Recombinant yeast cell expressing bacterial antigens intracellularly and on its extracellular surface and its vaccine application

Abstract

The present invention relates to a recombinant yeast cell genetically modified to express (i) at least one first bacterial antigen or an immunogenic fragment thereof intracellularly and (ii) at least one second bacterial antigen or an immunogenic fragment thereof on its extracellular surface, the at least one first bacterial antigen or an immunogenic fragment thereof being identical to or different from the at least one second bacterial antigen or an immunogenic fragment thereof. The present invention also concerns this recombinant yeast for use in vaccines and in treating or preventing bacteria-caused pathologies in livestock.

Description

[0001] RECOMBINANT YEAST CELL EXPRESSING BACTERIAL ANTIGENS INTRACELLULARLY AND ON ITS EXTRACELLULAR SURFACE AND ITS VACCINE APPLICATION

[0002] DESCRIPTION

[0003] TECHNICAL FIELD

[0004] The present invention relates to the technical field of vaccines and in particular to whole yeast vaccines.

[0005] More particularly, the present invention relates to recombinant yeast which has been genetically modified to express antigen(s) both intracellularly and on its extracellular surface. The present invention also concerns this recombinant yeast for use in vaccines and in treating or preventing bacteria-caused pathologies in livestock or farm animals such as, for example, mastitis, bovine tuberculosis and paratuberculosis.

[0006] STATE OF PRIOR ART

[0007] Epidemics among livestock pose significant challenges to agricultural communities, public health, and economies worldwide. Outbreaks of infectious diseases caused by bacteria such as, for example, paratuberculosis, bovine tuberculosis, metritis, lameness, respiratory infections and gastrointestinal infections can lead to widespread morbidity and mortality among animals, necessitating emergency control measures that often include culling of entire herds or flocks. Such events result in substantial economic losses to farmers due to death of animals, reduced productivity, and trade restrictions.

[0008] Even if mastitis that is an inflammatory response of the mammary gland, usually caused by bacterial infections does not cause mortality in affected animals, it is however a prevalent and economically significant condition affecting dairy cows, goats, sheep, and other milk-producing animals. Indeed this condition can result in reduced milk yield, altered milk composition, and can have severe welfare implications for the affected animals.

[0009] Treatment of these infectious diseases caused by bacteria often involves the use of antibiotics. In particular mastitis is the first cause of antibiotics use in dairy farming. However increasing concerns about antibiotic resistance are prompting the industry to explore alternative strategies. Vaccination of livestock serves as a proactive measure to build immunity within animal populations, thereby reducing the incidence and spread of infectious agents. Implementing widespread vaccination protocols can diminish the need for drastic measures like mass culling and the use of broad-spectrum antibiotics.

[0010] Great effort has been made in the last decades to develop vaccines for bacteria- caused disease in livestock such as for mastitis. However, the efficacy of available options is still controversial, as they fail to prevent mammary gland infection and promote only limited economic benefit in field trials [1], Commercial and candidate vaccines have been mostly conceived to elicit antibody production, but accumulating evidence shows that induction of strong cell-mediated immune mechanisms, particularly T-helper 1 (Thl) and Thl7-type immunity, is necessary for protection of the mammary gland (MG) [2],

[0011] Yeasts have been recently identified as promising workhorses for vaccine developments. Whole yeast-based vaccines represent an innovative approach to immunization that utilizes the entire yeast cell as a platform for vaccine development [3,4]. Indeed, due to the immunogenic nature of their conserved cell wall carbohydrates, whole yeast-based vaccines do not require adjuvants and proved to induce Thl, Thl7 and cytotoxic T-cell responses in vivo, surpassing the antibody-biased responses elicited by conventional adjuvanted vaccines [5].

[0012] Vaccine design has been largely focused on the induction of antibody responses. In this context, vaccine developers tend to favour antigen expression on the surface of the vaccine particle, as this approach might facilitate antigen recognition by memory B-cells and improve humoral responses.

[0013] Nevertheless, previous attempts to produce yeast-based vaccines exploring antigen expression on the surface neglected the limited capacity of yeast to display heterologous proteins on their surface. This lead to the production of vaccines carrying a low amount of antigen that is not adapted to vaccination of large animals. Experimental yeastbased vaccines have been tested in murine models at a dose of 1,5.109colony-forming units (CFU) (30 mg) per mice of approximately 25 g [6,7].

[0014] The present inventors have set themselves the goal of proposing an effective strategy for achieving efficient vaccination of livestock and in particular large livestock against infectious diseases caused by bacteria. DISCLOSURE OF THE INVENTION

[0015] The present invention enables the technical problems of vaccination processes of prior art such as those previously defined to be solved and the goal set by the inventors to be reached.

[0016] First of all, the present inventors have observed that a dose (2.109CFU) similar to the one implemented in [6,7] induces poor immune responses against the target antigen in bovine (see below experimental part). In this sense, the production of whole-cell yeast expressing antigens on their surface in sufficient quantity to immunise a large animal such as a dairy cow of approximately 700 Kg would represent an economical and technical handicap to potential manufacturers.

[0017] To that end, the inventors have explored for the first time a double expression system consisting of a surface display coupled to intracellular expression to produce a yeastbased vaccine.

[0018] Besides resulting in the distribution of antigens in two different yeast cell compartments, this strategy also enables to increase the amount of antigens produced per each yeast cell in significantly higher amount than yeast cell genetically modified to express antigens intracellularly and yeast cell genetically modified to express antigens on its extracellular surface (see Figure 7 below).

[0019] Consequently immunisation with yeast expressing a higher amount of antigens (on the surface and intracellularly) at a dose of 5.109CFU / immunisation is sufficient to induce cell- mediated immune mechanisms in bovine as demonstrated in the hereinafter experimental part.

[0020] At the time of the invention, this synergetic effect was not at all obvious for those skilled in the art. In addition the need for transforming yeast cells with two different expression vectors such as plasmids to produce antigens on the surface and in the cytoplasm would have discouraged those skilled in the art to implement such a strategy. The latter would have explored a simpler approach based on the transformation with a single plasmid enabling antigen expression only on yeast surface. Then, selecting transformants containing the two different expression vectors such as plasmids relies on the depletion of two amino acids from the culture medium at the same time such as, for example, tryptophan and leucine. Most of laboratories use less laborious approaches to screen transformants i.e. selection based only on one amino acid, such as tryptophan, depletion and culture of yeast in minimum medium with casamino acids as source of amino acids.

