WO2017108515A1 - Souches bactériennes à gram négatif modifiées et leurs utilisations - Google Patents

Souches bactériennes à gram négatif modifiées et leurs utilisations Download PDF

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WO2017108515A1
WO2017108515A1 PCT/EP2016/080933 EP2016080933W WO2017108515A1 WO 2017108515 A1 WO2017108515 A1 WO 2017108515A1 EP 2016080933 W EP2016080933 W EP 2016080933W WO 2017108515 A1 WO2017108515 A1 WO 2017108515A1
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gene coding
strain
autologous
formyltransferase
heterologous
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Inventor
Vilma Arce Gorvel
Raquel CONDE ÁLVAREZ
Jean-Pierre Gorvel
Sean HANNIFFY
Maite IRIARTE CILVETI
Ignacio MORIYÓN URÍA
Amaia ZÚÑIGA RIPA
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Universidad de Navarra
Aix Marseille Universite
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
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Universidad de Navarra
Aix Marseille Universite
Centre National de la Recherche Scientifique CNRS
Institut National de la Sante et de la Recherche Medicale INSERM
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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1014—Hydroxymethyl-, formyl-transferases (2.1.2)
    • 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
    • A61K39/098—Brucella
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Gram-negative bacteria
    • C07K16/1221—Gram-negative bacteria from Brucella (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • 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
    • 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
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • 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
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention is related to the fields of medicine and immunology.
  • the present invention provides a modified Gram negative bacterium strain useful as a vaccine as well as a tool for differentiating animals suffering an infection from those animals vaccinated with the modified strain.
  • LPS Lipopolysaccharide region
  • Antigen O (also so-called and referred hereinafter as ⁇ chain”) is a repetitive glycan polymer of LPS.
  • the O antigen is attached to the core oligosaccharide, and comprises the outermost domain of the LPS molecule. Consequently, it isa target for recognition by host antibodies.
  • the composition of the O chain varies depending on the bacterium and serotype. The presence or absence of O chains determines whether the LPS is termed smooth or rough,
  • polysaccharide i.e., they are rough mutants
  • are attenuated i.e., they are rough mutants
  • Brucella causes brucellosis, a zoonotic disease affecting mostly ruminants (B. melitensis and B. abortus) and swine (B. suis), having a significant impact on animal market and products derived therefrom (including milk, cheese, dairy products, etc.).
  • This disease can also be transmitted to human beings if infected products, such as unpasteurized milk or undercooked meat from infected animals, are ingested, or by contact with infected animals.
  • Yersinia is a gram-negative bacterial genus that includes several animal and human pathogens.
  • Y. enterocolitica which causes mild intestinal infections in animals and humans, includes several serotypes.
  • Y. enterocolitica serotypes differ in the O-chain of the LPS, and Y. enterocolitica serotype 0:9 carries an LPS with an O-chain that, like the O-chain of smooth Brucella, is a homopolymer of N- formylperosamine.
  • Animal vaccination is the most important tool in the control and eradication of the diseases caused by Brucella.
  • current vaccines such as B.
  • the modified Brucella carries a modified epitope in O chain that can give rise to antibodies not developed during infection by wild-type smooth Brucella (which carry an unmodified O-chain). Therefore, this modification can be used for
  • microorganism and can be readily differentiated from that resulting from infection.
  • further bacterial strains with the ability of eliciting an immune response which can be readily differentiated from the immune response due to the bacterial infection and which, at the same time, can be protective against the microorganism causing the infection.
  • the present inventors have developed a modified bacterium wherein (a) its authologous N-formyltransferase activity has been suppressed, and (b) a heterologous gene encoding a N-acyltransferase other than a N- formyltransferase enzyme is functionally expressed.
  • the resulting bacterium displays a LPS wherein the O chain is a homopolymer made of acylperosamine units, the "acyl" portion having been expressed by the heterologous gene.
  • the modified bacterium differs from the wild-type version in the composition of the O chain: in the present invention the O chain is a homopolymer of N- acylperosamine residues (other than N-formylperosamine) and in the wild-type version the O chain is a homopolymer of N-formylperosamine.
  • the present inventors have found that upon performing these modifications in two different Brucella strains, the resulting modified strains carrying a so markedly different antigen O are still able to elicit an effective specific immune response. And what is even more remarkable, the modified bacteria were found to be attenuated despite carrying an O chain (see FIG. 4).
  • FIG. 2 and 3 show that the bacterium of the invention elicits completely different specific antibodies when compared with other modified strains. Furthermore, from the data obtained using the Rose Bengal test (OIE - World Organisation for Animal Health recommended diagnostic test
  • the present invention provides in a first aspect a modified Gram negative bacterium strain characterized in that (a) its autologous N- formyltransferase activity is suppressed; and (b) it comprises a functional heterologous gene coding for a N-acyltransferase enzyme other than a N- formyltransferase enzyme.
  • the present invention provides a process for preparing a modified Gram negative bacterium strain as defined in the first aspect of the invention, comprising, in any order, the following steps:
  • the present invention provides a modified Gram negative bacterium strain obtainable by the process as defined in the second aspect of the invention.
  • the present invention provides a method for preparing a cell extract of the modified Gram negative bacterium strain as defined in the first or third aspect of the invention, which comprises the lysis of the strain.
  • the present invention provides a cell extract of the modified Gram negative bacterium strain as defined in the first or third aspect of the invention.
  • the present inventors have found that the antibodies elicited by the modified bacterium of the invention are clearly different from those disclosed in the prior art. From this experimental data, the inventors, without being bound to the theory, assert that these new epitopes are due to the absence of autologous N-formyltransferase activity and to the expression of the heterologous gene.
  • the present invention provides an antibody against the modified Gram negative bacterium strain as defined in the first or third aspect of the invention.
  • the present invention provides a method for preparing antibodies which comprises: (a) administer the modified Gram negative bacterium strain as defined in the first or third aspect of the invention or the cell extract of the invention to a non-human animal, and (b) isolate the antibodies.
  • the invention provides an antibody obtainable by the method of the sixth aspect of the invention.
  • either the modified bacterium, or the cell extract of the invention, or the antibody of the fifth or seventh aspect can be formulated in the form of a pharmaceutical or veterinary composition.
  • the invention provides a veterinary or pharmaceutical composition
  • a veterinary or pharmaceutical composition comprising a therapeutically effective amount of the modified Gram negative bacterium strain as defined in the first or third aspect of the invention or the antibody of aspects fifth or seventh, or the cell extract of the invention, together with one or more veterinary or pharmacologically acceptable carriers or vehicles.
  • the present invention provides a vaccine comprising a therapeutically effective amount of the modified Gram negative bacterium strain as defined in the first or third aspect of the invention or the antibody of the fifth or seventh aspect of the invention, or the cell extract of the invention, together with one or more pharmacologically or veterinary acceptable carriers or vehicles.
  • the present invention provides the modified Gram negative bacterium strain as defined in the first or third aspect of the invention or of the antibody as defined in the fifth or seventh aspect of the invention or the cell extract of the invention, for use as a medicament.
  • the present invention provides a modified Gram negative bacterium strain as defined in the first or third aspect of the invention or the antibody defined in the fifth or seventh aspect of the invention or the cell extract of the invention, for use as immunogen.
  • the present invention provides a modified Gram negative bacterium strain as defined in the first or third aspect, or of the antibody of the fifth or seventh aspect of the invention or the cell extract of the invention, for use in the treatment or prevention of an infection caused by a bacterium strain, such as a wild-type bacterium strain, belonging to the same genus as the modified bacterium.
