mA for 120 minutes through discontinuous polyacrylamide gels
(Laemmli, 1970 (6)). les bandes ont été visualisées par coloration à l'argent (Heukeshoven et Dernick, 1985 (4)). (Laemmli, 1970 (6)). the bands were visualized by silver staining (Heukeshoven and Dernick, 1985 (4)).
Pour le marquage de la surface cellulaire, on a centrifugé 400 ml d'une culture de 18 heures de L. monocytogenes; les bactéries ont été lavées à 3 reprises avec PBS à pH 7,4 et remises en suspension dans 8 ml de PBs PH 8,0 à 4º C.  For labeling the cell surface, 400 ml of an 18 hour culture of L. monocytogenes were centrifuged; the bacteria were washed 3 times with PBS at pH 7.4 and resuspended in 8 ml of pH PBs 8.0 at 4 ° C.
Les bactéries ont ensuite été traitées avec de la sulfosuccinimido biotine (sulfo-NHS-biotine; Pierce) à une concentration finale de 0,5 mg/ml pendant 2 minutes sous agitation modérée.  The bacteria were then treated with sulfosuccinimido biotin (sulfo-NHS-biotin; Pierce) at a final concentration of 0.5 mg / ml for 2 minutes with moderate agitation.
Les cellules ont été lavées à trois reprises avec PBS à pH 7,4 et extraites par extraction au SDS.  The cells were washed three times with PBS at pH 7.4 and extracted by extraction with SDS.
Les extraits correspondant à 7 μg de protéinés par couloir ont été déposés sur des gels SDS et transférés comme décrit par De Rycke et al., 1989 (2) sur nitrocellulose (BA 85, Schleicher et Schϋll. Les filtres de nitrocellulose ont été saturés pendant une nuit dans PBS à 0,5 % de gélatine et incubés pendant 1, 5 heure avec de la streptavidine conjuguée à de la peroxydase (Jackson) dans du PBS contenant 0,5 % de gélatine et 0,1 M de Tween 20. Après différents lavages dans le même tampon, les bandes réactives ont été révélées avec 0, 5 mg/ml de 4-chloro-1-naphtol (Biorad) et 0,03 % v/v d'H2O2 dans l'eau. Les analyses des gels d'électrophorèse montrent un bande de 90 kDa pour le type sauvage qui est absente chez la souche LUT 12. Cette bande est également retrouvée chez les mutants plcB et les mutants LUT 12 transformés par act A mentionnés ci-dessus. The extracts corresponding to 7 μg of protein per lane were deposited on SDS gels and transferred as described by De Rycke et al., 1989 (2) on nitrocellulose (BA 85, Schleicher and Schϋll. The nitrocellulose filters were saturated for overnight in PBS 0.5% gelatin and incubated for 1.5 hours with streptavidin conjugated to peroxidase (Jackson) in PBS containing 0.5% gelatin and 0.1 M Tween 20. After different washes in the same buffer, the reaction bands were revealed with 0.5 mg / ml of 4-chloro-1-naphthol (Biorad) and 0.03% v / v of H 2 O 2 in water. Analyzes of the electrophoresis gels show a 90 kDa band for the wild type which is absent in the LUT 12 strain. This band is also found in the plcB mutants and the LUT 12 mutants transformed by act A mentioned above.
Les analyses de marquage de surface par la sulfosuccinimido-biotine montrent de façon directe une protéine biotinylée de 90 kA chez les bactéries du type sauvage qui est absente chez la souche mutante LUT 12.  Surface labeling analyzes with sulfosuccinimido-biotin directly show a 90 kA biotinylated protein in wild type bacteria which is absent in the mutant strain LUT 12.
Pour identifier sans ambiguïté la protéine 90 kDA, la bande de 90 kDA a été isolée et la séquence des 6 aminoacides de l'extrémité NH2 a été déterminée et comparée avec la séquence d'aminoacides déduite de la séquence nucléotidique du gène act A. To unambiguously identify the 90 kDA protein, the 90 kDA band was isolated and the sequence of the 6 amino acids of the NH 2 end was determined and compared with the amino acid sequence deduced from the nucleotide sequence of the act A gene.
Les extraits sur SDS correspondant à 100 μg de protéines par canal ont été mis à bouillir dans un tampon d'échantillonage SDS contenant 7 % (p/v) d'urée avant de réaliser une électrophorèse sur des gels au SDS à 7,5 %.  The extracts on SDS corresponding to 100 μg of proteins per channel were boiled in an SDS sampling buffer containing 7% (w / v) of urea before performing electrophoresis on 7.5% SDS gels .
Les protéines séparées ont été transférées sur une membrane Problott (Applied Biosystems) dans du Tris 50 mM - borate 50 mM pendant 17 heures à 4 à 5 volts/cm. Les protéines ont été colorées pendant 5 secondes à l'aide de noir amido à 0,1 % dans une solution d'acide acétique à 1 % et de méthanol à 40 %, et rincées soigneusement à l'eau. Une bande de 90 kDa a été découpée dans plusieurs couloirs. Les protéines de la membrane ont été séquencées par dégradation selon Edman dans un séquenceur 740 A d'Applied Biosystems, avec, en ligne un analyseur HPLC PTH 120 A programmé par le fabricant pour la membrane Problott. Les séquences d'aminoacides ont été analysées sur un ordinateur Data General MV 10000 à l'Unité d' Informatique Scientifique de l'Institut Pasteur.  The separated proteins were transferred to a Problott membrane (Applied Biosystems) in 50 mM Tris - 50 mM borate for 17 hours at 4 to 5 volts / cm. The proteins were stained for 5 seconds using 0.1% amido black in a solution of 1% acetic acid and 40% methanol, and rinsed thoroughly with water. A 90 kDa strip was cut in several lanes. The membrane proteins were sequenced by degradation according to Edman in a 740 A sequencer from Applied Biosystems, with an on-line HPLC PTH 120 A analyzer programmed by the manufacturer for the Problott membrane. The amino acid sequences were analyzed on a Data General MV 10000 computer at the Scientific Computing Unit of the Institut Pasteur.