[0021] In addition, in the invention, the present inventors have exploited the immunogenic nature of yeast sugar-rich cell wall to facilitate phagocytosis by antigen presenting cells and improve T-cell priming / activation.

[0022] Thus, the present invention concerns a recombinant yeast cell genetically modified to express (i) at least one first bacterial antigen or an immunogenic fragment thereof intracellularly and (ii) at least one second bacterial antigen or an immunogenic fragment thereof on its extracellular surface, the at least one first bacterial antigen or an immunogenic fragment thereof being identical to or different from the at least one second bacterial antigen or an immunogenic fragment thereof.

[0023] In the present invention, the term « yeast » and the expression « yeast cell » are equivalent and can be used interchangeably.

[0024] In the present invention, the expressions « the at least one first bacterial antigen and the at least one second bacterial antigen » and « the bacterial antigens implemented in the invention » are equivalent and can be used interchangeably.

[0025] By « recombinant yeast », it is intended yeast not found in nature and which is genetically different from its equivalent in nature. The terms « equivalent in the nature », « non-modified yeast », « natural yeast » and « wild type yeast » are equivalent and usable interchangeably. The recombinant yeast according to the invention is modified by introduction, deletion and / or modification of genetic elements and in particular by introduction of bacterial genetic elements.

[0026] By way of examples of yeasts usable within the scope of the present invention, yeasts from the families Saccharomycetaceae, Pichiaceae, Schizosaccharomycetaceae and Yarrowia can be mentioned. Avantageously, the yeast of the present invention is selected from the group consisting of Schizosaccharomyces pombe, Saccharomyces cerevisiae, Saccharomyces boulardii, Hansenula polymorpha, Pichia pastoris, Candida boldmu, Kluyveromyces lactis and Yarrowia lipolytica. In particular, the yeast of the present invention is selected from the group consisting of Schizosaccharomyces pombe, Saccharomyces cerevisiae, Hansenula polymorpha, Pichia pastoris, Candida boldmu, Kluyveromyces lactis and Yarrowia lipolytica. More particularly, the yeast of the present invention is either Saccharomyces cerevisiae or Pichia pastoris. Even more particularly, the yeast in the present invention is Saccharomyces cerevisiae (5. cerevisiae).

[0027] The recombinant yeast cell of the present invention is genetically modified to express at least one exogenous gene. The recombinant yeast cell of the present invention is modified to express exogenous genes if these genes are introduced with all the elements enabling them to be expressed in this microorganism. Those skilled in the art know different modification, transformation or transfection methods of yeast with an exogenous gene. By way of example and not exhaustively, this method can be a conjugation; an electroporation; a lipofection; a micro-injection; a particle bombardment (or biolistic); a biological transformation of a plant using Agrobacterium tumefasciens; a transformation by a chemical permeabilisation; a transformation by the DEAE-dextran method or a transformation by lithium acetate / single-stranded carrier DNA / PEG method (LiAc SS carrier DNA / PEG method) [8].

[0028] By « exogenous gene », it is intended a gene which has been introduced in the yeast of the present invention, by means well known to those skilled in the art whereas this gene is not naturally found in this yeast. Exogenous genes can be integrated in one of the chromosome of the yeast or be expressed extra-chromosomally by means of expression vectors. In the present invention, the exogenous genes implemented are genes coding bacterial antigens and in particular genes coding bacterial antigens expressed extrachromosomally by means of expression vectors.

[0029] In a particular embodiment, the recombinant yeast cell of the invention comprises (or contains):

[0030] (i') a first expression vector comprising a first nucleotide sequence encoding at least one first bacterial antigen or an immunogenic fragment thereof operably linked to at least one first sequence controlling the intracellular expression of the at least one first bacterial antigen or an immunogenic fragment thereof and

[0031] (ii') a second expression vector comprising a nucleotide sequence encoding at least one second bacterial antigen or an immunogenic fragment thereof operably linked to at least one second sequence controlling the expression of the at least one second bacterial antigen or an immunogenic fragment thereof on the extracellular surface of this yeast cell.

[0032] By « expression vector », it is intended a nucleic acid adapted to express, either in the cytoplasm of a yeast cell or on the extracellular surface of this yeast cell, at least one bacterial antigen coded by a nucleotide sequence contained in this vector. Each expression vector implemented in the invention comprises, in addition to the nucleotide sequence coding for at least one bacterial antigen, one (or more) element(s) which enable(s) this nucleotide sequence to be expressed i.e. transcribed and translated and in particular to be expressed either intracellularly or on the cell surface.

[0033] The expression vectors implemented in the invention are advantageously either plasmids or cosmids. In particular, expression vectors implemented in the invention are plasmids.

[0034] More particularly, the expression vectors implemented in the invention are autonomously replicating vectors including elements enabling it to be maintained and replicated in the yeast such as a replication origin. Further, these expression vectors include elements enabling them to be selected in the yeast. These elements are also known as « selection markers ». Such expression vectors are well known to those skilled in the art and widely described in literature.

