WO2016193161A1 - Multivalent immunogenic composition for inducing an immune response against yersinia species - Google Patents

Multivalent immunogenic composition for inducing an immune response against yersinia species Download PDF

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WO2016193161A1
WO2016193161A1 PCT/EP2016/062047 EP2016062047W WO2016193161A1 WO 2016193161 A1 WO2016193161 A1 WO 2016193161A1 EP 2016062047 W EP2016062047 W EP 2016062047W WO 2016193161 A1 WO2016193161 A1 WO 2016193161A1
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yersinia
seq
lcrv
amino acid
antigen
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French (fr)
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Florent SEBBANE
Catherine Daniel
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Centre National de la Recherche Scientifique CNRS
Universite Lille 1 Sciences et Technologies
Universite Lille 2 Droit et Sante
Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
Institut Pasteur
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Centre National de la Recherche Scientifique CNRS
Universite Lille 1 Sciences et Technologies
Universite Lille 2 Droit et Sante
Institut Pasteur de Lille
Institut National de la Sante et de la Recherche Medicale INSERM
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
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0291—Yersinia
    • 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
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • 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
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • 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
    • A61K2039/70—Multivalent vaccine

Definitions

  • the present invention relates to immunogenic compositions and methods for inducing an immune response against Yersinia species.
  • Yersinia pseudotuberculosis (Ypst), Yersinia enterocolitica (Ye) and Yersinia pestis (Yp) species are bacterial pathogens in humans and animals (Butler T, Plenum Medical Book Company, New and London, 1983).
  • Y. pestis is responsible for plague, a fatal flea-borne disease.
  • the two other Yersinia pseudotuberculosis and Yersinia enterocolitica species cause a self-limiting bowel disease in humans having ingested contaminated food.
  • Yersinioses are thus considered to be significant human and veterinary health problems; unsurprisingly, research geared towards the production of anti-yersiniosis vaccines is now underway.
  • LcrV also referred to as "V antigen”
  • LcrV is a polymorphic protein within which the most effectively protective region encompasses residues 135 to 262 (Miller NC, Infection and immunity, 80, 2012: 1572-82; Motin VL, Microbial pathogenesis, 12, 1992: 165-75; Anisimov AP, Infect Genet Evol, 10, 2010: 137- 45; Schmidt A, Microbial pathogenesis, 26, 1999: 221-33; Vernazza C, Vaccine, 27, 2009: 2775- 80).
  • LcrV antigens have been classified into different types, subtypes and variants on the basis of (i) the length of the hypervariable region, (ii) the polymorphisms at residues 51 and
  • mice with rabbit anti-sera raised against purified V antigen from Y. pestis or with purified rabbit IgG raised against a recombinant V antigen from Y. pseudotuberculosis protected against plague and pseudotuberculosis (Motin VL, Infection and immunity, 62, 1994: 4192-201).
  • WO2006/110881 teaches that antisera raised against recombinant V antigen or a stable staphylococcal protein A-V antigen fusion peptide may provide statistically significant protection against Y. pestis and Y. pseudotuberculosis but not Y. enterocolitica.
  • WO2008/063240 reports multivalent vaccines, in the form of live bacterial vaccines, expressing a plurality of Yersinia antigens.
  • a vaccine carrier a Lactobacillus plantarum expressing a FI capsular antigen, Yersinia outer surface protein D (YopD), and a LcrV antigen (e.g. from Y pestis serotype 0:3), is disclosed.
  • the present invention relates to a multivalent immunogenic composition for inducing an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising :
  • the present invention relates to a multivalent immunogenic composition for inducing an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group consisting of: Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group consisting of :
  • LcrV antigens inducing an immune response against Yersinia enterocolitica.
  • said Yersinia LcrV antigens may be selected in a group comprising (or consisting of):
  • SEQ ID N°A-1, SEQ ID N°A-2 and SEQ ID N°A-3 mean an amino acid sequence having at least 80% amino acid identity, preferably at least 90%) amino acid identity, with the sequence SEQ ID N°2,
  • - CS-1 means an amino acid sequence of sequence SEQ ID N°8,
  • - CS-2 means an amino acid sequence selected from the group comprising the sequences SEQ ID N°8, 10, 11, 12, 13, 107, 108, 109, 110, 111, 112, 113, 114 and 115,
  • - CS-3 means an amino acid sequence selected from the group comprising the sequences SEQ ID N°14, 15, 16, 17 and 18,
  • SEQ ID N°B-1, SEQ ID N°B-2 and SEQ ID N°B-3 mean an amino acid sequence having at least 80%> amino acid identity, preferably at least 90 %> amino acid identity, with the sequence SEQ ID N°3,
  • - AA-1, AA-2 and AA-3 mean an amino acid selected from the group consisting of aspartic acid (or aspartate) and asparagine, preferably AA-1 represents asparagine, AA-2 represents aspartic acid and AA-3 represents aspartic acid, and - SEQ ID N°C-1, SEQ ID N°C-2 and SEQ ID N°C-3, one independently from the others, mean an amino acid sequence having at least 80% amino acid identity, preferably at least 90 % amino acid identity, with the sequence SEQ ID N°4.
  • the multivalent immunogenic composition according to the invention may be such as, when the first or the second Yersinia LcrV antigen induces an immune response against Yersinia pestis and Yersinia pseudotuberculosis, then said immunogenic composition:
  • - further comprises or expresses at least one additional Yersinia LcrV antigen(s) inducing an immune response against Yersinia pestis or Yersinia pseudotuberculosis, different from whichever of the first or the second Yersinia LcrV antigen is already present.
  • the Yersinia LcrV antigen inducing an immune response against Yersinia pestis and Yersinia pseudotuberculosis may be represented by the following formula (IV):
  • SEQ ID N°A-4 means an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %> amino acid identity, with the sequence SEQ ID N°2,
  • SEQ ID N°B-4 means an amino acid sequence having at least 80%) amino acid identity, preferably at least 90 %> amino acid identity, with the sequence SEQ ID N°3,
  • SEQ ID N°C-4 means an amino acid sequence having at least 80%> amino acid identity, preferably at least 90%> amino acid identity, with the sequence SEQ ID N°4.
  • This invention also pertains to a multivalent immunogenic composition according to the invention, for its use as a vaccine.
  • the invention pertains to this multivalent immunogenic composition according to the invention, for its use for the simultaneous vaccination of a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two, preferably three, Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species.
  • the invention pertains in particular to this multivalent immunogenic composition according to the invention, for its use for the simultaneous vaccination of a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two, preferably three, Yersinia species selected in a group consisting of, Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species.
  • the invention also concerns a method of immunizing a vertebrate, notably of the Mammalia and/or of the aves class(es), against disease(s) caused by Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species, said method comprising administering to the vertebrate at least one immunoprotective dose of a multivalent immunogenic composition according to the invention.
  • the invention also concerns a method of immunizing a vertebrate, notably of the Mammalia and/or of the aves class(es), against disease(s) caused by Yersinia species selected in a group consisting of Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species, said method comprising administering to the vertebrate at least one immunoprotective dose of a multivalent immunogenic composition according to the invention.
  • the invention also concerns a recombinant bacteria comprising or expressing at least two heterologous Yersinia LcrV recombinant antigens, each different from each other, said Yersinia LcrV recombinant antigens being selected in a group comprising:
  • a first Yersinia LcrV recombinant antigen being capable of raising an immune response against Yersinia pestis in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined
  • a second Yersinia LcrV recombinant antigen different from the first Yersinia LcrV antigen, being capable of raising an immune response against Yersinia pseudotuberculosis in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined, and
  • Yersinia LcrV recombinant antigen different from the first and the second Yersinia LcrV antigens, being capable of raising an immune response against Yersinia enterocolitica in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined.
  • the invention also concerns a recombinant bacteria comprising or expressing at least two heterologous Yersinia LcrV recombinant antigens, each different from each other, said Yersinia LcrV recombinant antigens being selected in a group consisting of :
  • a first Yersinia LcrV recombinant antigen being capable of raising an immune response against Yersinia pestis in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined
  • a second Yersinia LcrV recombinant antigen different from the first Yersinia LcrV antigen, being capable of raising an immune response against Yersinia pseudotuberculosis in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined, and
  • Yersinia LcrV recombinant antigen different from the first and the second Yersinia LcrV antigens, being capable of raising an immune response against Yersinia enterocolitica in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined.
  • the invention also concerns a device for detecting at least one Yersinia infection due to at least one Yersinia species selected from the group consisting of Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species, wherein said device comprises at least two Yersinia's LcrV-specific binding molecules selected among the group comprising:
  • the first Yersinia's LcrV-specific binding molecule being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pestis species;
  • the second Yersinia's LcrV-specific binding molecule different from the first Yersinia's LcrV-specific binding molecule, being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pseudotuberculosis species;
  • the third Yersinia's LcrV-specific binding molecule different from the first and the second Yersinia's LcrV-specific binding molecules, being capable of binding to at least one Yersinia LcrV antigen of the Yersinia enterocolitica species.
  • the invention also concerns a device for detecting at least one Yersinia infection due to at least one Yersinia species selected from the group consisting of Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species, wherein said device comprises at least two Yersinia's LcrV-specific binding molecules selected among the group consisting of:
  • the first Yersinia's LcrV-specific binding molecule being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pestis species;
  • the second Yersinia's LcrV-specific binding molecule different from the first Yersinia's LcrV-specific binding molecule, being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pseudotuberculosis species;
  • the third Yersinia's LcrV-specific binding molecule different from the first and the second Yersinia's LcrV-specific binding molecules, being capable of binding to at least one
  • the invention also concerns a device for detecting antibodies anti-Yersinia's LcrV in a vertebrate, notably of the Mammalia and/or of the aves class(es), wherein said device comprises at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising :
  • the invention also concerns a device for detecting antibodies anti- Yersinia's LcrV in a vertebrate, notably of the Mammalia and/or of the aves class(es), wherein said device comprises at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group consisting of :
  • the invention further concerns a kit comprising at least:
  • Figure 1 shows a graph illustrating the mouse survival for groups of mice previously immunized intranasally with L. lactis secreting (or not) the Y. pestis strain 195/P LcrV or the Y. pseudotuberculosis strain 2777 LcrV and then challenged intravenously with a lethal dose of Y. pseudotuberculosis strain 2777.
  • X-axis time in days; in ordinate, mouse survival, expressed in percentage.
  • Curve 1 mouse survival for the group immunized with L. lactis which does not secrete any LcrV
  • curve 2 mouse survival for the group immunized with L. lactis secreting the Y. pseudotuberculosis strain 2777 LcrV
  • curve 3 mouse survival for the group immunized with L. lactis secreting the Y. pestis strain 195 LcrV.
  • Figure 2 illustrates an alignment of a plurality of amino acid sequences of LcrV antigens (position 204 to 262/or 263).
  • Sequence 1 amino acid sequence of LcrV antigen of Y. pestis strain 195/P (i.e. Yp 195/P);
  • Sequence 2 amino acid sequence of LcrV antigen of Y. pseudotuberculosis strain 32777 (i.e. Ypst 32777);
  • Sequence 3 amino acid sequence of LcrV antigen of Y. pestis strain 195/P in which the conformational segment ("CS") had been exchanged by the CS of Y. pseudotuberculosis strain 32777 (i.e.
  • Sequence 4 amino acid sequence of LcrV antigen from the Y. pseudotuberculosis strain 32777 in which the conformational segment ("CS") had been exchanged by the CS of Y. pestis strain 195/P (i.e. Ypst CS Yp ); Sequence 5: amino acid sequence of LcrV antigen from the Y. pseudotuberculosis strain 32777 wherein the amino acid residue [AA] was replaced by the [AA] of Y. pestis strain 195/P (i.e. Ypst D255N ).
  • Figure 3 shows a graph illustrating the mouse survival for groups of mice previously immunized intranasally with L. lactis secreting (or not) the LcrV of Y. pestis strain 195/P, of Y. pseudotuberculosis strain 2777, of Y p CS Y P st 5 Y ps t cs"Yp and Ypst D255N and then challenged intravenously with a lethal dose of Y. pseudotuberculosis strain 2777.
  • X-axis time in days; in ordinate, mouse survival, expressed in percentage.
  • Curve 1 mouse survival for the group immunized with L.
  • pseudotuberculosis strain 32777 LcrV in which the conformational segment ("CS") had been exchanged by the CS of Y. pestis strain 195/P (i.e. Ypst CS Yp ); curve 5: mouse survival for the group immunized with L. lactis secreting the Y. pseudotuberculosis strain 32777 LcrV wherein the amino acid residue [AA] was replaced by the [AA] of Y. pestis strain 195/P (i.e. Ypst D255N ).
  • Figure 4 shows a graph illustrating the mouse survival for groups of mice previously immunized intranasally (or not) with L. lactis secreting the Y. pseudotuberculosis strain 2777 LcrV and then challenged with inoculation with the Y. pseudotuberculosis 2777, 2889 and AH strains.
  • X-axis time in days; in ordinate, mouse survival, expressed in percentage.
  • Curve 1 mouse survival for the group inoculated with Y. pseudotuberculosis strain 2777;
  • curve 2 mouse survival for the group inoculated with Y. pseudotuberculosis strain 2889;
  • curve 3 mouse survival for the group inoculated with Y.
  • pseudotuberculosis strain AH the entire curves (ie entitled “...a)” relates to a group which have been previously immunized; the dotted curves (ie entitled “...b)" relates to a group which have not been previously immunized.
  • Figure 5 shows a graph illustrating the mouse survival for groups of mice previously immunized intranasally with L. lactis secreting the Y. pestis strain 195 LcrV and then challenged intravenously with a lethal dose of Y. pseudotuberculosis strain 2843 (Clade C) or Y. pseudotuberculosis strain 2889 (Clade D2).
  • X-axis time in days; in ordinate, mouse survival, expressed in percentage.
  • Curve 1 mouse survival for the group inoculated with Y. pseudotuberculosis strain 2843
  • curve 2 mouse survival for the group inoculated with Y.
  • Figure 6A shows a graph of the mice's anti-V IgG titers of groups of mice previously immunized intranasally with L. lactis secreting (or not) the Y. pestis strain 195 LcrV or the Y. pseudotuberculosis strain 2777 LcrV and then challenged intravenously with a lethal dose of pseudotuberculosis strain 2777.
  • group 1 mice immunized with L. lactis secreting Y. pst strain 2777 LcrV
  • group 2 mice immunized with L. lactis secreting Y. p strain 195 LcrV
  • group 3 mice immunized with L. lactis which does not secrete any LcrV
  • anti-V IgG titers amount in ordinate, anti-V IgG titers amount.
  • Figure 6B shows a graph of the mice's anti-V IgG titers of groups of mice previously immunized intranasally with L. lactis secreting (or not) the plurality of Yersinia LcrVs displayed in above figure 2, and then challenged intravenously with a lethal dose of pseudotuberculosis strain 2777.
  • X-axis each of the considered groups of mice (group 1: mice immunized with L. lactis secreting Y. pst strain 2777 LcrV; group 2: mice immunized with L. lactis secreting Y. pst strain 32777 LcrV wherein the amino acid residue [AA] was replaced by the [AA] of Y.
  • Figure 6C shows a graph of the mice's anti-V IgG titers of groups of mice previously immunized with L. lactis secreting from Ypst 2777.
  • group 1 mice immunized with L. lactis secreting Y. pst strain 2777 LcrV
  • group 2 mice immunized with L. lactis which does not secrete any LcrV
  • anti-V IgG titers amount.
  • Figure 7 shows a phylogenetic table of several strains of Yersinia pseudotuberculosis , Yersinia pestis and Yersinia enterocolitica; this phylogenetic table has been established in considering the "conformational segment" (ie position 218 to 234 with respect to the LcrV sequence of Y pst 32777) and the amino acid residue [AA] of amino acids sequences of Lcrv respective to the considered strains.
  • Figure 8 illustrates an alignment of a plurality of amino acid sequences of LcrV antigens mainly based on the conformational segment and the amino acid residue [AA]; this alignment displays all clades of Yersinia herein considered.
  • Figure 9 shows a graph illustrating in A: the mouse survival (%) for the group inoculated with Y. pseudotuberculosis strain 2777 (clade B - white square) and with Y. pseudotuberculosis strain 2790 (clade D - black triangle).
  • B the mouse survival (%) for the group inoculated with Y. pseudotuberculosis strain 2781 (clade B - grey square).
  • X-axis time in days; in ordinate, mouse survival, expressed in percentage, the entire curves (ie entitled "...a)” relates to a group which have been previously vaccinated with V2777; the dotted curves (ie entitled “...b)” relates to a group which have not been previously vaccinated.
  • LcrV or V antigen are used interchangeably in the present description and refer to the protein found in bacterial species of the Yersinia genus, and notably Yersinia pestis. It forms part of the Yersinia virulence protein factors that also includes all Yop's, this used to stand for Yersinia outer protein, but the name has been kept out of convention.
  • microorganism refers to a bacterium which is not modified artificially.
  • the bacteria of the invention may be chosen among bacteria which expresses genes for expressing heterologous Yersinia LcrV recombinant antigens.
  • the bacteria of the invention may be also chosen among bacteria which already expresses genes for expressing an endogenous Yersinia LcrV antigen.
  • recombinant bacteria or "genetically modified bacteria”, as used herein, refers to a bacteria genetically modified or genetically engineered. It means, according to the usual meaning of these terms, that the bacteria is not found in nature and is modified either by introduction or by deletion or by modification of genetic elements from equivalent bacteria found in nature. It can also be modified by forcing the development and evolution of new metabolic pathways by combining directed mutagenesis and evolution under specific selection pressure (see for instance WO 2004/076659).
  • a bacteria may be modified to express exogenous genes if these genes are introduced into the bacteria with all the elements allowing their expression in the host bacteria.
  • a bacteria may be modified to modulate the expression level of an endogenous gene. The modification or "transformation" of bacteria with exogenous DNA is a routine task for those skilled in the art.
  • endogenous gene means that the gene was present in the bacteria before any genetic modification, in the wild-type strain.
  • Endogenous genes may be overexpressed by introducing heterologous sequences in addition to, or to replace endogenous regulatory elements, or by introducing one or more supplementary copies of the gene into the chromosome or a plasmid.
  • Endogenous genes may also be modified to modulate their expression and/or activity. For example, mutations may be introduced into the coding sequence to modify the gene product or heterologous sequences may be introduced in addition to or to replace endogenous regulatory elements.
  • Modulation of an endogenous gene may result in the up-regulation and/or enhancement of the activity of the gene product, or alternatively, in the down-regulation and/or attenuation of the activity of the endogenous gene product.
  • Another way to enhance expression of endogenous genes is to introduce one or more supplementary copies of the gene onto the chromosome or a plasmid.
  • exogenous gene means that the gene was introduced into a bacteria, by means well known by the man skilled in the art, whereas this gene is not naturally occurring in the wild-type microorganism, bacteria can express exogenous genes if these genes are introduced into the microorganism with all the elements allowing their expression in the host bacteria. Transforming microorganisms with exogenous DNA is a routine task for the man skilled in the art. Exogenous genes may be integrated into the host chromosome, or be expressed extra- chromosomally from plasmids or vectors. A variety of plasmids, which differ with respect to their origin of replication and their copy number in the cell, are all known in the art. The sequence of exogenous genes may be adapted for its expression in the host bacteria. Indeed, the man skilled in the art knows the notion of codon usage bias and how to adapt nucleic sequences for a particular codon usage bias without modifying the deduced protein.
  • heterologous gene means that the gene is derived from a species of organism, or at least from a strain of organism of a same species, different from the recipient bacteria that expresses it. It refers to a gene which is not naturally occurring in the bacteria.
  • heterologous antigen means that the antigen is derived from a species of organism, or at least from a strain of organism of a same species, different from the recipient bacteria that expresses it. It refers to a antigen which is not naturally occurring in the bacteria.
  • nucleic acid expression construct preferably comprises regulatory sequences, such as promoter and terminator sequences, which are operatively linked with the nucleic acid sequence coding for each of the LcrV antigens according to the present invention.
  • the nucleic acid expression construct may further comprise 5' and/or 3' recognition sequences and/or selection markers.
  • overexpression means that the expression of a gene is increased as compared to the non-modified microorganism. Increasing the expression of a protein is obtained by increasing the expression of a gene encoding said protein. Increasing the expression of a gene may be carried out by all techniques known by the one skilled in the art. In this regard, it may be notably cited the implementation of a strong promoter upstream the nucleic acid intended to be overexpressed or the introduction of several copies of the said nucleic acid between a promoter, especially a strong promoter, and a terminator.
  • the "activity" of a protein is used interchangeably with the term “function” and designates, in the context of the invention, the capacity of a LcrV antigen or of a LcrV-derived antigen to raise an immune response against at least one Yersinia species selected from the group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica in a vertebrate, notably of the Mammalia and/or of the aves class(es), when appropriately administered.
  • the "antigenicity" of a protein in the context of the invention, means the ability of a LcrV antigen or of a LcrV-derived antigen to raise an immune response against at least one Yersinia species selected from the group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica in a vertebrate, notably of the Mammalia and/or of the aves class(es), when appropriately administered.
  • LcrV and LcrV-derived antigen may be used interchangeably in the context of the invention.
  • a LcrV-derived antigen according to the present invention more particularly relates to a fragment of a LcrV antigen from at least one Yersinia species selected from the group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, said LcrV- derived antigen being such that it keeps a same or at least a similar ability to induce an immune response in the considered vertebrate, notably of the Mammalia and/or of the aves class(es), when appropriately administered.
  • encoding or “coding” refer to the process by which a polynucleotide, through the mechanisms of transcription and translation, produces an amino-acid sequence.
  • the gene(s) encoding the protein(s) considered in the present invention can be exogenous or endogenous according to the nature of the considered bacteria.
  • nucleic acid sequences comprising a coding sequence (e.g. a sequence coding for a LcrV antigen according to the invention).
  • a coding sequence e.g. a sequence coding for a LcrV antigen according to the invention
  • reference sequences are described herein.
  • the present description also encompasses nucleic acid sequences having specific percentages of nucleic acid identity, with a reference nucleic acid sequence.
  • amino acid sequences e.g. enzyme amino acid sequences
  • a specific nucleic acid sequence or a specific amino acid sequence which complies with, respectively, the considered nucleotide or amino acid identity should further lead to obtaining a protein (or enzyme) which displays the desired biological activity.
  • the "percentage of identity" between two nucleic acid sequences or between two amino acid sequences is determined by comparing both optimally aligned sequences through a comparison window.
  • the portion of the nucleotide or amino-acid sequence in the comparison window may thus include additions or deletions (for example "gaps") as compared to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment between both sequences.
  • additions or deletions for example "gaps”
  • the identity percentage is calculated by determining the number of positions at which an identical nucleic base, or an identical amino-acid residue, can be noted for both compared sequences, then by dividing the number of positions at which identity can be observed between both nucleic bases, or between both amino-acid residues, by the total number of positions in the comparison window, then by multiplying the result by hundred to obtain the percentage of nucleotide identity between the two sequences or the percentage of amino acid identity between the two sequences.
  • the comparison of the sequence optimal alignment may be effected by a computer using known algorithms.
  • an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence.
  • nucleic acid sequence having at least 80%) nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%), 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence.
  • the present invention relates to a multivalent immunogenic composition for inducing an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enter ocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising :
  • the present invention relates to a multivalent immunogenic composition for inducing an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group consisting of: Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group consisting of :
  • Yersinia LcrV antigens as defined above are considered. Accordingly, at least two Yersinia LcrV antigens, one distinct from each other, may be selected in a group consisting of:
  • LcrV antigen inducing an immune response against Yersinia enterocolitica
  • At least three Yersinia LcrV antigens may be selected in a group consisting of:
  • LcrV antigens which are suitable for the invention are further detailed here after. As shown in the examples herein, the inventors identified the residues composing the major protective epitopes in V antigen, and an assay other than that based on anti-V titer measurement and bacterial-mediated toxicity against macrophages to measure vaccine efficacy.