La séquence Ala-Thr-Asp-Ser-Glu-Asp de la protéine isolée correspond exactement aux amino-acides du site de clivage de la séquence signal de prédite d'après la séquence peptidique-prédite à partir du gène act A (fig. B). The Ala-Thr-Asp-Ser-Glu-Asp sequence of the isolated protein corresponds exactly to the amino acids of the cleavage site of the predicted signal sequence according to the peptide-predicted sequence from the act A gene (fig. B).
Par conséquent, le produit mature du gène act Therefore, the mature product of the act gene
A est une protéine de 610 aminoacides avec un poids moléculaire calculé de 67 kDA. Elle a un poids moléculaire apparent de 90 kDA et est exprimée à la surface de la bactérie. A is a protein of 610 amino acids with a calculated molecular weight of 67 kDA. It has an apparent molecular weight of 90 kDA and is expressed on the surface of the bacteria.
Cette protéine est nécessaire à l'assemblage de l'actine F et son absence conduit à une atténuation très importante de la virulence de Listeria monocytognes. Par conséquent, toute mutation affectant le gène act A ou son promoteur et modifiant sensiblement ou empêchant l'expression de son produit permettra l'obtention d'une souche atténuée non pathogène conformément à l'invention.  This protein is necessary for the assembly of actin F and its absence leads to a very significant attenuation of the virulence of Listeria monocytognes. Consequently, any mutation affecting the act A gene or its promoter and appreciably modifying or preventing the expression of its product will make it possible to obtain an attenuated non-pathogenic strain in accordance with the invention.
On rapportera ci-après les résultats obtenus in vivo avec la souche LUT 12 sur la protection de souris contre une infection par Listeria monocytogenes.  The results obtained in vivo with the LUT 12 strain on the protection of mice against infection by Listeria monocytogenes will be reported below.
IV - Effets, in vivo, de la souche LUT 12 : étude chez la souris IV - Effects, in vivo, of the LUT 12 strain: study in mice
A/ Multiplication du mutant actA dans le foie et la rate de souris infectées.  A / Multiplication of the actA mutant in the liver and spleen of infected mice.
Le comportement de LUT 12 a été étudié, après injection intraveineuse, dans la rate et le foie de souris, qui sont les principaux organes cibles où L. monocytogenes de type sauvage expriment leur pathogénicité. Les essais cliniques utilisés étaient les suivants : les foies et rates des souris infectées étaient récoltés à différents moments après l'infection, et homogénéisés pour permettre la libération de bactéries, et les bactéries vivantes étaient comptées in vitro.  The behavior of LUT 12 has been studied, after intravenous injection, in the spleen and liver of mice, which are the main target organs where L. monocytogenes of wild type express their pathogenicity. The clinical trials used were as follows: the livers and spleens of infected mice were harvested at various times after infection, and homogenized to allow the release of bacteria, and live bacteria were counted in vitro.
La DL50 du mutant actA LUT 12, après injection intraveineuse chez des souris C3H de lignée pure était plus élevée d'un facteur 3 log10 que celle de Listeria monocytogenes de type sauvage (2,5 × 107 contre 2,5 × 104). The LD 50 of the actA LUT 12 mutant, after intravenous injection in C3H pure line mice was a factor 3 log 10 higher than that of wild-type Listeria monocytogenes (2.5 × 10 7 versus 2.5 × 10 4 ).
Les cinétiques de croissance du mutant actA et de la souche virulente de type sauvage dans le foie et la rate ont été comparées. Après injection intraveineuse d'une dose maximum sublétale du mutant actA (1,5 × 107 organismes) ou de deux doses différentes de L. monocytogenes virulentes (7 × 103 ou 6 × 104), le nombre de bactéries dans le foie et dans la rate des souris infectées a été déterminé à des durées variables au cours de l'infection. The growth kinetics of the actA mutant and the virulent wild-type strain in the liver and spleen were compared. After intravenous injection of a maximum sublethal dose of the actA mutant (1.5 × 10 7 organisms) or two different doses of virulent L. monocytogenes (7 × 10 3 or 6 × 10 4 ), the number of bacteria in the liver and in the spleen of infected mice was determined at varying durations during infection.
Une augmentation du nombre de mutant actA a été observée dans la rate pendant les 24 premières heures, mais cette augmentation était limitée (1 log10) en comparaison avec l'augmentation d'un facteur 4 log10 observée avec la souche de type sauvage. A partir du jour 1, le nombre de bactéries mutantes actA a rapidement diminué et au jour 5, presqu'aucune bactérie ne pouvait être récoltée à partir de la rate. Au contraire, la souche sauvage de Listeria monocytogenes pouvait toujours être détectée dans cet organe aux jours 9 à 10 de l'infection. Dans le foie, le nombre de bactéries mutantes actA a persisté à un niveau stable jusqu'au jour 4, et après cela a diminué rapidement ; au contraire, le nombre de Listeria monocytogenes de type sauvage a augmenté d'un facteur de 2 log10 avant d'atteindre un plateau pendant 6 à 7 jours. An increase in the number of actA mutants was observed in the spleen during the first 24 hours, but this increase was limited (1 log 10 ) compared to the increase of a factor 4 log 10 observed with the wild type strain. From day 1, the number of actA mutant bacteria decreased rapidly and by day 5, almost no bacteria could be harvested from the spleen. On the contrary, the wild strain of Listeria monocytogenes could still be detected in this organ on days 9 to 10 of the infection. In the liver, the number of actA mutant bacteria persisted at a stable level until day 4, and after that decreased rapidly; on the contrary, the number of wild-type Listeria monocytogenes increased by a factor of 2 log 10 before reaching a plateau for 6 to 7 days.