[0035] By « selection marker », it is intended a marker usable in yeast such as a metabolism gene to be used with auxotrophic yeast, i.e. a selection gene which ensures the complementation with the respective gene deleted from the genome of the host yeast. By way of illustrating examples of selection metabolism genes, one can mention the gene trpl to be used with yeast trpl~ i.e. yeast lacking the phosphoribosylanthranilate isomerase enzyme; the gene Ieu2 to be used with yeast Ieu2~ i.e. yeast lacking the beta-isopropylmalate dehydrogenase enzyme; the gene his3 to be used with yeast his3~ i.e. yeast lacking the imidazoleglycerol-phosphate dehydratase enzyme; the gene ura3 to be used with yeast urcr i.e. yeast lacking the orotidine 5-phosphate decarboxylase enzyme; the tk gene to be used with yeast depleted of the thymidine kinase enzyme; the ada gene to be used with yeast depleted of the adenosine deaminase enzyme; the apt gene to be used with yeast depleted of the adenine phosphoribosyl-transferase enzyme and the Hprt gene to be used with yeast depleted of the Hypoxanthine-guanine phosphoribosyl-transferase enzyme. It is to be pointed out that the expression vector implemented in the invention may also contain a selectable marker which can be used in prokaryotes such as a bacterial gene having resistance to an antibiotic such as ampicillin, neomycin, hygromycin, geneticin, carboxin, nourseothricin or G418. Thanks to this bacterial selection marker, the expression vectors implemented in the invention can be cloned and amplified in bacteria before being used in yeast. By « promoter » in the invention is meant both a promoter, constitutive or inducible, adapted for any eukaryotic cell and in particular for yeast. A promoter adapted for any eukaryotic cell which can be used in the present invention is notably selected in the group consisting of the CMV promoter (CytoMegaloVirus) and, in particular, intron A of this promoter; the CYCl-TetO-7 promoter; the early SV40 promoter (Simian Virus 40); the HSV promoter (Herpes Simplex Virus); the GPD (TDH3) promoter (promoter from glyceraldehyde- 3-phosphate deshydrogenase); constitutive TEF1 promoter (promoter from the gene encoding translation elongation factor la)and TEV promoter (Tobacco Etch Virus). In addition, a yeast inducible promoter which can be used in the present invention can be the GALI promoter inducible by galactose, the AOX1 promoter inducible by methanol, the ADH-2 promoter inducible by glucose depletion, the MET15 promoter inducible by methionine depletion or the CUP1 promoter inducible by copper ions. In the expression vector implemented in the present invention, the promoter can be associated with one or more transcriptional regulation sequences i.e. enhancers.

[0036] Advantageously, each expression vector implemented in the invention comprises, operationally linked together, a promoter, a nucleotide sequence encoding at least one bacterial antigen and a transcription terminating signal comprising a cleavage site and / or polyA signal. By « operationally linked together » in the invention is meant elements linked together so that the functioning of one of the elements is affected by the functioning of the other. For example, a promoter is operationally linked to a coding sequence when it is capable of affecting the expression thereof. The elements regulating the transcription, translation and maturation of peptides which may be contained in the vector are known to those skilled in the art who are capable of choosing from among these in relation to yeast in which expression or cloning are to be performed.

[0037] In the second expression vector implemented in the invention for bacteria antigen(s) or immunogen fragment(s) thereof to be expressed on the extracellular surface of the recombinant yeast, the nucleotide sequence encoding at least one second bacterial antigen or an immunogenic fragment thereof is typically a nucleotide sequence encoding a fusion protein comprising at least one second bacterial antigen or an immunogenic fragment thereof.

[0038] By « fusion protein » is meant a protein comprising at least two polypeptides from one same source or from different sources functionally linked to each other whereby each polypeptide of the fusion protein maintains its own function or activity. The two polypeptides of the fusion protein can be directly linked to each other via a peptide bond, or indirectly via a spacer arm (or link arm or junction agent) separating the two polypeptides. The 2ndpolypeptide may be directly or indirectly bound to the C-terminal or N-terminal end of the 1stpolypeptide. If the bond is a direct bond, the nucleotide sequences encoding each of the polypeptides are linked to one another in 5'-3' direction whereby the translation frame of the encoded polypeptides is not deteriorated.

[0039] In the fusion protein implemented in the invention, one of these two polypeptides is the at least one second bacterial antigen or an immunogenic fragment thereof and the other one is a yeast extracellular surface protein such as, for example, A-agglutinin-binding subunit Aga2p, A-agglutinin-binding subunit Agalp, cell wall protein 1 (Cwplp), cell wall protein 2 (Cwp2p), Tiplp (Temperature shock-lnducible 1 protein), Flolp (FLOcculation 1 protein), Sedlp (Suppression of Exponential Defect 1), YCR89W (or FIG2 for Factor-Induced gene 2 protein), and Tirl (Tipi-Related). Advantageously the sequence of the gene coding the at least one second bacterial antigen or an immunogenic fragment thereof does not contain any sequence coding for a peptide signal.

[0040] In other words, in this embodiment, the nucleotide sequence encoding at least one second bacterial antigen or an immunogenic fragment thereof is a nucleotide sequence encoding a fusion protein comprising at least one second bacterial antigen or an immunogenic fragment thereof and a yeast extracellular surface protein.

[0041] As examples of expression vectors implemented in the invention for bacteria antigen(s) to be expressed on the surface of the recombinant yeast, one can cite plasmids pYDl and pCTcon2 sold by Addgene and plasmid pPIC9AGA2 disclosed in Jacobs et al, 2008 [9].

[0042] In the first expression vector implemented in the invention for bacteria antigen(s) to be expressed intracellularly in the recombinant yeast, the gene coding the at least one first bacterial antigen or an immunogenic fragment thereof is under the control of a strong constitutive yeast promoter such as, for example, the GPD and TEF1 promoters or the GAL inducible promoter. In addition the sequence of the gene coding the at least one first bacterial antigen or an immunogenic fragment thereof does not contain any sequence coding for a peptide signal. As examples of expression vectors implemented in the invention for bacteria antigen(s) to be expressed in the cytoplasm of the recombinant yeast, one can cite plasmid p425-GPD (ATCCW87359), p425-TEF (ATCCW87367), p423-GDP (ATCCW87355), p423-TEF (ATCCW87363), p415-GPD (ATCCW87358) and p413-GPD (ATCCW87354).

[0043] In a particular embodiment, the recombinant yeast cell according to the invention is inactivated. First of all, the inactivation of the yeast cell after genetically modifying it and before using it as immunogen in a vaccine minimizes eventual risks regarding vaccine biosafety. In addition using an inactivated whole yeast is a way to generate vaccine particles unable to enter lymphatic vessels by direct diffusion and exclusively shuttled to draining lymph-nodes by antigen presenting cells, which favours T-cell priming . Any method known to those skilled in the art to inactivate yeasts can be used in the present invention. Typically, this inactivation involves a heat treatment at a temperature ranging from 54°C to 95°C for a duration ranging from 15 min to 2h. Sometimes, this heat treatment may be followed by a lyophilisation step. Alternatively, this inactivation involves a chemical treatment such as a treatment with 4% formaldehyde at room temperature (23°C ± 5°C) for a duration ranging from 5 min to lh.