  • the multivalent immunogenic composition according to the invention comprises or expresses at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising (or consisting of) :
  • the second Yersinia LcrV antigen is represented by the following formula (II): NH 2 -[SEQ ID N°A-2]-[CS-2]-[SEQ ID N°B-2]-[AA-2]-[SEQ ID N°C-2]-COOH (II),
  • SEQ ID N°A-1, SEQ ID N°A-2 and SEQ ID N°A-3 mean an amino acid sequence having at least 80% amino acid identity, preferably at least 90 % amino acid identity, with the sequence SEQ ID N°2 ,
  • - CS-1 means an amino acid sequence of sequence SEQ ID N°8,
  • - CS-2 means an amino acid sequence selected from the group comprising the sequences SEQ ID N°8, 10, 11, 12, 13, 107, 108, 109, 110, 111, 112, 113, 114 and 115,
  • - CS-3 means an amino acid sequence selected from the group comprising the sequences SEQ ID N°14, 15, 16, 17 and 18,
  • SEQ ID N°B-1, SEQ ID N°B-2 and SEQ ID N°B-3 mean an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %> amino acid identity, with the sequence SEQ ID N°3,
  • AA-1, AA-2 and AA-3 mean an amino acid selected from the group consisting of aspartic acid (or aspartate) and asparagine, preferably AA-1 represents asparagine, AA-2 represents aspartic acid and AA-3 represents aspartic acid, and
  • the composition according to the invention when the first or the second Yersinia LcrV antigen induces an immune response against Yersinia pestis and Yersinia pseudotuberculosis, then said immunogenic composition:
  • - further comprises or expresses at least one additional Yersinia LcrV antigen(s) inducing an immune response against Yersinia pestis or Yersinia pseudotuberculosis, different from whichever of the first or the second Yersinia LcrV antigen is already present.
  • the composition according to the invention may comprise or express a Yersinia LcrV antigen inducing an immune response against Yersinia pestis and Yersinia pseudotuberculosis which is represented by the following formula (IV):
  • SEQ ID N°A-4 means an amino acid sequence having at least 80% amino acid identity, preferably at least 90 % amino acid identity, with the sequence SEQ ID N°2,
  • SEQ ID N°B-4 means an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %> amino acid identity, with the sequence SEQ ID N°3,
  • SEQ ID N°C-4 means an amino acid sequence having at least 80%) amino acid identity, preferably at least 90 %> amino acid identity, with the sequence SEQ ID N°4.
  • the second Yersinia LcrV antigen(s), in a composition according to the invention may have the following formula (II):
  • - CS-2 means an amino acid sequence selected from a group comprising sequences SEQ ID N° 107, 108, 109, 110, 111, 112, 113, 114 and 115, and AA-2 represents aspartic acid,
  • - CS-2 means an amino acid sequence of sequence SEQ ID N° 10 and AA-2 represents aspartic acid (i.e. strain NT);
  • - CS-2 means an amino acid sequence of sequence SEQ ID N° 11 and AA-2 represents aspartic acid (i.e. clades Bl and B2);
  • - CS-2 means an amino acid of sequence SEQ ID N° 12 and AA-2 represents aspartic acid (i.e. clade Dl);
  • - CS-2 means an amino acid sequence of sequence SEQ ID N° 13 and AA-2 represents aspartic acid (i.e. clade D2);
  • - CS-2 means an amino acid sequence of sequence SEQ ID N° 8 and AA-2 represents asparagine (i.e. clade C);
  • the third Yersinia LcrV antigen(s) in a composition according to the invention may have the following formula (III):
  • - CS-3 means an amino acid sequence of sequence SEQ ID N° 14 and AA-3 represents aspartic acid (i.e. clade F);
  • - CS-3 means an amino acid sequence of sequence SEQ ID N° 15 and AA-3 represents aspartic acid (i.e. clade G);
  • - CS-3 means an amino acid sequence of sequence SEQ ID N° 16 and AA-3 represents asparagine (i.e. strain 8081);
  • - CS-3 means an amino acid sequence of sequence SEQ ID N° 17 and AA-3 represents aspartic acid (i.e. clade H);
  • - CS-3 means an amino acid sequence of sequence SEQ ID N° 18 and AA-3 represents aspartic acid (i.e. strain E40);
  • SEQ ID N°B-4 one independently from the others, mean an amino acid sequence SEQ ID N°5, wherein:
  • - X2 means an amino acid selected from the group comprising aspartic acid, glycine and asparagine,
  • - X3 means an amino acid selected from the group comprising glycine and glutamic acid
  • - X4 means an amino acid selected from the group comprising asparagine and lysine, and preferably mean:
  • SEQ ID N°B-1 and SEQ ID N°B-2 mean an amino acid sequence having at least 90% amino acid identity with the amino acid sequence SEQ ID N°6;
  • SEQ ID N°B-3 means an amino acid sequence having at least 90%) amino acid identity with the amino acid sequence SEQ ID N°7;
  • SEQ ID N°B-4 means an amino acid sequence having at least 90%> amino acid identity with the amino acid sequence SEQ ID N°23.
  • SEQ ID N°A-4 one independently from the others, may be represented by an amino acid sequence of the following formula (V):
  • SEQ ID N°Aa one independently from the other, mean an amino acid sequence having at least 80%) amino acid identity, preferably at least 90 %>, with the sequence SEQ ID N°19, and
  • SEQ ID N°Ab one independently from the other, means an amino acid sequence SEQ ID N°20, wherein X5 means an amino acid selected from the group comprising glutamic acid and lysine.
  • sequences SEQ ID N°C-1, SEQ ID N°C-2, SEQ ID N°C-3 and SEQ ID N°C-4, one independently from the others may be represented by an amino acid sequence of SEQ ID N°22 or SEQ ID N°21 , wherein ⁇ means an amino acid selected from the group comprising threonine and alanine, and preferably of SEQ ID N°21.
  • composition according to the invention may comprise or express:
  • Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis species one independently from the other, and notably selected among the group such as above-defined, and/or
  • Yersinia enterocolitica species one independently from the other, notably selected among the group such as above-defined.
  • composition according to the invention may be such that:
  • the first Yersinia LcrV antigen(s) inducing an immune response against Yersinia pestis is/are selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80% amino acid identity, preferably at least 90%) amino acid identity, and still better of sequences SEQ ID N°74 and 75;
  • the second Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis is/are selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80%) amino acid identity, preferably at least 90%o amino acid identity, and still better of sequences SEQ ID N°25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 72, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, and combination thereof ;
  • the third Yersinia LcrV antigen(s) inducing an immune response against Yersinia enterocolitica is/are selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80%o amino acid identity, preferably at least 90%o amino acid identity, and still better of sequences SEQ ID N°45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 99 and 100, and combination thereof; and
  • SEQ ID N°A-1 represents an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %, with the sequence SEQ ID N°2 ,- CS-1 represents an amino acid sequence of sequence SEQ ID N°8,
  • SEQ ID N°B-1 represents an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %, with the sequence SEQ ID N°3,
  • SEQ ID N°C-1 represents an amino acid sequence having at least 80%) amino acid identity, preferably at least 90 %>, with the sequence SEQ ID N°4.
  • sequence represented by the above formula (I) is defined by the amino acid sequence SEQ ID N° 75 or 76.
  • SEQ ID N°A-2 represents an amino acid sequence having at least 80%> amino acid identity, preferably at least 90 %>, with the sequence SEQ ID N°2,
  • - CS-2 represents an amino acid sequence of sequence SEQ ID N°l 1
  • SEQ ID N°B-2 represents an amino acid sequence having at least 80%> amino acid identity, preferably at least 90 %>, with the sequence SEQ ID N°3,
  • SEQ ID N°C-2 represents an amino acid sequence having at least 80%> amino acid identity, preferably at least 90 %, with the sequence SEQ ID N°4. More particularly, the sequence represented by the above formula (II) is defined by the amino acid sequences selected from the group comprising SEQ ID N° 86.
  • SEQ ID N°A-3 represents an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %, with the sequence SEQ ID N°2,
  • - CS-3 represents an amino acid sequence of sequence SEQ ID N°14
  • SEQ ID N°B-3 represents an amino acid sequence having at least 80%) amino acid identity, preferably at least 90%>, with the sequence SEQ ID N°3,
  • SEQ ID N°C-3 represents an amino acid sequence having at least 80%> amino acid identity, preferably at least 90%>, with the sequence SEQ ID N°4.
  • sequence represented by the above formula (III) is defined by the amino acid sequence SEQ ID N° 47.
  • this first illustrative embodiment relates to a multivalent immunogenic composition according to the invention which comprises or expresses:
  • This embodiment relates to a multivalent immunogenic composition wherein the first or the second Yersinia LcrV antigen induces an immune response against Yersinia pestis and Yersinia pseudotuberculosis .
  • said immunogenic composition comprises or expresses:
  • SEQ ID N°A-4 represents an amino acid sequence having at least 80%> amino acid identity, preferably at least 90 %>, with the sequence SEQ ID N°2,
  • - CS-4 represents an amino acid sequence of sequence SEQ ID N°8, - SEQ ID N°B-4 represents an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %, with the sequence SEQ ID N°3,
  • SEQ ID N°C-4 represents an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %, with the sequence SEQ ID N°4.
  • sequence represented by the above formula (IV) is defined by the amino acid sequence SEQ ID N° 75.
  • SEQ ID N°A-3 represents an amino acid sequence having at least 80%) amino acid identity, preferably at least 90 %>, with the sequence SEQ ID N°2,
  • - CS-3 represents an amino acid sequence of sequence SEQ ID N°14
  • SEQ ID N°B-3 represents an amino acid sequence having at least 80%> amino acid identity, preferably at least 90%>, with the sequence SEQ ID N°3,
  • SEQ ID N°C-3 represents an amino acid sequence having at least 80%> amino acid identity, preferably at least 90%>, with the sequence SEQ ID N°4.
  • sequence represented by the above formula (III) is defined by the amino acid sequence SEQ ID N° 47.
  • this second illustrative embodiment relates to a multivalent immunogenic composition according to the invention which comprises or expresses:
  • each above-mentioned Yersinia LcrV antigens may be independently selected from the group consisting of:
  • the different considered Yersinia LcrV antigens may be independently selected from the group consisting of:
  • a recombinant bacteria comprising and/or expressing the said Yersinia LcrV antigen, or a combination thereof.
  • a recombinant bacteria implemented in a composition of the invention may comprise and/or express only one specific Yersinia LcrV antigen, such as herein defined.
  • composition of the invention may thus comprise as many different recombinant bacteria as desired/required LcrV antigens.
  • a recombinant bacteria implemented in a composition of the invention may comprise and/or express at least two different desired/required LcrV antigens, such as herein defined.
  • composition of the invention may thus comprise only one type of recombinant bacteria or at least two different recombinant bacteria.
  • a composition according to the invention may notably comprise and/or express Yersinia LcrV antigens of different strains of a same Yersinia species selected from a group comprising Y. pestis, Y. pseudotuberculosis and Y. enterocolitica are herein described.
  • each above-mentioned Yersinia LcrV antigens may be present in a composition according to the invention in the form of a purified antigen.
  • composition according to the invention may further comprise at least a pharmaceutically acceptable carrier, a diluent and/or an immuno- adjuvant.
  • Suitable adjuvants include, but are not limited to, aluminium salts (aluminium phosphate or aluminium hydroxide), squalene mixtures (SAF-1), muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, non-ionic block copolymer surfactants, Quil A, cholera toxin B subunit, polphosphazene and derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al. (1990) Nature 344:873-875.
  • aluminium salts aluminium phosphate or aluminium hydroxide
  • SAF-1 squalene mixtures
  • muramyl peptide saponin derivatives
  • mycobacterium cell wall preparations monophosphoryl lipid A
  • mycolic acid derivatives mycolic acid derivatives
  • non-ionic block copolymer surfactants non-ionic block
  • a composition according to the invention may further comprise at least one additional active agent against Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, preferably said additional active agent being selected in a group comprising Mab 7.3, sulfonamides, fluoroquinolones such as ciprofloxacine, levofloxacin, streptomycin, gentamicin, tetracyclines, doxycycline, Oxytetracycline, ampicillin, cephalosporins, aminoglycosides, chloramphenicol, trimethroprime-sulfamethoxazole, amoxicilline/clavulanic acid, cefotaxime, cotrimoxazole, imipeneme, aztreonam, latamoxef and mixtures thereof
  • a composition according to the invention may be buffered at, or adjusted to, between pH 7.0 and 8.0, pH 7.2 and 7.6 or around or exactly pH 7.4.
  • a composition according to the invention may be lyophilised, optionally in presence of a stabilizing agent, for example a polyol such as sucrose or trehalose.
  • a stabilizing agent for example a polyol such as sucrose or trehalose.
  • a composition according to the invention may be encapsulated.
  • an encapsulation within liposomes is described by Fullerton, U.S. Pat. No. 4,235,877.
  • the immunologically effective amounts of the immunogens must be determined empirically. Factors to be considered include the immunogenicity, whether or not the immunogen will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier, route of administrations and the number of immunising dosages to be administered. Such factors are known in the vaccine art and it is well within the skill of immunologists to make such determinations without undue experimentation.
  • the Yersinia LcrV antigens can be present in varying concentrations in a composition of the invention.
  • the minimum concentration of the substance is an amount necessary to achieve its intended use, while the maximum concentration is the maximum amount that will remain in solution or homogeneously suspended within the initial mixture.
  • the minimum amount of a therapeutic agent is optionally one which will provide a single therapeutically effective dosage.
  • the minimum concentration is an amount necessary for bioactivity upon reconstitution and the maximum concentration is at the point at which a homogeneous suspension cannot be maintained.
  • the amount is that of a single therapeutic application.
  • each dose will comprise 1-100 ⁇ g of Yersinia LcrV antigens, optionally 5-50 ⁇ g or 5-25 ⁇ g.
  • the concentration in bioactive substances should not be harmful for the considered vertebrate wherein said bioactive substances are administered.
  • the present invention also encompasses a multivalent immunogenic composition for inducing an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia strains of a same Yersinia species selected in a group comprising (or consisting of) Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica; said immunogenic composition comprising (or consisting of) or expressing at least two Yersinia LcrV antigens wherein: - the first Yersinia LcrV antigen induces an immune response against a first Yersinia strain selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enter ocolitica, and
  • the second Yersinia LcrV antigen different from the first Yersinia LcrV antigen, induces an immune response against a second Yersinia strain, different from the first Yersinia strain, but selected in the same Yersinia species than the first Yersinia LcrV antigen.
  • a composition according to the invention may be administered in considering all the conventional route of administration in the field of immunogenic compositions or vaccines, including notably:
  • the enteral route including the oral, the nasal and the rectal route
  • parenteral route including intravenous, intra-arterial, intraosseous infusion, intramuscular, intracerebral, intracerebroventricular, intrathecal and subcutaneous route .
  • composition according to the invention is administered by topical route, by oral route or by nasal route.
  • a composition according to the invention induces an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enter ocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising :
  • Yersinia LcrV antigen inducing an immune response against Yersinia pestis species, preferably said Yersinia LcrV antigen being represented by the above-mentioned formula
  • [CS-1] means the amino acid sequence SEQ ID N° 8 and [AA-1] amino acid is asparagine
  • Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis species one independently from the other, preferably said Yersinia LcrV antigen(s) being represented by the above-mentioned formula (II) wherein the [CS-2] amino acid sequence and the [AA-2] amino acid is/are selected from the group comprising (or consisting of):
  • - CS-2 means an amino acid sequence selected from a group comprising sequences SEQ ID N° 107, 108, 109, 110, 11 1, 1 12, 1 13, 1 14 and 115 and AA-2 represents aspartic acid, x representing an integer ranging from 0 to 8 (i.e. clade A);
  • - CS-2 means an amino acid of sequence SEQ ID N° 10 and AA-2 represents aspartic acid (i.e. strain NT);
  • - CS-2 means an amino acid of sequence SEQ ID N° 11 and AA-2 represents aspartic acid (i.e. clades Bl and B2);
  • - CS-2 means an amino acid of sequence SEQ ID N° 12 and AA-2 represents aspartic acid (i.e. clade Dl);
  • - CS-2 means an amino acid of sequence SEQ ID N° 13 and AA-2 represents aspartic acid (i.e. clade D2);
  • - CS-2 means an amino acid of sequence SEQ ID N° 8 and AA-2 represents asparagine (i.e. clade C);
  • Yersinia LcrV antigen(s) inducing an immune response against Yersinia enterocolitica species one independently from the other, preferably said Yersinia LcrV antigen(s) being represented by the above-mentioned formula (III) wherein the [CS-3] amino acid sequence and the [AA-3] amino acid is/are selected from the group comprising (or consisting of):
  • - CS-3 means an amino acid of sequence SEQ ID N° 14 and AA-3 represents aspartic acid (i.e. clade F);
  • - CS-3 means an amino acid of sequence SEQ ID N° 15 and AA-3 represents aspartic acid (i.e. clade G);
  • - CS-3 means an amino acid of sequence SEQ ID N° 16 and AA-3 represents asparagine (i.e. strain 8081);
  • - CS-3 means an amino acid of sequence SEQ ID N° 17 and AA-3 represents aspartic acid (i.e. clade H);
  • - CS-3 means an amino acid of sequence SEQ ID N° 18 and AA-3 represents aspartic acid (i.e. strain E40);
  • a composition according to the invention induces an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising (or consisting of) Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising (or consisting of) :
  • Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis species one independently from the other, said Yersinia LcrV antigen(s) being selected from the group comprising sequence SEQ ID N°25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 72, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and combination thereof;
  • Yersinia LcrV antigen(s) inducing an immune response against Yersinia enterocolitica species, one independently from the other, said Yersinia LcrV antigen(s) being selected from the group comprising sequence SEQ ID N°45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 99, 100, and combination thereof; and
  • This invention also pertains to a multivalent immunogenic composition according to the invention and such as above-defined, for its use as a vaccine.
  • a multivalent immunogenic composition according to the invention and such as above-defined may be used for inducing a simultaneous immune response in, and preferably a simultaneous vaccination of, a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising (or consisting of) Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species.
  • a multivalent immunogenic composition according to the invention when intended to induce an immune response in a non-human vertebrate, notably of the aves class, then said composition comprises or expresses at least two Yersinia LcrV antigens, different from each other, for inducing an immune response against Yersinia pseudotuberculosis and Yersinia enterocolitica species.
  • a multivalent immunogenic composition according to the invention when intended to induce an immune response in a Mammalia, especially in a human, then said composition comprises or expresses at least two Yersinia LcrV antigens, different from each other, for inducing an immune response against Yersinia pestis and Yersinia pseudotuberculosis species, and preferably at least three Yersinia LcrV antigens, each different from each other, for inducing an immune response against Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica.
  • a composition according to the invention induces a simultaneous immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising (or consisting of) :
  • Yersinia LcrV antigen(s) inducing an immune response against Yersinia pestis being selected in the group comprising Yersinia LcrV antigens of amino acid sequences having at least 80% amino acid identity with, preferably at least 90% amino acid identity with, and still better of, sequences SEQ ID N°74 and 75;
  • Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis being selected in the group comprising Yersinia LcrV antigens of amino acid sequences having at least 80%) amino acid identity with, preferably at least 90%> amino acid identity with, and still better of, sequences SEQ ID N°25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 72, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and combination thereof ;
  • Yersinia LcrV antigen(s) inducing an immune response against Yersinia enterocolitica being selected in the group comprising Yersinia LcrV antigens of amino acid sequences having at least 80%> amino acid identity with, preferably at least 90%> amino acid identity with, and still better of, sequences SEQ ID N°45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 99 and 100, and combination thereof; and
  • a multivalent immunogenic composition according to the invention may be intended to induce an immune response in a non-human vertebrate, notably of the aves class.
  • said composition preferably comprises or expresses at least two Yersinia LcrV antigens, each different from each other, for inducing an immune response against Yersinia pseudotuberculosis and Yersinia enterocolitica species, said Yersinia LcrV antigens being selected in a group comprising (or consisting of):
  • Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis being selected in the group comprising Yersinia LcrV antigens of amino acid sequences having at least 80% amino acid identity with, preferably at least 90% amino acid identity with, and still better of, sequences SEQ ID N°25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 72, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and combination thereof ;
  • Yersinia LcrV antigen(s) inducing an immune response against Yersinia enterocolitica being selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80%) amino acid identity with, preferably at least 90%> amino acid identity with, and still better of, sequences SEQ ID N°45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 99 and 100, and combination thereof; and
  • a multivalent immunogenic composition according to the invention may be intended to induce an immune response in a Mammalia, in particular in a human.
  • said composition preferably comprises or expresses at least two Yersinia LcrV antigens, different from each other, for inducing an immune response against Yersinia pestis and Yersinia pseudotuberculosis species, said Yersinia LcrV antigens being selected in a group comprising:
  • Yersinia LcrV antigen(s) inducing an immune response against Yersinia pestis being selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80%> amino acid identity with, preferably at least 90%> amino acid identity with, and still better of, sequences SEQ ID N°74 and 75;
  • Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis being selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80%> amino acid identity with, preferably at least 90%> amino acid identity with, and still better of, sequences SEQ ID N°25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 72, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and combination thereof ; and
  • This invention also pertains to a recombinant bacteria expressing at least two Yersinia LcrV antigens, different from each other, said Yersinia LcrV antigens being selected in a group comprising (or consisting of):
  • Yersinia LcrV antigen being capable of raising an immune response against Yersinia pestis in a vertebrate, notably of the Mammalia and/or of the aves class(es), said Yersinia
  • LcrV antigen(s) being notably such as above-defined
  • Yersinia LcrV antigen different from the first Yersinia LcrV antigen, being capable of raising an immune response against Yersinia pseudotuberculosis in a vertebrate, notably of the Mammalia and/or of the aves class(es), said Yersinia LcrV antigen(s) being notably such as above-defined, and
  • Yersinia LcrV antigen different from the first and the second Yersinia LcrV antigens, being capable of raising an immune response against Yersinia enterocolitica in a vertebrate, notably of the Mammalia and/or of the aves class(es), said Yersinia LcrV antigen(s) being notably such as above-defined.
  • the invention also concerns a recombinant bacteria comprising or expressing at least two heterologous Yersinia LcrV recombinant antigens, each different from each other, said Yersinia LcrV recombinant antigens being selected in a group consisting of :
  • a first Yersinia LcrV recombinant antigen being capable of raising an immune response against Yersinia pestis in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined
  • a second Yersinia LcrV recombinant antigen different from the first Yersinia LcrV antigen, being capable of raising an immune response against Yersinia pseudotuberculosis in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined, and/or
  • Yersinia LcrV recombinant antigen different from the first and the second Yersinia LcrV antigens, being capable of raising an immune response against Yersinia enterocolitica in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined.
  • the above-mentioned Yersinia is notably capable of raising an immune response against Yersinia enterocolitica in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined. According to the nature of the considered bacteria, the above-mentioned Yersinia
  • LcrV antigens may be endogenous or heterologous Yersinia LcrV antigens. However, and for obvious reasons, at least one, preferably at least two, of the considered Yersinia LcrV antigens is/are necessarily heterologous. According to a first embodiment wherein the recombinant bacteria is of the Yersinia species, said recombinant bacteria may take advantage of the LcrV antigen naturally expressed by said bacteria of Yersinia species.
  • all Yersinia LcrV antigens expressed by a recombinant bacteria of the invention are not necessarily heterologous.
  • a Yersinia LcrV antigen not naturally expressed by a bacterium may be also designated by the term "Yersinia LcrV recombinant antigen".
  • the invention relates to a recombinant bacteria such as above-mentioned in the genome of which has been inserted at least one, and preferably at least two, exogenous nucleic acids.
  • the invention relates to a recombinant bacteria such as above-mentioned in the genome of which has been inserted at least one, preferably at least two, Yersinia LcrV recombinant antigen(s) selected among a group comprising (or consisting of):
  • nucleic acids encoding a first Yersinia recombinant LcrV antigen(s) capable of raising an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against Yersinia pestis species, and notably said nucleic acid encoding an amino acid sequence such as above-defined ;
  • nucleic acids encoding a second Yersinia recombinant LcrV antigen(s) capable of raising an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against Yersinia pseudotuberculosis species, and notably said nucleic acid encoding an amino acid sequence such as above-defined; and/or
  • nucleic acids encoding a third Yersinia recombinant LcrV antigen(s) capable of raising an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against Yersinia enterocolitica species, and notably at said nucleic acid encoding an amino acid sequence such as above-defined.