  • This aspect can also be formulated as the use of a modified Gram negative bacterium strain as defined in the first or third aspect of the invention, or of the antibody as defined in the fifth or seventh aspect of the invention or the cell extract of the invention, for the manufacture of a medicament for the treatment or prevention of an infection caused by a bacterium strain, such as a wild-type bacterium strain, belonging to the same genus as the modified bacterium.
  • a modified Gram negative bacterium strain as defined in the first or third aspect of the invention, or of the antibody as defined in the fifth or seventh aspect of the invention or the cell extract of the invention, for the manufacture of a medicament for the treatment or prevention of an infection caused by a bacterium strain, such as a wild-type bacterium strain, belonging to the same genus as the modified bacterium.
  • This aspect can also be formulated as a method for the treatment or prevention of an infection, the method comprising the step of administering an therapeutically effective amount of a modified Gram negative bacterium strain as defined in the first or third aspect of the invention, or of the antibody of the fifth or seventh aspect of the invention or the cell extract of the invention, in a subject in need thereof, and wherein the infection is caused by a bacterium strain, such as a wild-type bacterium strain, belonging to the same genus as the modified bacterium.
  • a bacterium strain such as a wild-type bacterium strain
  • the present invention provides the use of a modified Gram negative bacterium strain as defined in the first or third aspect of the invention, the antibodies of the fifth or seventh aspect or the cell extract of the invention, for use in diagnostics.
  • the present invention provides the use of a modified Gram negative bacterium strain as defined in the first or third aspect of the invention, or of the antibody of the fifth or seventh aspect of the invention or of the cell extract of the invention, for differentiating infected from vaccinated animals; particularly for differentiating an animal vaccinated with the modified bacterium of the first or third aspect of the invention from an animal infected with a bacterium strain, such as a wild-type bacterium strain, belonging to the same genus as the modified bacterium.
  • the present invention provides an in vitro method for differentiating an animal vaccinated with the modified Gram negative bacterium strain as defined in the first or third aspect of the invention from an animal infected with a bacterium strain, such as a wild-type bacterium strain, belonging to the same genus as the modified bacterium, the method
  • the present invention provides a kit comprising the modified Gram negative bacterium strain as defined in the first or third aspect of the invention, and/or the antibody of the fifth or seventh aspect of the invention, and/or the cell extract of the invention.
  • the present invention also provides the use of a kit for differentiating an animal vaccinated with the modified Gram negative bacterium as defined in the first or third aspect of the invention, from an animal infected by a
  • bacterium strain such as a wild-type bacterium strain, belonging to the same genus as the modified bacterium, the kit being one as above defined or one comprising:
  • the bacterium such as the wild-type bacterium strain, belonging to the same genus as the modified bacterium of the first aspect of the invention, or a variant of the bacterium, such as the wild-type bacterium strain, which retains the autologous N-formyltransferase activity, and/or
  • the present invention provides an in vitro method of differentiating an animal vaccinated with a modified Brucella strain from one infected with
  • kits for differentiating an animal vaccinated with a modified Brucella 5 strain from an animal infected with Brucella comprising one or more of the following components: (i) the bacterium strain of the invention as defined above, (ii) the antibodies raised against the bacterium of the invention; (iii) means for detecting the presence of the heterologous gene and the absence of the autologous gene; and/or (iv) the cell extract of the invention
  • FIG. 1 Reactivity of (A) Ba-parental LPS, and (B) Ba::Tn7wbdRAwbkC with sera form rabbit immunized with Ba-pwbR (black triangles) or with this sera5 absorbed with Ba-parental (black circles) or Ba::Tn7wbdRAwbkC (white circles).
  • FIG. 2 Reactivity in ELISA of (A) Ba-parental, and (B) Ba::Tn7wbdRAwbkC with sera from mice infected with Ba-parental (black triangles), or
  • FIG. 3 Reactivity in ELISA of (A) Ba-parental LPS, and (B)
  • the present invention provides a modified bacterium as defined in the first aspect of the invention.
  • the N-formyltransferase enzyme acts in the LPS biosynthesis pathway catalyzing the conversion of perosamine (GDP-4-NH-4,6dideoxymannose) to formylperosamine:
  • this N-formyltransferase enzyme is only expressed in Brucella and Yersinia enterolitica serotype 0:9 strains.
  • the expression "its autologous N-formyltransferase activity is suppressed” means that the modified bacterium (either from
  • Brucella or Yersinia genus is not able, when compared to the wild-type bacterium of the same genus, of catalyzing the conversion of perosamine (GDP-4-NH-4,6dideoxymannose) to N-formylperosamine.
  • the lack of the autologous N-formyltransferase activity can be due to mutations in the gene encoding the N-formyltransferase that can stop either the transcription to RNA or the translation to protein or negatively affect the activity of the protein, so that it becomes inactive. Alternatively, it can be due to the removal of part or all the autologous gene coding for the enzyme.
  • the modified bacterium lacks the autologous gene coding for the N-formyltransferase enzyme.
  • the inventors achieved the deletion of the gene performing, in a first step, a PCR overlap strategy.
  • the skilled person can design the appropriate pairs of primers for performing the PCR on the basis of the gene sequence and using available bioinformatics tools.
  • the inventors designed primers with sequences SEQ ID NO: 1 to 4 for performing the PCR.
  • the resulting fragment could be cloned in an appropriate vector system.
  • there are other alternative ways for deleting the autologous gene (Sambrook, J. et al., (2001 ). Molecular cloning Chapter 13. Mutagenesis. Pagesl 3.1 -13.62, Cold spring harbor laboratory press New York).
  • the N-acyltransferase encoded by the heterologous gene transfers an acyl group, other than the N-formyl, to the 4-amine group of perosamine.
  • the acyl group transferred by the heterologous enzyme is selected from the group consisting of: acetyl group, 3-deoxy-L- glycerotethronyl group, 3-hydroxypropionyl group, S(+)-2-hydroxypropionyl group, and R(-)2- hydroxypropionyl group.
  • the acyl group transferred by the heterologous enzyme is an acetyl group.
  • the heterologous gene codes for a N-acetyltransferase or a variant able of transferring an acetyl group to 4-amine perosamine position.
  • This heterologous gene can be isolated from Escherichia coli O157:H7, Escherichia hermanii, Vibrio cholerae Hakata, Salmonella's N group, Stenotrophomonas maltophila, Citrobacter gillenii, Citrobacter youngae, or Caulobacter crescentus.
  • the heterologous gene is wbdR gene coding for the N- acetyltransferase in E.coli 0157:H7 (NCBI; 16.12.2010. ID: 962088; locus tag: z3192; ORF in SEQ ID NO: 15)
  • heterologous gene coding for the N-acyltransferase can be isolated from any of the microorganisms listed above by cloning the gene from the wild-type bacteria.
  • the heterologous gene can code for a variant of the wild-type gene isolated from the microorganism, provided that it keeps the property of transferring the acyl group.
  • the following general strategy can be followed: (a) to identify bacteria with a O chain made of a N- acylperosamine with a group in N position different from N-formyl; (b) to identify the enzyme responsible for the substitution; (c) to clone the enzyme and include the resulting construct to the bacterium host (the one to be modified); and (d) to analyze the properties of the modified LPS.
  • the heterologous gene forms part of an expression construct or transcriptional unit, which allows the transcription and production of the heterologous enzyme in the host bacterium, comprising: (a) a regulatory-promoter region that drives and regulates the transcription; (b) a starting transcription signal; (c) the gene coding the enzyme; and (d) a stop transcription signal.
  • the bacterium of the first or third aspect of the invention is characterized by lacking the autologous gene expressing the N- formyltransferase and by expressing a heterologous gene coding for a N- acyltransferase other than a N-formyltransferase. In another embodiment, the bacterium of the first or third aspect of the invention is characterized by lacking the autologous gene expressing the N-formyltransferase and by expressing a heterologous gene coding for a N-acetyltransferase.