[0044] According to the usual definition, an antigen is a natural or synthetic macromolecule which, when recognized by antibodies or cells of an organism's immune system, can trigger an immune response. An immunogenic fragment of an antigen is a part of the antigen directly involved in triggering the immune response.

[0045] « Immune response » as used herein, refers to a response of a cell of the immune system, such as a B cell, T cell, dendritic cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen or vaccine. An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate and / or adaptive immune response.

[0046] Typically, the bacterial antigens implemented in the present invention are selected from the group consisting of bacterial proteins, bacterial protein fragments, bacterial polypeptides, bacterial peptides and synthetic polypeptides comprising at least two bacterial peptides identical or different, from identical or different bacterial strains. As examples of bacterial protein fragments, one can mention bacterial protein depleted of its N-terminus peptide signal, bacterial protein depleted of its 10, 20, 30, 40 or 50 first amino acids, protein fragments and / or domains not conserved between bacteria and animals and protein fragments and / or domains not conserved between pathogenic and commensal bacteria.

[0047] The expressions "at least one first bacterial antigen" and the expression "at least one second bacterial antigen" clearly mean that one or more bacterial antigens are expressed intracellularly and that one or more bacterial antigens are expressed at the extracellular surface of the yeast cell. When several bacterial antigens are expressed either intracellularly or at the extracellular surface of the yeast cell, they are expressed as fusion proteins. What has already been explained for fusion protein also applies to the expression of at least two different bacterial antigens mutatis mutandis.

[0048] In addition, the yeast cell of the present invention can comprise one or more first expression vector(s) as previously defined and one or more second expression vector(s) as previously defined.

[0049] In a particular embodiment, the at least one first bacterial antigen and the at least one second bacterial antigen expressed in the recombinant yeast cell according to the invention are independently selected from the group consisting of antigens from Escherichia coli, antigens from Klebsiella spp., antigens from Staphylococcus aureus, antigens from coagulase negative Staphylococcus sp., antigens from Streptococcus uberis, antigens from Streptococcus agalactiae, antigens from Mycobacterium bovis, antigens from Mycobacterium avium subspecies paratuberculosis (MAP), antigens from Trueperella pyogenes, antigens from Prevotella sp., antigens from Treponema sp. and antigens from Fusobacterium sp..

[0050] The bacterial antigens to be implemented in the invention can be selected amongst metabolic pathway enzymes, bacterial virulence factors and bacterial antigens expressed by bacteria in milk, in the mammary gland, in the lungs, in feces and / or in the guts.

[0051] In a particular embodiment, the at least one first bacterial antigen and the at least one second bacterial antigen are independently selected from the group consisting of Enterobacteriaceae cell division protein FtsA (NCBI Reference WP_000588474.1), Enterobacteriaceae rod shape-determining protein MreB (NCBI Reference WP_000913396.1), Enterobacteriaceae phosphoporin PhoE (NCBI Reference WP_000749881.1^, Enterobacteriaceae cysteine synthase A (NCBI Reference WP_000034402.1), Enterobacteriaceae porin OmpF (NCBI Reference WP_000977905), E. coli Outer membrane protein A hereinafter "OmpA" (GenBank protein ID WWN84339.1), 5. aureus Enolase hereinafter "Enolase" (GenBank protein ID VDZ34627.1), 5. aureus Elongation Factor Tu hereinafter "EloTu" (GenBank protein ID BCD42744.1), 5. aureus 50S ribosomal protein L6 (GenBank protein ID BCD44412.1), 5. aureus Bifunctional pyr operon transcriptional regulator / uracil phosphoribosyltransferase hereinafter "PyrR" (GenBank ID BCD43304.1), 5. aureus Universal stress protein hereinafter "UspA" (GenBank protein ID QKE58197.1), 5. aureus Ferritin-like protein 2 (GenBank protein ID VDZ35843.1), 5. aureus Galactose-6- phosphate isomerase, LacB subunit (GenBank ID VDZ36124.1), 5. aureus

[0052] Phosphoribosylformylglycinamide synthase (GenBank protein ID VDZ34869.1), 5. aureus Immunodominant staphylococcal antigen IsaB (GenBank protein ID WP_046463258.1), 5. aureus Asp23 / Gls24 family envelope stress response (GenBank ID BCD44345.1), 5. aureus Alpha / beta hydrolase hereinafter "Lipase" (GenBank ID WPA54171.1), 5. aureus

[0053] Staphylocoagulase hereinafter "Coagulase" (GenBank ID WPA42669.1), 5. uberis staphylokinase domain-containing protein (NCBI Reference WP_015912075.1), 5. uberis surface-displayed alpha-enolase (NCBI Reference WP_012658173.1), 5. uberis sortase (NCBI Reference WP_012658383.1), 5. uberis cell division protein FtsZ (NCBI Reference WP_012658764.1), 5. uberis cell division protein FtsA (NCBI Reference WP_012658765.1), 5. uberis cell division protein FtsL (NCBI Reference WP_015911740.1), 5. uberis septation ring formation regulator EzrA (NCBI Reference WP_012658169.1), 5. uberis ubericin A precursor peptide (GenBank ID ABQ23939.1), 5. uberis ubericin A immunity protein (GenBank ID ABQ23940.1), 5. uberis circular bacteriocin uberolysin (GenBank ID ABG48503), M. bovis ESAT- 6 like proteins (

[0010] ), M. bovis CFP-10 (

[0010] ), Mycobacterium avium subsp. Paratuberculosis (MAP) antigen complex 85B (

[0011] ), MAP antigen complex 85C (

[0011] ), MAP superoxide dismutase (

[0011] ), MAP polyprotein 74F (

[0011] ) and homologues thereof.