La persistance du mutant actA à un niveau stable pendant 4 jours dans le foie peut refléter soit un équilibre entre la multiplication bactérienne et la mort bactérienne, ou une survie des bactéries sans multiplication. Pour faire la différence entre ces deux possibilités, les courbes de croissance bactérienne dans le foie et dans la rate de souris traitées par l'ampicil- une ont été comparées à celles de souris témoins. L'ampicilline inhibe la synthèse du peptidoglycane et est bactéricide sur des bactéries en phase de multiplication active. Les souris infectées ont été traitées à deux reprises avec 15 mg d'ampicilline par voie intrapéritonéale à partir du jour 1, 2 ou 3 de l'infection ; le foie et la rate ont été prélevés un jour plus tard, et le nombre de bactéries restantes a été déterminé et comparé avec celui obtenu à partir de souris non traitées par l'antibiotique. The persistence of the actA mutant at a stable level for 4 days in the liver may reflect either a balance between bacterial multiplication and bacterial death, or survival of the bacteria without multiplication. To differentiate between these two possibilities, the bacterial growth curves in the liver and spleen of mice treated with ampicillin one were compared to those of control mice. Ampicillin inhibits the synthesis of peptidoglycan and is bactericidal on bacteria in the active multiplication phase. The infected mice were treated twice with 15 mg of ampicillin intraperitoneally from day 1, 2 or 3 of the infection; the liver and spleen were removed a day later, and the number of remaining bacteria was determined and compared with that obtained from mice not treated with the antibiotic.
Après un tel traitement, le nombre de bactéries actA a diminué brutalement dans la rate au jour 2, et dans le foie aux jours 2 et 3, mais aucune différence entre la courbe de croissance témoin n'a été trouvée aux jours 3 et 4 dans la rate, ou au jour 4 dans le foie.  After such treatment, the number of actA bacteria decreased abruptly in the spleen on day 2, and in the liver on days 2 and 3, but no difference between the control growth curve was found on days 3 and 4 in the spleen, or on day 4 in the liver.
Ces résultats suggèrent que la persistance du mutant actA dans le foie est due à un équilibre entre la multiplication bactérienne et la mort. Etant donné que les mutants actA sont déficients en ce qui concerne la dissémination de cellule à cellule, in vitro, la persistante de actA dans le foie est vraisemblablement due à une infection de cellules voisines après lyse des premières cellules hôtes infectées ; par conséquent, Listeria monocytogenes peut être exposée à des effecteurs bactéricides présents dans un milieu extracellulaire et sa capacité de dissémination locale peut être diminuée.  These results suggest that the persistence of the actA mutant in the liver is due to a balance between bacterial multiplication and death. Since actA mutants are deficient in cell-to-cell dissemination in vitro, the persistence of actA in the liver is likely due to infection of neighboring cells after lysis of the first infected host cells; consequently, Listeria monocytogenes can be exposed to bactericidal effectors present in an extracellular medium and its capacity for local dissemination can be reduced.
En outre, si des Listeria monocytogenes extra-cellulaires sont phagocytées par des macrophages activés par l'interféron γ, elles peuvent être incapables d'atteindre le cytosol et de poursuivre leur cycle intracellulaire.  In addition, if extra-cellular Listeria monocytogenes are phagocytosed by macrophages activated by interferon γ, they may be unable to reach the cytosol and to continue their intracellular cycle.
Finalement, les mutants actA ont été éliminés de la rate et du foie plus tôt que la souche de type sauvage, suggérant que les effecteurs protecteurs de l'hôte sont rapidement induits chez la souris infectée avec le mutant actA. Ultimately, the actA mutants were eliminated from the spleen and liver sooner than the wild-type strain, suggesting that the protective effectors of the host are rapidly induced in mice infected with the actA mutant.
B/ Effets d'une infection unique avec le mutant actA sur l'induction d'une immunité persistante.  B / Effects of a single infection with the actA mutant on the induction of persistent immunity.
L'existence d'une résistance non spécifique due à l'activation des macrophages est un phénomène bien connu, survenant rapidement et de manière transitoire chez des souris "récupérant" d'une infection sublétale.  The existence of non-specific resistance due to the activation of macrophages is a well-known phenomenon, occurring rapidly and transiently in mice "recovering" from a sublethal infection.
Dans le but d'éviter de détecter simultanément des effets de l'immunité non spécifique et spécifique, les inventeurs ont déterminé à quel moment la résistance non spécifique cessait de s'exprimer.  In order to avoid simultaneously detecting the effects of non-specific and specific immunity, the inventors determined when the non-specific resistance stopped expressing.
Ils ont injecté par voie intraveineuse un pathogène intra-cellulaire non apparenté, Yersinia enterocolitica Ye8081 0:8 (16,17) soit chez des souris naïves, soit chez des souris infectées avec le mutant actA, 4, 6,5, et 8,5 semaines avant l'injection. Ils ont comparé le nombre de bactéries dans la rate et dans le foie dans ces deux groupes de souris. Aucune différence n'a été observée entre les deux groupes à chaque moment du test. Ils ont alors réalisé les expériences suivantes, They injected intravenously an unrelated intracellular pathogen, Yersinia enterocolitica Ye8081 0: 8 (16,17) either in naive mice or in mice infected with the mutant actA, 4, 6.5, and 8, 5 weeks before the injection. They compared the number of bacteria in the spleen and in the liver in these two groups of mice. No difference was observed between the two groups at any time during the test. They then performed the following experiments,
6 semaines ou plus après l'infection avec le mutant actA. 6 weeks or more after infection with the actA mutant.