  • a recombinant bacteria according to the invention may be selected from the group comprising (or consisting of) Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Escherichia, Streptococcus, Agrobacterium, Bacillus, Corynebacterium, Clostridium, Gluconobacter, Citrobacter, Enterobacter, Klebsiella, Aerobacter, Methylobacter , Salmonella, Streptomyces, Yersinia and Pseudomonas.
  • a recombinant bacteria according to the invention is a probiotic lactic acid bacteria, in particular of the Lactococcus genus, and more particularly of the Lactococcus lactis species.
  • the invention also pertains to a recombinant bacteria according to the invention, for its use for inducing a simultaneous immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising (or consisting of) Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species.
  • said recombinant bacteria may be used for inducing a simultaneous immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two different strains of a same Yersinia species selected in a group comprising (or consisting of) Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species.
  • the above-mentioned exogenous nucleic acid preferably comprises regulatory sequences, such as promoter and terminator sequences, which are operatively linked with the considered exogenous nucleic acid sequence coding for a particular LcrV antigen according to the present invention.
  • exogenous nucleic acids considered in the present invention is under the control of a promoter, identical or different.
  • each of nucleic acids considered in the present invention is linked to a transcription terminator (which may be also termed “terminator” herein), identical or different.
  • an exogenous nucleic acid may further comprise 5' and/or 3' recognition sequences and/or selection markers.
  • recombinant bacteria according to the invention may thus advantageously comprise one or more selectable markers useful for the selection of transformed bacteria.
  • said selectable markers are comprised in the integrated nucleic acid construct intended to allow expressing the amino acid sequences according to the present invention.
  • the selectable marker is an antibiotic resistance marker.
  • antibiotic resistance markers include, but are not limited to the, NAT1 , AUR1-C, HPH, DSDA, KAN ⁇ R>, and SH BLE gene products; the NAT 1 gene product from S.
  • noursei confers resistance to nourseothricin
  • the AUR1-C gene product from Saccharomyces cerevisiae confers resistance to Auerobasidin A (AbA)
  • the HPH gene product of Klebsiella pneumonia confers resistance to Hygromycin B
  • the DSDA gene product of E. coli allows cells to grow on plates with D-serine as the sole nitrogen source
  • the KAN ⁇ R> gene of the Tn903 transposon confers resistance to G418
  • the SH BLE gene product from Streptoalloteichus hindustanus confers resistance to Zeocin (bleomycin).
  • the antibiotic resistance marker is deleted after the genetically modified microbial cell of the invention is isolated.
  • the man skilled in the art is able to choose suitable marker in specific genetic context.
  • the selectable marker rescues an auxotrophy (e.g., a nutritional auxotrophy) in the genetically modified microbial cell.
  • a parent microbial cell comprises a functional disruption in one or more gene products that function in an amino acid or nucleotide biosynthetic pathway, such as, for example, the HIS3, LEU2, LYS 1 , LYS2, MET 15, TRP1 , ADE2, and URA3 gene products in yeast, which renders the parent microbial cell incapable of growing in media without supplementation with one or more nutrients (auxotrophic phenotype).
  • the auxotrophic phenotype can then be rescued by transforming the parent microbial cell with a chromosomal integration encoding a functional copy of the disrupted gene product (NB: the functional copy of the gene can originate from close species, such as Kluveromyces, Candida, etc.) and the genetically modified microbial cell generated can be selected for based on the loss of the auxotrophic phenotype of the parent microbial cell.
  • NB the functional copy of the gene can originate from close species, such as Kluveromyces, Candida, etc.
  • the "culture" of a recombinant bacteria according to the invention is generally conducted in an appropriate culture medium adapted to the bacteria being cultivated, containing at least one simple carbon source, and if necessary co-substrates.
  • the microorganism strains used as production hosts preferably have a high rate of carbohydrate utilization. These characteristics may be conferred by mutagenesis and selection, genetic engineering, or may be natural.
  • a conventional "culture medium" for bacteria may notably contain glucose, and in particular may contain at least about 10 g/L of glucoseAdditional carbon substrates may include but are not limited to monosaccharides such as fructose, mannose, xylose and arabinose, oligosaccharides such as lactose maltose, galactose, or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate cornsteep liquor, sugar beet molasses, and barley malt.
  • Other carbon substrates may include glycerol.
  • the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.
  • preferred carbon substrates are glucose, fructose, and sucrose, or mixtures of these with C5 sugars such as xylose and/or arabinose for microorganisms modified to use C5 sugars, and more particularly glucose.
  • the culture media may contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the pathway necessary for the production of the desired LcrV antigen(s).
  • the conditions to consider in the present invention may be anaerobic or aerobic conditions.
  • anaerobic conditions refer to conditions under which the oxygen concentration in the culture medium is too low for the microorganism to be used as terminal electron acceptor. Anaerobic conditions may be achieved either by sparging the culture medium with an inert gas such as nitrogen until oxygen is no longer available to the microorganism or by the consumption of the available oxygen by the microorganism.
  • aerobic conditions refers to concentrations of oxygen in the culture medium that are sufficient for an aerobic or facultative anaerobic microorganism to use as a terminal electron acceptor.
  • Microaerobic condition refers to a culture medium in which the concentration of oxygen is less than that in air, i.e. oxygen concentration up to 6% (1 ⁇ 4.
  • An "appropriate culture medium” designates a medium (e.g. a sterile, liquid medium) comprising nutrients essential or beneficial to the maintenance and/or growth of the cell such as carbon sources or carbon substrate, nitrogen sources, for example, peptone, yeast extracts, meat extracts, malt extracts, urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; phosphorus sources, for example, monopotassium phosphate or dipotassium phosphate; trace elements (e.g., metal salts), for example magnesium salts, cobalt salts and/or manganese salts; as well as growth factors such as amino acids, vitamins, growth promoters, and the like.
  • a medium e.g. a sterile, liquid medium
  • nutrients essential or beneficial to the maintenance and/or growth of the cell such as carbon sources or carbon substrate, nitrogen sources, for example, peptone, yeast extracts, meat extracts, malt extracts, urea, am
  • carbon source or “carbon substrate” or “source of carbon” according to the present invention denotes any source of carbon that can be used by those skilled in the art to support the normal growth of a microorganism, including hexoses (such as glucose, galactose or lactose), pentoses, monosaccharides, oligosaccharides, disaccharides (such as sucrose, cellobiose or maltose), molasses, starch or its derivatives, cellulose, hemicelluloses and combinations thereof.
  • hexoses such as glucose, galactose or lactose
  • pentoses monosaccharides, oligosaccharides, disaccharides (such as sucrose, cellobiose or maltose), molasses, starch or its derivatives, cellulose, hemicelluloses and combinations thereof.
  • monosaccharides such as glucose, galactose or lactose
  • oligosaccharides such as sucrose, cell
  • an "appropriate culture medium” may be the Ml 7 medium supplemented with glucose, and especially 0,5% glucose.
  • the invention also pertains to a device for detecting at least one Yersinia infection due to at least one Yersinia species selected from the group consisting of Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species, wherein said device comprises at least two Yersinia's LcrV-specific binding molecules selected among the group comprising (or consisting of):
  • At least one first Yersinia's LcrV-specific binding molecule(s) being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pestis species;
  • said device may:
  • - further comprise at least one additional Yersinia's LcrV-specific binding molecule(s) being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pestis or Yersinia pseudotuberculosis, different from whichever of the first or the second Yersinia's LcrV-specific binding molecule is already present.
  • Said detection device may be also appropriate for detecting at least one Yersinia infection due to at least one specific Yersinia pestis, Yersinia pseudotuberculosis or Yersinia enterocolitica strain.
  • a such device may be subject to any adequate adaptations which belong to the knowledge of a man skilled in the art, notably in view of the teaching displayed in the present description.
  • each Yersinia's LcrV-specific binding molecules may be selected from the group consisting of:
  • a device for detecting at least one Yersinia infection may be based on the device described in R. Bianucci et ah, C. R. Biologies 330 (2007) and Chanteau et ah, 2000 a ; 2003 a et b.
  • a device for detecting at least one Yersinia infection may be also based on the device commercialized under the name recomLine Yersinia IgG 2.0 by the company MIKROGEN.
  • the invention further relates to the above-mentioned devices for use for detecting at least one Yersinia species selected from the group consisting of: Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species
  • a companion diagnostic test device is notably intended to monitor the response to a treatment with a particular therapeutic product for the purpose of adjusting treatment to achieve improved safety or effectiveness.
  • a companion diagnostic test device is intended to monitor the presence of an immune response of a vertebrate, notably of the Mammalia and/or of the aves class(es), to at least one previous administration of at least one multivalent immunogenic composition according to the invention and such as above-defined.
  • this companion diagnostic test device allows assessing the presence of an immune response and, the case arising, the level of this immune response.
  • the invention also pertains to a device for detecting antibodies anti- Yersinia's LcrV in a vertebrate, notably of the Mammalia and/or of the aves class(es), wherein said device comprises at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising (or consisting of) :
  • a device for detecting at least one Yersinia infection may be based on the device described in situ et ah, Vaccine, 2008, 26(13): 1616-25.
  • kits comprising at least :
  • - a device for detecting antibodies anti- Yersinia's LcrV in a vertebrate, notably of the Mammalia and/or of the aves class(es), and notably such as above-defined.
  • the invention also relates to the above-mentioned devices and kits for use for monitoring the presence of an immune response of a vertebrate, notably of the Mammalia and/or of the aves class(es), to at least one previous administration of at least one multivalent immunogenic composition according to the invention and such as above-defined.
  • the invention also relates to the above-mentioned devices and kits, for use for detecting antibodies anti- Yersinia's LcrV in a vertebrate, notably of the Mammalia and/or of the aves class(es).
  • At least one should be understood as comprising, if applicable, "at least one”, “at least two”, “at least three”, “at least four", “at least five” and so on.
  • Bacterial strains, plasmids and growth conditions Y. pseudotuberculosis strains, L. lactis strain MG1363, and the lactococcal pNZYR expression vector (conferring resistance to chloramphenicol) are used.
  • Y. pseudotuberculosis strains and L. lactis were respectively cultured in lysogeny broth at 28°C and M17 medium (Difco) supplemented with 0.5% glucose at 30°C.
  • the growth media were supplemented with chloramphenicol (10 ⁇ g/ml) when required.
  • Custom pNZYR vectors (Eurogentec) containing the LcrVs of interest (translationally fused with the L. lactis usp45 secretion signal sequence and under the control of the Pusp45 promoter) were generated as described in Daniel C et ah, Vaccine, 27, 2009: 1141-4.
  • mice were inoculated intravenously with 300 ⁇ of PBS containing 10 3 Y. pseudotuberculosis .
  • mice's survival was monitored daily for three weeks.
  • DNA from above-mentioned Y. pseudotuberculosis strains was purified using the Nucleospin kit (Macherey-Nagel) and then used to amplify LcrV by PCR (using the primer sets 5 ' -TCACCGCGCAAAATTATTGC-3 ' (i.e. SEQ ID N°101) ; 5'-TTGTCTGCGATAAGCTCTTG-3 ' (i.e. SEQ ID N°102)).
  • Amplicons were purified using a PCR purification kit (Qiagen) and sequenced with the primers used for amplification and the following primers : 5 ' -CCTAGCTTATTTTCTACCCG-3 ' (i.e. SEQ ID N°103) ; 5'-GAACCGGGGCGTTGGGTAATC-3' (i.e. SEQ ID N°104) ; 5'-GTTGGTTGTCATAATGACCGCC-3' (i.e. SEQ ID N°105) ; 5'- CTAACCAAGTCGTTGAGCGG-3 ' (i.e. SEQ ID N°106). Nucleotide sequences were translated using the ExPaSy Translate tool (http://web.expasy.org/translate/).
  • a phylogenic tree based on LcrV sequences may be generated using the http://phylogeny.lirmm.fr/phylo_cgi/index.cgi web server (Dereeper A, Nucleic acids research, 36, 2008: W465-9) (see figure 7).
  • Statistical analysis The Mann- Whitney U test, the log-rank test and Fisher's exact probability test were used to compare antibody titers, survival curves and the degree of protection conferred by the various recombinant vaccine strains.
  • Example 1 Yersinia pseudotuberculosis escapes from the immune response against the V antigen from Y. pestis
  • mice were immunized with L. lactis producing LcrV from Y. pseudotuberculosis strain 2777 (VYpst).
  • mice Two weeks after the end of the immunization protocol, mice were challenged intravenously with a lethal dose of Y. pseudotuberculosis strain 2777. The proportion of animals developing fatal pseudotuberculosis was similar in mice vaccinated with VYp and in unvaccinated mice ( Figure 1). This contrasted with the higher survival rate observed in animals vaccinated with VYpst ( Figure 1).
  • mice's anti-V IgG titers did not appear to depend on the V antigen used ( Figure 6A).
  • the present data shown that the LcrV sequence polymorphism affects cross- protective immunity.
  • the sequences of the LcrV proteins from Y. pseudotuberculosis strain 2777 and Y. pestis strain 195/P differ in the region considered to contain a plague-protective epitope (Motin VL, Infection and immunity, 62, 1994: 4192-201; Hill J, Infection and immunity, 65, 1997: 4476- 82; Vernazza C, Vaccine, 27, 2009:2775-80).
  • the Y. pestis strain 195/P LcrV region lacks a threonine and has three residue substitutions (E205A, V226M, and N255D) ( Figure 2).
  • the missing threonine and the substituted residue 226 are located within the 16-amino acid LcrV segment (residues 218 to 234), which is referred to here as the "conformational segment".
  • Example 3 assessment of the immune escape from vaccination against LcrV due to the polymorphism in LcrV's conformational segment
  • strains Y.pst 2889 and Y.pst AH from clade D2 were selected.
  • LcrV from both strains 2889 and AH had an Asp in amino acid position [AA] like Y. pseudotuberculosis strain 2777.
  • sequences of the LcrV conformational segment in strains 2889 and AH differ from that of V2777 (ie clade Bl) ( Figure 8).
  • mice was immunized with L. lactis secreting V2777 and the animals' survival was measured after inoculation with the 2777, 2889 and AH strains.
  • figure 9 shows that L.lactis secreting clade B V antigen affords protection against a Y .pseudotuberculosis strain producing clade B (but not clade D) V antigen.
  • Example 4 LcrV from Y. pestis confers protection against specific Y. pseudotuberculosis strains
  • LcrV proteins from this particular clade C have the same conformational segment sequence and the same residue (an asparagine) at amino acid position [AA] as LcrV from Y. pestis ( Figure 8 (see Clades CC and CC-bis)).
  • mice vaccinated with L. lactis secreting VYp were challenged with a lethal dose of Y. pseudotuberculosis strains 2843 or 2889.
  • Strain 2843 belongs to clade C (which includes Y. pestis LcrV) while the 2889 strain belongs to clade D2 ( Figure 8).
  • mice The vaccinated mice were protected against pseudotuberculosis caused by strain 2843
  • LcrV from Y. pestis confers protection against Y. pseudotuberculosis strains belonging to clade C.
  • Example 5 As a control, different Y. pseudotuberculosis strains used in this study have been shown to produce similar amounts of V antigen, by immunoblot of whole-cell lysates of Y. pseudotuberculosis strains (2777, 2781 , AH, 2790, 2889 and 2843) grown under conditions that induce the production of V antigen. The relative density value (expressed as a percentage), was calculated by dividing the density of the band of interest by the sum of the density of all the bands shown on the blot. Results are detailed hereafter:
  • LcrV is a polymorphic antigen within which the major protective epitope encompasses residues 135 to 275 and more particularly is characterized by the above-identified conformational segment ("CS") and amino acid residue [AA].
  • CS conformational segment
  • AA amino acid residue
  • V-based vaccine optionally using a bacteria, and more particularly L. lactis, as antigen vehicle, comprising multiple V antigens of a plurality a strains of Yersinia is particularly interesting in that it leads to an immune response, and thus a protection, against a wide number of Yersinia strains (whether they belong to the same species or not).
  • - X2 means an amino acid selected from the group comprising aspartic acid, glycine and asparagine,
  • - X3 means an amino acid selected from the group comprising glycine and glutamic acid
  • KMPQTTTIQMDGSEKKI SEQ ID N°12 ( CS-2; from LcrV of Y. pseudotuberculosis Clade Dl)
  • Xs means an amino acid selected from the group comprising glutamic acid and lysine

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Abstract

The invention concerns a multivalent immunogenic composition for inducing an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising : - at least a first Yersinia LcrV antigen inducing an immune response against Yersinia pestis; - at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, inducing an immune response against Yersinia pseudotuberculosis; and - at least a third Yersinia LcrV antigen, different from the first and second Yersinia LcrV antigens, inducing an immune response against Yersinia enterocolitica.

Description

MULTIVALENT IMMUNOGENIC COMPOSITION FOR INDUCING AN IMMUNE
RESPONSE AGAINST YERSINIA SPECIES
FIELD OF THE INVENTION
The present invention relates to immunogenic compositions and methods for inducing an immune response against Yersinia species.
BACKGROUND OF THE INVENTION
Yersinia pseudotuberculosis (Ypst), Yersinia enterocolitica (Ye) and Yersinia pestis (Yp) species are bacterial pathogens in humans and animals (Butler T, Plenum Medical Book Company, New and London, 1983).
Y. pestis is responsible for plague, a fatal flea-borne disease. The two other Yersinia pseudotuberculosis and Yersinia enterocolitica species cause a self-limiting bowel disease in humans having ingested contaminated food.
However, some patients with enteric yersiniosis also develop immunopathological complications, such as reactive polyarthritis and erythema nodosum (Kaasch AJ, Infection, 40, 2012: 185-90; Drummond N, Foodborne pathogens and disease, 9, 2012: 179-89).
Furthermore, fatal hematogenous spreading of Y. enterocolitica and Y. pseudotuberculosis is often reported in immunocompromised individuals or patients suffering from hemochromatosis (Kaasch AJ, Infection, 40, 2012: 185-90; Bergmann TK, Scandinavian journal of gastroenterology, 36, 2001 : 891-5; Piroth L, La Revue de medicine interne, 18, 1997: 932-8).
Although plague outbreaks are reported in both hemispheres, outbreaks of human enteric yersiniosis occur mostly in the northern hemisphere (i.e. northern Europe, the United States, Canada, Russia and Japan). It is noteworthy that human enteric yersiniosis is among the five economic burden of foodborne illnesses worldwide.
In addition to the diseases' impact on human health, all three yersinioses are associated with a major socio-economic burden because they cause recurrent, fatal epidemics among livestock and in zoological and wildlife parks (Brown CC, Journal of comparative pathology, 101, 1989: 463-6; Gaydos JK, Journal of wildlife diseases, 45, 2009: 1182-6).
Yersinioses are thus considered to be significant human and veterinary health problems; unsurprisingly, research geared towards the production of anti-yersiniosis vaccines is now underway.
Today's anti- Yersinia vaccine development programs are focused on the major virulence factor LcrV (also referred to as "V antigen") (Demeure CE, Caister Academic Press, Northfolk, UK 2010: 143-68; Smiley ST, Expert review of vaccines, 7, 2008: 209-21; Williamson ED, Clinical and experimental immunology, 172, 2013: 1-8).
LcrV is a polymorphic protein within which the most effectively protective region encompasses residues 135 to 262 (Miller NC, Infection and immunity, 80, 2012: 1572-82; Motin VL, Microbial pathogenesis, 12, 1992: 165-75; Anisimov AP, Infect Genet Evol, 10, 2010: 137- 45; Schmidt A, Microbial pathogenesis, 26, 1999: 221-33; Vernazza C, Vaccine, 27, 2009: 2775- 80).
Most amino-acid variations occur in the second half-part of the protein, within which a hypervariable region (both in terms of composition and length) has been identified (Roggenkamp A, Infection and immunity, 65, 1997: 446-51).
The residues at positions 51 and 63 are known to be highly polymorphic (Sing A, Proceedings of the National Academy of Sciences of the United States of America, 102, 2005: 16049-54).
Indeed, LcrV antigens have been classified into different types, subtypes and variants on the basis of (i) the length of the hypervariable region, (ii) the polymorphisms at residues 51 and
63 and (iii) other subtle sequence differences (Motin VL, Microbial pathogenesis, 12, 1992: 165-
75; Anisimov AP, Infect Genet Evol, 10, 2010: 137-45; Roggenkamp A, Infection and immunity,
65, 1997: 446-51; Sing A, Proceedings of the National Academy of Sciences of the United States of America, 102, 2005: 16049-54).
However, the exact nature of the residues composing the major epitope have not been identified per se (Miller NC, Infection and immunity, 80, 2012: 1572-82; Schmidt A, Microbial pathogenesis, 26, 1999: 221-33).
What is more, there is debate as to whether immunization using a single V type, subtype or even variant confers cross-protection against challenge with a Yersinia strain producing another V type, subtype or variant (Miller NC, Infection and immunity, 80, 2012:
1572-82; Schmidt A, Microbial pathogenesis, 26, 1999: 221-33; Roggenkamp A, Infection and immunity, 65, 1997: 446-51; Daniel C, Vaccine, 27, 2009: 1141-4; Motin VL, Infection and immunity, 62, 1994: 4192-201).
Accordingly, there is a lack of understanding of the absence of cross-protection after active immunization, despite the fact that LcrV sequences from both Yersinia species share more than 95% identity.
For instance, passive immunization of mice with rabbit anti-sera raised against purified V antigen from Y. pestis or with purified rabbit IgG raised against a recombinant V antigen from Y. pseudotuberculosis protected against plague and pseudotuberculosis (Motin VL, Infection and immunity, 62, 1994: 4192-201).
Also, WO2006/110881 teaches that antisera raised against recombinant V antigen or a stable staphylococcal protein A-V antigen fusion peptide may provide statistically significant protection against Y. pestis and Y. pseudotuberculosis but not Y. enterocolitica.
In contrast, it has been reported that active immunization with Lactococcus lactis secreting the V antigen from Y. pseudotuberculosis confers protection against pseudotuberculosis but not against plague (Daniel C, Vaccine, 27, 2009: 1 141-4). This absence of cross-protection after active immunization with an LcrV-producing L. lactis is not determined.
WO2008/063240 reports multivalent vaccines, in the form of live bacterial vaccines, expressing a plurality of Yersinia antigens. A vaccine carrier, a Lactobacillus plantarum expressing a FI capsular antigen, Yersinia outer surface protein D (YopD), and a LcrV antigen (e.g. from Y pestis serotype 0:3), is disclosed.
Thus, there is a need in the art for methods and products for immunizing non-human and human organisms against infections by Yersinia species as well as for diagnosing such infections.
In particular, there is a need in the art for methods and products for immunizing non- human and human organisms against infections by a plurality of Yersinia species.
Thus, there is still a need in the art for immunogenic compositions which provide cross-protection against a plurality of Yersinia species.
There is also a need in the art for tools, such as devices and kits, for for monitoring the presence of an immune response to Yersinia species.
SUMMARY OF THE INVENTION
The present invention relates to a multivalent immunogenic composition for inducing an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising :
- at least a first Yersinia LcrV antigen inducing an immune response against Yersinia pestis;
- at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, inducing an immune response against Yersinia pseudotuberculosis; and
- at least a third Yersinia LcrV antigen, different from the first and second Yersinia LcrV antigens, inducing an immune response against Yersinia enterocolitica. Accordingly, the present invention relates to a multivalent immunogenic composition for inducing an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group consisting of: Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group consisting of :
- at least a first Yersinia LcrV antigen inducing an immune response against Yersinia pestis;
- at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, inducing an immune response against Yersinia pseudotuberculosis; and
- at least a third Yersinia LcrV antigen, different from the first and second Yersinia
LcrV antigens, inducing an immune response against Yersinia enterocolitica.