[0054] In a particular embodiment, the at least one first bacterial antigen and the at least one second bacterial antigen are independently selected from the group consisting of E. coli Outer membrane protein A hereinafter "OmpA" (GenBank protein ID WWN84339.1), 5. aureus Enolase hereinafter "Enolase" (GenBank protein ID VDZ34627.1), 5. aureus Elongation Factor Tu hereinafter "EloTu" (GenBank protein ID BCD42744.1), 5. aureus Bifunctional pyr operon transcriptional regulator / uracil phosphoribosyltransferase hereinafter "PyrR" (GenBank ID BCD43304.1), 5. aureus Universal stress protein hereinafter "UspA" (GenBank protein ID QKE58197.1), 5. aureus Alpha / beta hydrolase hereinafter "Lipase" (GenBank ID WPA54171.1), 5. aureus Staphylocoagulase hereinafter "Coagulase" (GenBank ID WPA42669.1) and homologues thereof. In a more particular embodiment, the at least one first bacterial antigen and the at least one second bacterial antigen are independently selected from the group consisting of E. coli Outer membrane protein A hereinafter "OmpA" (GenBank protein ID WWN84339.1), 5. aureus Bifunctional pyr operon transcriptional regulator / uracil phosphoribosyltransferase hereinafter "PyrR" (GenBank ID BCD43304.1), 5. aureus Universal stress protein hereinafter "UspA" (GenBank protein ID QKE58197.1), 5. aureus Staphylocoagulase hereinafter "Coagulase" (GenBank ID WPA42669.1) and homologues thereof.

[0055] In the list of antigens of this more particular embodiment, can be added

[0056] - 5. aureus Elongation Factor Tu hereinafter "EloTu" (GenBank protein ID BCD42744.1), or

[0057] - 5. aureus Alpha / beta hydrolase hereinafter "Lipase" (GenBank ID WPA54171.1), or

[0058] - 5. aureus Enolase hereinafter "Enolase" (GenBank protein ID VDZ34627.1), or

[0059] - 5. aureus Elongation Factor Tu hereinafter "EloTu" (GenBank protein ID BCD42744.1) and 5. aureus Alpha / beta hydrolase hereinafter "Lipase" (GenBank ID WPA54171.1), or

[0060] - 5. aureus Elongation Factor Tu hereinafter "EloTu" (GenBank protein ID BCD42744.1) and 5. aureus Enolase hereinafter "Enolase" (GenBank protein ID VDZ34627.1), or

[0061] - 5. aureus Alpha / beta hydrolase hereinafter "Lipase" (GenBank ID WPA54171.1), and 5. aureus Enolase hereinafter "Enolase" (GenBank protein ID VDZ34627.1).

[0062] By « protein homologue », it is intended a protein homologous to a reference protein i.e. a protein having a similar function and / or a similar structure as the reference protein. Thus, when the reference protein is an enzyme, a protein homologous to this reference protein catalyses the same enzymatic reaction.

[0063] By using references given in databases of amino acid or nucleotide sequences such as Genbank or NCBI BioProject for known genes or proteins, those skilled in the art are capable of determining genes or proteins which are homologous i.e. equivalent in other bacterial strains. This routine work is advantageously made by using consensus sequences identified by sequence alignments with genes or proteins, derived from other bacterial strains.

[0064] Typically, a protein (or a gene) homologous to a reference protein (or a reference gene) has at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% and / or at least 99% identity respectively with the amino acid sequence of the reference protein (or the nucleotide sequence of the reference gene). By "identity percentage" between two amino acid sequences (or between two nucleotide sequences), it is intended, within the scope of the present invention, a percentage of amino acid residues (or nucleotides) that are identical between both compared sequences, this percentage being obtained after implementing the best alignment (optimum alignment) between both sequences. Those skilled in the art know different techniques enabling such an identity percentage to be obtained and involving homology algorithms or computer programs such as the BLAST program.

[0065] In another particular embodiment, in the recombinant yeast of the invention, the expression of at least one gene involved in the synthesis or assembly of cell wall sugar components is altered. In other words, the expression of at least one gene involved in the synthesis or assembly of cell wall sugar components can be suppressed, decreased or increased whereby the nature and / or the amount of cell wall sugar components differ from the nature and / or the amount of cell wall sugar components present in the same yeast before this gene alteration.

[0066] Within the scope of the invention, gene expression can be decreased or suppressed by (ii) modifying the promoter, the regulatory regions and / or the ribosome binding site so as to achieve a reduced expression of the gene, (i i i) modifying the sequence of the gene so as to achieve a reduced expression of the gene and / or (i ii i) inactivating the gene in particular by total or partial deletion of said gene, by total or partial deletion of the promoter preventing any expression of the gene and / or by inserting an external gene element in the coding region of the gene or in the promoter region.

[0067] Within the scope of the invention, gene expression can be increased by (iz) increasing the number of copies of the gene in the yeast and / or (iiz) modifying the promoter, the regulatory regions and / or the ribosome binding site so as to achieve an increase expression of the gene.

[0068] In a particular embodiment, at least one gene involved in the synthesis or assembly of a particular cell wall sugar component is deleted in the recombinant yeast cell of the invention such as 5. cerevisiae and notably by using the KanMX4 resistance marker. In a particular embodiment, the at least one gene involved in the synthesis or assembly of cell wall sugar components the expression of which is altered is selected from the group consisting of genes gasl, krel, Ias21, mnn2, chs3, kre6, exgl,fksl and ccwl2.

[0069] In a more particular embodiment, the at least one gene involved in the synthesis or assembly of cell wall sugar components the expression of which is altered is kre6.

[0070] The gene gasl (for « Glycophospholipid-Anchored Surface protein ») codes for a beta-1, 3-glucanosyltransferase required for cell wall assembly. The gene krel (for « Killer toxin Resistant 1 ») codes for a cell wall glycoprotein involved in beta-glucan assembly. The gene Ias21 (for « Local Anestheticum Sensitive 21 ») codes for a mannose-ethanolamine phosphotransferase; involved in biosynthesis of the glycosylphosphatidylinositol (GPI) core structure. The gene mnn2 (for « MaNNosylTransferase 2 ») codes for an Alpha-1, 2- mannosyltransferase responsible for addition of the first alpha-1, 2-linked mannose to form the branches on the mannan backbone of oligosaccharides. The gene chs3 (for « Chitin Synthase-related 3 ») codes for a chitin synthase III that catalyzes the transfer of N- acetylglucosamine (GIcNAc) to chitin and that is required for synthesis of the majority of cell wall chitin. The gene kre6 (for « Killer toxin Resistant 6 ») codes for a glucosyl hydrolase required for beta-1, 6-glucan biosynthesis. The gene exgl (for « EXo-l,3-beta-glucanase 1 ») codes for the major exo-l,3-beta-glucanase of the cell wall involved in cell wall beta-glucan assembly. The gene fksl (for « FK5O6 Sensitivity 1 ») codes for the catalytic subunit of 1,3- beta-D-glucan synthase. The gene ccwl2 (for « Covalently linked Cell Wall protein 12 ») codes for a cell wall mannoprotein.