Premièrement, la DL50 de L. monocytogenes de type sauvage a été déterminée chez des souris infectées 6 semaines auparavant avec le mutant actA, et chez des souris témoins : une différence d'un ordre de 100 a été observée entre les deux groupes (2,2 × 106 et 2,5 × 104 respectivement). First, the LD 50 of wild-type L. monocytogenes was determined in mice infected 6 weeks previously with the actA mutant, and in control mice: a difference of around 100 was observed between the two groups (2 , 2 × 10 6 and 2.5 × 10 4 respectively).
Deuxièmement, les courbes de croissance de L. monocytogenes de type sauvage ont été comparées dans le foie et la rate de souris naïves et de souris immunisées depuis 6 semaines, ce pendant les 3 premiers jours de l'infection. Un ralentissement significatif de la croissance bactérienne a été observé à partir du jour 1 dans la rate, et à partir des jours 2 à 3 dans le foie. Troisièmement, cette inhibition spécifique de la croissance de L. monocytogenes de type sauvage était encore efficace 8,5 semaines après l'infection avec le mutant actA (diminution de 4,01 log10 dans la numération bactérienne de la rate 48 heures après un inoculum bactérien de 5 × 104). Second, the growth curves of wild-type L. monocytogenes were compared in the liver and spleen of naive mice and of mice immunized for 6 weeks, during the first 3 days of infection. A significant slowdown in bacterial growth was observed from day 1 in the spleen, and from days 2 to 3 in the liver. Third, this specific inhibition of the growth of wild-type L. monocytogenes was still effective 8.5 weeks after infection with the actA mutant (decrease of 4.01 log 10 in the bacterial count of the spleen 48 hours after an inoculum 5 × 10 4 ).
Ces résultats montrent qu'une infection unique avec le mutant actA atténué est suffisante pour induire une immunité contre L. monocytogenes de type sauvage.  These results show that a single infection with the attenuated actA mutant is sufficient to induce immunity against wild-type L. monocytogenes.
C/ Génération de lymphocytes T CD8+ protecteurs contre Listeria. C / Generation of CD8 + T lymphocytes protecting against Listeria.
Un transfert de protection a été effectué chez des "récepteurs" syngéniques naïfs en utilisant des cellules de rate récoltées 7 jours après une injection intra-veineuse de 1,5 × 107 bactéries mutantes actA. Les "récepteurs" ont été exposés à une infection intraveineuse avec une dose létale de L. monocytogenes de type sauvage pendant 1 heure, et le nombre de bactéries a été déterminé dans le foie et dans la rate des "récepteurs" deux jours après l'infection. A protection transfer was carried out in naive syngeneic "receptors" using spleen cells harvested 7 days after an intravenous injection of 1.5 × 10 7 actA mutant bacteria. The "receptors" were exposed to intravenous infection with a lethal dose of wild-type L. monocytogenes for 1 hour, and the number of bacteria was determined in the liver and in the spleen of the "receptors" two days after infection.
Premièrement, l'injection de splénocytes de souris infectées avec le mutant actA a provoqué une diminution importante du nombre de L. monocytogenes de type sauvage récoltées à partir de la rate des "récepteurs" ayant reçu les cellules, et cet effet était dosedépendant. La diminution du nombre de bactéries a également été observée dans le foie des "récepteurs", mais à un degré moindre (diminution de 1,00 ± 0,45 log10, n = 4, pour des transferts de 5 × 107 à 2,5 × 10e cellules, P < 0,02). First, the injection of splenocytes from mice infected with the actA mutant caused a significant reduction in the number of wild-type L. monocytogenes harvested from the spleen of "receptors" which received the cells, and this effect was dependent. The decrease in the number of bacteria was also observed in the liver of "receptors", but to a lesser degree (decrease of 1.00 ± 0.45 log 10 , n = 4, for transfers from 5 × 10 7 to 2 , 5 × 10 th cells, P <0.02).
Dans le but de caractériser le phénotype des cellules de rate protectrices, on a soustrait dans la population cellulaire splénique immunitaire, soit des lymphocytes Thy-1+, soit CD4% soit CD8\ avant le transfert passif (Tableau 1 ci-dessous). Le transfert des splénocytes non soustraits a résulté en une réduction de 3 à 4 log10 du nombre de bactéries dans la rate. Cette protection était transférée par les lymphocytes T, car une déplétion en lymphocytes Thy-1+ a aboli la diminution de la charge des bactéries dans la rate. Le niveau élevé de protection conférée par des splénocytes immuns après 7 jours n'était que peu affecté par la déplétion de la sous-population CD4. La majorité de l'effet protecteur conféré par les splénocytes immuns après 7 jours était sensible à une déplétion de la sous-population de CD8, mais ne pouvait pas être mis sur le compte uniquement de la sous-population de lymphocytes CD8+. In order to characterize the phenotype of the protective spleen cells, the immune splenic cell population, namely Thy-1 + lymphocytes, either CD4% or CD8 \ before passive transfer (Table 1 below). The transfer of untreated splenocytes resulted in a 3-4 log 10 reduction in the number of bacteria in the spleen. This protection was transferred by T lymphocytes because a depletion of Thy-1 + lymphocytes abolished the reduction in the burden of bacteria in the spleen. The high level of protection conferred by immune splenocytes after 7 days was only slightly affected by the depletion of the CD4 subpopulation. The majority of the protective effect conferred by the immune splenocytes after 7 days was sensitive to depletion of the CD8 subpopulation, but could not be attributed solely to the CD8 + lymphocyte subpopulation.