  • the bacterium of the first or third aspect of the invention is characterized by lacking the autologous gene expressing the N- formyltransferase and by expressing a heterologous gene coding for a N- acetyltransferase isolated from E. coli 0157:H7.
  • the O chain of the modified bacterium is a homopolymer totally lacking
  • the genetic modification could be extended to tag vaccines strains, such as the known smooth Brucella vaccines (i.e. the classical B. abortus S19 and B. melitensis Rev1 ), or LPS and/or metabolic
  • the bacterium of the first or third aspect of the invention is a vaccine strain in nature.
  • the expression "vaccine strain” means a strain inducing a protective immune response when inoculated in animals.
  • vaccine strains There are well-known vaccine strains in the state of the
  • the modified bacterium includes other modifications that have been previously disclosed as suitable for developing vaccine strains.
  • the modified bacterium is of the genus Brucella.
  • the bacterium of the first or third aspect of the invention is of the genus Brucella and is characterized by the lack of the autologous gene expressing the N-formyltransferase and by the expression of 25 a heterologous gene coding for a N-acyltransferase other than a N- formyltransferase.
  • the bacterium of the first or third aspect of the invention is of the genus Brucella and is characterized by the lack the autologous gene expressing the N-formyltransferase and by the expression of a heterologous gene coding for a N-acetyltransferase.
  • the bacterium of the first or third aspect of the invention is of the genus Brucella and is characterized by the lack of the autologous gene expressing the N-formyltransferase and by the expression of a heterologous gene coding for a N-acetyltransferase obtained from E. coli O157:H7.
  • the O chain of the modified bacterium is a 35 100% N-acetylperosamine homopolymer.
  • the wbkC gene (NCBI-GenelD: 3787272) codes for protein of sequence SEQ ID NO: 15.
  • the bacterium of the first or third aspect of the invention is from Brucella genus and of the specie selected from the group consisting of: Brucella melitensis, Brucella abortus, Brucella suis, Brucella pinnipedialis, Brucella ceti, Brucella microti, Brucella canis, and Brucella ovis.
  • the modified bacterium of the first or third aspect of the invention is from Brucella genus and of the specie selected from the group consisting of: Brucella melitensis, Brucella abortus, Brucella suis, Brucella pinnipedialis, Brucella ceti and Brucella microti.
  • the modified bacterium of the first or third aspect of the invention is from Brucella genus and of the specie selected from the group consisting of: Brucella melitensis and Brucella abortus.
  • the modified bacterium is of the genus Brucella and it includes other genetic modifications previously disclosed in the prior art as responsible for conferring to the strain a vaccine profile, i.e., a protective immune response (Moriyon I. et al., "Rough vaccines in animal brucellosis: Structural and genetic basis and present status", 2004, Vet. Res., vol. 35, pages 1-38; Gonzalez D.
  • the modified bacterium of the first or third aspect of the invention further comprises one or more genetic modifications which suppresses the activity of one or more enzymes selected from the group consisting of: i) the glycosyltransferase enzymes involved in the synthesis of the core of the LPS of said Gram negative bacterium;
  • the suppression of the enzymatic activity can be due to the insertion, deletion or replacement of one or more nucleotides that gives rise to a frame-shift or to a change in the open reading frame.
  • a construction carrying an antibiotic resistance gene can be inserted in the gene coding for the enzyme, which can disrupt the original reading frame, thus inactivating the gene.
  • the promoter region of the gene coding for the enzyme can be 5 partially or completely deleted. Routine methods can be followed to achieve this suppression (Conde-Alvarez R. et al., "Synthesis of phosphatidylcholine, a typical eukaryotic phospholipid, is necessary for full virulence of the
  • the inactivation of any of the enzymes is due to the deletion of all or part of the gene(s) coding thereof.
  • the enzymatic activity suppression is performed deleting5 all or part of a gene encoding a glycosyltransferase enzyme, the enzyme being selected from the group consisting of: WadA (glycosyltransferase), WadB (glycosyltransferase), WadC (mannosyl Itransferase), and WadD (glycosyltransferase).
  • WadA glycosyltransferase
  • WadB glycosyltransferase
  • WadC mannosyl Itransferase
  • WadD glycosyltransferase
  • glycosyltransferase enzyme involved in the synthesis of the core of the LPS, o is performed and comprises the deletion of all or part of a gene coding for the WadC mannosyl Itransferase.
  • the suppression of the enzymatic activity comprises the deletion of all or part of a gene coding for an enzyme involved in the
  • the enzyme being selected from the group consisting of: Wzm (ABC transporter), Wzt (ABC transporter), and WbkF (undecaprenyl- glyscosyltransferase).
  • Wzm ABSC transporter
  • Wzt ABSC transporter
  • WbkF undecaprenyl- glyscosyltransferase
  • the suppression of the enzymatic activity comprises the deletion of all or part of a gene coding for an enzyme involved in the bacterial metabolism, the enzyme being selected from the group consisting of: Pyruvate phosphate dikinase (PpdK), Malic enzyme (Mae), Fba (Fructose5 bisphosphate aldolase), EryA (erythritol kinase), and RpiB (D-erythrose-4- phosphate isomerase).
  • PpdK Pyruvate phosphate dikinase
  • Malic enzyme Mae
  • Fba Feructose5 bisphosphate aldolase
  • EryA erythritol kinase
  • RpiB D-erythrose-4- phosphate isomerase
  • glycosyltransferase enzymes comprises the deletion of all or part of a gene encoding a glycosyltransferase enzyme which is selected from the group consisting of: WadA (SEQ ID NO: 16), WadB (SEQ ID NO: 17), WadC (SEQ ID NO: 18), and WadD (SEQ ID NO: 19).
  • the inactivation of the gene encoding a glycosyltransferase involved in the synthesis of the core of the LPS comprises the deletion of all or part of a gene coding for the WadC mannosyl transferase of sequence SEQ ID NO: 18.
  • the suppression in the activity of one or more enzymes involved in the transport of the O chain is performed and comprises the deletion of all or part of a gene coding for a protein selected from the group consisting of: Wzm (SEQ ID NO: 20), Wzt (SEQ ID NO: 21 ), and WbkF (SEQ ID NO: 22).
  • the suppression of the activity of an enzyme involved in the transport of the O chain is performed and consists in the deletion of the gene coding for protein Wzm (SEQ ID NO: 20).
  • the suppression of the activity of one or more enzymes involved in the metabolism of said Gram negative bacterium is performed and comprises the deletion of all or part of a gene coding for a protein selected from the group consisting of: PpdK (SEQ ID NO: 23), Mae (SEQ ID NO: 24), Fba (SEQ ID NO: 25), Erya (SEQ ID NO: 26), and Rpib (SEQ ID NO: 27).
  • the suppression of the activity of an enzyme involved in the metabolism of said Gram negative bacterium is performed and comprises the deletion of the gene coding for enzyme Ppdk (SEQ ID NO: 23).