[0071] The present invention concerns a vaccine comprising as immunogen at least one recombinant yeast cell as previously defined.

[0072] Indeed, the recombinant yeast cell as previously defined and in particular the antigen fragments it expresses intracellularly and on its extracellular surface presents an immunogenic activity. The term « immunogenic activity » refers to the ability of this recombinant yeast cell to elicit an immunological response in a subject and in particular a mammal. An immunological response to the recombinant yeast cell is the development in a subject and in particular a mammal of a cellular and / or antibody-mediated immune response to the recombinant yeast cell. Usually, an immunological response includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, suppressor T cells and / or cytotoxic T cells, directed to an epitope or epitopes of the recombinant yeast cell as previously defined and in particular of the antigen fragments it expresses. The term « epitope » refers to the site on an antigen to which specific B cells and / or T cells respond so that antibody is produced.

[0073] The immunogenic activity of the recombinant yeast cell as previously defined and in particular the antigen fragments it expresses may be protective thanks to a protective immune response. In the present invention, a « protective immune response » refers to an immune response that protects an animal and in particular a mammal from bacterial infection i.e. that prevents bacterial infection or prevents the development of disease(s) associated with bacterial infection. Methods for measuring immune responses are well known in the art and include, for example, measuring proliferation and / or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation and antibody production.

[0074] In the present invention, « prevention », refers to prophylaxis, avoidance of disease manifestation, a delay of onset, and / or reduction in frequency and / or severity of one or more symptoms of a bacterial infection. In some embodiments, prevention is assessed on a population basis such that an agent is considered to "prevent" a particular bacterial infection if a statistically significant decrease in the development, frequency, and / or intensity of one or more symptoms of the bacterial infection is observed in a population susceptible to the bacterial infection.

[0075] Thus the present invention also concerns a vaccine composition comprising at least one recombinant yeast cell as previously defined and a pharmaceutically acceptable excipient.

[0076] The expression "at least one recombinant yeast cell" implemented for the vaccine or the vaccine composition of the invention clearly means that the vaccine or the vaccine composition may comprise one or more recombinant yeast cells as previously defined. When the vaccine or the vaccine composition comprises several yeast cells, each yeast cell differ from the other one(s) by at least one bacterial antigen expressed intracellularly or at the surface of this cell.

[0077] The vaccine composition of the invention can be prepared in a wide variety of conventional dosage forms such as, for example, injectable formulations, granules, tablets, pills, powders, suppositories, capsules, solutions, emulsions, syrups, suspensions and sprays.

[0078] Typically, the vaccine compositions of the invention contains at least one recombinant yeast cell as a previously defined combined with a pharmaceutically acceptable excipient. A pharmaceutically acceptable excipient comprises a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable diluent such as, for example, water, saline, phosphate buffered saline, glycerol and ethanol and / or auxiliary substances such as, for example, lubricants, dyes, coatings, wetting agents, isotonisers, emulsifying agents and pH buffering substances.

[0079] A pharmaceutical acceptable carrier does not itself induce the deleterious effects on the subject receiving the vaccine composition of the invention. Suitable carriers which can be implemented in the preparation of vaccine composition are well-known to those skilled in the art. As examples of carriers usable in the vaccine composition of the invention, one can mention large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acid, polyglycolic acid, amino acid polymers, amino acid copolymers and lipid aggregates such as oil droplets or liposomes.

[0080] Usually, vaccine compositions contain adjuvants that enhance the effect of the immunological composition. These adjuvants include, for example, alum, oil adjuvant, saponin adjuvant, Freund complete adjuvant, Freund's incomplete adjuvant, cytokines, chemokines, interferons, macrophage colony stimulating factor and tumor necrosis factor (TNF).

[0081] Even if the vaccine composition of the invention may contain at least one of such adjuvants, there is no need of adjuvants since, as already explained, this vaccine composition implements a whole yeast. Advantageously, the vaccine composition of the invention is free of adjuvant.

[0082] The vaccine of the invention or vaccine composition of the invention contains an immunologically effective amount of recombinant yeast cells as previously defined.

[0083] An « immunologically effective amount » means that the amount administered to a subject as a single dose or as part of a continuous agent is effective for treatment or prophylaxis. The amount can vary depending on the health and physiology of the subject to be treated, the type, age and weight of the subject to be treated, the ability of the subject's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine or of the vaccine composition and the medical physician's assessment of medical condition. It is expected that this amount will be within a relatively wide range and can be determined by routine experimentation.

[0084] In a particular embodiment, the effective amount of the vaccine or of the vaccine composition administrated as a single dose or as several doses will be from 0.001 mg / kg to 1000 mg / kg, advantageously 0.01 mg / kg to 100 mg / kg of body weight of the subject to be treated. Alternatively, the effective amount of the vaccine or of the vaccine composition administrated as a single dose or as several doses may be defined as mg of dry weight of recombinant yeast cells administrated to the subject to be treated or as number of recombinant yeast cells administrated to the subject to be treated. In these conditions, the effective amount of the vaccine composition may be

[0085] - from 0.3 mg of dry weight of recombinant yeast cells / kg to 3 g of dry weight of recombinant yeast cells / kg of body weight of the subject to be treated, advantageously from 3 mg of dry weight of recombinant yeast cells / kg to 1.5 g of dry weight of recombinant yeast cells / kg of body weight of the subject to be treated and / or

[0086] - from 107CFU to 1011CFU administrated subject to be treated, advantageously from 109CFU to 5.1010CFU administrated subject to be treated.

[0087] In a particular embodiment, the vaccine composition of the invention can be formulated as an oral preparation.

[0088] In another particular embodiment, the vaccine or the vaccine composition of the invention can be formulated as an injectable formulation, for example as a liquid solution or suspension; it can also be prepared as a solid form suitable for administration as a solution or suspension, by adding a liquid excipient prior to injection.