Dans la mesure où la déplétion en Thy-1 a supprimé la protection, et où la déplétion en CD4 n'a eu qu'un effet marginal, on peut considérer qu'une partie du rôle protecteur non CD8 dépendant est due aux lymphocytes T doublement négatifs, comme cela a déjà été observé par DUNN et NORTH ( 18). Des expériences publiées auparavant de déplétion/protection sur des cellules lymphoïdes isolées de souris récupérant d'une infection avec L. monocytogenes de type sauvage ont montré que L. monocytogenes de type sauvage était capable d' induire une protection qui était presque exclusivement due à des lymphocytes CD8+, et que ces lymphocytes CD8+ protégeaient sans la participation de lymphocytes CD4+. Insofar as the Thy-1 depletion suppressed the protection, and since the CD4 depletion had only a marginal effect, it can be considered that part of the non-CD8 dependent protective role is due to doubly T lymphocytes. negative, as already noted by DUNN and NORTH (18). Previously published depletion / protection experiments on lymphoid cells isolated from mice recovering from infection with wild-type L. monocytogenes have shown that wild-type L. monocytogenes is capable of inducing protection which was almost exclusively due to CD8 + lymphocytes, and that these CD8 + lymphocytes protected without the participation of CD4 + lymphocytes.
Le mutant actA de L. monocytogenes est ainsi capable d'induire la génération de lymphocytes CD8+ spécifiques, protégeant contre Listeria. The actA mutant of L. monocytogenes is thus capable of inducing the generation of specific CD8 + lymphocytes, protecting against Listeria.
Le mutant actA possède un gène de la listériolysine-O fonctionnel qui lui permet de s'échapper du phagosome et d'entrer dans le cytosol ; il est probable que le mutant actA est capable de stimuler la production de lymphocytes T CD8+ protecteurs contre Listeria, recon- naissant des peptides naturels de L. monocytogenes. En outre, la capacité du mutant actA à se multiplier de manière transitoire dans les organes des souris infectées et de sécréter une quantité suffisante de protéines bactériennes est probablement critique pour permettre une production efficace des lymphocytes CD8+ protecteurs. The actA mutant has a functional listeriolysin-O gene which allows it to escape from the phagosome and enter the cytosol; it is likely that the actA mutant is capable of stimulating the production of CD8 + T lymphocytes which protect against Listeria, recognized arising from natural peptides of L. monocytogenes. In addition, the ability of the actA mutant to transiently multiply in the organs of infected mice and to secrete a sufficient amount of bacterial proteins is probably critical to allow efficient production of protective CD8 + lymphocytes.
Le transposon Tn917-lac s'est inséré dans actA, le second gène de l'opéron lécithinase, et a un effet polaire sur l'expression du gène plcB codant pour une lécithinase. Les résultats obtenus dans la présente invention indiquent que la lécithinase ne joue pas un rôle essentiel dans l'induction d 'une immunité protectrice contre L. monocytogenes.  The transposon Tn917-lac is inserted into actA, the second gene of the operon lecithinase, and has a polar effect on the expression of the plcB gene coding for a lecithinase. The results obtained in the present invention indicate that lecithinase does not play an essential role in the induction of protective immunity against L. monocytogenes.
En conclusion, les résultats de la présente invention montrent que des mutants atténués actA sont capables d'induire des lymphocytes T CD8+ protecteurs contre Listeria, et qu'un état d'immunité protectrice contre L. monocytogenes de type sauvage peut être établi par une infection unique. Comme les mutants actA pénètrent dans le cytosol des cellules infectées et se multiplient dans ce compartiment, on peut considérer leur utilisation comme vecteur vivant pour délivrer des protéines heterologues dans le cytosol et favoriser la production de lymphocytes T CD8+ ; de tels vecteurs vivants capables de se multiplier de façon transitoire sont supposés délivrer une charge suffisante de protéines heterologues dans le cytosol, ce pendant une durée de temps courte. En plus de leur utilisation potentielle comme modèle pour le développement de vaccins mettant en oeuvre des vecteurs vivants, ces Listeria de virulence atténuée peuvent être utiles pour le criblage et la caractérisation de peptides bactériens ou parasitaires spécifiques d'allèles du locus codant pour les molécules de classe 1 du CMH. En effet, certaines bactéries et certains parasites résident dans les compartiments vacuo- laires ; en outre, la cinétique de croissance peut être faible. Ainsi, pour les espèces de Leishmania ou les espèces de Mycobacterium, cet outil pourrait être très utile pour définir la spécificité des lymphocytes CD8+ qui sont produits en réponse à l'infection. In conclusion, the results of the present invention show that actA attenuated mutants are capable of inducing protective CD8 + T lymphocytes against Listeria, and that a state of protective immunity against wild-type L. monocytogenes can be established by a single infection. As the actA mutants enter the cytosol of infected cells and multiply in this compartment, we can consider their use as a living vector to deliver heterologous proteins in the cytosol and promote the production of CD8 + T lymphocytes; such living vectors capable of transiently multiplying are supposed to deliver a sufficient charge of heterologous proteins in the cytosol, for a short period of time. In addition to their potential use as a model for the development of vaccines using live vectors, these Listeria of attenuated virulence can be useful for the screening and characterization of bacterial or parasitic peptides specific for alleles of the locus encoding the molecules of CMH class 1. Some bacteria and parasites reside in the vacuo- laires; in addition, the growth kinetics may be low. Thus, for Leishmania species or Mycobacterium species, this tool could be very useful for defining the specificity of CD8 + lymphocytes which are produced in response to infection.