According to a particular embodiment, in the multivalent immunogenic composition according to the invention, said Yersinia LcrV antigens may be selected in a group comprising (or consisting of):
- the first Yersinia LcrV antigen is represented by the following formula (I):
NH2-[SEQ ID N°A-1]-[CS-1]-[SEQ ID N°B-1]-[AA-1]-[SEQ ID N°C-l]-COOH (I),
- the second Yersinia LcrV antigen is represented by the following formula (II):
NH2-[SEQ ID N°A-2]-[CS-2]-[SEQ ID N°B-2]-[AA-2]-[SEQ ID N°C-2]-COOH (II),
- the third Yersinia LcrV antigen is represented by the following formula (III):
NH2-[SEQ ID N°A-3]-[CS-3]-[SEQ ID N°B-3]-[AA-3]-[SEQ ID N°C-3]-COOH (III), wherein:
- SEQ ID N°A-1, SEQ ID N°A-2 and SEQ ID N°A-3, one independently from the others, mean an amino acid sequence having at least 80% amino acid identity, preferably at least 90%) amino acid identity, with the sequence SEQ ID N°2,
- CS-1 means an amino acid sequence of sequence SEQ ID N°8,
- CS-2 means an amino acid sequence selected from the group comprising the sequences SEQ ID N°8, 10, 11, 12, 13, 107, 108, 109, 110, 111, 112, 113, 114 and 115,
- CS-3 means an amino acid sequence selected from the group comprising the sequences SEQ ID N°14, 15, 16, 17 and 18,
- SEQ ID N°B-1, SEQ ID N°B-2 and SEQ ID N°B-3, one independently from the others, mean an amino acid sequence having at least 80%> amino acid identity, preferably at least 90 %> amino acid identity, with the sequence SEQ ID N°3,
- AA-1, AA-2 and AA-3, one independently from the others, mean an amino acid selected from the group consisting of aspartic acid (or aspartate) and asparagine, preferably AA-1 represents asparagine, AA-2 represents aspartic acid and AA-3 represents aspartic acid, and - SEQ ID N°C-1, SEQ ID N°C-2 and SEQ ID N°C-3, one independently from the others, mean an amino acid sequence having at least 80% amino acid identity, preferably at least 90 % amino acid identity, with the sequence SEQ ID N°4.
According to another particular embodiment, the multivalent immunogenic composition according to the invention may be such as, when the first or the second Yersinia LcrV antigen induces an immune response against Yersinia pestis and Yersinia pseudotuberculosis, then said immunogenic composition:
- is devoid of the second or the first Yersinia LcrV antigen; or
- further comprises or expresses at least one additional Yersinia LcrV antigen(s) inducing an immune response against Yersinia pestis or Yersinia pseudotuberculosis, different from whichever of the first or the second Yersinia LcrV antigen is already present.
According to this particular embodiment, the Yersinia LcrV antigen inducing an immune response against Yersinia pestis and Yersinia pseudotuberculosis may be represented by the following formula (IV):
NH2-[SEQ ID N°A-4]-[CS-4]-[SEQ ID N°B-4]-[AA-4]-[SEQ ID N°C-4]-COOH (IV), and wherein:
- SEQ ID N°A-4 means an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %> amino acid identity, with the sequence SEQ ID N°2,
- CS-4 means an amino acid sequence of sequence SEQ ID N° 8,
- SEQ ID N°B-4 means an amino acid sequence having at least 80%) amino acid identity, preferably at least 90 %> amino acid identity, with the sequence SEQ ID N°3,
- AA-4 represents asparagine, and
- SEQ ID N°C-4 means an amino acid sequence having at least 80%> amino acid identity, preferably at least 90%> amino acid identity, with the sequence SEQ ID N°4.
This invention also pertains to a multivalent immunogenic composition according to the invention, for its use as a vaccine.
In particular, the invention pertains to this multivalent immunogenic composition according to the invention, for its use for the simultaneous vaccination of a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two, preferably three, Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species.
Accordingly, the invention pertains in particular to this multivalent immunogenic composition according to the invention, for its use for the simultaneous vaccination of a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two, preferably three, Yersinia species selected in a group consisting of, Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species.
The invention also concerns a method of immunizing a vertebrate, notably of the Mammalia and/or of the aves class(es), against disease(s) caused by Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species, said method comprising administering to the vertebrate at least one immunoprotective dose of a multivalent immunogenic composition according to the invention.
Accordingly, the invention also concerns a method of immunizing a vertebrate, notably of the Mammalia and/or of the aves class(es), against disease(s) caused by Yersinia species selected in a group consisting of Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species, said method comprising administering to the vertebrate at least one immunoprotective dose of a multivalent immunogenic composition according to the invention.
The invention also concerns a recombinant bacteria comprising or expressing at least two heterologous Yersinia LcrV recombinant antigens, each different from each other, said Yersinia LcrV recombinant antigens being selected in a group comprising:
- at least a first Yersinia LcrV recombinant antigen being capable of raising an immune response against Yersinia pestis in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined,
- at least a second Yersinia LcrV recombinant antigen, different from the first Yersinia LcrV antigen, being capable of raising an immune response against Yersinia pseudotuberculosis in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined, and
- at least a third Yersinia LcrV recombinant antigen, different from the first and the second Yersinia LcrV antigens, being capable of raising an immune response against Yersinia enterocolitica in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined.
Accordingly, the invention also concerns a recombinant bacteria comprising or expressing at least two heterologous Yersinia LcrV recombinant antigens, each different from each other, said Yersinia LcrV recombinant antigens being selected in a group consisting of :
- at least a first Yersinia LcrV recombinant antigen being capable of raising an immune response against Yersinia pestis in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined,
- at least a second Yersinia LcrV recombinant antigen, different from the first Yersinia LcrV antigen, being capable of raising an immune response against Yersinia pseudotuberculosis in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined, and
- at least a third Yersinia LcrV recombinant antigen, different from the first and the second Yersinia LcrV antigens, being capable of raising an immune response against Yersinia enterocolitica in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined.
The invention also concerns a device for detecting at least one Yersinia infection due to at least one Yersinia species selected from the group consisting of Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species, wherein said device comprises at least two Yersinia's LcrV-specific binding molecules selected among the group comprising:
- the first Yersinia's LcrV-specific binding molecule being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pestis species;
- the second Yersinia's LcrV-specific binding molecule, different from the first Yersinia's LcrV-specific binding molecule, being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pseudotuberculosis species; and/or
- the third Yersinia's LcrV-specific binding molecule, different from the first and the second Yersinia's LcrV-specific binding molecules, being capable of binding to at least one Yersinia LcrV antigen of the Yersinia enterocolitica species.
Accordingly, the invention also concerns a device for detecting at least one Yersinia infection due to at least one Yersinia species selected from the group consisting of Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species, wherein said device comprises at least two Yersinia's LcrV-specific binding molecules selected among the group consisting of:
- the first Yersinia's LcrV-specific binding molecule being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pestis species;
- the second Yersinia's LcrV-specific binding molecule, different from the first Yersinia's LcrV-specific binding molecule, being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pseudotuberculosis species; and/or
- the third Yersinia's LcrV-specific binding molecule, different from the first and the second Yersinia's LcrV-specific binding molecules, being capable of binding to at least one
Yersinia LcrV antigen of the Yersinia enterocolitica species.
The invention also concerns a device for detecting antibodies anti-Yersinia's LcrV in a vertebrate, notably of the Mammalia and/or of the aves class(es), wherein said device comprises at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising :
- at least a first Yersinia LcrV antigen being the LcrV antigen of Yersinia pestis species;
- at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, being the LcrV antigen of Yersinia pseudotuberculosis species; and
- at least a third Yersinia LcrV antigen, different from the first and second Yersinia LcrV antigens, being the LcrV antigen of Yersinia enterocolitica species.
Accordingly, the invention also concerns a device for detecting antibodies anti- Yersinia's LcrV in a vertebrate, notably of the Mammalia and/or of the aves class(es), wherein said device comprises at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group consisting of :
- at least a first Yersinia LcrV antigen being the LcrV antigen of Yersinia pestis species;
- at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, being the LcrV antigen of Yersinia pseudotuberculosis species; and
- at least a third Yersinia LcrV antigen, different from the first and second Yersinia LcrV antigens, being the LcrV antigen of Yersinia enterocolitica species.
The invention further concerns a kit comprising at least:
- a multivalent immunogenic composition according to the invention and such as above- defined, and
- a device for detecting antibodies anti- Yersinia's LcrV in a vertebrate, notably of the
Mammalia and/or of the aves class(es), and notably such as above-defined.
BRIEF DESCRIPTION OF THE DRAWING
Figure 1 shows a graph illustrating the mouse survival for groups of mice previously immunized intranasally with L. lactis secreting (or not) the Y. pestis strain 195/P LcrV or the Y. pseudotuberculosis strain 2777 LcrV and then challenged intravenously with a lethal dose of Y. pseudotuberculosis strain 2777. In X-axis: time in days; in ordinate, mouse survival, expressed in percentage. Curve 1: mouse survival for the group immunized with L. lactis which does not secrete any LcrV; curve 2: mouse survival for the group immunized with L. lactis secreting the Y. pseudotuberculosis strain 2777 LcrV; curve 3: mouse survival for the group immunized with L. lactis secreting the Y. pestis strain 195 LcrV.
Figure 2 illustrates an alignment of a plurality of amino acid sequences of LcrV antigens (position 204 to 262/or 263). Sequence 1: amino acid sequence of LcrV antigen of Y. pestis strain 195/P (i.e. Yp 195/P); Sequence 2: amino acid sequence of LcrV antigen of Y. pseudotuberculosis strain 32777 (i.e. Ypst 32777); Sequence 3: amino acid sequence of LcrV antigen of Y. pestis strain 195/P in which the conformational segment ("CS") had been exchanged by the CS of Y. pseudotuberculosis strain 32777 (i.e. YpCS YPst) ; Sequence 4: amino acid sequence of LcrV antigen from the Y. pseudotuberculosis strain 32777 in which the conformational segment ("CS") had been exchanged by the CS of Y. pestis strain 195/P (i.e. YpstCS Yp); Sequence 5: amino acid sequence of LcrV antigen from the Y. pseudotuberculosis strain 32777 wherein the amino acid residue [AA] was replaced by the [AA] of Y. pestis strain 195/P (i.e. YpstD255N).
Figure 3 shows a graph illustrating the mouse survival for groups of mice previously immunized intranasally with L. lactis secreting (or not) the LcrV of Y. pestis strain 195/P, of Y. pseudotuberculosis strain 2777, of Yp CS YPst 5 Ypstcs"Yp and YpstD255N and then challenged intravenously with a lethal dose of Y. pseudotuberculosis strain 2777. In X-axis: time in days; in ordinate, mouse survival, expressed in percentage. Curve 1: mouse survival for the group immunized with L. lactis which does not secrete any LcrV; curve 2: mouse survival for the group immunized with L. lactis secreting the Y. pseudotuberculosis strain 2777 LcrV; curve 3: mouse survival for the group immunized with L. lactis secreting the Y. pestis strain 195/P LcrV in which the conformational segment ("CS") had been exchanged by the CS of Y. pseudotuberculosis strain 32777 (i.e. YpCS YPst); curve 4: mouse survival for the group immunized with L. lactis secreting the Y. pseudotuberculosis strain 32777 LcrV in which the conformational segment ("CS") had been exchanged by the CS of Y. pestis strain 195/P (i.e. YpstCS Yp); curve 5: mouse survival for the group immunized with L. lactis secreting the Y. pseudotuberculosis strain 32777 LcrV wherein the amino acid residue [AA] was replaced by the [AA] of Y. pestis strain 195/P (i.e. YpstD255N).
Figure 4 shows a graph illustrating the mouse survival for groups of mice previously immunized intranasally (or not) with L. lactis secreting the Y. pseudotuberculosis strain 2777 LcrV and then challenged with inoculation with the Y. pseudotuberculosis 2777, 2889 and AH strains. In X-axis: time in days; in ordinate, mouse survival, expressed in percentage. Curve 1: mouse survival for the group inoculated with Y. pseudotuberculosis strain 2777; curve 2: mouse survival for the group inoculated with Y. pseudotuberculosis strain 2889; curve 3: mouse survival for the group inoculated with Y. pseudotuberculosis strain AH; the entire curves (ie entitled "...a)" relates to a group which have been previously immunized; the dotted curves (ie entitled "...b)" relates to a group which have not been previously immunized.
Figure 5 shows a graph illustrating the mouse survival for groups of mice previously immunized intranasally with L. lactis secreting the Y. pestis strain 195 LcrV and then challenged intravenously with a lethal dose of Y. pseudotuberculosis strain 2843 (Clade C) or Y. pseudotuberculosis strain 2889 (Clade D2). In X-axis: time in days; in ordinate, mouse survival, expressed in percentage. Curve 1: mouse survival for the group inoculated with Y. pseudotuberculosis strain 2843; curve 2: mouse survival for the group inoculated with Y. pseudotuberculosis strain 2889; the entire curves (ie entitled "...a)" relates to a group which have been previously immunized; the dotted curves (ie entitled "...b)" relates to a group which have not been previously immunized.
Figure 6A shows a graph of the mice's anti-V IgG titers of groups of mice previously immunized intranasally with L. lactis secreting (or not) the Y. pestis strain 195 LcrV or the Y. pseudotuberculosis strain 2777 LcrV and then challenged intravenously with a lethal dose of pseudotuberculosis strain 2777. In X-axis: each of the considered groups of mice (group 1: mice immunized with L. lactis secreting Y. pst strain 2777 LcrV; group 2: mice immunized with L. lactis secreting Y. p strain 195 LcrV; group 3: mice immunized with L. lactis which does not secrete any LcrV); in ordinate, anti-V IgG titers amount.
Figure 6B shows a graph of the mice's anti-V IgG titers of groups of mice previously immunized intranasally with L. lactis secreting (or not) the plurality of Yersinia LcrVs displayed in above figure 2, and then challenged intravenously with a lethal dose of pseudotuberculosis strain 2777. In X-axis: each of the considered groups of mice (group 1: mice immunized with L. lactis secreting Y. pst strain 2777 LcrV; group 2: mice immunized with L. lactis secreting Y. pst strain 32777 LcrV wherein the amino acid residue [AA] was replaced by the [AA] of Y. pestis strain 195/P (i.e. YpstD255N); group 3: mice immunized with L. lactis secreting Y. pst strain 32777 LcrV in which the conformational segment ("CS") had been exchanged by the CS of Y. pestis strain 195/P (i.e. YpstCS Yp); group 4: mice immunized with L. lactis secreting Y. pestis strain 195/P LcrV in which the conformational segment ("CS") had been exchanged by the CS of Y. pst strain 32777 (i.e. YpCS YPst); group 5: mice immunized with L. lactis which does not secrete any LcrV) ; in ordinate, anti-V IgG titers amount.
Figure 6C shows a graph of the mice's anti-V IgG titers of groups of mice previously immunized with L. lactis secreting from Ypst 2777. In X-axis: each of the considered groups of mice (group 1: mice immunized with L. lactis secreting Y. pst strain 2777 LcrV; group 2: mice immunized with L. lactis which does not secrete any LcrV) ; in ordinate, anti-V IgG titers amount.
Figure 7 shows a phylogenetic table of several strains of Yersinia pseudotuberculosis , Yersinia pestis and Yersinia enterocolitica; this phylogenetic table has been established in considering the "conformational segment" (ie position 218 to 234 with respect to the LcrV sequence of Y pst 32777) and the amino acid residue [AA] of amino acids sequences of Lcrv respective to the considered strains. Figure 8 illustrates an alignment of a plurality of amino acid sequences of LcrV antigens mainly based on the conformational segment and the amino acid residue [AA]; this alignment displays all clades of Yersinia herein considered.
Figure 9 shows a graph illustrating in A: the mouse survival (%) for the group inoculated with Y. pseudotuberculosis strain 2777 (clade B - white square) and with Y. pseudotuberculosis strain 2790 (clade D - black triangle). In B: the mouse survival (%) for the group inoculated with Y. pseudotuberculosis strain 2781 (clade B - grey square). In X-axis: time in days; in ordinate, mouse survival, expressed in percentage, the entire curves (ie entitled "...a)" relates to a group which have been previously vaccinated with V2777; the dotted curves (ie entitled "...b)" relates to a group which have not been previously vaccinated.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
The terms LcrV or V antigen are used interchangeably in the present description and refer to the protein found in bacterial species of the Yersinia genus, and notably Yersinia pestis. It forms part of the Yersinia virulence protein factors that also includes all Yop's, this used to stand for Yersinia outer protein, but the name has been kept out of convention.
The term "microorganism" or "bacteria", as used herein, refers to a bacterium which is not modified artificially. The bacteria of the invention may be chosen among bacteria which expresses genes for expressing heterologous Yersinia LcrV recombinant antigens. The bacteria of the invention may be also chosen among bacteria which already expresses genes for expressing an endogenous Yersinia LcrV antigen.
The term "recombinant bacteria" or "genetically modified bacteria", as used herein, refers to a bacteria genetically modified or genetically engineered. It means, according to the usual meaning of these terms, that the bacteria is not found in nature and is modified either by introduction or by deletion or by modification of genetic elements from equivalent bacteria found in nature. It can also be modified by forcing the development and evolution of new metabolic pathways by combining directed mutagenesis and evolution under specific selection pressure (see for instance WO 2004/076659).
A bacteria may be modified to express exogenous genes if these genes are introduced into the bacteria with all the elements allowing their expression in the host bacteria. A bacteria may be modified to modulate the expression level of an endogenous gene. The modification or "transformation" of bacteria with exogenous DNA is a routine task for those skilled in the art.
The term "endogenous gene" means that the gene was present in the bacteria before any genetic modification, in the wild-type strain. Endogenous genes may be overexpressed by introducing heterologous sequences in addition to, or to replace endogenous regulatory elements, or by introducing one or more supplementary copies of the gene into the chromosome or a plasmid. Endogenous genes may also be modified to modulate their expression and/or activity. For example, mutations may be introduced into the coding sequence to modify the gene product or heterologous sequences may be introduced in addition to or to replace endogenous regulatory elements. Modulation of an endogenous gene may result in the up-regulation and/or enhancement of the activity of the gene product, or alternatively, in the down-regulation and/or attenuation of the activity of the endogenous gene product. Another way to enhance expression of endogenous genes is to introduce one or more supplementary copies of the gene onto the chromosome or a plasmid.
The term "exogenous gene" means that the gene was introduced into a bacteria, by means well known by the man skilled in the art, whereas this gene is not naturally occurring in the wild-type microorganism, bacteria can express exogenous genes if these genes are introduced into the microorganism with all the elements allowing their expression in the host bacteria. Transforming microorganisms with exogenous DNA is a routine task for the man skilled in the art. Exogenous genes may be integrated into the host chromosome, or be expressed extra- chromosomally from plasmids or vectors. A variety of plasmids, which differ with respect to their origin of replication and their copy number in the cell, are all known in the art. The sequence of exogenous genes may be adapted for its expression in the host bacteria. Indeed, the man skilled in the art knows the notion of codon usage bias and how to adapt nucleic sequences for a particular codon usage bias without modifying the deduced protein.
The term "heterologous gene" means that the gene is derived from a species of organism, or at least from a strain of organism of a same species, different from the recipient bacteria that expresses it. It refers to a gene which is not naturally occurring in the bacteria.
The term "heterologous antigen" means that the antigen is derived from a species of organism, or at least from a strain of organism of a same species, different from the recipient bacteria that expresses it. It refers to a antigen which is not naturally occurring in the bacteria.
In bacteria, nucleic acid expression construct preferably comprises regulatory sequences, such as promoter and terminator sequences, which are operatively linked with the nucleic acid sequence coding for each of the LcrV antigens according to the present invention.
The nucleic acid expression construct may further comprise 5' and/or 3' recognition sequences and/or selection markers.
The term "overexpression" means that the expression of a gene is increased as compared to the non-modified microorganism. Increasing the expression of a protein is obtained by increasing the expression of a gene encoding said protein. Increasing the expression of a gene may be carried out by all techniques known by the one skilled in the art. In this regard, it may be notably cited the implementation of a strong promoter upstream the nucleic acid intended to be overexpressed or the introduction of several copies of the said nucleic acid between a promoter, especially a strong promoter, and a terminator.
The "activity" of a protein is used interchangeably with the term "function" and designates, in the context of the invention, the capacity of a LcrV antigen or of a LcrV-derived antigen to raise an immune response against at least one Yersinia species selected from the group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica in a vertebrate, notably of the Mammalia and/or of the aves class(es), when appropriately administered.
The "antigenicity" of a protein, in the context of the invention, means the ability of a LcrV antigen or of a LcrV-derived antigen to raise an immune response against at least one Yersinia species selected from the group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica in a vertebrate, notably of the Mammalia and/or of the aves class(es), when appropriately administered.
The "LcrV" and "LcrV-derived antigen" may be used interchangeably in the context of the invention. A LcrV-derived antigen according to the present invention more particularly relates to a fragment of a LcrV antigen from at least one Yersinia species selected from the group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, said LcrV- derived antigen being such that it keeps a same or at least a similar ability to induce an immune response in the considered vertebrate, notably of the Mammalia and/or of the aves class(es), when appropriately administered.
The terms "encoding" or "coding" refer to the process by which a polynucleotide, through the mechanisms of transcription and translation, produces an amino-acid sequence.
The gene(s) encoding the protein(s) considered in the present invention can be exogenous or endogenous according to the nature of the considered bacteria.
For each of the nucleic acid sequences comprising a coding sequence (e.g. a sequence coding for a LcrV antigen according to the invention), reference sequences are described herein. The present description also encompasses nucleic acid sequences having specific percentages of nucleic acid identity, with a reference nucleic acid sequence.
For each or the amino acid sequences of interest, reference sequences are described herein. The present description also encompasses amino acid sequences (e.g. enzyme amino acid sequences), having specific percentages of amino acid identity, with a reference amino acid sequence. For obvious reasons, in all the present description, a specific nucleic acid sequence or a specific amino acid sequence which complies with, respectively, the considered nucleotide or amino acid identity, should further lead to obtaining a protein (or enzyme) which displays the desired biological activity. As used herein, the "percentage of identity" between two nucleic acid sequences or between two amino acid sequences is determined by comparing both optimally aligned sequences through a comparison window.
The portion of the nucleotide or amino-acid sequence in the comparison window may thus include additions or deletions (for example "gaps") as compared to the reference sequence (which does not include these additions or these deletions) so as to obtain an optimal alignment between both sequences.
The identity percentage is calculated by determining the number of positions at which an identical nucleic base, or an identical amino-acid residue, can be noted for both compared sequences, then by dividing the number of positions at which identity can be observed between both nucleic bases, or between both amino-acid residues, by the total number of positions in the comparison window, then by multiplying the result by hundred to obtain the percentage of nucleotide identity between the two sequences or the percentage of amino acid identity between the two sequences.
The comparison of the sequence optimal alignment may be effected by a computer using known algorithms.
Most preferably, the sequence identity percentage is determined using the CLUSTAL W software (version 1.82) the parameters being set as follows: (1) CPU MODE=ClustalW mp; (2) ALIGNMENT="full"; (3) OUTPUT FORMAT="aln w/numbers"; (4) OUTPUT ORDER="aligned"; (5) COLOR ALIGNMENT="no"; (6) KTUP (word size)="default"; (7) WINDOW LENGTH- 'default"; (8) SCORE TYPE="percent"; (9) TOPDIAG="default"; (10) PAIRGAP="default"; (11) PHYLOGENETIC TREE/TREE TYPE="none"; (12) MATRIX="default"; (13) GAP OPEN="default"; (14) END GAPS="default"; (15) GAP EXTENSION="default"; (16) GAP DISTANCES="default"; (17) TREE TYPE="cladogram" and (18) TREE GRAP DISTANCES- 'hide".
As described herein, an amino acid sequence having at least 80% amino acid identity with a reference amino acid sequence encompasses amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity with the said reference amino acid sequence.
As described herein, a nucleic acid sequence having at least 80%) nucleotide identity with a reference nucleic acid sequence encompasses nucleic acid sequences having at least 81%), 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% nucleotide identity with the said reference nucleic acid sequence.
MULTIVALENT IMMUNOGENIC COMPOSITION
As above-mentioned, the present invention relates to a multivalent immunogenic composition for inducing an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enter ocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising :
- at least a first Yersinia LcrV antigen inducing an immune response against Yersinia pestis;
- at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, inducing an immune response against Yersinia pseudotuberculosis; and
- at least a third Yersinia LcrV antigen, different from the first and second Yersinia LcrV antigens, inducing an immune response against Yersinia enter ocolitica.