  • the Gram negative bacterium is selected from the group consisting of:
  • B. abortus S79-wbdRAwbkC corresponds to the B. abortus S19 strain which lacks the autologous wbkC gene coding for N-formyltransferase and comprises a heterologous wbdR gene coding for the N- acetyltransferase
  • B.melitensis Rev1- wbdRAwbkC corresponds to the B. melitensis Rev1 strain which lacks the autologous wbkC gene coding for N- formyltransferase and comprises a heterologous wbdR gene coding for the N-acetyltransferase;
  • B.melitensis Rev2-wbdRAwbkC corresponds to the B. melitensis Rev2 strain which lacks the autologous wbkC gene coding for N- formyltransferase and comprises a heterologous wbdR gene coding for the N-acetyltransferase;
  • B. aborfus-wbdRAwbkC corresponds to a B. abortus strain which lacks the autologous wbkC gene coding for N-formyltransferase, and comprises a heterologous wbdR gene coding for the N- acetyltransferase;
  • B. aborfus-AwadCwbdRAwbkC corresponds to a B. abortus strain which lacks the autologous wbkC gene coding for N-formyltransferase and the autologous wadC gene coding for mannosyl transferase, and comprises a heterologous wbdR gene coding for the N- acetyltransferase;
  • B. aborfus-AppdKwbdRAwbkC corresponds to a B. abortus strain which lacks the autologous wbkC gene coding for N-formyltransferase and the autologous ppdK gene coding for phosphoenolpyruvate dikinase, and comprises a heterologous wbdR gene coding for the N- acetyltransferase;
  • B. aboffus-AwadCAppdKwbdRAwbkC corresponds to a B. abortus strain which lacks the autologous wbkC gene coding for N- formyltransferase, the autologous wadC gene coding for mannosyl transferase, and the autologous ppdK gene coding for
  • phosphoenolpyruvate dikinase and comprises a heterologous wbdR gene coding for the N-acetyltransferase
  • B. aboffus-AwznnwbdRAwbkC corresponds to a B. abortus strain which lacks the autologous wbkC gene coding for N-formyltransferase and the autologous wzm gene coding for the ABC transporter, and comprises a heterologous wbdR gene coding for the N- acetyltransferase;
  • B. me//iens/s-wbdRAwbkC corresponds to a B.
  • melitensis strain which lacks the autologous wbkC gene coding for N-formyltransferase gene, and comprises a heterologous wbdR gene coding for the N- acetyltransferase;
  • - B. me//iens/s-AwadCwbdRAwbkC corresponds to a B. melitensis strain which lacks the autologous wbkC gene coding for N-formyltransferase and the autologous wadC gene coding for a mannosyl transferase, and comprises a heterologous wbdR gene coding for the N- acetyltransferase;
  • - B. /77e//tens/s-AppdKwbdRAwbkC corresponds to a B. melitensis strain which lacks the autologous wbkC gene coding for N-formyltransferase and the autologous wadC gene coding for mannosyl transferase, and comprises a heterologous wbdR gene coding for the N- acetyltransferase;
  • melitensis strain which lacks the autologous wbkC gene coding for N- formyltransferase, the autologous wadC gene coding for a mannosyl transferase, and the autologous ppdK gene coding for a
  • phosphoenolpyruvate dikinase comprises a heterologous wbdR gene coding for the N-acetyltransferase
  • - B. me//iens/s-AwzmwbdRAwbkC corresponds to a B. melitensis strain which lacks the autologous wbkC gene coding for N-formyltransferase and the autologous wzm gene coding for the ABC transporter, and comprises a heterologous wbdR gene coding for the N- acetyltransferase;
  • - B. su/s-AwadCwbdRAwbkC corresponds to a B.
  • B. su/s-wbdRAwbkC corresponds to a B. suis strain which lacks the autologous wbkC gene coding for N-formyltransferase and comprises a heterologous wbdR gene coding for the N-acetyltransferase;
  • B.su/s-AppdKwbdRAwbkC corresponds to a B. suis strain which lacks the autologous wbkC gene coding for N-formyltransferase and the autologous ppdK gene coding for phosphoenolpyruvate dikinase, and comprises a heterologous wbdR gene coding for the N- acetyltransferase;
  • B. su/s-AwadCAppdKwbdRAwbkC corresponds to a B. suis strain which lacks the autologous wbkC gene coding for N-formyltransferase wbkC, the autologous wadC gene coding for a mannosyl transferase, and the autologous ppdK gene coding for a phosphoenolpyruvate dikinase, and comprises a heterologous wbdR gene coding for the N- acetyltransferase;
  • B. su/s-AwzmwbdRAwbkC corresponds to a B. suis strain which lacks the autologous wbkC gene coding for N-formyltransferase and the autologous wzm gene coding for the ABC transporter and comprises a heterologous wbdR gene coding for the N-acetyltransferase;
  • B. suis Tn7wbdRAwbkC corresponds to a B. suis strain which lacks the autologous wbkC gene coding for N-formyltransferase, and comprises a heterologous wbdR gene coding for the N- acetyltransferase, and the miniTn7 transposon;
  • B. abortus- 7n7wbdRAwbkC corresponds to a B. abortus strain which lacks the autologous wbkC gene coding for N-formyltransferase, and comprises a heterologous wbdR gene coding for the N- acetyltransferase, and the miniTn7 transposon;
  • B. melitensis-Tn 7wbdRAwbkC corresponds to a B. melitensis strain which lacks the autologous wbkC gene coding for N-formyltransferase, and comprises a heterologous wbdR gene coding for the N- acetyltransferase, and the miniTn7 transposon.
  • melitensis correspond to the wild-type strains.
  • B. abortus S19 and B. melitensis Rev1 are vaccines (described in Nicoletti P.L: et al., 1990, and Elberg and Fraunce, 1957, respectively) that are currently marketed and are available in authorized brucellosis laboratories and culture collections worldwide.
  • B. melitensis Rev2 is obtained as described by Mancilla, M., et al., "Deletion of the GI-2 integrase and the wbkA flanking transposase improves the stability of Brucella melitensis Rev 1 vaccine", Vet. Res., vol. 44, pages1-12.
  • strain of the first aspect of the invention is selected from the group consisting of:
  • abortus- 7n7wbdRAwbkC corresponds to a B. abortus strain which lacks the autologous wbkC gene coding for N-formyltransferase, and comprises the heterologous wbdR gene coding for the N- acetyltransferase, and the miniTn7 transposon;
  • B. me/Ztens/s- 7n7wbdRAwbkC corresponds to a B. melitensis strain which lacks the autologous wbkC gene coding for N-formyltransferase, and comprises the heterologous wbdR gene coding for the N- acetyltransferase, and the miniTn7 transposon;
  • - B. aboffus-wbdRAwbkC corresponds to a B. abortus strain which lacks the autologous wbkC gene coding for N-formyltransferase, and comprises the heterologous wbdR gene coding for the N- acetyltransferase
  • - B. me//tens/s-wbdRAwbkC corresponds to a B. melitensis strain which lacks the autologous wbkC gene coding for N-formyltransferase, and comprises the heterologous wbdR gene coding for the N- acetyltransferase.
  • the N-acetyltransferase gene of the strains referred in any of the previous lists is from E. coli 0157:H7.
  • the present invention provides a process for preparing the bacterium strain of the first aspect of the invention.
  • the step of suppressing the autologous N-formyltransferase activity comprises suppressing the expression of the autologous gene.
  • the process comprises, in any order, the steps of:
  • the step of suppression of the expression of the autologous gene is due to a mutation, the mutation negatively affecting the ability of transcribing its mRNA, the translation to the protein or the activity of the enzyme.
  • Protocols for performing these methods are well-known for the skilled in the art (Sambrook, J. and Russell, D. W. (2001 ). Molecular cloning. Chapter 13, Mutagenesis, pages 13.1 to 13.62, Cold spring harbor laboratory press New York).
  • the absence of autologous N-formyltransferase activity is caused by the deletion of part or the entire autologous gene coding for the N- fomyltransferase enzyme.
  • the method comprises, in any order, the steps of: - deleting all or part of the autologous gene coding for N- formyltransferase enzyme; and
  • the expression "deletion of part of the autologous gene coding for the N-formyltransferase enzyme” means that the modified bacterium of the invention lacks a region of the gene responsible for its transcription to RNA or its translation to protein.
  • the deletion of part of the gene means that about 1 to 99% of the whole sequence of the gene is deleted. In one embodiment, the deletion of part of the gene means that about 20-95% of the whole gene sequence is deleted.