Figure imgf000027_0001
Les cellules spléniques ont été isolées à partir de souris C3H au jour 7 après injection intraveineuse de 1,5 × 107 mutants actA. Les cellules Thy-1+, CD4+ ou CD8+ ont été soustraites in vitro avant le transfert adoptif. Les anticorps monoclonaux utilisés ici étaient Anti-Thy-1, 2 J1j (ATCC TIB 184), anti-CD4 RL1724 (Ceredig, R. Lowenthal , J.W. Nabholz M. and MacDonald, H.R. , 1985, Nature 314 98 ) , et anti-CD8 31M ( Sarmiento M. Glasebrook. A. L. et Fitch F.W. , 1980 , J. Immunol . 125-2665) . La déplétion était < 90% après chaque traitement cytotoxique par des anticorps monoclonaux (résultats non représentés). Les populations cellulaires non déplétées [complément (C)] ou déplétées ont été transférées chez des "récepteurs" naïfs syngéniques 1 heure après injection de 4 à 8 × 104 L. monocytogenes de type sauvage. Les groupes témoins comprenaient des récepteurs de cellules non déplétées (c'est-à-dire incubées seulement avec C) et des récepteurs recevant seulement une injection de L. monocytogenes de type sauvage (c'est-à-dire pas de cellules). 48 heures après l'injection, les bactéries ont été comptées (moyenne + ET) dans la rate des "récepteurs" (3 à 5 souris par groupe). La signification statistique (test de Student) est rapportée pour l'efficacité du transfert (cellules traitées contre pas de cellules) et pour l'effet du traitement de déplétion sur l'efficacité du transfert (cellules déplétées contre cellules traitées par C').
Figure imgf000027_0001
Spleen cells were isolated from C3H mice on day 7 after intravenous injection of 1.5 × 10 7 actA mutants. Thy-1 + , CD4 + or CD8 + cells were removed in vitro before adoptive transfer. The monoclonal antibodies used here were Anti-Thy-1, 2 J1j (ATCC TIB 184), anti-CD4 RL1724 (Ceredig, R. Lowenthal, JW Nabholz M. and MacDonald, HR, 1985, Nature 314 98), and anti- CD8 31M (Sarmiento M. Glasebrook. AL and Fitch FW, 1980, J. Immunol. 125-2665). Depletion was <90% after each cytotoxic treatment with monoclonal antibodies (results not shown). The non-depleted [complement (C)] or depleted cell populations were transferred to naive syngeneic "receptors" 1 hour after injection of 4 to 8 × 10 4 L. wild-type monocytogenes. Control groups included receptors for non-depleted cells (i.e. incubated only with C) and receptors receiving only an injection of wild type L. monocytogenes (i.e., no cells). 48 hours after the injection, the bacteria were counted (average + TE) in the spleen of the "receptors" (3 to 5 mice per group). Statistical significance (Student's test) is reported for the efficiency of transfer (cells treated against no cells) and for the effect of depletion treatment on the efficiency of transfer (depleted cells against cells treated with C ').

Claims

REFERENCES BIBLIOGRAPHIQUES BIBLIOGRAPHICAL REFERENCES
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2. De Rycke, J., Phan-Thanh, L. and Bernard, S. (1989). Immunochemical identification and biological characetrization of cytotoxic necrotizing factor from Escherichia coli. J. Clin. Microbiol. 27, 983-988. 3. Domann, E., Leimeister-Wâchter, M. Goebel, W. and Chakraborty, T. (1991). Molecular cloning, sequencing, and identification of a métalloprotease gène from listeria monocytogenes that is species spécifie and physically linked to the listeriolysin gène. Infect. Immun. 59, 65-72. 2. De Rycke, J., Phan-Thanh, L. and Bernard, S. (1989). Immunochemical identification and biological characetrization of cytotoxic necrotizing factor from Escherichia coli. J. Clin. Microbiol. 27, 983-988. 3. Domann, E., Leimeister-Wâchter, M. Goebel, W. and Chakraborty, T. (1991). Molecular cloning, sequencing, and identification of a metalloprotease gene from listeria monocytogenes that is species specifies and physically linked to the listeriolysin gene. Infect. Immun. 59, 65-72.
4. Heukeshoven, J. and Dernick, R. (1985). Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6, 103-112. 4. Heukeshoven, J. and Dernick, R. (1985). Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6, 103-112.
5. Kuhn, M., Prévost, M.-C, Mounier, J. and Sansonetti, P.J. (1990). A nonvirulent mutant of Listeria monocytogenes does not move intracellularly but still induces polymérisation of actin. Infect, immun. 58, 3477-3486. 5. Kuhn, M., Prévost, M.-C, Mounier, J. and Sansonetti, P.J. (1990). A nonvirulent mutant of Listeria monocytogenes does not move intracellularly but still induces polymerization of actin. Infect, immune. 58, 3477-3486.
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8. Mengaud, J., Geoffroy, C. and Cossart, P. (1991b). Identification of a novel operon involved in virulence of Listeria monocytogenes; its first gène encodes a protein homologous to bacterial metalloproteases. Infect. Immun. 59, 1043-1049. 8. Mengaud, J., Geoffroy, C. and Cossart, P. (1991b). Identification of a novel operon involved in virulence of Listeria monocytogenes; its first gene encodes a protein homologous to bacterial metalloproteases. Infect. Immun. 59, 1043-1049.
9. Racz, P., Tenner, K. and Szivessy, K. (1970). Electron microscopic studies in expérimental keratoconjunctivitis listeriosa. I. Pénétration of Listeria monocytogenes into corneal epithelial cells. Acta Microbiol. Acad. Sci. Hung. 17, 221-236. 9. Racz, P., Tenner, K. and Szivessy, K. (1970). Electron microscopic studies in experimental keratoconjunctivitis listeriosa. I. Penetration of Listeria monocytogenes into corneal epithelial cells. Acta Microbiol. Acad. Sci. Hung. 17, 221-236.