Accordingly, the present invention relates to a multivalent immunogenic composition for inducing an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group consisting of: Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group consisting of :
- at least a first Yersinia LcrV antigen inducing an immune response against Yersinia pestis;
- at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, inducing an immune response against Yersinia pseudotuberculosis; and
- at least a third Yersinia LcrV antigen, different from the first and second Yersinia LcrV antigens, inducing an immune response against Yersinia enterocolitica.
In the sense of the invention, combinations of any of the recited Yersinia LcrV antigens as defined above are considered. Accordingly, at least two Yersinia LcrV antigens, one distinct from each other, may be selected in a group consisting of:
(i) at least a first Yersinia LcrV antigen inducing an immune response against Yersinia pestis; and at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, inducing an immune response against Yersinia pseudotuberculosis; or (ii) at least a first Yersinia LcrV antigen inducing an immune response against Yersinia pestis; and at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, inducing an immune response against Yersinia enterocolitica; or
(iii) at least a first Yersinia LcrV antigen inducing an immune response against Yersinia pseudotuberculosis; and at least a second Yersinia LcrV antigen, different from the first Yersinia
LcrV antigen, inducing an immune response against Yersinia enterocolitica; or
(iv) at least a first Yersinia LcrV antigen inducing an immune response against Yersinia pestis and Yersinia pseudotuberculosis; and at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, inducing an immune response against Yersinia enterocolitica
Accordingly, at least three Yersinia LcrV antigens, one distinct from each other, may be selected in a group consisting of:
- at least a first Yersinia LcrV antigen inducing an immune response against Yersinia pestis;
- at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, inducing an immune response against Yersinia pseudotuberculosis; and
- at least a third Yersinia LcrV antigen, different from the first and second Yersinia LcrV antigens, inducing an immune response against Yersinia enterocolitica.
Particular LcrV antigens which are suitable for the invention are further detailed here after. As shown in the examples herein, the inventors identified the residues composing the major protective epitopes in V antigen, and an assay other than that based on anti-V titer measurement and bacterial-mediated toxicity against macrophages to measure vaccine efficacy.
Indeed, results of previous vaccination studies using purified recombinant V antigen produced in E. coli suggested that protection against all types of yersiniosis does not depend on the type, subtype or variant of V antigen used for vaccination (Schmidt A et ah, Microbial pathogenesis 1999; 26:221-33; Hill J et ah, Vaccine 2009; 27:7073-9). The present invention is particularly interesting in that it does not support the latter conclusion, as shown in the hereinafter examples.
According to certain embodiments, the multivalent immunogenic composition according to the invention comprises or expresses at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising (or consisting of) :
- the first Yersinia LcrV antigen is represented by the following formula (I):
NH2-[SEQ ID N°A-1]-[CS-1]-[SEQ ID N°B-1]-[AA-1]-[SEQ ID N°C-l]-COOH (I),
- the second Yersinia LcrV antigen is represented by the following formula (II): NH2-[SEQ ID N°A-2]-[CS-2]-[SEQ ID N°B-2]-[AA-2]-[SEQ ID N°C-2]-COOH (II),
- the third Yersinia LcrV antigen is represented by the following formula (III):
NH2-[SEQ ID N°A-3]-[CS-3]-[SEQ ID N°B-3]-[AA-3]-[SEQ ID N°C-3]-COOH (III), wherein:
- SEQ ID N°A-1, SEQ ID N°A-2 and SEQ ID N°A-3, one independently from the others, mean an amino acid sequence having at least 80% amino acid identity, preferably at least 90 % amino acid identity, with the sequence SEQ ID N°2 ,
- CS-1 means an amino acid sequence of sequence SEQ ID N°8,
- CS-2 means an amino acid sequence selected from the group comprising the sequences SEQ ID N°8, 10, 11, 12, 13, 107, 108, 109, 110, 111, 112, 113, 114 and 115,
- CS-3 means an amino acid sequence selected from the group comprising the sequences SEQ ID N°14, 15, 16, 17 and 18,
- SEQ ID N°B-1, SEQ ID N°B-2 and SEQ ID N°B-3, one independently from the others, mean an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %> amino acid identity, with the sequence SEQ ID N°3,
- AA-1, AA-2 and AA-3, one independently from the others, mean an amino acid selected from the group consisting of aspartic acid (or aspartate) and asparagine, preferably AA-1 represents asparagine, AA-2 represents aspartic acid and AA-3 represents aspartic acid, and
- SEQ ID N°C-1, SEQ ID N°C-2 and SEQ ID N°C-3, one independently from the others, mean an amino acid sequence having at least 80%) amino acid identity, preferably at least 90 %> amino acid identity, with the sequence SEQ ID N°4.
According to a particular embodiment, the composition according to the invention, when the first or the second Yersinia LcrV antigen induces an immune response against Yersinia pestis and Yersinia pseudotuberculosis, then said immunogenic composition:
- is devoid of the second or the first Yersinia LcrV antigen; or
- further comprises or expresses at least one additional Yersinia LcrV antigen(s) inducing an immune response against Yersinia pestis or Yersinia pseudotuberculosis, different from whichever of the first or the second Yersinia LcrV antigen is already present.
This particular embodiment is notably met when the Yersinia LcrV antigen inducing an immune response against Yersinia pseudotuberculosis belongs to the hereinafter Clade C (see notably the figure 8). According to this particular embodiment, the composition according to the invention may comprise or express a Yersinia LcrV antigen inducing an immune response against Yersinia pestis and Yersinia pseudotuberculosis which is represented by the following formula (IV):
NH2-[SEQ ID N°A-4]-[CS-4]-[SEQ ID N°B-4]-[AA-4]-[SEQ ID N°C-4]-COOH (IV), and wherein:
- SEQ ID N°A-4 means an amino acid sequence having at least 80% amino acid identity, preferably at least 90 % amino acid identity, with the sequence SEQ ID N°2,
- CS-4 means an amino acid sequence of sequence SEQ ID N° 8,
- SEQ ID N°B-4 means an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %> amino acid identity, with the sequence SEQ ID N°3,
- AA-4 represents asparagine, and
- SEQ ID N°C-4 means an amino acid sequence having at least 80%) amino acid identity, preferably at least 90 %> amino acid identity, with the sequence SEQ ID N°4.
According to certain embodiments, the second Yersinia LcrV antigen(s), in a composition according to the invention, may have the following formula (II):
NH2-[SEQ ID N°A-2]-[CS-2]-[SEQ ID N°B-2]-[AA-2]-[SEQ ID N°C-2]-COOH (II), wherein the [CS-2] amino acid sequence and the [AA-2] amino acid are selected from the group comprising:
- CS-2 means an amino acid sequence selected from a group comprising sequences SEQ ID N° 107, 108, 109, 110, 111, 112, 113, 114 and 115, and AA-2 represents aspartic acid,
- CS-2 means an amino acid sequence of sequence SEQ ID N° 10 and AA-2 represents aspartic acid (i.e. strain NT);
- CS-2 means an amino acid sequence of sequence SEQ ID N° 11 and AA-2 represents aspartic acid (i.e. clades Bl and B2);
- CS-2 means an amino acid of sequence SEQ ID N° 12 and AA-2 represents aspartic acid (i.e. clade Dl);
- CS-2 means an amino acid sequence of sequence SEQ ID N° 13 and AA-2 represents aspartic acid (i.e. clade D2);
- CS-2 means an amino acid sequence of sequence SEQ ID N° 8 and AA-2 represents asparagine (i.e. clade C); and
- the combinations thereof.
According to certain embodiments, the third Yersinia LcrV antigen(s) in a composition according to the invention may have the following formula (III):
NH2-[SEQ ID N°A-3]-[CS-3]-[SEQ ID N°B-3]-[AA-3]-[SEQ ID N°C-3]-COOH (III), wherein the [CS-3] amino acid sequence and the [AA-3] amino acid are selected from the group comprising:
- CS-3 means an amino acid sequence of sequence SEQ ID N° 14 and AA-3 represents aspartic acid (i.e. clade F); - CS-3 means an amino acid sequence of sequence SEQ ID N° 15 and AA-3 represents aspartic acid (i.e. clade G);
- CS-3 means an amino acid sequence of sequence SEQ ID N° 16 and AA-3 represents asparagine (i.e. strain 8081);
- CS-3 means an amino acid sequence of sequence SEQ ID N° 17 and AA-3 represents aspartic acid (i.e. clade H);
- CS-3 means an amino acid sequence of sequence SEQ ID N° 18 and AA-3 represents aspartic acid (i.e. strain E40); and
- the combinations thereof.
Advantageously, the sequences SEQ ID N°B-1, SEQ ID N°B-2, SEQ ID N°B-3 and
SEQ ID N°B-4, one independently from the others, mean an amino acid sequence SEQ ID N°5, wherein:
- X2 means an amino acid selected from the group comprising aspartic acid, glycine and asparagine,
- X3 means an amino acid selected from the group comprising glycine and glutamic acid, and
- X4 means an amino acid selected from the group comprising asparagine and lysine, and preferably mean:
- SEQ ID N°B-1 and SEQ ID N°B-2 mean an amino acid sequence having at least 90% amino acid identity with the amino acid sequence SEQ ID N°6;
- SEQ ID N°B-3 means an amino acid sequence having at least 90%) amino acid identity with the amino acid sequence SEQ ID N°7; and
- SEQ ID N°B-4 means an amino acid sequence having at least 90%> amino acid identity with the amino acid sequence SEQ ID N°23.
Advantageously, the sequences SEQ ID N°A-1, SEQ ID N°A-2, SEQ ID N°A-3 and
SEQ ID N°A-4, one independently from the others, may be represented by an amino acid sequence of the following formula (V):
NH2-[SEQ ID N°Aa]-[SEQ ID N°Ab]- (V)
wherein:
- SEQ ID N°Aa, one independently from the other, mean an amino acid sequence having at least 80%) amino acid identity, preferably at least 90 %>, with the sequence SEQ ID N°19, and
- SEQ ID N°Ab, one independently from the other, means an amino acid sequence SEQ ID N°20, wherein X5 means an amino acid selected from the group comprising glutamic acid and lysine. Advantageously, the sequences SEQ ID N°C-1, SEQ ID N°C-2, SEQ ID N°C-3 and SEQ ID N°C-4, one independently from the others, may be represented by an amino acid sequence of SEQ ID N°22 or SEQ ID N°21 , wherein Χβ means an amino acid selected from the group comprising threonine and alanine, and preferably of SEQ ID N°21.
Advantageously, the composition according to the invention may comprise or express:
- at least one Yersinia LcrV antigen inducing an immune response against Yersinia pestis species,
- at least one, preferably at least two, preferably at least three, in particular at least four, more particularly at least five, preferably at least six, and even at least seven Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis species, one independently from the other, and notably selected among the group such as above-defined, and/or
- at least one, preferably at least two, preferably at least three, in particular at least four, and more particularly at least five, Yersinia LcrV antigen(s) inducing an immune response against
Yersinia enterocolitica species, one independently from the other, notably selected among the group such as above-defined.
Advantageously, the composition according to the invention may be such that:
- the first Yersinia LcrV antigen(s) inducing an immune response against Yersinia pestis is/are selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80% amino acid identity, preferably at least 90%) amino acid identity, and still better of sequences SEQ ID N°74 and 75;
- the second Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis is/are selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80%) amino acid identity, preferably at least 90%o amino acid identity, and still better of sequences SEQ ID N°25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 72, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, and combination thereof ;
- the third Yersinia LcrV antigen(s) inducing an immune response against Yersinia enterocolitica is/are selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80%o amino acid identity, preferably at least 90%o amino acid identity, and still better of sequences SEQ ID N°45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 99 and 100, and combination thereof; and
- the combinations thereof. Herein after are presented some illustrative embodiments of a composition according to the present invention.
First illustrative embodiment
This embodiment relates to a multivalent immunogenic composition according to the invention which comprises or expresses:
- a first Yersinia LcrV antigen represented by the following formula (I):
NH2-[SEQ ID N°A-1]-[CS-1]-[SEQ ID N°B-1]-[AA-1]-[SEQ ID N°C-l]-COOH (I),
- a second Yersinia LcrV antigen represented by the following formula (II):
NH2-[SEQ ID N°A-2]-[CS-2]-[SEQ ID N°B-2]-[AA-2]-[SEQ ID N°C-2]-COOH (II), and
- a third Yersinia LcrV antigen represented by the following formula (III):
NH2-[SEQ ID N°A-3]-[CS-3]-[SEQ ID N°B-3]-[AA-3]-[SEQ ID N°C-3]-COOH (III).
Regarding the formula (I):
- SEQ ID N°A-1 represents an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %, with the sequence SEQ ID N°2 ,- CS-1 represents an amino acid sequence of sequence SEQ ID N°8,
- SEQ ID N°B-1 represents an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %, with the sequence SEQ ID N°3,
- AA- 1 represents an asparagine, and
- SEQ ID N°C-1 represents an amino acid sequence having at least 80%) amino acid identity, preferably at least 90 %>, with the sequence SEQ ID N°4.
More particularly, the sequence represented by the above formula (I) is defined by the amino acid sequence SEQ ID N° 75 or 76.
Regarding the formula (II):
- SEQ ID N°A-2 represents an amino acid sequence having at least 80%> amino acid identity, preferably at least 90 %>, with the sequence SEQ ID N°2,
- CS-2 represents an amino acid sequence of sequence SEQ ID N°l 1,
- SEQ ID N°B-2 represents an amino acid sequence having at least 80%> amino acid identity, preferably at least 90 %>, with the sequence SEQ ID N°3,
- AA-2 represents an aspartic acid, and
- SEQ ID N°C-2 represents an amino acid sequence having at least 80%> amino acid identity, preferably at least 90 %, with the sequence SEQ ID N°4. More particularly, the sequence represented by the above formula (II) is defined by the amino acid sequences selected from the group comprising SEQ ID N° 86.
Regarding the formula (III):
- SEQ ID N°A-3 represents an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %, with the sequence SEQ ID N°2,
- CS-3 represents an amino acid sequence of sequence SEQ ID N°14,
- SEQ ID N°B-3 represents an amino acid sequence having at least 80%) amino acid identity, preferably at least 90%>, with the sequence SEQ ID N°3,
- AA-3 represents an aspartic acid, and
- SEQ ID N°C-3 represents an amino acid sequence having at least 80%> amino acid identity, preferably at least 90%>, with the sequence SEQ ID N°4.
More particularly, the sequence represented by the above formula (III) is defined by the amino acid sequence SEQ ID N° 47.
More particularly, this first illustrative embodiment relates to a multivalent immunogenic composition according to the invention which comprises or expresses:
- a first Yersinia LcrV antigen represented by the sequence SEQ ID N° 75,
- a second Yersinia LcrV antigen represented by the sequence SEQ ID N° 86, and
- a third Yersinia LcrV antigen represented by the sequence SEQ ID N° 47.
Second illustrative embodiment
This embodiment relates to a multivalent immunogenic composition wherein the first or the second Yersinia LcrV antigen induces an immune response against Yersinia pestis and Yersinia pseudotuberculosis .
According to this particular embodiment, said immunogenic composition comprises or expresses:
- a Yersinia LcrV antigen inducing an immune response against Yersinia pestis and Yersinia pseudotuberculosis represented by the following formula (IV):
NH2-[SEQ ID N°A-4]-[CS-4]-[SEQ ID N°B-4]-[AA-4]-[SEQ ID N°C-4]-COOH (IV), and - a third Yersinia LcrV antigen represented by the following formula (III):
NH2-[SEQ ID N°A-3]-[CS-3]-[SEQ ID N°B-3]-[AA-3]-[SEQ ID N°C-3]-COOH (III).
Regarding the formula (IV):
- SEQ ID N°A-4 represents an amino acid sequence having at least 80%> amino acid identity, preferably at least 90 %>, with the sequence SEQ ID N°2,
- CS-4 represents an amino acid sequence of sequence SEQ ID N°8, - SEQ ID N°B-4 represents an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %, with the sequence SEQ ID N°3,
- AA-4 represents an asparagine, and
- SEQ ID N°C-4 represents an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %, with the sequence SEQ ID N°4.
More particularly, the sequence represented by the above formula (IV) is defined by the amino acid sequence SEQ ID N° 75.
Regarding the formula (III):
- SEQ ID N°A-3 represents an amino acid sequence having at least 80%) amino acid identity, preferably at least 90 %>, with the sequence SEQ ID N°2,
- CS-3 represents an amino acid sequence of sequence SEQ ID N°14,
- SEQ ID N°B-3 represents an amino acid sequence having at least 80%> amino acid identity, preferably at least 90%>, with the sequence SEQ ID N°3,
- AA-3 represents an aspartic acid, and
- SEQ ID N°C-3 represents an amino acid sequence having at least 80%> amino acid identity, preferably at least 90%>, with the sequence SEQ ID N°4.
More particularly, the sequence represented by the above formula (III) is defined by the amino acid sequence SEQ ID N° 47.
More particularly, this second illustrative embodiment relates to a multivalent immunogenic composition according to the invention which comprises or expresses:
- a first Yersinia LcrV antigen represented by the sequence SEQ ID N° 75, and
- a second Yersinia LcrV antigen represented by the sequence SEQ ID N° 47.
Advantageously, each above-mentioned Yersinia LcrV antigens may be independently selected from the group consisting of:
- a purified antigen,
- a live attenuated strain comprising and/or expressing the said Yersinia LcrV antigen, - an inactivated or a killed strain comprising and/or expressing the said Yersinia LcrV antigen, or
- a recombinant bacteria comprising and/or expressing the said Yersinia LcrV antigen.
In a composition according to the invention, the different considered Yersinia LcrV antigens may be independently selected from the group consisting of:
- a purified antigen, - a live attenuated strain comprising and/or expressing the said Yersinia LcrV antigen,
- an inactivated or a killed strain comprising and/or expressing the said Yersinia LcrV antigen, or
- a recombinant bacteria comprising and/or expressing the said Yersinia LcrV antigen, or a combination thereof.
In a first variant of realization, a recombinant bacteria implemented in a composition of the invention may comprise and/or express only one specific Yersinia LcrV antigen, such as herein defined.
According to this first variant, a composition of the invention may thus comprise as many different recombinant bacteria as desired/required LcrV antigens.
In a second variant of realization, a recombinant bacteria implemented in a composition of the invention may comprise and/or express at least two different desired/required LcrV antigens, such as herein defined.
According to this second variant, a composition of the invention may thus comprise only one type of recombinant bacteria or at least two different recombinant bacteria.
A composition according to the invention may notably comprise and/or express Yersinia LcrV antigens of different strains of a same Yersinia species selected from a group comprising Y. pestis, Y. pseudotuberculosis and Y. enterocolitica are herein described.
Alternatively, each above-mentioned Yersinia LcrV antigens may be present in a composition according to the invention in the form of a purified antigen.
According to a particular embodiment, a composition according to the invention may further comprise at least a pharmaceutically acceptable carrier, a diluent and/or an immuno- adjuvant.
Suitable adjuvants include, but are not limited to, aluminium salts (aluminium phosphate or aluminium hydroxide), squalene mixtures (SAF-1), muramyl peptide, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, non-ionic block copolymer surfactants, Quil A, cholera toxin B subunit, polphosphazene and derivatives, and immunostimulating complexes (ISCOMs) such as those described by Takahashi et al. (1990) Nature 344:873-875.
According to another particular embodiment, a composition according to the invention may further comprise at least one additional active agent against Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, preferably said additional active agent being selected in a group comprising Mab 7.3, sulfonamides, fluoroquinolones such as ciprofloxacine, levofloxacin, streptomycin, gentamicin, tetracyclines, doxycycline, Oxytetracycline, ampicillin, cephalosporins, aminoglycosides, chloramphenicol, trimethroprime-sulfamethoxazole, amoxicilline/clavulanic acid, cefotaxime, cotrimoxazole, imipeneme, aztreonam, latamoxef and mixtures thereof
According to a particular embodiment, a composition according to the invention may be buffered at, or adjusted to, between pH 7.0 and 8.0, pH 7.2 and 7.6 or around or exactly pH 7.4.
According to a particular embodiment, a composition according to the invention may be lyophilised, optionally in presence of a stabilizing agent, for example a polyol such as sucrose or trehalose.
According to a particular embodiment, a composition according to the invention may be encapsulated. For illustrative purposes, an encapsulation within liposomes is described by Fullerton, U.S. Pat. No. 4,235,877.
As with all immunogenic compositions or vaccines, the immunologically effective amounts of the immunogens must be determined empirically. Factors to be considered include the immunogenicity, whether or not the immunogen will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier, route of administrations and the number of immunising dosages to be administered. Such factors are known in the vaccine art and it is well within the skill of immunologists to make such determinations without undue experimentation.
The Yersinia LcrV antigens can be present in varying concentrations in a composition of the invention. Typically, the minimum concentration of the substance is an amount necessary to achieve its intended use, while the maximum concentration is the maximum amount that will remain in solution or homogeneously suspended within the initial mixture. For instance, the minimum amount of a therapeutic agent is optionally one which will provide a single therapeutically effective dosage. For bioactive substances, the minimum concentration is an amount necessary for bioactivity upon reconstitution and the maximum concentration is at the point at which a homogeneous suspension cannot be maintained. In the case of single-dosed units, the amount is that of a single therapeutic application. Generally, it is expected that each dose will comprise 1-100 μg of Yersinia LcrV antigens, optionally 5-50 μg or 5-25 μg.
For obvious reasons, the concentration in bioactive substances should not be harmful for the considered vertebrate wherein said bioactive substances are administered.
According to a particular embodiment, the present invention also encompasses a multivalent immunogenic composition for inducing an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia strains of a same Yersinia species selected in a group comprising (or consisting of) Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica; said immunogenic composition comprising (or consisting of) or expressing at least two Yersinia LcrV antigens wherein: - the first Yersinia LcrV antigen induces an immune response against a first Yersinia strain selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enter ocolitica, and
- the second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, induces an immune response against a second Yersinia strain, different from the first Yersinia strain, but selected in the same Yersinia species than the first Yersinia LcrV antigen.
A composition according to the invention may be administered in considering all the conventional route of administration in the field of immunogenic compositions or vaccines, including notably:
- the topical route,
- the enteral route, including the oral, the nasal and the rectal route,
- the inhalation or pulmonary route, or
- the parenteral route, including intravenous, intra-arterial, intraosseous infusion, intramuscular, intracerebral, intracerebroventricular, intrathecal and subcutaneous route .
Preferably, a composition according to the invention is administered by topical route, by oral route or by nasal route.
Preferably, a composition according to the invention induces an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enter ocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising :
- at least one Yersinia LcrV antigen inducing an immune response against Yersinia pestis species, preferably said Yersinia LcrV antigen being represented by the above-mentioned formula
(I) wherein [CS-1] means the amino acid sequence SEQ ID N° 8 and [AA-1] amino acid is asparagine,
- at least one, preferably at least two, preferably at least three, in particular at least four, more particularly at least five, preferably at least six, and even at least seven, Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis species, one independently from the other, preferably said Yersinia LcrV antigen(s) being represented by the above-mentioned formula (II) wherein the [CS-2] amino acid sequence and the [AA-2] amino acid is/are selected from the group comprising (or consisting of): - CS-2 means an amino acid sequence selected from a group comprising sequences SEQ ID N° 107, 108, 109, 110, 11 1, 1 12, 1 13, 1 14 and 115 and AA-2 represents aspartic acid, x representing an integer ranging from 0 to 8 (i.e. clade A);
- CS-2 means an amino acid of sequence SEQ ID N° 10 and AA-2 represents aspartic acid (i.e. strain NT);
- CS-2 means an amino acid of sequence SEQ ID N° 11 and AA-2 represents aspartic acid (i.e. clades Bl and B2);
- CS-2 means an amino acid of sequence SEQ ID N° 12 and AA-2 represents aspartic acid (i.e. clade Dl);
- CS-2 means an amino acid of sequence SEQ ID N° 13 and AA-2 represents aspartic acid (i.e. clade D2);
- CS-2 means an amino acid of sequence SEQ ID N° 8 and AA-2 represents asparagine (i.e. clade C); and
- a combination thereof,
- at least one, preferably at least two, preferably at least three, in particular at least four, and more particularly at least five, Yersinia LcrV antigen(s) inducing an immune response against Yersinia enterocolitica species, one independently from the other, preferably said Yersinia LcrV antigen(s) being represented by the above-mentioned formula (III) wherein the [CS-3] amino acid sequence and the [AA-3] amino acid is/are selected from the group comprising (or consisting of):
- CS-3 means an amino acid of sequence SEQ ID N° 14 and AA-3 represents aspartic acid (i.e. clade F);
- CS-3 means an amino acid of sequence SEQ ID N° 15 and AA-3 represents aspartic acid (i.e. clade G);
- CS-3 means an amino acid of sequence SEQ ID N° 16 and AA-3 represents asparagine (i.e. strain 8081);
- CS-3 means an amino acid of sequence SEQ ID N° 17 and AA-3 represents aspartic acid (i.e. clade H);
- CS-3 means an amino acid of sequence SEQ ID N° 18 and AA-3 represents aspartic acid (i.e. strain E40); and
- a combination thereof.