[0089] The vaccine or the vaccine composition of the invention may be prophylactic or therapeutic i.e. useful to treat a disease caused by bacteria.

[0090] In addition, the vaccine or the vaccine composition of the invention may be a monovalent or multivalent vaccine.

[0091] Once formulated into the vaccine or the vaccine composition of the invention, the recombinant yeast cells as previously defined can be administered directly to the subject. The subject to be treated may be a mammal, advantageously a livestock and in particular a bovine, an ovine, a caprine, a swine and an equine.

[0092] The vaccine or the vaccine composition of the invention can be administered before, during, and / or after exposure to bacteria causing a disease, and / or to the development of one or more symptoms.

[0093] The vaccine or the vaccine composition of the invention is usually administered using the same route of administration as conventional vaccines and / or mimicking the bacteria infection. Routes of administration of the vaccine or the vaccine composition of the invention include, but are not limited to intramuscular (IM), intramammary (IMM), subcutaneous, intradermal, intrapulmonary, intravenous, intraarterial, intraperitoneal, intrathecal, nasal, oral or other parenteral routes of administration. If desired, the route of administration may be combined or adjusted according to the condition of the disease. Vaccine composition of the invention can be administered in a single dose or in multiple doses, and can include the administration of booster doses to elicit and / or maintain immunity. The present invention also concerns a recombinant yeast cell as previously defined, a vaccine as previously defined or a vaccine composition as previously defined for use in protecting a mammal and in particular a bovine, an ovine, a caprine, a swine and an equine from a disease caused by bacteria. In particular, the disease is selected from the group consisting of mastitis, bovine / ovine / caprine paratuberculosis, bovine tuberculosis, metritis and lameness.

[0094] In a particular embodiment, the present invention also concerns a recombinant yeast cell as previously defined, a vaccine as previously defined or a vaccine composition as previously defined for use in protecting a mammal and in particular a bovine, an ovine or a caprine from bovine / ovine / caprine paratuberculosis and / or bovine tuberculosis. In this particular embodiment, the recombinant yeast cell as previously defined or the vaccine as previously defined or the vaccine composition as previously defined is advantageously administrated by nasal or oral route.

[0095] In a particular embodiment, the present invention concerns a recombinant yeast cell as previously defined, a vaccine as previously defined or a vaccine composition as previously defined for use in protecting a mammal and in particular a bovine, an ovine or a caprine from mastitis.

[0096] Advantageously, in this particular embodiment, the at least one first bacterial antigen and the at least one second bacterial antigen are independently selected from the group consisting of Enterobacteriaceae cell division protein FtsA (NCBI Reference WP_000588474.1), Enterobacteriaceae rod shape-determining protein MreB (NCBI Reference WP_000913396.1), Enterobacteriaceae phosphoporin PhoE (NCBI Reference WP_000749881.1^, Enterobacteriaceae cysteine synthase A (NCBI Reference WP_000034402.1), Enterobacteriaceae porin OmpF (NCBI Reference WP_000977905), E. coli Outer membrane protein A (OmpA) (GenBank protein ID WWN84339.1), E. coli Outer membrane protein A (OmpC) (GenBank protein ID CAC01403.1), E. coli enolase (GenBank protein ID AAC75821.1), E. coli GTP-binding tubulin-like cell division protein (GenBank ID BAB96663.2), 5. aureus Enolase (GenBank protein ID VDZ34627.1 ), S. aureus Elongation Factor Tu (GenBank protein ID BCD42744.1), 5. aureus 50S ribosomal protein L6 (GenBank protein ID BCD44412.1), 5. aureus Bifunctional pyr operon transcriptional regulator / uracil phosphoribosyltransferase PyrR (GenBank ID BCD43304.1), 5. aureus Universal stress protein UspA (GenBank protein ID QKE58197.1), 5. aureus Ferritin-like protein 2 ( GenBank protein ID VDZ35843.1), 5. aureus Galactose-6-phosphate isomerase, LacB subunit (GenBank ID VDZ36124.1), 5. aureus Phosphoribosylformylglycinamide synthase (GenBank protein ID VDZ34869.1), 5. aureus Immunodominant staphylococcal antigen IsaB (GenBank protein ID WP_046463258.1), 5. aureus Asp23 / Gls24 family envelope stress response (GenBank ID BCD44345.1), 5. aureus Alpha / beta hydrolase (GenBank ID WPA54171.1), 5. aureus Staphylocoagulase (GenBank ID WPA42669.1), 5. uberis staphylokinase domain-containing protein (NCBI Reference WP_015912075.1), 5. uberis surface-displayed alpha-enolase (NCBI Reference WP_012658173.1), 5. uberis sortase (NCBI Reference WP_012658383.1), 5. uberis cell division protein FtsZ (NCBI Reference WP_012658764.1), 5. uberis cell division protein FtsA (NCBI Reference WP_012658765.1), 5. uberis cell division protein FtsL (NCBI Reference WP_015911740.1), 5. uberis septation ring formation regulator EzrA (NCBI Reference WP_012658169.1), 5. uberis ubericin A precursor peptide (GenBank ID ABQ23939.1), 5. uberis ubericin A immunity protein (GenBank ID ABQ23940.1), 5. uberis circular bacteriocin uberolysin (GenBank ID ABG48503) and homologues thereof.

[0097] In a particular embodiment, the at least one first bacterial antigen and the at least one second bacterial antigen are independently selected from the group consisting of E. coli Outer membrane protein A hereinafter "OmpA" (GenBank protein ID WWN84339.1), 5. aureus Enolase hereinafter "Enolase" (GenBank protein ID VDZ34627.1), 5. aureus Elongation Factor Tu hereinafter "EloTu" (GenBank protein ID BCD42744.1), 5. aureus Bifunctional pyr operon transcriptional regulator / uracil phosphoribosyltransferase hereinafter "PyrR" (GenBank ID BCD43304.1), 5. aureus Universal stress protein hereinafter "UspA" (GenBank protein ID QKE58197.1), 5. aureus Alpha / beta hydrolase hereinafter "Lipase" (GenBank ID WPA54171.1), 5. aureus Staphylocoagulase hereinafter "Coagulase" (GenBank ID WPA42669.1) and homologues thereof.