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12. Vazquez-Boland, J.-A., Kocks, C, Dramsi, S., Ohayon, H., Geoffroy, C, Mengaud J., and Cossart, P. (). Nucleotide séquence of the lécithinase operon of Listeria monocytogenes and possible rôle of lécithinase in cell-to-cell spread. Infect. Immun., Jan. 1992, p. 219-230. 12. Vazquez-Boland, J.-A., Kocks, C, Dramsi, S., Ohayon, H., Geoffroy, C, Mengaud J., and Cossart, P. (). Nucleotide sequence of the lecithinase operon of Listeria monocytogenes and possible role of lecithinase in cell-to-cell spread. Infect. Immun., Jan. 1992, p. 219-230.
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LISTE DES SEQUENCES LIST OF SEQUENCES
- - - - - - - - - - - - - - - - - - - I - INFORMATION GENERALE  - - - - - - - - - - - - - - - - - - - I - GENERAL INFORMATION
(1) DEMANDEUR : INSTITUT PASTEUR (1) APPLICANT: INSTITUT PASTEUR
(2) TITRE DE L'INVENTION :  (2) TITLE OF THE INVENTION:
Mutant atténué de Listeria monocytogenes.  Attenuated mutant of Listeria monocytogenes.
souche recombinante de Listeria monocytogenes. utilisation comme vecteurs heterologues d'antigène vaccinal et utilisation comme vaccin ou composition diagnostique.  recombinant strain of Listeria monocytogenes. use as heterologous vectors of vaccine antigen and use as vaccine or diagnostic composition.
(3) NOMBRE DE SEQUENCES : 1 INFORMATION POUR SEQ ID N° 1  (3) NUMBER OF SEQUENCES: 1 INFORMATION FOR SEQ ID N ° 1
CARACTERISTIQUES DES SEQUENCES TYPE : protéine  CHARACTERISTICS OF THE TYPE SEQUENCES: protein
LONGUEUR : 610 aminoacides  LENGTH: 610 amino acids
TYPE DE MOLECULE : protéine de surface  TYPE OF MOLECULE: surface protein
ORIGINE ORIGIN
ORGANISME : Listeria monocytogenes LIGNEE CELLULAIRE : LO 28 ORGANISM: Listeria monocytogenes CELL LINE: LO 28
CARACTERISTIQUE FEATURE
NOM DE LA PROTEINE : produit du gène Act A PROTEIN NAME: Act A gene product
Figure imgf000032_0001
SYMBOLES DES ACIDES AMINES - - - - - - - - - - - - - - - - - - - - - - - - - -
Figure imgf000032_0001
SYMBOLS OF AMINO ACIDS - - - - - - - - - - - - - - - - - - - - - - - - - -
A Ala alanine To Ala alanine
C Cys citéine  C Cys city
D Asp acide aspartique D Asp aspartic acid
E Glu acide glutamiqueE Glu glutamic acid
F Phe phénylalanineF Phe phenylalanine
G Gly glycine G Gly glycine
H His histidine  H His histidine
I Ile isoleucine  I isoleucine island
K Lys lysine  K lysine lysine
L Leu leucine  L Leu leucine
M Met méthionine  M Met methionine
N Asn asparagine  N Asn asparagine
P Pro proline  P Pro proline
Q Gin glutamine  Q Gin glutamine
R Arg arginine  R Arg arginine
S Ser serine  S Ser Serine
T Thr thréonine  T Threonine Thr
V Val valine  V Val valine
W Trp tryptophane  W Trp tryptophan
Y Tyr tyrosine Y Tyr tyrosine
REVENDICATIONS
1. Mutant atténué de Listeria monocytogenes comportant, dans le gène act A ou dans le promoteur de celui-ci, une mutation apte à bloquer ou modifier sensiblement l'expression de la protéine codée par le gène act A.  1. Attenuated mutant of Listeria monocytogenes comprising, in the act A gene or in the promoter thereof, a mutation capable of blocking or substantially modifying the expression of the protein encoded by the act A gene.
2. Mutant atténué de Listeria monocytogenes selon la revendication 1, caractérisé en ce que la mutation consiste en une insertion, une délétion ou une mutation par mutagénèse dirigée.  2. Attenuated mutant of Listeria monocytogenes according to claim 1, characterized in that the mutation consists of an insertion, a deletion or a mutation by site-directed mutagenesis.
3. Mutant atténué de Listeria monocytogenes selon la revendication 1 ou la revendication 2, caractérisé en ce que la mutation consiste en l'insertion d'un transposon stable.  3. Attenuated mutant of Listeria monocytogenes according to claim 1 or claim 2, characterized in that the mutation consists in the insertion of a stable transposon.
4. Mutant atténué de Listeria monocytogenes selon la revendication 3, caractérisé en ce que le transposon stable est le transposon Tn917-lac.  4. Attenuated mutant of Listeria monocytogenes according to claim 3, characterized in that the stable transposon is the Tn917-lac transposon.
5. Mutant atténué de Listeria monocytogenes selon l'une quelconque des revendications précédentes, caractérisé en ce que la mutation est effectuée dans le fragment d'ADN codant pour la séquence peptidique à motifs répétés comprise entre les aminoacides 235 à 315, 350 à 360, 367 à 385 et 389 à 393 de la séquence SEQ ID n°1.  5. Attenuated mutant of Listeria monocytogenes according to any one of the preceding claims, characterized in that the mutation is carried out in the DNA fragment coding for the peptide sequence with repeating motifs comprised between amino acids 235 to 315, 350 to 360, 367 to 385 and 389 to 393 of the sequence SEQ ID No. 1.