According to a preferably embodiment, a composition according to the invention induces an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising (or consisting of) Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising (or consisting of) :
- at least one Yersinia LcrV antigen of sequence SEQ ID N°74 or 75 (= LcrV 135-275 de
Y.p.);
- at least one, preferably at least two, preferably at least three, in particular at least four, more particularly at least five, preferably at least six, and even at least seven, Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis species, one independently from the other, said Yersinia LcrV antigen(s) being selected from the group comprising sequence SEQ ID N°25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 72, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and combination thereof;
- at least one, preferably at least two, preferably at least three, in particular at least four, and more particularly at least five, Yersinia LcrV antigen(s) inducing an immune response against Yersinia enterocolitica species, one independently from the other, said Yersinia LcrV antigen(s) being selected from the group comprising sequence SEQ ID N°45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 99, 100, and combination thereof; and
- combination thereof. USE AS VACCINE
This invention also pertains to a multivalent immunogenic composition according to the invention and such as above-defined, for its use as a vaccine.
More particularly, a multivalent immunogenic composition according to the invention and such as above-defined may be used for inducing a simultaneous immune response in, and preferably a simultaneous vaccination of, a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising (or consisting of) Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species.
According to a first variant, when a multivalent immunogenic composition according to the invention is intended to induce an immune response in a non-human vertebrate, notably of the aves class, then said composition comprises or expresses at least two Yersinia LcrV antigens, different from each other, for inducing an immune response against Yersinia pseudotuberculosis and Yersinia enterocolitica species.
According to a second variant, when a multivalent immunogenic composition according to the invention is intended to induce an immune response in a Mammalia, especially in a human, then said composition comprises or expresses at least two Yersinia LcrV antigens, different from each other, for inducing an immune response against Yersinia pestis and Yersinia pseudotuberculosis species, and preferably at least three Yersinia LcrV antigens, each different from each other, for inducing an immune response against Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica.
MOST PREFERRED EMBODIMENTS
According to a preferred embodiment, a composition according to the invention induces a simultaneous immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising (or consisting of) :
- at least one Yersinia LcrV antigen(s) inducing an immune response against Yersinia pestis being selected in the group comprising Yersinia LcrV antigens of amino acid sequences having at least 80% amino acid identity with, preferably at least 90% amino acid identity with, and still better of, sequences SEQ ID N°74 and 75;
- at least one Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis being selected in the group comprising Yersinia LcrV antigens of amino acid sequences having at least 80%) amino acid identity with, preferably at least 90%> amino acid identity with, and still better of, sequences SEQ ID N°25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 72, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and combination thereof ;
- at least one Yersinia LcrV antigen(s) inducing an immune response against Yersinia enterocolitica being selected in the group comprising Yersinia LcrV antigens of amino acid sequences having at least 80%> amino acid identity with, preferably at least 90%> amino acid identity with, and still better of, sequences SEQ ID N°45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 99 and 100, and combination thereof; and
- the combinations thereof.
According to a first variant, a multivalent immunogenic composition according to the invention may be intended to induce an immune response in a non-human vertebrate, notably of the aves class.
According to this first variant, said composition preferably comprises or expresses at least two Yersinia LcrV antigens, each different from each other, for inducing an immune response against Yersinia pseudotuberculosis and Yersinia enterocolitica species, said Yersinia LcrV antigens being selected in a group comprising (or consisting of):
- at least one Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis being selected in the group comprising Yersinia LcrV antigens of amino acid sequences having at least 80% amino acid identity with, preferably at least 90% amino acid identity with, and still better of, sequences SEQ ID N°25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 72, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and combination thereof ;
- at least one Yersinia LcrV antigen(s) inducing an immune response against Yersinia enterocolitica being selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80%) amino acid identity with, preferably at least 90%> amino acid identity with, and still better of, sequences SEQ ID N°45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 99 and 100, and combination thereof; and
- the combinations thereof.
According to a second variant, a multivalent immunogenic composition according to the invention may be intended to induce an immune response in a Mammalia, in particular in a human.
According to this second variant, said composition preferably comprises or expresses at least two Yersinia LcrV antigens, different from each other, for inducing an immune response against Yersinia pestis and Yersinia pseudotuberculosis species, said Yersinia LcrV antigens being selected in a group comprising:
- at least one Yersinia LcrV antigen(s) inducing an immune response against Yersinia pestis being selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80%> amino acid identity with, preferably at least 90%> amino acid identity with, and still better of, sequences SEQ ID N°74 and 75;
- at least one Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis being selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80%> amino acid identity with, preferably at least 90%> amino acid identity with, and still better of, sequences SEQ ID N°25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 72, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and combination thereof ; and
- the combinations thereof.
RECOMBINANT BACTERIA This invention also pertains to a recombinant bacteria expressing at least two Yersinia LcrV antigens, different from each other, said Yersinia LcrV antigens being selected in a group comprising (or consisting of):
- at least a first Yersinia LcrV antigen being capable of raising an immune response against Yersinia pestis in a vertebrate, notably of the Mammalia and/or of the aves class(es), said Yersinia
LcrV antigen(s) being notably such as above-defined,
- at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, being capable of raising an immune response against Yersinia pseudotuberculosis in a vertebrate, notably of the Mammalia and/or of the aves class(es), said Yersinia LcrV antigen(s) being notably such as above-defined, and
- at least a third Yersinia LcrV antigen, different from the first and the second Yersinia LcrV antigens, being capable of raising an immune response against Yersinia enterocolitica in a vertebrate, notably of the Mammalia and/or of the aves class(es), said Yersinia LcrV antigen(s) being notably such as above-defined.
Accordingly, the invention also concerns a recombinant bacteria comprising or expressing at least two heterologous Yersinia LcrV recombinant antigens, each different from each other, said Yersinia LcrV recombinant antigens being selected in a group consisting of :
- at least a first Yersinia LcrV recombinant antigen being capable of raising an immune response against Yersinia pestis in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined,
- at least a second Yersinia LcrV recombinant antigen, different from the first Yersinia LcrV antigen, being capable of raising an immune response against Yersinia pseudotuberculosis in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined, and/or
- at least a third Yersinia LcrV recombinant antigen, different from the first and the second Yersinia LcrV antigens, being capable of raising an immune response against Yersinia enterocolitica in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as above-defined. According to the nature of the considered bacteria, the above-mentioned Yersinia
LcrV antigens may be endogenous or heterologous Yersinia LcrV antigens. However, and for obvious reasons, at least one, preferably at least two, of the considered Yersinia LcrV antigens is/are necessarily heterologous. According to a first embodiment wherein the recombinant bacteria is of the Yersinia species, said recombinant bacteria may take advantage of the LcrV antigen naturally expressed by said bacteria of Yersinia species.
Therefore, in said embodiment, all Yersinia LcrV antigens expressed by a recombinant bacteria of the invention are not necessarily heterologous.
According to a second embodiment wherein the recombinant bacteria is different from the Yersinia species, then all the Yersinia LcrV antigens expresses by the considered recombinant bacteria are thus necessarily heterologous.
A Yersinia LcrV antigen not naturally expressed by a bacterium may be also designated by the term "Yersinia LcrV recombinant antigen".
More particularly, and in view of the above, the invention relates to a recombinant bacteria such as above-mentioned in the genome of which has been inserted at least one, and preferably at least two, exogenous nucleic acids.
Hence, the invention relates to a recombinant bacteria such as above-mentioned in the genome of which has been inserted at least one, preferably at least two, Yersinia LcrV recombinant antigen(s) selected among a group comprising (or consisting of):
- one or more nucleic acids encoding a first Yersinia recombinant LcrV antigen(s) capable of raising an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against Yersinia pestis species, and notably said nucleic acid encoding an amino acid sequence such as above-defined ;
- one or more nucleic acids encoding a second Yersinia recombinant LcrV antigen(s) capable of raising an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against Yersinia pseudotuberculosis species, and notably said nucleic acid encoding an amino acid sequence such as above-defined; and/or
- one or more nucleic acids encoding a third Yersinia recombinant LcrV antigen(s) capable of raising an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against Yersinia enterocolitica species, and notably at said nucleic acid encoding an amino acid sequence such as above-defined.
A recombinant bacteria according to the invention may be selected from the group comprising (or consisting of) Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Escherichia, Streptococcus, Agrobacterium, Bacillus, Corynebacterium, Clostridium, Gluconobacter, Citrobacter, Enterobacter, Klebsiella, Aerobacter, Methylobacter , Salmonella, Streptomyces, Yersinia and Pseudomonas. Preferably, a recombinant bacteria according to the invention is a probiotic lactic acid bacteria, in particular of the Lactococcus genus, and more particularly of the Lactococcus lactis species.
In view of the above, the invention also pertains to a recombinant bacteria according to the invention, for its use for inducing a simultaneous immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising (or consisting of) Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species.
Also, said recombinant bacteria may be used for inducing a simultaneous immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two different strains of a same Yersinia species selected in a group comprising (or consisting of) Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species.
In a recombinant bacteria according to the invention, the above-mentioned exogenous nucleic acid preferably comprises regulatory sequences, such as promoter and terminator sequences, which are operatively linked with the considered exogenous nucleic acid sequence coding for a particular LcrV antigen according to the present invention.
The man skilled in the art knows a variety of promoters which exhibit different strengths and which promoter to use for a weak genetic expression.
Each of exogenous nucleic acids considered in the present invention is under the control of a promoter, identical or different.
Said promoters, identical or different, allowing the constitutive over-expression of a given gene, may be found in literature (velculescu et al. (1997) Cell 88, 243-251).
Synthetic promoters as described in Blazeck & Alper (2013) Biotechnol. J. 8 46-58 can also be used.
Also, each of nucleic acids considered in the present invention is linked to a transcription terminator (which may be also termed "terminator" herein), identical or different.
Said transcription terminators, identical or different, may be found in literature Yamanishi et al., (2013) ACS synthetic biology 2, 337-347.
As above-mentioned, an exogenous nucleic acid may further comprise 5' and/or 3' recognition sequences and/or selection markers.
In some embodiments, recombinant bacteria according to the invention may thus advantageously comprise one or more selectable markers useful for the selection of transformed bacteria. Preferably, said selectable markers are comprised in the integrated nucleic acid construct intended to allow expressing the amino acid sequences according to the present invention. In some embodiments, the selectable marker is an antibiotic resistance marker. Illustrative examples of antibiotic resistance markers include, but are not limited to the, NAT1 , AUR1-C, HPH, DSDA, KAN<R>, and SH BLE gene products; the NAT 1 gene product from S. noursei confers resistance to nourseothricin; the AUR1-C gene product from Saccharomyces cerevisiae confers resistance to Auerobasidin A (AbA); the HPH gene product of Klebsiella pneumonia confers resistance to Hygromycin B; the DSDA gene product of E. coli allows cells to grow on plates with D-serine as the sole nitrogen source; the KAN<R> gene of the Tn903 transposon confers resistance to G418; and the SH BLE gene product from Streptoalloteichus hindustanus confers resistance to Zeocin (bleomycin).
In some embodiments, the antibiotic resistance marker is deleted after the genetically modified microbial cell of the invention is isolated. The man skilled in the art is able to choose suitable marker in specific genetic context.
In some embodiments, the selectable marker rescues an auxotrophy (e.g., a nutritional auxotrophy) in the genetically modified microbial cell. In such embodiments, a parent microbial cell comprises a functional disruption in one or more gene products that function in an amino acid or nucleotide biosynthetic pathway, such as, for example, the HIS3, LEU2, LYS 1 , LYS2, MET 15, TRP1 , ADE2, and URA3 gene products in yeast, which renders the parent microbial cell incapable of growing in media without supplementation with one or more nutrients (auxotrophic phenotype). The auxotrophic phenotype can then be rescued by transforming the parent microbial cell with a chromosomal integration encoding a functional copy of the disrupted gene product (NB: the functional copy of the gene can originate from close species, such as Kluveromyces, Candida, etc.) and the genetically modified microbial cell generated can be selected for based on the loss of the auxotrophic phenotype of the parent microbial cell.
The "culture" of a recombinant bacteria according to the invention is generally conducted in an appropriate culture medium adapted to the bacteria being cultivated, containing at least one simple carbon source, and if necessary co-substrates.
For maximal growth, the microorganism strains used as production hosts preferably have a high rate of carbohydrate utilization. These characteristics may be conferred by mutagenesis and selection, genetic engineering, or may be natural. A conventional "culture medium" for bacteria may notably contain glucose, and in particular may contain at least about 10 g/L of glucoseAdditional carbon substrates may include but are not limited to monosaccharides such as fructose, mannose, xylose and arabinose, oligosaccharides such as lactose maltose, galactose, or sucrose, polysaccharides such as starch or cellulose or mixtures thereof and unpurified mixtures from renewable feedstocks such as cheese whey permeate cornsteep liquor, sugar beet molasses, and barley malt. Other carbon substrates may include glycerol. Hence, it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.
Although it is contemplated that all of the above-mentioned carbon substrates and mixtures thereof are suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose, or mixtures of these with C5 sugars such as xylose and/or arabinose for microorganisms modified to use C5 sugars, and more particularly glucose.
In addition to an appropriate carbon source, the culture media may contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the pathway necessary for the production of the desired LcrV antigen(s).
The conditions to consider in the present invention may be anaerobic or aerobic conditions.
The terms "anaerobic conditions" refer to conditions under which the oxygen concentration in the culture medium is too low for the microorganism to be used as terminal electron acceptor. Anaerobic conditions may be achieved either by sparging the culture medium with an inert gas such as nitrogen until oxygen is no longer available to the microorganism or by the consumption of the available oxygen by the microorganism.
The terms "aerobic conditions" refers to concentrations of oxygen in the culture medium that are sufficient for an aerobic or facultative anaerobic microorganism to use as a terminal electron acceptor.
"Microaerobic condition" refers to a culture medium in which the concentration of oxygen is less than that in air, i.e. oxygen concentration up to 6% (¼.
An "appropriate culture medium" designates a medium (e.g. a sterile, liquid medium) comprising nutrients essential or beneficial to the maintenance and/or growth of the cell such as carbon sources or carbon substrate, nitrogen sources, for example, peptone, yeast extracts, meat extracts, malt extracts, urea, ammonium sulfate, ammonium chloride, ammonium nitrate and ammonium phosphate; phosphorus sources, for example, monopotassium phosphate or dipotassium phosphate; trace elements (e.g., metal salts), for example magnesium salts, cobalt salts and/or manganese salts; as well as growth factors such as amino acids, vitamins, growth promoters, and the like. The term "carbon source" or "carbon substrate" or "source of carbon" according to the present invention denotes any source of carbon that can be used by those skilled in the art to support the normal growth of a microorganism, including hexoses (such as glucose, galactose or lactose), pentoses, monosaccharides, oligosaccharides, disaccharides (such as sucrose, cellobiose or maltose), molasses, starch or its derivatives, cellulose, hemicelluloses and combinations thereof.
According to a particular embodiment, when the recombinant bacteria implemented is a probiotic lactic acid bacteria, notably of the Lactococcus genus, an "appropriate culture medium" may be the Ml 7 medium supplemented with glucose, and especially 0,5% glucose.
Methods implemented to allow expressing in a bacteria of a specific exogenous nucleic acid construct belong to the general knowledge of a man skilled in the art. A related method is described in more details in the herein after examples. DETECTION DEVICE
The invention also pertains to a device for detecting at least one Yersinia infection due to at least one Yersinia species selected from the group consisting of Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species, wherein said device comprises at least two Yersinia's LcrV-specific binding molecules selected among the group comprising (or consisting of):
- at least one first Yersinia's LcrV-specific binding molecule(s) being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pestis species;
- at least one second Yersinia's LcrV-specific binding molecule(s), different from the first Yersinia's LcrV-specific binding molecule(s), being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pseudotuberculosis species; and/or
- at least one third Yersinia's LcrV-specific binding molecule(s), different from the first and the second Yersinia's LcrV-specific binding molecules, being capable of binding to at least one Yersinia LcrV antigen of the Yersinia enterocolitica species.
According to a particular embodiment, namely when the first or the second Yersinia's LcrV-specific binding molecule is capable of binding to Yersinia LcrV antigens of Yersinia pestis and Yersinia pseudotuberculosis , then said device may:
- be devoid of the second or the first Yersinia's LcrV-specific binding molecule; or
- further comprise at least one additional Yersinia's LcrV-specific binding molecule(s) being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pestis or Yersinia pseudotuberculosis, different from whichever of the first or the second Yersinia's LcrV-specific binding molecule is already present.
Said detection device may be also appropriate for detecting at least one Yersinia infection due to at least one specific Yersinia pestis, Yersinia pseudotuberculosis or Yersinia enterocolitica strain. A such device may be subject to any adequate adaptations which belong to the knowledge of a man skilled in the art, notably in view of the teaching displayed in the present description.
In particular, in a device according to the invention and such as above-mentioned, each Yersinia's LcrV-specific binding molecules, one independently from the others, may be selected from the group consisting of:
- an antibody,
- a nucleic acids or a peptide aptamer, and
- a mixture thereof.
Subject to adequate adaptations, a device for detecting at least one Yersinia infection may be based on the device described in R. Bianucci et ah, C. R. Biologies 330 (2007) and Chanteau et ah, 2000 a ; 2003 a et b.
Subject to adequate adaptations, a device for detecting at least one Yersinia infection may be also based on the device commercialized under the name recomLine Yersinia IgG 2.0 by the company MIKROGEN.
Accordingly, the invention further relates to the above-mentioned devices for use for detecting at least one Yersinia species selected from the group consisting of: Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species
COMPANION DIAGNOSTIC TEST DEVICE
A companion diagnostic test device is notably intended to monitor the response to a treatment with a particular therapeutic product for the purpose of adjusting treatment to achieve improved safety or effectiveness.
In the context of the present invention, a companion diagnostic test device is intended to monitor the presence of an immune response of a vertebrate, notably of the Mammalia and/or of the aves class(es), to at least one previous administration of at least one multivalent immunogenic composition according to the invention and such as above-defined.
More particularly, this companion diagnostic test device allows assessing the presence of an immune response and, the case arising, the level of this immune response.
Thus, the invention also pertains to a device for detecting antibodies anti- Yersinia's LcrV in a vertebrate, notably of the Mammalia and/or of the aves class(es), wherein said device comprises at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising (or consisting of) :
- at least a first Yersinia LcrV antigen being the LcrV antigen of Yersinia pestis species;
- at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, being the LcrV antigen of Yersinia pseudotuberculosis species; and - at least a third Yersinia LcrV antigen, different from the first and second Yersinia LcrV antigens, being the LcrV antigen of Yersinia enterocolitica species.
Subject to adequate adaptations, a device for detecting at least one Yersinia infection may be based on the device described in Zauberman et ah, Vaccine, 2008, 26(13): 1616-25.
Besides, the invention further pertains to a kit comprising at least :
- a multivalent immunogenic composition such as above-defined, and
- a device for detecting antibodies anti- Yersinia's LcrV in a vertebrate, notably of the Mammalia and/or of the aves class(es), and notably such as above-defined.
Accordingly, the invention also relates to the above-mentioned devices and kits for use for monitoring the presence of an immune response of a vertebrate, notably of the Mammalia and/or of the aves class(es), to at least one previous administration of at least one multivalent immunogenic composition according to the invention and such as above-defined.
Accordingly, the invention also relates to the above-mentioned devices and kits, for use for detecting antibodies anti- Yersinia's LcrV in a vertebrate, notably of the Mammalia and/or of the aves class(es).
The terms "between... and..." and "ranging from... to..." should be understood as being inclusive of the limits, unless otherwise specified.
The terms "comprising", "comprise" and "comprises" herein are intended by the inventors to be optionally substitutable with the terms "consisting of, "consist of and "consists of, respectively, in every instance.
The term "at least one" should be understood as comprising, if applicable, "at least one", "at least two", "at least three", "at least four", "at least five" and so on.
All references or patent applications cited within this patent specification are incorporated by reference herein.
The examples and figures which follow are presented by way of illustration and without implied limitation of the invention.
EXAMPLES
The protocol implemented to make a recombinant bacteria expressing a LcrV antigen specific of a Yersinia strain is the same as the protocol described in the publication Daniel C, Vaccine, 27, 2009: 1141-4 which is thus incorporated by reference herein.
For a better clarity is however hereinafter displayed some information/parameters regarding this protocol. Material and methods
Bacterial strains, plasmids and growth conditions: Y. pseudotuberculosis strains, L. lactis strain MG1363, and the lactococcal pNZYR expression vector (conferring resistance to chloramphenicol) are used.
Y. pseudotuberculosis strains and L. lactis were respectively cultured in lysogeny broth at 28°C and M17 medium (Difco) supplemented with 0.5% glucose at 30°C. The growth media were supplemented with chloramphenicol (10 μg/ml) when required.
Custom pNZYR vectors (Eurogentec) containing the LcrVs of interest (translationally fused with the L. lactis usp45 secretion signal sequence and under the control of the Pusp45 promoter) were generated as described in Daniel C et ah, Vaccine, 27, 2009: 1141-4.
Evaluation of the efficacy of immunization: Groups of 8-week-old, female BALB/c mice (Charles River) were immunized intranasally with sterile, phosphate-buffered saline (PBS) containing 109 CFU of L. lactis producing (or not) the V antigen of interest, as described in Daniel C ei « ., Vaccine, 27, 2009: 1141-4.
Immunization was repeated on days 2, 22, 23, 43 and 44. One week after the final boost, serum was collected from animals and the anti-V titers were measured in an ELISA, as also described in Daniel C et ah, Vaccine, 27, 2009: 1141-4.
One week later, the mice were inoculated intravenously with 300 μΐ of PBS containing 103 Y. pseudotuberculosis .
The mice's survival was monitored daily for three weeks.
Sequencing, translation and phylogenetic analysis: DNA from above-mentioned Y. pseudotuberculosis strains was purified using the Nucleospin kit (Macherey-Nagel) and then used to amplify LcrV by PCR (using the primer sets 5 ' -TCACCGCGCAAAATTATTGC-3 ' (i.e. SEQ ID N°101) ; 5'-TTGTCTGCGATAAGCTCTTG-3 ' (i.e. SEQ ID N°102)). Amplicons were purified using a PCR purification kit (Qiagen) and sequenced with the primers used for amplification and the following primers : 5 ' -CCTAGCTTATTTTCTACCCG-3 ' (i.e. SEQ ID N°103) ; 5'-GAACCGGGGCGTTGGGTAATC-3' (i.e. SEQ ID N°104) ; 5'-GTTGGTTGTCATAATGACCGCC-3' (i.e. SEQ ID N°105) ; 5'- CTAACCAAGTCGTTGAGCGG-3 ' (i.e. SEQ ID N°106). Nucleotide sequences were translated using the ExPaSy Translate tool (http://web.expasy.org/translate/). A phylogenic tree based on LcrV sequences may be generated using the http://phylogeny.lirmm.fr/phylo_cgi/index.cgi web server (Dereeper A, Nucleic acids research, 36, 2008: W465-9) (see figure 7). Statistical analysis: The Mann- Whitney U test, the log-rank test and Fisher's exact probability test were used to compare antibody titers, survival curves and the degree of protection conferred by the various recombinant vaccine strains. Example 1: Yersinia pseudotuberculosis escapes from the immune response against the V antigen from Y. pestis
A strain of L. lactis that secreted LcrV from Y. pestis (VYp) strain 195/P was generated and then it was assessed whether this new strain afforded protection against Y. pseudotuberculosis strain 2777. To this end, groups of mice were immunized intranasally with L. lactis secreting (or not) VYp, according to the protocol reported in Daniel C et ah, Vaccine, 27, 2009: 1141-4.