  • the step of suppressing the autologous N-formyltransferase activity comprises the complete deletion of the autologous gene.
  • There are well-known techniques for deleting the autologous gene (Sambrook, J. and Russell, D. W. (2001 ). Chapter 13. Mutagenesis. Pagesl 3.1 -13.62. Molecular cloning. Cold spring harbor laboratory press New York), such as the one disclosed by Conde-Alvarez R. et al., 2006. Briefly, the autologous gene is replaced by a mutated gene with an internal deletion. Replacement of the wild type gene for the mutated gene takes place by homologous recombination.
  • the inventors achieved the deletion of the gene performing, in first step, a PCR overlap strategy.
  • the skilled person can design the appropriate pairs of primers and conditions for performing the PCR on the basis of the gene sequence and using available bioinformatics tools.
  • the inventors designed primers with sequences SEQ ID NO: 1 to 4 for performing the PCR.
  • the resulting fragment was cloned in an appropriate vector system.
  • the step of including the functional heterologous gene comprises transferring an expression construct comprising the functional heterologous gene.
  • the heterologous gene can be included in any appropriate system for the correct expression of the gene without negatively affecting the host. Following well-known techniques such as the transposon site-directed insertion in the host bacterium it is possible to perform the insertion of the heterologous gene.
  • the insertion can be performed using an expression vector derived from mini-Tn7, such as mini-Tn7TpUC18T-Gm (Choi KH et al., 2005; Choi, K.-H.
  • the process comprises, in the specified order: (a) including a functional heterologous gene coding for a N-acyltransferase other than a N-formyltransferase; and (b) deleting all or part of the autologous gene coding for N-formyltransferase enzyme.
  • the process comprises the following steps in the specified order: (1 ) including the functional heterologous gene coding for a N-acetyltransferase enzyme; and (2) deleting part or the entire autologous N-formyltransferase gene.
  • the present invention provides a method for preparing an extract from the bacterium of the invention.
  • the method comprises a step of cell lysis.
  • the cell envelope can be broken by viral, enzymatic, physical or osmotic mechanisms. It is well-established which conditions can be used to lyse one cell. The skilled person, therefore, can routinely adjust the most appropriate conditions to lyse the bacterium cell of the invention.
  • the present invention provides an antibody against the bacterium strain as defined in the first or third aspect of the invention. There are well known means in the state of the art for preparing and
  • polyclonal antibodies characterizing antibodies.
  • Methods for generating polyclonal antibodies are well known in the prior art. Briefly, one prepares polyclonal antibodies by immunizing an animal with the bacterium of the first or third aspect of the invention; then, serum from the immunized animal is collected and the antibodies isolated.
  • a wide range of animal species can be used for the production of the antiserum. Typically the animal used for production of antisera can be a rabbit, mouse, rat, hamster, guinea pig or goat.
  • monoclonal antibodies (MAbs) can be prepared using well-known techniques. Typically, the procedure involves immunizing a suitable animal with the bacterium of the first or third aspect of the invention.
  • the immunizing composition can be administered in an amount effective to stimulate antibody producing cells.
  • Methods for preparing monoclonal antibodies are initiated generally following the same lines as the polyclonal antibody preparation.
  • the immunogen bacteria
  • the antigen may be mixed with adjuvants such as complete or incomplete Freund's adjuvant. At intervals of two weeks, approximately, the immunization is repeated with the same antigen.
  • the present invention provides a pharmaceutical or veterinary composition
  • a pharmaceutical or veterinary composition comprising a therapeutically effective amount of the bacterium as defined in the first or third aspect of the invention or the antibody as defined in the fifth or seventh aspect, or the cell extract of the invention, together with one or more veterinary or pharmaceutically acceptable carriers or vehicles.
  • therapeutically effective amount refers to the amount of either the bacterium or antibody that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the symptoms of the disease which is addressed.
  • the particular dose administered according to this invention will of course be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and similar considerations.
  • the term "pharmaceutically acceptable vehicles or carriers” refers to pharmaceutically acceptable materials, compositions or excipients. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity or other problems or complications commensurate with a reasonable benefit/risk ratio. Likewise, the term “veterinary acceptable” means suitable for use in contact with a non-human animal.
  • the present invention provides the modified Gram negative bacterium of the first or third aspect of the invention or the antibody of the firth or seventh aspect of the invention or the cell extract of the invention, for use as a medicament. In one embodiment of the tenth aspect of the invention, the medicament is a vaccine.
  • pharmaceutical or veterinary composition can be understood as a biological agent (either the modified bacterium of the first or third aspect of the invention or the antibody of the fifth or seventh aspect of the invention) capable of providing a protective response in an animal to which the vaccine has been delivered and that is incapable of causing severe disease.
  • the vaccine stimulates antibody production or cellular immunity against the pathogen causing the disease; administration of the vaccine thus results in immunity to the disease.
  • excipients commonly present in vaccine preparations are: aluminum salts or gels; oil-based adjuvants; formaldehyde, monosodium glutamate (MSG), or 2-phenoxyethanol, among others.
  • the present invention provide uses of the bacterium or of antibody of the invention or of the cell extract of the invention for the prevention or treatment of an infection caused by a bacterium strain, such as a wild-type bacterium strain, belonging to the same genus as the modified bacterium, and for differentiating infected from vaccinated animals.
  • a bacterium strain such as a wild-type bacterium strain
  • the term "vaccinated animal” means an animal vaccinated with a modified Gram negative bacterium strain encompassing both the strain of the invention as well as other bacterium strains disclosed in the prior art which are modified in its genome.
  • the animal has been vaccinated with a modified bacterium strains as disclosed, for example, in WO2012131 128.
  • the animal has been vaccinated with the strain of the present invention. In one embodiment of these
  • the modified Gram negative bacterium of the first and third aspect of the invention is of the Brucella genus and the animal is infected with Brucella.
  • the present invention provides the use of a modified Gram negative bacterium strain as defined in the first or third aspect of the invention, the antibodies of the fifth or seventh aspect or the cell extract of the invention, for use in diagnostics.
  • the present invention provides the use of a modified Gram negative bacterium strain as defined in the first or third aspect of the invention, the antibodies of the fifth or seventh aspect or the cell extract of the invention, for use in diagnosing brucellosis.
  • This embodiment can be alternatively formulated as a method for diagnosing brucellosis, the method comprising contacting an isolated sample from a subject with the modified Gram negative bacterium strain as defined in the first or third aspect of the invention, the antibodies of the fifth or seventh aspect or the cell extract of the invention.
  • the present invention provides the use of the bacterium or of antibody of the invention or of the cell extract of the invention for
  • a bacterium strain such as a wild-type bacterium strain, belonging to the same genus as the modified bacterium.
  • the present invention provides an in vitro method for differentiating an animal vaccinated with the modified Gram negative bacterium strain as defined in the first or third aspect of the invention from an animal infected by a bacterium strain, such as a wild-type bacterium strain, belonging to the same genus as the modified bacterium, the process comprising the step of determining, in an isolated sample: (a) the presence of the modified Gram negative bacterium strain as defined in the first or third aspect of the invention (by culture or direct detection with specific
  • the modified Gram negative bacterium strain as defined in the first or third aspect is of the genus Brucella, and the animal is infected by Brucella.
  • strain and antibodies of the invention are useful as reagents in
  • immunoassays to detect antibodies against the bacterium strain, such as a wild-type bacterium strain, belonging to the same genus as the modified bacterium.
  • the immunoassay procedures suitable include enzyme-linked immunosorbent assays (ELISA), enzyme immunodot assay, agglutination assay, antibody- antigen-antibody sandwich assay, antigen-antibody-antigen sandwich assay, immunocromatography or other immunoassay formats well-known to the ordinarily skilled artisan. These immunoassay formats and procedures have been described in many standard immunology manuals and texts.