6. Mutant atténué selon l'une des revendications 1 à 4, caractérisé en ce que la mutation consiste en une insertion entre les aminoacides 61 et 62 de la séquence peptidique SEQ ID n° 1.  6. Attenuated mutant according to one of claims 1 to 4, characterized in that the mutation consists of an insertion between amino acids 61 and 62 of the peptide sequence SEQ ID No. 1.
7. Mutant atténué de Listeria monocytogenes selon la revendication 6, dénommé LUT 12, déposé à la 7. Attenuated mutant of Listeria monocytogenes according to claim 6, called LUT 12, deposited at the
CNCM le 30 janvier 1992 sous le n° 1-1167. CNCM on January 30, 1992 under number 1-1167.
8. Vaccin humain ou vétérinaire, caractérisé en ce qu'il comprend en tant que composant actif une souche mutante atténuée de Listeria monocytogenes selon l'une des revendications précédentes. 8. Human or veterinary vaccine, characterized in that it comprises, as active component, an attenuated mutant strain of Listeria monocytogenes according to one of the preceding claims.
9. Souche recombinante de Listeria monocytogenes. caractérisée en ce qu'elle comporte un ADN hétérologue, soit inséré dans le génome d'un mutant atténué selon l'une des revendications précédentes, soit porté par un plasmide qui se réplique dans le mutant atténué. 9. Recombinant strain of Listeria monocytogenes. characterized in that it comprises a heterologous DNA, either inserted into the genome of an attenuated mutant according to one of the preceding claims, or carried by a plasmid which replicates in the attenuated mutant.
10. Souche recombinante selon la revendication 9, caractérisée en ce que l'ADN hétérologue consiste en un gène hétérologue codant pour un antigène protecteur cible de lymphocytes T de la sous-classe CD8.  10. Recombinant strain according to claim 9, characterized in that the heterologous DNA consists of a heterologous gene coding for a target protective antigen of T lymphocytes of the CD8 subclass.
11. Souche recombinante selon la revendication 10, caractérisée en ce que l'antigène est un antigène bactérien, notamment de mycobactéries.  11. Recombinant strain according to claim 10, characterized in that the antigen is a bacterial antigen, in particular of mycobacteria.
12. Souche recombinante selon la revendication 10, caractérisée en ce que l'antigène est un antigène parasitaire, notamment de Leishmania. de Trypanosoma ou de Toxoplasma. Theileria.  12. Recombinant strain according to claim 10, characterized in that the antigen is a parasitic antigen, in particular of Leishmania. Trypanosoma or Toxoplasma. Theileria.
13. Souche recombinante selon la revendication 10, caractérisée en ce que l'antigène est un antigène viral, notamment du VIH, du virus de la chorioméningite lymphocytaire ou du virus de la grippe.  13. Recombinant strain according to claim 10, characterized in that the antigen is a viral antigen, in particular of HIV, of the lymphocytic choriomeningitis virus or of the influenza virus.
14. Souche recombinante selon la revendication 13, caractérisée en ce que l'antigène est l'antigène gag et/ou l'antigène nef du VIH et/ou tout ou partie de l'enveloppe gp 120 du VIH1 ou gp 140 du VIH2.  14. Recombinant strain according to claim 13, characterized in that the antigen is the gag antigen and / or the HIV nave antigen and / or all or part of the gp 120 envelope of HIV1 or gp 140 of HIV2.
15. Souche recombinante l'une des revendications 9 à 14, caractérisée en ce qu'elle comporte un promoteur de Listeria en amont de l'ADN hétérologue.  15. Recombinant strain one of claims 9 to 14, characterized in that it comprises a Listeria promoter upstream of the heterologous DNA.
16. Souche recombinante selon la revendication 15, caractérisée en ce que le promoteur est le promoteur hly.  16. Recombinant strain according to claim 15, characterized in that the promoter is the hly promoter.
17. Souche recombinante selon la revendication 16, caractérisée en ce que l'ADN hétérologue est fusionné avec le début du gène hly, de manière à utiliser la séquence signal de la listeriolysine O pour sécréter le produit de l'ADN hétérologue dans le cytoplasme de la cellule hôte. 17. Recombinant strain according to claim 16, characterized in that the heterologous DNA is fused with the start of the hly gene, so as to use the signal sequence of listeriolysin O to secrete the product of the heterologous DNA in the cytoplasm of the host cell.
18. Vaccin humain ou vétérinaire recombinant, caractérisé en ce qu'il comprend en tant que composant actif une souche recombinante selon l'une des revendications 9 à 17.  18. Recombinant human or veterinary vaccine, characterized in that it comprises, as active component, a recombinant strain according to one of claims 9 to 17.
19. Composition de diagnostic comprenant une souche recombinante de Listeria monocytogenes selon l'une des revendications 10 à 17, pour le contrôle de l'état de protection d'un hôte humain ou animal contre une infection provoquée par un microorganisme comprenant un antigène sensiblement identique à celui codé par le gène hétérologue inséré dans la souche mutante recombinante ou porté par un plasmide se répliquant dans la souche mutante recombinante.  19. A diagnostic composition comprising a recombinant strain of Listeria monocytogenes according to one of claims 10 to 17, for monitoring the protective state of a human or animal host against an infection caused by a microorganism comprising a substantially identical antigen to that encoded by the heterologous gene inserted into the recombinant mutant strain or carried by a plasmid replicating in the recombinant mutant strain.
PCT/FR1993/000105 1992-01-31 1993-02-01 Attenuated mutant of listeria monocytogenes; recombinant strain of listeria monocytogenes, use as heterologous vaccinal antigene vectors and use as vaccine or diagnostic composition WO1993015212A1 (en)

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