As an additional control, a group of mice was immunized with L. lactis producing LcrV from Y. pseudotuberculosis strain 2777 (VYpst).
Two weeks after the end of the immunization protocol, mice were challenged intravenously with a lethal dose of Y. pseudotuberculosis strain 2777. The proportion of animals developing fatal pseudotuberculosis was similar in mice vaccinated with VYp and in unvaccinated mice (Figure 1). This contrasted with the higher survival rate observed in animals vaccinated with VYpst (Figure 1).
However, the mice's anti-V IgG titers did not appear to depend on the V antigen used (Figure 6A). Thus, the present data shown that the LcrV sequence polymorphism affects cross- protective immunity.
Example 2: role of the amino acid residue [AA] of LcrV in active immunization against pseudotuberculosis
The sequences of the LcrV proteins from Y. pseudotuberculosis strain 2777 and Y. pestis strain 195/P differ in the region considered to contain a plague-protective epitope (Motin VL, Infection and immunity, 62, 1994: 4192-201; Hill J, Infection and immunity, 65, 1997: 4476- 82; Vernazza C, Vaccine, 27, 2009:2775-80).
In particular, the Y. pestis strain 195/P LcrV region lacks a threonine and has three residue substitutions (E205A, V226M, and N255D) (Figure 2). In this figure 2, the missing threonine and the substituted residue 226 are located within the 16-amino acid LcrV segment (residues 218 to 234), which is referred to here as the "conformational segment".
To assay the impact of the residue substitutions in the conformational segment and/or at position relative to the amino acid [AA] with respect to the absence of cross-protective immunity, L. lactis strains secreting a hybrid Y. pestis strain 195/P IY. pseudotuberculosis strain 2777 LcrV protein had been generated, in which the conformational segments had been exchanged (yielding VYpstCS-Yp and VYpCS-Ypst) or with an Asp to Asn substitution at amino acid position [AA] in VYpst (yielding VYpstD255N) (Figure 2).
After immunization such as described above (see example 1), the degree of protection conferred against a lethal intravenous challenge of Y. pseudotuberculosis strain 2777 in the various groups of mice had been assessed.
All vaccinated mice had similar anti-V IgG titers (Figure 6B).
All mice immunized with L. lactis alone or L. lactis secreting VYpCS-Ypst and VYpst D225N died, furthermore, the time courses of mortality were similar (p>0.05) in the three groups (Figure 3). Hence, VYpCS-Ypst and VYpstD225N did not confer any protection against pseudotuberculosis.
In contrast, immunization with L. lactis secreting VYpst and VYpstCS-Yp afforded protection (Figure 3). However, the survival rate was higher in mice immunized with VYpst than with VYpstCS-Yp (-75% and -35%, respectively). A similar difference between the respective survival rates was obtained in a second, independent experiment.
However, this difference in protection conferred by VYpstCS-Yp against Y. pseudotuberculosis strain 2777 in both experiments was not statistically significant (p>0.07 in a Gehan-Wilcoxon test and Fisher's exact test).
Taken as a whole, these data indicate that LcrV polymorphism at amino acid residue [AA] (but not in the conformational segment) is sufficient for the escape from anti-V immunity.
Example 3: assessment of the immune escape from vaccination against LcrV due to the polymorphism in LcrV's conformational segment
Phylogenetic and sequence analyses were performed, using LcrV (solely the above mentioned conformational sequence (CS) and the amino acid residue [AA] of 7 Y. pseudotuberculosis strains (included in sequences SEQ ID N° 26, 43, 91, 98, 78, 86 and 72), 1 Y. pestis strains (included in sequence SEQ ID N° 75) and 5 Y. enterocolitica strains (included in sequences notably SEQ ID N°47, 53, 71, 67 and 99).
The results are displayed in figure 7.
These results indicated that Y. pseudotuberculosis LcrV clusters into seven major clades (A, strain NT, Bl, B2, Dl, D2 and C) (Figure 7). Six clades had an Asp in amino acid position [AA] and except for the clades Bl and B2, each clades had its own distinct conformational segment (Figure 8). These results further indicated that Y. enterocolitica LcrV clusters into five major clades (F, G, strain 8081, H and strain E40) (Figure 7). Four clades had an Asp in amino acid position [AA] and each of the five clades had its own distinct conformational segment (Figure 8).
These results further indicated that Y. pestis LcrV clusters into a sole clade (C-bis) (Figure 7). This clade had an Asn in amino acid position [AA] (Figure 8).
The strains Y.pst 2889 and Y.pst AH from clade D2 were selected.
LcrV from both strains 2889 and AH had an Asp in amino acid position [AA] like Y. pseudotuberculosis strain 2777. However, the sequences of the LcrV conformational segment in strains 2889 and AH (see sequence SEQ ID N° 13) differ from that of V2777 (ie clade Bl) (Figure 8).
Hence, mice was immunized with L. lactis secreting V2777 and the animals' survival was measured after inoculation with the 2777, 2889 and AH strains.
Even though the vaccinated mice mounted a strong anti-V response (Figure 6C), the vaccination neither significantly decreased the mortality rate nor affected the kinetics of mortality in mice inoculated with strains 2889 and AH (Figure 4). In contrast, all the mice infected with the strain 2777 survived (Figure 4). This absence of cross-protection did not appear to be serotype- dependent, since 2889 and AH are distinct serotypes.
Lastly, this total absence of cross-protection between VYpst variants supports the fact that whereby the polymorphic residues in the conformational segment (i.e. CS region) influence protective immunity.
Following a similar protocol, figure 9 shows that L.lactis secreting clade B V antigen affords protection against a Y .pseudotuberculosis strain producing clade B (but not clade D) V antigen.
Example 4: LcrV from Y. pestis confers protection against specific Y. pseudotuberculosis strains
A phylogenetic analysis of two Y. pestis strains (C092 and 195/P) and Y. pseudotuberculosis strains revealed that Y. pseudotuberculosis LcrV clade C was closely related to Y. pestis LcrV (Figure 7).
Further sequence analysis showed that LcrV proteins from this particular clade C have the same conformational segment sequence and the same residue (an asparagine) at amino acid position [AA] as LcrV from Y. pestis (Figure 8 (see Clades CC and CC-bis)).
This contrasts with the other clades of Y. pseudotuberculosis, in which (i) the conformational segments differ from Y. pestis and from each other and (ii) the amino acid residue [AA] is an aspartate and not an asparagine (Figure 8). In other words, the sequence of the LcrV from some Y. pseudotuberculosis strains (i.e. belonging to the clade C) differs from that of the Y. pestis LcrV only in regions distinct from the conformational segment sequence and the amino acid [AA] .
Hence, the protection against Y. pseudotuberculosis strains producing one of the seven LcrV clades had been assessed with a vaccination using L. lactis secreting an LcrV from Y. pestis.
Thus, mice vaccinated with L. lactis secreting VYp (from the strain 195/P) were challenged with a lethal dose of Y. pseudotuberculosis strains 2843 or 2889. Strain 2843 belongs to clade C (which includes Y. pestis LcrV) while the 2889 strain belongs to clade D2 (Figure 8).
The vaccinated mice were protected against pseudotuberculosis caused by strain 2843
(clade C) but not against that caused by strain 2889 (clade D2) (Figure 5). Lastly, it is noteworthy that L. lactis secreting VYp (clade C) did not confer protection against a lethal challenge of Y. pseudotuberculosis strain 2777 (Figure 1), which belongs to clade B l (Figure 7).
Thus, LcrV from Y. pestis confers protection against Y. pseudotuberculosis strains belonging to clade C.
Example 5: As a control, different Y. pseudotuberculosis strains used in this study have been shown to produce similar amounts of V antigen, by immunoblot of whole-cell lysates of Y. pseudotuberculosis strains (2777, 2781 , AH, 2790, 2889 and 2843) grown under conditions that induce the production of V antigen. The relative density value (expressed as a percentage), was calculated by dividing the density of the band of interest by the sum of the density of all the bands shown on the blot. Results are detailed hereafter:
- 2777 strain: 16%
- 2781 strain: 15%
- AH strain: 16%
- 2790 strain: 17%
- 2889 strain: 19%
- 2843 strain: 17% CONCLUSIONS
The above-examples confirms that LcrV is a polymorphic antigen within which the major protective epitope encompasses residues 135 to 275 and more particularly is characterized by the above-identified conformational segment ("CS") and amino acid residue [AA]. The above-examples further allow concluding that polymorphism in the LcrV, and more particularly in the conformational segment and the amino acid residue [AA] enables escape from an active immune response against LcrV (Figures 3, 4 and 5).
The results of previous vaccination studies using purified recombinant V antigen produced in E. coli suggested that protection against all types of yersiniosis does not depend on the type, subtype or variant of V antigen used for vaccination (Schmidt A, Microbial pathogenesis, 26, 1999: 221-33 ; Hill J, Vaccine, 27, 2009: 7073-9).
The data displayed in the above-examples do not support this conclusion, since vaccination using LcrV-secreting L. lactis did not provide cross-protective immunity between different V antigen variants or even between different V subtypes and types.
The above-mentioned data thus indicate that V-based vaccine, optionally using a bacteria, and more particularly L. lactis, as antigen vehicle, comprising multiple V antigens of a plurality a strains of Yersinia is particularly interesting in that it leads to an immune response, and thus a protection, against a wide number of Yersinia strains (whether they belong to the same species or not).
SEQUENCE LISTING
SEQ ID N°l (= amino acid sequence full-lenght of the LcrVof Ypst 32777)
MIRAYEQNPQHFIEDLEKVRVEQLTGHGSSVLEELVQLVKDKNIDISIKYDPRKDSEFFAN RVITDDIELLKKILAYFLPEDAILKGGHYDNQLQNGIKRVKEFLESSPNTQWELRAFMAVI HFSLTADRIDDDILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTI NIHDKSINLMDKNLYGYTDEAIFKASAEYKILEKMPQTTTIQMDGSEKKIVSIKDFLGSEN KRTGALGNLKDSYSYNKDNNELSHFATTCSDKSRPLNDLVSQKTTQLSDITSRFNSAIEAL NRFIQKYD S VMQRLLDDT S AR SEQ ID N°2 (=SEQ ID N°A ;from LcrVof Ypst 32777)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDK NLYGYTDEAIFKASAEYKILE
SEQ ID N°3 (=SEQ ID N°B ;from LcrVof Ypst 32777)
VSIKDFLGSENKRTGALGNLK
SEQ ID N°4 (=SEQ ID N°C ;from LcrVof Ypst 32777)
SYSYNKDNNELSHFATTCSD
SEQ ID N°5 (=SEQ ID N°B variable ;from LcrVof Ypst 32777)
VSIKX2FLX3SEX4KRTGALGNLK
wherein:
- X2 means an amino acid selected from the group comprising aspartic acid, glycine and asparagine,
- X3 means an amino acid selected from the group comprising glycine and glutamic acid, and
- X4 means an amino acid selected from the group comprising asparagine and lysine, SEQ ID N°6 (=SEQ ID N°B;from LcrVof pestis)
VSIKDFLGSENKRTGALGNLK
SEQ ID N°7 (=SEQ ID N°B;from LcrVof Y enterocolitica)
VSIKNFLESENKRTGALGNLK
SEQ ID N°8 (=CS-l;from LcrVof Y. pestis)
(CS-2; from LcrV of Y. pseudotuberculosis Clade C)
KMPQT-TIQVDGSEKKI
SEQ ID N°10 (=CS-2; from LcrV of Y. pseudotuberculosis strain NT)
KMPQTNTTQVDGSEKKI
SEQ ID N°l l (=CS-2; from LcrV of Y. pseudotuberculosis Clade Bl)
(=CS-2; from LcrV of Y. pseudotuberculosis Clade B2)
KMPQTTTIQMDGSEKKI SEQ ID N°12 (=CS-2; from LcrV of Y. pseudotuberculosis Clade Dl)
KM--T-TIQEGETEKKI
SEQ ID N°13 (=CS-2; from LcrV of Y. pseudotuberculosis Clade D2)
KMPQT-TIQEGETEKKI
SEQ ID N°14 (=CS-3;from LcrV of Y. enterocolitica Clade F)
KMPQT-TIKEGETEKKI
SEQ ID N°15 (=CS-3;from LcrV of Y. enterocolitica Clade G)
KMS QT-TIKEGETEKKI
SEQ ID N°16 (=CS-3;from LcrV of Y. enterocolitica strain 8081)
KMS QT-TIKEGETEKKI
SEQ ID N°17 (=CS-3;from LcrV of Y. enterocolitica Clade H)
KMPQTTIKDDELHEVGVIAGAEKQI
SEQ ID N°18 (=CS-3;from LcrV of Y. enterocolitica strain E40)
KMPQTTFPPIPGNGNGSEKKI
SEQ ID N°19 (=from LcrVof Ypst 32777)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSrNLMDK NLYGYTDEA
SEQ ID N°20 (=from LcrVof Ypst 32777)
IFKASAEYKILXs
wherein Xs means an amino acid selected from the group comprising glutamic acid and lysine
SEQ ID N°21 (=from LcrVof Ypst 32777)
SYSYNKDNNELSHFATX6CSD
wherein Xemeans an amino acid selected from the group comprising threonine and alanine SEQ ID N°22 (=from LcrV of Ye Q56879, Y108P et Y96P)
SYSYNKDNNELSHLGTTCSD
SEQ ID N°23 (=SEQ ID N°B;from LcrV of Yp/Ypst Clade C)
VSIKDFLGSENKRTGALGNLK
SEQ ID N°24 (=from LcrV of strain Y.pst 165_89)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSrNLMDK
NLYGYTDEEIFKASAEYKILEKMTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSYSY
NKDNNELSHFATTCSD
SEQ ID N°25 (=from LcrV of the strain Y.pst 297 _89)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSrNLMDK
NLYGYTDEEIFKASAEYKILEKMTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSYSY
NKDNNELSHFATTCSD SEQ ID N°26 (=from LcrVofthe strain Y.pst 300_89)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSYSY
NKDNNELSHFATTCSD
SEQ ID N°27 (=from LcrVofthe strain Y.pst 1432_94)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK ILYGYTDEEIFKASAEYKILEKMTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSYSYN KDNNELSHFATTCSD
SEQ ID N°28 (=from LcrVofthe strain Y.pst PBlxxx45)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°29 (=from LcrVofthe strain Y.pst 2953xx44)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°30 (=from LcrVofthe strain Y.pst STxxxx43)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSrNLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°31 (=from LcrVofthe strain Y.pst KMxxxx42)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°32 (=from LcrVofthe strain Y.pst 3255_93)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°33 (=from LcrVofthe strain Y.pst 32861x40)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°34 (=from LcrVofthe strain Y.pst 32984x39) ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°35 (=from LcrVofthe strain Y.pst 32977x38)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°36 (=from LcrVofthe strain Y.pst YPIIIx37)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAErNKHLSSGGTINIHDKSrNLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°37 (=from LcrVofthe strain Y.pst YPTLVx36)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°38 (=from LcrVofthe strain Y.pst WE27_93)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°39 (=from LcrVofthe strain Y.pst 2853xx34)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°40 (=from LcrVofthe strain Y.pst 2790xx33)
ILKVIVD SMNHHGD ARSKLREELAELTAELKIYS VIQ AEINKHL S S GGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°41 (=from LcrVofthe strain Y.pst 2783xx32)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°42 (=from LcrVofthe strain Y.pst 2666xx27)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD SEQ ID N°43 (=from LcrVofthe strain Y.pst 2889xx26)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°44 (=from LcrVofthe strain Y.pst AHxxxx28)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSGGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQEGETEKKIVSIKNFLESEKKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°45 (=from LcrVofthe strain Y.e GD_Y34)
ILKVIVDSMNHHGDARGKLREELAELTAELKIYSVIQAEINKHLSNSGTINIHDKSINLMD KNLYGYTDEEIFKASAEYKILEKMPQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDS YSYNKDNNELSHFATTCSD
SEQ ID N°46 (=from LcrVofthe strain Y.e 1055Rx50)
ILKVIVDSMNHHGDARGKLREELAELTAELKIYSVIQAEINKHLSNSGTINIHDKSINLMD KNLYGYTDEEIFKASAEYKILEKMPQTTIKEGETEKKIVSIKNFLESENKRTGALGSLKDS YSYNKDNNELSHFATTCSD
SEQ ID N°47 (=from LcrVofthe strain Y.e W22703)
ILKVIVDSMNHHGDARGKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMD KNLYGYTDEEIFKASAEYKILEKMPQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDS YSYNKDNNELSHFATTCSD
SEQ ID N°48 (=from LcrVofthe strain Y.e GD_L1135)
ILKVIVDSMNHHGDARGKLREELAELTAELKrYSVIQAEINKHLSNSDTINIHDKSrNLMD KNLYGYTDEEIFKASAEYKILEKMPQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDS YSYNKDNNELSHFATTCSD
SEQ ID N°49 (=from LcrVofthe strain Y.e GD_L1681)
ILKVIVDSMNHHGDARGKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMD KNLYGYTDEEIFKASAEYKILEKMPQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDS YSYNKDNNELSHFATTCSD
SEQ ID N°50 (=from LcrVofthe strain Y.e GD_Yax54)
ILKVIVDSMNHHGDARGKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMD
KNLYGYTDEEIFKASAEYKILEKMPQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDS
YSYNKDNNELSHFATTCSD
SEQ ID N°51 (=from LcrVofthe strain Y.e GD_Y38) ILKVIVDSMNHHGDARGKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMD KNLYGYTDEEIFKASAEYKILEKMPQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDS YSYNKDNNELSHFATTCSD
SEQ ID N°52 (=from LcrVofthe strain Y.e GD_Y29)
ILKVIVDSMNHHGDARGKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMD
KNLYGYTDEEIFKASAEYKILEKMPQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDS
YSYNKDNNELSHFATTCSD
SEQ ID N°53 (=from LcrVofthe strain Y.e GD_Y30)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSrNLMDK
NLYGYTNEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°54 (=from LcrVofthe strain Y.e GD_Y33)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSrNLMDK
NLYGYTNEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°55 (=from LcrVofthe strain Y.e GD_Y31)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMDK
NLYGYTNEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°56 (=from LcrVofthe strain Y.e GD_L183)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSrNLMDK
NLYGYTNEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°57 (=from LcrVofthe strain Y.e GD_L4035)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMDK
NLYGYTNEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°58 (=from LcrVofthe strain Y.e GD_Y39)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMDK
NLYGYTNEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°59 (=from LcrVofthe strain Y.e GDL1491)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMDK
NLYGYTNEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDSY
SYNKDNNELSHFATTCSD SEQ ID N°60 (=from LcrVofthe strain Y.e GD_L1996)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMDK
NLYGYTNEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°61 (=from LcrVofthe strain Y.e GD_Y41)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMDK
NLYGYTNEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°62 (=from LcrVofthe strain Y.e YE149x66)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSrNLMDK
NLYGYTNEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°63 (=from LcrVofthe strain Y.e YEPlxx67)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMDK
NLYGYTNEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDSY
SYNKDNNELSHFATTCSD
SEQ ID N°64 (=from LcrVofthe strain Y.e Q56879)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMDK
NLYGYTNEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDSY
SYNKDNNELSHLGTTCSD
SEQ ID N°65 (=from LcrVofthe strain Y.e Y108Px69)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMDK
NLYGYTNEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDSY
SYNKDNNELSHLGTTCSD
SEQ ID N°66 (=from LcrVofthe strain Y.e Y96Pxx70)
ILKVIVDSMNHHGDARGKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMD
KNLYGYTDEEIFKASAEYKILEKMPQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDS
YSYNKDNNELSHLGTTCSD
SEQ ID N°67 (=from LcrVofthe strain Y.e GD_Y37)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHEKSrNLMDK
NLYGYTDEEIFKASAEYKILKKMPQTTIKDDELHEVGVIAGAEKQIVSIKNFLESENKRTG
ALGNLKDSYSYNKDNNELSHFATACSD
SEQ ID N°68 (=from LcrVofthe strain Y.e NCTCxx74) ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHEKSINLMDK
NLYGYTDEEIFKASAEYKILKKMPQTTIKDDELHEVGVIAGAEKQIVSIKNFLESENKRTG
ALGNLKDSYSYNKDNNELSHFATACSD
SEQ ID N°69 (=from LcrVofthe strain Y.eA12790)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHEKSINLMDK
NLYGYTDEEIFKASAEYKILKKMPQTTIKDDELHEVGVIAGAEKQIVSIKNFLESENKRTG
ALGNLKDSYSYNKDNNELSHFATACSD
SEQ ID N°70 (=from LcrVofthe strain Ye WA314x73)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHEKSrNLMDK
NLYGYTDEEIFKASAEYKILKKMPQTTIKDDELHEVGVIAGAEKQIVSIKNFLESENKRTG
ALGNLKDSYSYNKDNNELSHFATACSD
SEQ ID N°71 (=from LcrVofthe strain Ye E40xxx49)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTFPPIPGNGNGSEKKIVSIKNFLESENKRTGALGNL
KDSYSYNKDNNELSHFATTCSD
SEQ ID N°72 (=from LcrVofthe strain Y.pst NT)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDK
NLYGYTDEAIFKASAEYKILEKMPQTNTTQVDGSEKKIVSIKDFLGSEKKRTGALGNLKD
SYSYNKDNNELSHFATTCSD
SEQ ID N°74 (=from LcrVofthe strain Y.p 195Pxxl4)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDK NLYGYTDEEIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNSY SYNKDNNELSHFATTCSD
SEQ ID N°75 (=from LcrVofthe strain Y.p C092xxl5)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDK
NLYGYTDEEIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNSY
SYNKDNNELSHFATTCSD
SEQ ID N°76 (=from LcrVofthe strain Y.pst_2892)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDESrNLMDK
NLYGYTDDDIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNS
YSYNKDNNELSHFATTCSD
SEQ ID N°77 (=from LcrVofthe strain Y.pst_2515)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDESrNLMDK
NLYGYTDDDIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNS
YSYNKDNNELSHFATTCSD SEQ ID N°78 (=from LcrVofthe strain Y.pst_2843)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDESINLMDK
NLYGYTDDDIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNS
YSYNKDNNELSHFATTCSD
SEQ ID N°79 (=from LcrVofthe strain Y.pst_2929)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDESINLMDK
NLYGYTDDDIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNS
YSYNKDNNELSHFATTCSD
SEQ ID N°80 (=from LcrVofthe strain Y.pst_2926)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDESrNLMDK
NLYGYTDDDIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNS
YSYNKDNNELSHFATTCSD
SEQ ID N°81 (=from LcrVofthe strain Y.pst_WS25_91)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDESrNLMDK
NLYGYTDDDIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNS
YSYNKDNNELSHFATTCSD
SEQ ID N°82 (=from LcrVofthe strain Y.pst_199_90)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDESrNLMDK
NLYGYTDDDIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNS
YSYNKDNNELSHFATTCSD
SEQ ID N°83 (=from LcrVofthe strain Y.pst_99_91)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDESrNLMDK
NLYGYTDDDIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNS
YSYNKDNNELSHFATTCSD
SEQ ID N°84 (=from LcrVofthe strain Y.pst_1119)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDESrNLMDK
NLYGYTDDDIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNS
YSYNKDNNELSHFATTCSD
SEQ ID N°85 (=from LcrVofthe strain Y.pst_MA)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDESrNLMDK
NLYGYTDDDIFKASAEYKILEKMPQTTIQVDGSEKKIVSIKDFLGSENKRTGALGNLKNS
YSYNKDNNELSHFATTCSD
SEQ ID N°86 (=from LcrVofthe strain Y.pst 32777x6) ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDK NLYGYTDEAIFKASAEYKILEKMPQTTTIQMDGSEKKIVSIKDFLGSENKRTGALGNLKDS YSYNKDNNELSHFATTCSD