  • the preferred immunoassay formats are ELISA, agglutinations, such as a plaque- agglutination assay or a tube-agglutination assay, the most preferred tests are
  • the immunochromatography tests are based on the immunological capture of antibodies against acetyl group.
  • the LPS (or O-chain) of the modified Gram negative bacterium will be adsorbed to a chromatographic strip on which serum components and anti-immunoglobulin conjugate (usually colloidal gold) are developed.
  • the in vitro immunoassay method comprises the step of coating a solid phase with:
  • the immunoassay method is selected from ELISA, plaque-agglutination assay, tube-agglutination assay, and
  • the immunoassay method is an ELISA or an agglutination test.
  • Another aspect of the present invention is directed to a kit for differentiating an animal vaccinated with the modified bacterium of the invention or the antibody of the invention or the cell extract of the invention from one having the disease.
  • the kit can comprise one or more of the following: - the modified bacterium of the first or third aspect of the invention;
  • the bacterium such as a wild-type bacterium, causing the infection;or a cell extract thereof;
  • the kit When analyzing the sample of the animal with a kit comprising the modified bacterium or the antibodies of the invention or the cell extract of the invention, there will be binding reaction if the animal is vaccinated (positive reaction). If the kit comprises the wild-type bacterium or the antibodies raised by the wild- type bacterium or the cell extract of the wild-type bacterium, there will not be binding reaction (negative reaction) if the animal is vaccinated with the modified bacterium or the antibodies of the invention. And if it is detected the presence of the heterologous gene and the absence of the autologous gene, this will be indicative of the fact that the animal is vaccinated.
  • the means for detecting the presence of the heterologous gene and the absence of the autologous gene can be primers specially designed for amplifying the target sequences.
  • the primers have sequences flanking the deleted or inserted genes, such as primers wbkC-F (SEQ ID NO: 1 ) and wbkC-R (SEQ ID NO: 4) for the wbkC deletion (which amplify a fragment of 827 bp in the mutant and a fragment of 1373 bp in the parental strain) and primers wbdR Fw: (SEQ ID NO: 5) and wbdR Rv: (SEQ ID NO: 6) for the wbdR insertion (which amplify a 1000 bp fragment).
  • the kit of the invention can be compartmentalized to receive a first container adapted to contain each one of the components, including the modified bacterium of the invention or a cell extract thereof or an antibody against it.
  • the kit of this invention is an ELISA or an agglutination test kit for detecting of antibodies and thereby differentiating an animal infected by a wild-type bacterium strain belonging to the same genus as the modified bacterium.
  • the kit contains (a) a container (e.g., a 96- well plate) having a solid phase coated with (i) the modified Gram negative bacterium of the first or third aspect, or a cell extract thereof; or (b) the wild- type bacterium strain which causes the infection disease or a variant thereof which retains the autologous N-formyltransferase activity, or cell extract thereof; (b) a negative control sample; (c) a positive control sample; and (d) specimen diluent.
  • a container e.g., a 96- well plate having a solid phase coated with (i) the modified Gram negative bacterium of the first or third aspect, or a cell extract thereof; or (b) the wild- type bacterium strain which causes the infection disease or a variant thereof which retains the autologous N-formyltransferase activity, or cell extract thereof; (b) a negative control sample; (c) a positive control sample; and (d) specimen diluent.
  • the kit comprises (a) a test card or microscope slide, (b) the bacterium strain of the invention as agglutinating agent and (c) means for visual detection (latex particles, gelatin beads, colloidal particles, among others) as separate components.
  • kits of the present invention a sample of body fluid to be tested, diluted in sample diluent if necessary, is placed in contact with the modified bacterium of the first or third aspect of the invention, or a cell extract thereof, on a coated solid phase for a time and under conditions for any antibodies present in the body fluid to bind to the modified bacterium of the first or third aspect of the invention, or a cell extract thereof.
  • the secondary complex is contacted with labeled antibodies to species-specific IgG or labeled protein A, protein G, or protein A G.
  • the reporter molecule can be an enzyme, radioisotope, fluorophore, bioluminescent molecule, chemiluminescent molecule, biotin, avidin, streptavidin or the like.
  • the reporter molecule is preferably an enzyme.
  • a suspension of the bacterial strain of the invention is contacted with an isolated test serum sample of the animal. If the animal has been vaccinated with the strain of the invention, agglutination will be detected, whereas if the animal is infected, no agglutination will be detected.
  • the modified Gram negative bacterium of the first and third aspect of the invention is of the genus Brucella and the animal is infected with Brucella.
  • the present invention provide an in vitro method of differentiating an animal vaccinated with a modified Brucella strain from one infected with Brucella, as well as the use of the bacterium of the invention or the kit of the invention for performing the differentiation between an animal vaccinated with a modified Brucella strain from an animal infected with
  • Brucella strain encompasses both the strain of the invention as well as other Brucella strains disclosed in the prior art which are modified in its genome and show a protective effect. Modified Brucella strains are disclosed, for example, in WO2012131 128.
  • Bacteria were routinely grown in standard tryptic soy broth or agar either plain or supplemented with anamycin at 50 pg/ml, or/and A/a/idixic at 25 pg/ml, chloramphenicol at 20 g/ml or/and 5% sucrose. All strains were stored in skim milk at -80°C.
  • Plasmid and chromosomal DNA were extracted with Qiaprep spin Miniprep (Qiagen GmbH, Hilden, Germany), and Ultradean Microbial DNA Isolation kit
  • B. abortus wbkC non polar mutant BAB1_0540
  • BaAwbkC BAB1_0540
  • B. abortus 2308 is a reference strain available in authorized brucellosis laboratories worldwide and culture collections, including the "Culture Colection of the Department of Microbiology, University of Navarra" . Primers were designed based on the available sequence of the corresponding genes in B. abortus 2308.
  • ACCTTGATTGGCATGGGCAGATGGTCGGAAGTCCAGATT - 3'; SEQ ID NO: 3 and wbkC -R4 (5 ' - TCTGAACTCGGCTGGATGAC -3 ' ; SEQ ID NO: 4) were used to amplify a 434-bp fragment including codons 212 to 259 of the wbkC ORF and 287-bp downstream of the wbkC stop codon.
  • Both fragments were ligated by overlapping PCR using oligonucleotides wbkC -F1 (SEQ ID NO: 1 ) and wbkC -R4 (SEQ ID NO: 4) for amplification, and the complementary regions between wbkC -R2 (SEQ ID NO: 2) and wbkC -F3 (SEQ ID NO: 3) for overlapping.
  • wbkC -F1 SEQ ID NO: 1
  • wbkC -R4 SEQ ID NO: 4
  • Typical conditions for PCR amplification are:
  • the first recombination integration of the suicide vector in the chromosome
  • the second recombination excision of the mutator plasmid leading to construction of the mutant by allelic exchange
  • was selected by Nal and sucrose resistance and Kan sensitivity Conde-Alvarez et al. 2006.
  • the resulting colonies were screened by PCR with primers wbkC-F (SEQ ID NO: 1 ) and wbkC-RA (SEQ ID NO: 4) which amplify a fragment of 827 bp in the mutant and a fragment of 1373 bp in the parental strain.
  • the mutant was called BaAwbkC. Typical conditions are those described above.
  • Gene wbdR (NCBI Reference Sequence: WP_001055391 .1 Gl:446978135) encoding a perosamine acetyltransferase (SEQ ID NO: 15) with the region upstream containing its promoter was amplified from E. coli 0157:H7 using primers wbdR Fw: 5 ' TTCCCCGGGGGAGAAGTTCGCCACAGTAAATCGAA 3 ' (SEQ ID NO: 5) and wbdR Rv: 5 '
  • the miniTn7 vector carrying wbdR with its own promoter was inserted in B. abortus 2308 (Ba-parental) chromosome using a method previously described by Choi and Schweizer (2006) with some modifications.
  • Brucella we first introduced pUC18R6KT-miniTn7T-KmR-Pwi c/R in E. coli S17.1 Apir. The resulting plasmid was then transferred to Brucella strain using a four-parental mating among E. coli S17.1 Apir (pUC18R6KT-miniTn7T-KmR- PwbdR), E. coli HB101 (pRK2013), E.
  • TATATTCTGGCGAGCGATCC 3 ' TATATTCTGGCGAGCGATCC 3 '
  • GlmS_B SEQ ID NO: 7
  • RecG SEQ ID NO: 10
  • Strain Ba::Tn7wi c/R is resistant to kanamycin (Km).
  • Km kanamycin
  • Two PCR fragments were firstly generated: oligonucleotides Km-F1 (5 ' - AGGAAGCGGAACACGTAGAA-3 ' ; SEQ ID NO: 1 1 ) and Km-R2 (5 ' -AATCATGCGAAACGATCCTC -3 ' ; SEQ ID NO: 12) were used to amplify a 318-bp fragment including codons 1 to 2 of the Km ORF, as well as 312 bp upstream of the Km start codon, and
  • Both fragments were ligated by overlapping PCR using oligonucleotides Km - F1 (SEQ ID NO: 1 1 ) and Km -R4 (SEQ ID NO: 14) for amplification, and the complementary regions between Km -R2 (SEQ ID NO: 12) and Km -F3 (SEQ ID NO: 13) for overlapping.
  • This suicide plasmid was used to delete the kanamycin gene of Ba ::Tn7 wi c/R using the allelic exchange by double recombination described above for wbkC mutation. Deletion of Km resistance gene was checked with oligonucleotides Km-F1 (SEQ ID NO: 1 1 ) and Km-R4 (SEQ ID NO: 14). Sensitivity to km was confirmed by plating
  • the mutator plasmid pJQKAwbkC (obtained as disclosed above) was introduced into strain Ba::Tn7w c/RAKm. After allelic exchange, the double mutant was selected as described above using primers wbkC-n (SEQ ID NO: 1 ) and wbkC-RA (SEQ ID NO: 4).
  • Total S-LPS was obtained by methanol precipitation of the phenol phase of a phenol-water extract (Leong, D et al 1970). This fraction (10 mg/ml in 175 mM NaCI, 0.05% NaN3, 0.1 M Tris-HCI [pH 7.0]) was then purified by digestion with nucleases (50 ⁇ g/ml each of DNase-ll type V, and RNase [Sigma, St. Louis, Missouri, U.S.A.] 30 min at 37 °C) and three times with proteinase K (Sigma, 50 ⁇ / ⁇ , 3 hours at 55°C), and ultracentrifuged (6h, 100,000 x g) (Velasco et al 2000).
  • nucleases 50 ⁇ g/ml each of DNase-ll type V, and RNase [Sigma, St. Louis, Missouri, U.S.A.] 30 min at 37 °C
  • proteinase K Sigma, 50 ⁇ / ⁇ , 3 hours at 55°C
  • mice Female BALB/c mice (Charles River, France) were kept in cages with water and food ad libitum, and accommodated under P3 biosafety containment conditions 2 weeks before and during the experiments, in the facilities of the "CIMA" (registration code ES31 2010000132). The animal handling and other procedures were in accordance with the current European (directive
  • RBT Rose Bengal Test
  • DIVAT Differentiation Infected Vaccinated Agglutination test
  • Tn7 wbdRAwbkC cells stained with a mixture of crystal violet and brilliant green (Diagnostic procedures and reagents. In Coleman, HA (ed.), Techniques for the laboratory diagnosis and control of communicable diseases. Chapter 12. American Public Health Association Inc., New York. 1963). The stained cells were resuspended in the same buffer used in the RBT commercial test
  • Rabbits were immunized following the protocol previously described by Diaz et al., (1967). Briefly, rabbits were inoculated intravenously with 1 mg/ml of Ba-pwbdR cells (Gil-Ramirez, Y. 201 1 . Thesis with the title: Role of ethanolamine-phosphate transferase and a glycosyltransferase in the synthesis of a lipopolysaccharide of Brucella and use of an acetyltransferase for the epitope modification of the O chain, pages 1 19-139. University of Navarra) inactivated with phenol, lyophilized and resuspended in saline. Two and 4 days later, a similar dose was administrated intraperitoneally. Three weeks later the animals were bled and euthanized.
  • the staphylococci were prepared and sensitized with the corresponding antiserum as described by Kronvall (Kronvall, G. "A rapid slide-agglutination method for typing pneumococci by means of specific antibody adsorbed to protein A-containing staphylococci", 1973, J. Med. Microbiol., vol. 6, pages 187-190).
  • Several colonies were resuspended in 25 ⁇ saline solution on a glass slide and the suspension was mixed with an equal amount of the sensitized staphylococci.
  • 96-well ELISA plates (Thermo scientific) were coated with LPS (2,5 ⁇ g/ml) from wild-type (Ba-parental LPS) or (Ba::Tn7wbdRAwbkC-LPS) overnight at 4°C. Plates were then washed extensively with phosphate-buffered saline (PBS)-Tween 20, and incubated with serial dilutions of the sera from immunized rabbit, absorbed or not (see above) at 37°C for 5h. Then, the plates were washed extensively with PBS-Tween 20 and bound antibodies were detected with a protein G- peroxidase-conjugated (Nordic Immunological laboratories, Tilbrug, Netherlands).
  • PBS phosphate-buffered saline
  • BaAwbkC and Ba::Tn7 wbdRAwbkC were grown on TSA and the plates were covered with crystal violet dye. The results showed that BaAwbkC colonies retained crystal violet whereas in the case of classical rough strains,
  • BaAwbkC but not Ba::Tn7 wbdRAwbkC, agglutinated in acriflavine solutions.
  • the epitopic structure of Ba::Tn 7 wi c/R and Ba..Tn7 wbdRAwbkC was first analyzed by coagglutination with staphylococci sensitized with sera from rabbits immunized with Ba pwbdR (that contain antibodies against N-acetyl and N-formyl perosamine related epitopes), or with the same sera absorbed with either Ba-parental (to remove the N-formyl-perosamine-related antibodies characteristic of infections by wild-type smooth Brucella) or
  • Ba..Tn7 wbdRAwbkC indicates that the latter lacks the epitopes related to N- formyl-perosamine (characteristic of Ba-parental and still present in Ba- pwbdR).
  • the experiments in Table 1 also show some degree of reactivity of the sera to Ba parental with Ba::Tn7wbdRAwi / C.
  • the antibodies involved were removed by absorption with a per mutant. Since this mutant lacks the O-chain and keeps the core oligosaccharide and lipid A of LPS, the absorption removes the antibodies to these last two sections but not against the O chain. Therefore, this result demonstrates (a) that the positive coagglutination with the anti-Ba-parental serum is caused by core-lipid A epitopes, and (b) the lack of cross-reactivity at O chain level between Ba-parental and
  • Ba..Tn7 wbdRAwbkC LPS were extracted and purified, and their reactivity was analyzed by ELISA with sera from rabbits immunized with Ba-pwbdR and with the same sera absorbed with either Ba-parental or Ba::Tn7 wbdRAwbkC.
  • the results are shown in FIG. 1 .
  • the two LPSs reacted with sera from rabbits immunized with Ba-pwbdR, and absorption with Ba-parental eliminated the reactivity with Ba parental LPS (Fig. 1 , panel A) but not with Ba Tn7 wbdRdwbkC LPS (Fig. 1 panel B), confirming that the latter LPS carries epitopes not present in wild-type strains and derived from N-acetyl-perosamine. These results are in agreement with those observed in the coagglutination tests.