SEQ ID N°87 (=from LcrVofthe strain Y.pst YPTllx7)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDK NLYGYTDEAIFKASAEYKILEKMPQTTTIQMDGSEKKIVSIKDFLGSENKRTGALGNLKDS YSYNKDNNELSHFATTCSD
SEQ ID N°88 (=from LcrVofthe strain Y.pst 278 lxx8)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSrNLMDK NLYGYTDEAIFKASAEYKILEKMPQTTTIQMDGSEKKIVSIKDFLGSENKRTGALGNLKDS YSYNKDNNELSHFATTCSD
SEQ ID N°89 (=from LcrVofthe strain Y.pst WS32_92)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDK NLYGYTDEAIFKASAEYKILEKMPQTTTIQMDGSEKKIVSIKDFLGSENKRTGALGNLKDS YSYNKDNNELSHFATTCSD
SEQ ID N°90 (=from LcrVofthe strain Y.pst WE417_94)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDK NLYGYTDEAIFKASAEYKILEKMPQTTTIQMDGSEKKIVSIKDFLGSENKRTGALGNLKDS YSYNKDNNELSHFATTCSD
SEQ ID N°91 (=from LcrVofthe strain Y.pst LP32275)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDK NLYGYTDEAIFKASAEYKILEKMPQTTTIQMDGSEKKIVSIKGFLGSENKRTGALGNLKDS YSYNKDNNELSHFATTCSD
SEQ ID N°92 (=from LcrVofthe strain Y.pst 2775xxl3)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSSSGTINIHDKSINLMDK NLYGYTDEAIFKASAEYKILEKMPQTTTIQMDGSEKKIVSIKGFLGSENKRTGALGNLKDS YSYNKDNNELSHFATTCSD
SEQ ID N°93 (=from LcrVofthe strain Y.pst YPTl_xl)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSGTINISDGSIDLMNK
TLYGYTDEAIFKASAEYKILEKIPKTTTKNGNGNGNGKKIVSIKDFLGSENKRTGALGNLK
DSYSYNKDNNELSHFATTCSD
SEQ ID N°94 (=from LcrVofthe strain Y.pst 1216_93
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSGTINISDGSIDLMNK
TLYGYTDEAIFKASAEYKILEKIPKTTTKNGNGNGNGNGKKIVSIKDFLGSENKRTGALG
NLKDSYSYNKDNNELSHFATTCSD SEQ ID N°95 (=from LcrVofthe strain Y.pst 304_89
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINISDGSIDLMNK TLYGYTDEAIFKASAEYKILEKIPKTTTKNGNGNGNGNGNGNGNGNGNGKKIVSIKDFLG SENKRTGALGNLKDSYSYNKD NELSHFATTCSD SEQ ID N°96 (=from LcrV of the strain Y.pst 32975 J
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSGTINISDGSIELMNK TLYGYTDEAIFKASAEYKILEKISKTTTKNGNGNGNGNGNGNGNGNGNGNGNGKKIVSI KDFLGSENKRTGALGNLKDSYSYNKD NELSHFATTCSD SEQ ID N°97 (=from LcrVofthe strain Y.pst 2887_x5)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSGTINISDRSIDLMNK TLYGYTDEAIFKASAEYKILEKIPKTTTKNGNGNGNGNGNGNGNGNGNGNGNGKKIVSK KDFLGSENKRTGALGNLKDSYSYNKD NELSHFATSCSD SEQ ID N°98 (=from LcrVofthe strain Y.pst YPTV_xO)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSGTINISDGSIDLMNK TLYGYTDEAIFKASAEYKILEKIPKTTTKNGNGNGNGNGNGNGNGNGNGNGKKIVSIKD FLGSENKRTGALGNLKDSYSYNKD NELSHFATTCSD SEQ ID N°99 (=from LcrVofthe strain Y.e 8081)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKYLSNSGTINIHDKSINLMDK NLYGYTDEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKNSY SYNKD NELSHFATACSD
SEQ ID N°100 (=from LcrVofthe strain Y.e 97/88)
ILKVIVDSMNHHGDARSKLREELAELTAELKIYSVIQAEINKHLSNSDTINIHDKSINLMDK
NLYGYTNEEIFKASAEYKILEKMSQTTIKEGETEKKIVSIKNFLESENKRTGALGNLKDSY
SYNKD NELSHFATTCSD SEQ ID N° 101 (= primer set la)
TCACCGCGCAAAATTATTGC
SEQ ID N°102 (= primer set lb)
TTGTCTGCGATAAGCTCTTG
SEQ ID N° 103 (= primer set 2a)
CCTAGCTTATTTTCTACCCG
SEQ ID N°104 (= primer set 2b)
GAACCGGGGCGTTGGGTAATC
SEQ ID N° 105 (= primer set 2c)
GTTGGTTGTCATAATGACCGCC SEQ ID N° 106 (= primer set 2d) CTAACCAAGTCGTTGAGCGG
SEQ ID N°107 (=CS-2; from LcrVof Y. pseudotuberculosis Clade A) KIPKTTTKNGNGNGNGKKI
(ue. SEQ ID N°9 when y = 0)
SEQ ID N°108 (=CS-2;from LcrVof Y. pseudotuberculosis Clade A) KIPKTTTKNGNGNGNGNGKKI
(ue. SEQ ID N°9 when y = l)
SEQ ID N°109 (=CS-2;from LcrV of Y. pseudotuberculosis Clade A) KIPKTTTKNGNGNGNGNGNGKKI
(i. e. SEQ ID N°9 when y = 2)
SEQ ID N°l 10 (=CS-2;from LcrVof Y. pseudotuberculosis Clade A)
KIPKTTTKNGNGNGNGNGNGNGKKI
( e. SEQ ID N°9 when y = 3)
SEQ ID N°l 11 (=CS-2;from LcrVof Y. pseudotuberculosis Clade A) KIPKTTTK GNGNGNGNGNGNGNGK I
(i.e. SEQ ID N°9 when y = 4)
SEQ ID N°l 12 (=CS-2;from LcrVof Y. pseudotuberculosis Clade A) KIPKTTTKNGNGNGNGNGNGNGNGNGKKI
(ue. SEQ ID N°9 when y = 5)
SEQ ID N°l 13 (=CS-2;from LcrVof Y. pseudotuberculosis Clade A) KIPKTTTKNGNGNGNGNGNGNGNGNGNGKKI
(ue. SEQ ID N°9 when y = 6)
SEQ ID N°l 14 (=CS-2;from LcrVof Y. pseudotuberculosis Clade A) KIPKTTTKNGNGNGNGNGNGNGNGNGNGNGKKI
(i. e. SEQ ID N°9 when y = 7)
SEQ ID N°l 15 (=CS-2;from LcrVof Y. pseudotuberculosis Clade A)
KIPKTTTKNGNGNGNGNGNGNGNGNGNGNGNGKKI
(ue. SEQ ID N°9 when y = 8)

Claims

1. A multivalent immunogenic composition for inducing an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, the said immunogenic composition comprising or expressing at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising :
- at least a first Yersinia LcrV antigen inducing an immune response against Yersinia pestis;
- at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, inducing an immune response against Yersinia pseudotuberculosis; and
- at least a third Yersinia LcrV antigen, different from the first and second Yersinia LcrV antigens, inducing an immune response against Yersinia enterocolitica.
2. The composition according to claim 1, wherein, when the first or the second Yersinia LcrV antigen induces an immune response against Yersinia pestis and Yersinia pseudotuberculosis, then said immunogenic composition:
- is devoid of the second or the first Yersinia LcrV antigen; or
- further comprises or expresses at least one additional Yersinia LcrV antigen(s) inducing an immune response against Yersinia pestis or Yersinia pseudotuberculosis, different from whichever of the first or the second Yersinia LcrV antigen is already present.
3. The multivalent immunogenic composition according to claim 1, wherein:
- the first Yersinia LcrV antigen is represented by the following formula (I):
NH2-[SEQ ID N°A-1]-[CS-1]-[SEQ ID N°B-1]-[AA-1]-[SEQ ID N°C-l]-COOH (I),
- the second Yersinia LcrV antigen is represented by the following formula (II):
NH2-[SEQ ID N°A-2]-[CS-2]-[SEQ ID N°B-2]-[AA-2]-[SEQ ID N°C-2]-COOH (II),
- the third Yersinia LcrV antigen is represented by the following formula (III):
NH2-[SEQ ID N°A-3]-[CS-3]-[SEQ ID N°B-3]-[AA-3]-[SEQ ID N°C-3]-COOH (III), wherein:
- SEQ ID N°A-1, SEQ ID N°A-2 and SEQ ID N°A-3, one independently from the others, mean an amino acid sequence having at least 80% amino acid identity, preferably at least 90% amino acid identity, with the sequence SEQ ID N°2,
- CS-1 means an amino acid sequence of sequence SEQ ID N°8, - CS-2 means an amino acid sequence selected from the group comprising the sequences SEQ ID N°8, 10, 11, 12, 13, 107, 108, 109, 110, 111, 112, 113, 114 and 115,
- CS-3 means an amino acid sequence selected from the group comprising the sequences SEQ ID N°14, 15, 16, 17 and 18,
- SEQ ID N°B-1, SEQ ID N°B-2 and SEQ ID N°B-3, one independently from the others, mean an amino acid sequence having at least 80% amino acid identity, preferably at least 90 % amino acid identity, with the sequence SEQ ID N°3,
- AA-1, AA-2 and AA-3, one independently from the others, mean an amino acid selected from the group consisting of aspartic acid (or aspartate) and asparagine, preferably AA-1 represents asparagine , AA-2 represents aspartic acid and AA-3 represents aspartic acid , and
- SEQ ID N°C-1, SEQ ID N°C-2 and SEQ ID N°C-3, one independently from the others, mean an amino acid sequence having at least 80% amino acid identity, preferably at least 90 % amino acid identity, with the sequence SEQ ID N°4.
4. The composition according to claim 2, wherein the Yersinia LcrV antigen inducing an immune response against Yersinia pestis and Yersinia pseudotuberculosis is represented by the following formula (IV):
NH2-[SEQ ID N°A-4]-[CS-4]-[SEQ ID N°B-4]-[AA-4]-[SEQ ID N°C-4]-COOH (IV), and wherein:
- SEQ ID N°A-4 means an amino acid sequence having at least 80% amino acid identity, preferably at least 90% amino acid identity, with the sequence SEQ ID N°2,
- CS-4 means an amino acid sequence of sequence SEQ ID N° 8,
- SEQ ID N°B-4 means an amino acid sequence having at least 80% amino acid identity, preferably at least 90 % amino acid identity, with the sequence SEQ ID N°3,
- AA-4 represents asparagine, and
- SEQ ID N°C-4 means an amino acid sequence having at least 80% amino acid identity, preferably at least 90 % amino acid identity, with the sequence SEQ ID N°4.
5. The composition according to claim 3, wherein the second Yersinia LcrV antigen(s) has/have the following formula (II):
NH2-[SEQ ID N°A-2]-[CS-2]-[SEQ ID N°B-2]-[AA-2]-[SEQ ID N°C-2]-COOH (II), wherein the [CS-2] amino acid sequence and the [AA-2] amino acid are selected from the groupcomprising:
- CS-2 means an amino acid sequence selected from a group comprising sequences SEQ ID N° 107, 108, 109, 110, 111, 112, 113, 114 and 115, and AA-2 represents aspartic acid, - CS-2 means an amino acid sequence of sequence SEQ ID N° 10 and AA-2 represents aspartic acid;
- CS-2 means an amino acid sequence of sequence SEQ ID N° 11 and AA-2 represents aspartic acid;
- CS-2 means an amino acid of sequence SEQ ID N° 12 and AA-2 represents aspartic acid;
- CS-2 means an amino acid sequence of sequence SEQ ID N° 13 and AA-2 represents aspartic acid;
- CS-2 means an amino acid sequence of sequence SEQ ID N° 8 and AA-2 represents asparagine; and
- the combinations thereof.
6. The composition according to anyone of claims 3 to 5, wherein the third Yersinia LcrV antigen(s) has/have the following formula (III):
NH2-[SEQ ID N°A-3]-[CS-3]-[SEQ ID N°B-3]-[AA-3]-[SEQ ID N°C-3]-COOH (III), wherein the [CS-3] amino acid sequence and the [AA-3] amino acid are selected from the groupcomprising:
- CS-3 means an amino acid sequence of sequence SEQ ID N° 14 and AA-3 represents aspartic acid;
- CS-3 means an amino acid sequence of sequence SEQ ID N° 15 and AA-3 represents aspartic acid;
- CS-3 means an amino acid sequence of sequence SEQ ID N° 16 and AA-3 represents asparagine;
- CS-3 means an amino acid sequence of sequence SEQ ID N° 17 and AA-3 represents aspartic acid ;
- CS-3 means an amino acid sequence of sequence SEQ ID N° 18 and AA-3 represents aspartic acid; and
- the combinations thereof.
7. The composition according to anyone of claims 3 to 6, wherein SEQ ID N°B-1, SEQ ID N°B-2, SEQ ID N°B-3 and SEQ ID N°B-4, one independently from the others, mean an amino acid sequence SEQ ID N°5, wherein:
- X2 means an amino acid selected from the group comprising aspartic acid, glycine and asparagine,
- X3 means an amino acid selected from the group comprising glycine and glutamic acid, and - X4 means an amino acid selected from the group comprising asparagine and lysine, and preferably mean:
- SEQ ID N°B-1 and SEQ ID N°B-2 mean an amino acid sequence having at least 90% amino acid identity with the amino acid sequence SEQ ID N°6;
- SEQ ID N°B-3 means an amino acid sequence having at least 90% amino acid identity with the amino acid sequence SEQ ID N°7; and
- SEQ ID N°B-4 means an amino acid sequence having at least 90% amino acid identity with the amino acid sequence SEQ ID N°23.
8. The composition according to anyone of claims 3 to 7, wherein SEQ ID N°A-1, SEQ ID N°A-2, SEQ ID N°A-3 and SEQ ID N°A-4, one independently from the others, is an amino acid sequence of the following formula (V):
NH2-[SEQ ID N°Aa]-[SEQ ID N°Ab]- (V)
wherein:
- SEQ ID N°Aa, one independently from the other, mean an amino acid sequence having at least 80% amino acid identity, preferably at least 90 %, with the sequence SEQ ID N°19 , and
- SEQ ID N°Ab, one independently from the other, means an amino acid sequence SEQ ID N°20, wherein Xs means an amino acid selected from the group comprising glutamic acid and lysine.
9. The composition according to anyone of claims 3 to 8, wherein SEQ ID N°C-1, SEQ ID N°C-2, SEQ ID N°C-3 and SEQ ID N°C-4, one independently from the others, is an amino acid sequence of SEQ ID N°22 or SEQ ID N°21, wherein Χβ means an amino acid selected from the group comprising threonine and alanine, and preferably of SEQ ID N°21.
10. The composition according to anyone of the preceding claims, wherein said composition comprises or expresses:
- at least one Yersinia LcrV antigen inducing an immune response against Yersinia pestis species,
- at least one, preferably at least two, preferably at least three, in particular at least four, more particularly at least five, preferably at least six, and even at least seven Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis species, one independently from the other, and notably selected among the group as defined in the claim 5, and/or
- at least one, preferably at least two, preferably at least three, in particular at least four, and more particularly at least five, Yersinia LcrV antigen(s) inducing an immune response against Yersinia enterocolitica species, one independently from the other, notably selected among the group as defined in the claim 6.
11. The composition according to anyone of the preceding claims, wherein:
- at least one Yersinia LcrV antigen(s) inducing an immune response against Yersinia pestis is/are selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80% amino acid identity with, preferably at least 90% amino acid identity with, and still better of, sequences SEQ ID N°74 and 75;
- at least one Yersinia LcrV antigen(s) inducing an immune response against Yersinia pseudotuberculosis is/are selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80% amino acid identity with, preferably at least 90% amino acid identity with, and still better of, sequences SEQ ID N°25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 72, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and combination thereof ;
- at least one Yersinia LcrV antigen(s) inducing an immune response against Yersinia enterocolitica is/are selected in the group comprising Yersinia LcrV antigen of amino acid sequences having at least 80% amino acid identity with, preferably at least 90% amino acid identity with, and still better of, sequences SEQ ID N°45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 99 and 100, and combination thereof; and
- the combinations thereof.
12. The composition according to anyone of the preceding claims, wherein each Yersinia LcrV antigens are independently selected from the group consisting of:
- a purified antigen,
- a live attenuated strain comprising and/or expressing the said Yersinia LcrV antigen, - an inactivated or a killed strain comprising and/or expressing the said Yersinia LcrV antigen, or
- a recombinant bacteria comprising and/or expressing the said Yersinia LcrV antigen.
13. The composition according to anyone of the preceding claims, wherein said composition further comprises at least a pharmaceutically acceptable carrier, a diluent and/or an immuno-adjuvant.
14. The composition according to anyone of the preceding claims, wherein said composition further comprises at least one additional active agent against Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica, preferably said additional active agent being selected in a group comprising Mab 7.3, sulfonamides, fluoroquinolones such as ciprofloxacine, levofloxacin, streptomycin, gentamicin, tetracyclines, doxycycline, Oxytetracycline, ampicillin, cephalosporins, aminoglycosides, chloramphenicol, trimethroprime-sulfamethoxazole, amoxicilline/clavulanic acid, cefotaxime, cotrimoxazole, imipeneme, aztreonam, latamoxef and mixtures thereof.
15. A composition according to anyone of the preceding claims, for its use as a vaccine.
16. The composition according to the preceding claim, for its use for inducing a simultaneous immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two, preferably three, Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species.
17. The composition according to the preceding claim, wherein, when said composition is intended to induce an immune response in a non-human vertebrate, notably of the aves class, then said composition comprises or expresses at least two Yersinia LcrV antigens, each different from each other, for inducing an immune response against Yersinia pseudotuberculosis and Yersinia enterocolitica species.
18. The composition according to claiml6, wherein, when said composition is intended to induce an immune response in a Mammalia, especially in a human, then said composition comprises or expresses at least two Yersinia LcrV antigens, each different from each other, for inducing an immune response against Yersinia pestis and Yersinia pseudotuberculosis species, and preferably at least three Yersinia LcrV antigens, each different from each other, for inducing an immune response against Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica.
19. The composition according to claim 17 or 18, wherein the Yersinia LcrV antigens are such as defined in claims 3 to 12.
20. The composition according to anyone of the preceding claims, wherein said composition is administered:
- by topical route,
- by enteral route, including the oral, the nasal and the rectal route ,
- by inhalation or pulmonary route, or
- by parenteral route, including intravenous, intra-arterial, intraosseous infusion, intra-muscular, intracerebral, intracerebroventricular, intrathecal and subcutaneous route.
21. A recombinant bacteria comprising or expressing at least two Yersinia LcrV antigens, each different from each other, said Yersinia LcrV antigens being selected in a group comprising: - at least a first Yersinia LcrV antigen being capable of raising an immune response against Yersinia pestis in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as defined in claims 3, 4, 7, 8, 9, 11 and 12,
- at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, being capable of raising an immune response against Yersinia pseudotuberculosis in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as defined in claims 3, 4, 5, 7, 8, 9, 10, 11 and 12, and
- at least a third Yersinia LcrV antigen, different from the first and the second Yersinia LcrV antigens, being capable of raising an immune response against Yersinia enterocolitica in a vertebrate, notably of the Mammalia and/or of the aves class(es), and is notably such as defined in claims 3, 6, 7, 8, 9, 10, 11 and 12,
at least one, preferably at least two, of said first, second and/or third Yersinia LcrV antigens being a heterologous Yersinia LcrV recombinant antigen.
22. The recombinant bacteria according to the preceding claim, in the genome of which has been inserted at least one, preferably at least two, Yersinia LcrV recombinant antigen(s) selected among a group comprising:
- one or more nucleic acids encoding a first Yersinia LcrV recombinant antigen(s) capable of raising an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against Yersinia pestis, and notably at least one nucleic acid encoding an amino acid sequence such as defined in claims 3, 4, 7, 8, 9 and 11 ;
- one or more nucleic acids encoding a second Yersinia LcrV recombinant antigen(s) capable of raising an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against Yersinia pseudotuberculosis and notably at least one nucleic acid encoding an amino acid sequence such as defined in claims 3, 4, 5, 7, 8, 9 and 11; and/or
- one or more nucleic acids encoding a third Yersinia LcrV recombinant antigen(s) capable of raising an immune response in a vertebrate, notably of the Mammalia and/or of the aves class(es), against Yersinia enterocolitica and notably at least one nucleic acid encoding an amino acid sequence such as defined in claims 3, 6, 7, 8, 9 and 11.
23. The recombinant bacteria according to anyone of claims 21 to 22, wherein said bacteria is selected from the group comprising Lactobacillus, Leuconostoc, Pediococcus,
Lactococcus, Streptococcus, Escherichia, Streptococcus, Agrobacterium, Bacillus, Corynebacterium, Clostridium, Gluconobacter, Citrobacter, Enterobacter, Klebsiella, Aerobacter, Methylobacter, Salmonella, Streptomyces, Yersinia and Pseudomonas. genus, in particular is a probiotic lactic acid bacteria, and preferably is the Lactococcus genus, and more particularly is Lactococcus lactis species.
24. A recombinant bacteria according to anyone of claims 21 to 23, for its use for the simultaneous vaccination of a vertebrate, notably of the Mammalia and/or of the aves class(es), against at least two Yersinia species selected in a group comprising Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species.
25. A device for detecting at least one Yersinia infection due to at least one Yersinia species selected from the group consisting of Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica species, wherein said device comprises at least two Yersinia's LcrV-specific binding molecules selected among the group comprising:
- at least a first Yersinia's LcrV-specific binding molecule(s) being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pestis species;
- at least a second Yersinia's LcrV-specific binding molecule(s), different from the first Yersinia's LcrV-specific binding molecule(s), being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pseudotuberculosis species; and/or
- at least a third Yersinia's LcrV-specific binding molecule(s), different from the first and the second Yersinia's LcrV-specific binding molecules, being capable of binding to at least one Yersinia LcrV antigen of the Yersinia enterocolitica species
26. The device according to the preceding claim, wherein, when the first or the second Yersinia's LcrV-specific binding molecule is capable of binding to Yersinia LcrV antigens of Yersinia pestis and Yersinia pseudotuberculosis , then said device:
- is devoid of the second or the first Yersinia's LcrV-specific binding molecule; or
- further comprises at least one additional Yersinia's LcrV-specific binding molecule(s) being capable of binding to at least one Yersinia LcrV antigen of the Yersinia pestis or Yersinia pseudotuberculosis, different from whichever of the first or the second Yersinia's LcrV-specific binding molecule is already present.
27. The device according to claim 25 or 26, wherein each Yersinia's LcrV-specific binding molecules, one independently from the others, are selected from the group consisting of:
- an antibody,
- a nucleic acids or a peptide aptamer, and
- a mixture thereof.
28. A device for detecting antibodies anti- Yersinia's LcrV in a vertebrate, notably of the Mammalia and/or of the aves class(es), wherein said device comprises at least two Yersinia LcrV antigens, one distinct from each other, said Yersinia LcrV antigens being selected in a group comprising :
- at least a first Yersinia LcrV antigen being the LcrV antigen of Yersinia pestis species;
- at least a second Yersinia LcrV antigen, different from the first Yersinia LcrV antigen, being the LcrV antigen of Yersinia pseudotuberculosis species; and
- at least a third Yersinia LcrV antigen, different from the first and second Yersinia LcrV antigens, being the LcrV antigen of Yersinia enterocolitica species.
29. A kit comprising at least:
- a multivalent immunogenic composition such as defined in anyone of claims 1 to 14, and
- a device for detecting antibodies anti- Yersinia's LcrV in a vertebrate, notably of the Mammalia and/or of the aves class(es), and notably such as defined in claim 28.
PCT/EP2016/062047 2015-05-29 2016-05-27 Multivalent immunogenic composition for inducing an immune response against yersinia species Ceased WO2016193161A1 (en)

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