  • mice were inoculated intraperitoneally with 5x10 4 CFU/mouse Ba-parental, Ba::Tn7wi c/R or Ba::Tn7 wbdRAwbkC and bled by intracardiac puncture 2 and 8 weeks after infection.
  • Sera from infected animals were tested in classical RBT and in DIVAT test that use
  • Ba..Tn7 wbdRAwbkC is used as antigen in the AAT agglutination test, all mice infected with Ba::Tn7 wbdRAwbkC or Bme:: Tn7 wbdRAwbkC become positive, while those infected with Ba-parental or Bme-parental are negative (only 3 out of 20 mice give a very weak and not clear agglutination).
  • the classical indirect ELISA used for brucellosis diagnosis would differentiate animals infected by a field strain (positive reaction) from animals vaccinated with a vaccine genetically modified to express a N-acetyl-perosamine O-chain (i.e. a Tn7 wbdRAwbkC construct).
  • FIG. 2 panel B shows that in such an ELISA the sera from animals infected by field strains give only a very week response, significantly lower than that to Ba..Tn7 wbdRAwbkC, which would unable to differentiate animals vaccinated with the latter constructs.
  • Bmevln fwbdRAwbkC carrying a complete LPS with N-acetyl-perosamine was attenuated at weeks 2 and 8 postinoculation. This result was unexpected because Bmevln fwbdRAwbkC carries a complete O-chain LPS, a feature that thus far has been shown to be essential in virulence, but no defect in critical virulence genes.
  • the attenuation is linked to Tn7wbdRAwbkC (i.e. full acetylation and new epitope(s)) and not to the mere presence of wbdR because the single Bme::Tn7wbdR was able to reach the chronic infection phase (i.e.
  • Ba::Tn7kvbci «A m B. abortus with miniTn? transposon, carrying the Construction described in the text gene from E coll, inserted in the chromosome. It
  • Bme::Tn7wW « B. melitensis with miniTn? transposon, carrying the Construction on a B. melitensis 16M wbdR gene from £. col), inserted in the chromosome background as described for its f . abortus
  • wbdR gene from E. coli, inserted in the chromosome. background as described for its B, abortus
  • BmeAwodC&wbkC carryin the wbdR gene from £ co/i, Construction on a BmebwadC background inserted in the chromosome. as described for its B. abortus 2308
  • HB101 F - hsdSIO recAU ara-14 proA2 lacVl galK2 Culture Collection of the Department of rpst20 xyl-5 rntt-1 sup£4 Microbiology. University of Navarra pRK2013: KmR, oriT helper ( provided by prof Schweeer; see Choi
  • Tc u KmR ( pir> Microbiology, University of Navarra pTNS2: ApR; helper piasmid encoding the site-specific (provided by prof Schweizer; see Choi TnsASCD Tn7 transposition pathway 2005).
  • Simon R. et al. "A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria", 1983, Nat Biotechnol, 1 , 784e91 .
  • Zuhiga-Ripa, A. et al. "Brucella abortus depends on pyruvate phosphate dikinase and malic enzyme but not on Fbp and GlpX fructose-1 ,6- bisphosphatases for full virulence in laboratory models", 2014 J Bacteriol 196, 3045-3057.
  • Gil-Ramirez, Y. 201 1 Thesis with the title: Role of ethanolamine-phosphate transferase and a glycosyltransferase in the synthesis of a lipopolysaccharide of Brucella and use of an acetyltransferase for the epitope modification of the O chain, pages 1 19-139. University of Navarra. Moriyon I. et al., "Rough vaccines in animal brucellosis: Structural and genetic basis and present status", 2004, Vet. Res., vol. 35, pages 1-3.

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Abstract

Cette invention concerne une bactérie modifiée caractérisée en ce qu'elle ne présente pas son activité de N-formyltransférase autologue et en ce qu'elle comprend un gène hétérologue fonctionnel codant pour une enzyme N-acyltransférase autre qu'une enzyme N-formyltransférase. L'invention concerne également des procédés pour la produire, des compositions pharmaceutiques et vétérinaires la comprenant et son utilisation dans le traitement ou la prévention de maladies, ainsi que dans la différenciation entre des animaux infectés et des animaux vaccinés. La souche de bactérie modifiée est atténuée et induit une réponse immunitaire spécifique avec des anticorps dirigés contre de nouveaux épitopes du lipopolysaccharide, qui permet une différenciation nette entre des animaux infectés et des animaux vaccinés.
PCT/EP2016/080933 2015-12-21 2016-12-14 Souches bactériennes à gram négatif modifiées et leurs utilisations Ceased WO2017108515A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019101993A1 (fr) 2017-11-24 2019-05-31 Consejo Superior De Investigaciones Científicas (Csic) Souche vaccinale modifiée de brucella pour le traitement de la brucellose
KR20200044548A (ko) * 2018-10-19 2020-04-29 대한민국(관리부서 질병관리본부장) 신속 면역크로마토그래피법을 이용한 콜레라균 진단·탐지 키트 및 이를 위한 특이항체와 항체생산세포주

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WO2011033129A1 (fr) * 2009-09-21 2011-03-24 INSERM (Institut National de la Santé et de la Recherche Médicale) Bactéries gram-négatives modifiées pour une utilisation comme vaccins
WO2012131128A1 (fr) * 2011-03-25 2012-10-04 Universidad De Navarra Méthode diva de différenciation d'animaux vaccinés contre la brucellose

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WO2011033129A1 (fr) * 2009-09-21 2011-03-24 INSERM (Institut National de la Santé et de la Recherche Médicale) Bactéries gram-négatives modifiées pour une utilisation comme vaccins
WO2012131128A1 (fr) * 2011-03-25 2012-10-04 Universidad De Navarra Méthode diva de différenciation d'animaux vaccinés contre la brucellose

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FABRICE GODFROID ET AL: "Genetic organisation of the lipopolysaccharide O-antigen biosynthesis region of Brucella melitensis 16M (wbk)", RESEARCH IN MICROBIOLOGY, vol. 151, no. 8, 1 October 2000 (2000-10-01), NL, pages 655 - 668, XP055347516, ISSN: 0923-2508, DOI: 10.1016/S0923-2508(00)90130-X *
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THAÍS LOURDES SANTOS LACERDA ET AL: "Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice", VACCINE, vol. 28, no. 34, 1 August 2010 (2010-08-01), AMSTERDAM, NL, pages 5627 - 5634, XP055347507, ISSN: 0264-410X, DOI: 10.1016/j.vaccine.2010.06.023 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019101993A1 (fr) 2017-11-24 2019-05-31 Consejo Superior De Investigaciones Científicas (Csic) Souche vaccinale modifiée de brucella pour le traitement de la brucellose
US11298416B2 (en) 2017-11-24 2022-04-12 Consejo Superior De Investigaciones Científicas (Csic) Modified Brucella vaccine strain for the treatment of brucellosis
US11707515B2 (en) 2017-11-24 2023-07-25 Consejo Superior De Investigaciones Científicas (Csic) Modified Brucella vaccine strain for the treatment of brucellosis
KR20200044548A (ko) * 2018-10-19 2020-04-29 대한민국(관리부서 질병관리본부장) 신속 면역크로마토그래피법을 이용한 콜레라균 진단·탐지 키트 및 이를 위한 특이항체와 항체생산세포주
KR102124260B1 (ko) 2018-10-19 2020-06-26 대한민국(관리부서 질병관리본부장) 신속 면역크로마토그래피법을 이용한 콜레라균 진단·탐지 키트 및 이를 위한 특이항체와 항체생산세포주

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