[0098] In a more particular embodiment, the at least one first bacterial antigen and the at least one second bacterial antigen are independently selected from the group consisting of E. coli Outer membrane protein A hereinafter "OmpA" (GenBank protein ID WWN84339.1), 5. aureus Bifunctional pyr operon transcriptional regulator / uracil phosphoribosyltransferase hereinafter "PyrR" (GenBank ID BCD43304.1), 5. aureus Universal stress protein hereinafter "UspA" (GenBank protein ID QKE58197.1), 5. aureus Staphylocoagulase hereinafter "Coagulase" (GenBank ID WPA42669.1) and homologues thereof.

[0099] In the list of antigens of this more particular embodiment, can be added

[0100] - 5. aureus Elongation Factor Tu hereinafter "EloTu" (GenBank protein ID BCD42744.1), or

[0101] - 5. aureus Alpha / beta hydrolase hereinafter "Lipase" (GenBank ID WPA54171.1), or

[0102] - 5. aureus Enolase hereinafter "Enolase" (GenBank protein ID VDZ34627.1), or

[0103] - 5. aureus Elongation Factor Tu hereinafter "EloTu" (GenBank protein ID BCD42744.1) and 5. aureus Alpha / beta hydrolase hereinafter "Lipase" (GenBank ID WPA54171.1), or

[0104] - 5. aureus Elongation Factor Tu hereinafter "EloTu" (GenBank protein ID BCD42744.1) and 5. aureus Enolase hereinafter "Enolase" (GenBank protein ID VDZ34627.1), or

[0105] - 5. aureus Alpha / beta hydrolase hereinafter "Lipase" (GenBank ID WPA54171.1), and 5. aureus Enolase hereinafter "Enolase" (GenBank protein ID VDZ34627.1).

[0106] In this particular embodiment, the recombinant yeast cell, the vaccine or the vaccine composition is administered in at least two doses. These at least two doses may be (i) at least two doses administered intramuscularly, (ii) at least two doses administered subcutaneously in the mammary gland, (iii) at least two doses administered subcutaneously in an area different from the mammary gland or (iv) the first dose administered intramuscularly and the second one subcutaneously in the mammary gland or via the intramammary route.

[0107] Further characteristics and advantages of the present invention will further appear to those skilled in the art upon reading examples given below by way of illustrating and nolimiting purposes, in reference to the appended figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0108] Figure 1: Stimulation of bovine cells and mammary tissue with 5. cerevisiae. A. Blood from 8 cows was stimulated in vitro with heat-killed 5. cerevisiae (strain EBY1OO) or heat-killed Staphylococcus aureus and Escherichia coli. B. Evaluation of cytokine release by ELISA. C. In vitro and ex vivo models used to estimate the inflammatory potential of 5. cerevisiae to bovine mammary gland. Stimulations were carried out as described in A. D-E. Evaluation of cytokine release by ELISA. Asterisks denote statistically significant difference (treated versus control).

[0109] Figure 2: In vivo evaluation of a model yeast-based vaccine against mastitis. A. Diagram depicting pYDl and pYDl-OVA constructs. B. Representation of Aga2p-OVA expression by yeast surface display. C. Flow-cytometry analysis of OVA surface expression by EBYIOO-OVA after galactose (GAL) induction and heat-inactivation. EBYIOO-OVA noninactivated or grown in raffinose (RAF) were used as controls. D. Wester-blot analysis of OVA expression by EBYIOO-OVA. E. Scheme of immunization strategy (see vaccination with formulation #1 in the section Materials and Methods). F. Monitoring of rectal temperature after prime. G. Rectal temperature and mammary gland evaluation after booster. H-l. PBMC from vaccinated animals were isolated in the indicated days and stimulated in vitro with empty EBY1OO or OVA. IFNy (H) and IL-17 (I) release in supernatants was measured by ELISA. J. Analysis of serum total antibodies anti-EBYlOO and anti-OVA by ELISA. K. Scheme depicting intramammary stimulations with recombinant OVA. L. Somatic cells count in mammary gland secretion before and after intramammary stimulations. M. Scheme of immunization with adjuvanted-recombinant OVA. N-O. PBMC from OVA (N) or EBYIOO-OVA (O) immunized cows were stimulated in vitro with empty EBY1OO, EBYIOO-OVA or OVA. IFNy and IL-17 release was analysed by ELISA. Asterisks denote statistically significant difference (indicated time points versus day 0).

[0110] Figure 3: Immunogenicity test of a prototype vaccine based on 5. cerevisiae yeast (EBY100). A. Vaccination schedule (see vaccination with formulation #2 in the section Materials and Methods). Six dairy cows at drying off were immunized via the IM and IMM routes with EBY100 expressing OVA and OmpA on surface and intracellularly with 15 days between the prime and the boost vaccination. Then, 58 days after prime administration (which corresponds to 43 days after IMM booster), the animals were stimulated via the IMM route with the vaccine antigens (OVA and OmpA). B. ELISA measurement of IFNy and IL-17 after ex vivo stimulation of blood samples with OVA or OmpA before and after immunization.

[0111] Analysed time points are indicated in the graph.

[0112] Figure 4: Analysis of the mammary gland response at day 58 after prime administration (which corresponds to 43 days after IMM booster) upon IMM challenge with vaccine antigens OVA and OmpA (vaccination was performed as described in Figure 3A). A-C. The percentage of neutrophils (A) and T lymphocytes producing IFNy (B) or IL-17 (C) in mammary secretions was analyzed by flow cytometry before and after stimulation with a mixture of OVA and OmpA. An antigen-specific neutrophilic inflammation and an increase in cells producing the two protective cytokines of the mammary gland were observed. D. Ninety days after IMM boost, mammary secretion samples were collected and stimulated in vitro with the vaccine antigens OVA or OmpA. Subsequently, the production of IFNy and IL-17 was assessed by ELISA.

[0113] Figure 5: Bovine blood cells response to the vaccine antigen (OVA) after vaccination with two prototype vaccines based on the yeast 5. cerevisiae. Whole blood samples were collected at the indicated time points. Left graphs: 0 to 91 days after vaccination via the intramuscular and intramammary routes with Formulation #1 with 60 days between prime and boost (see vaccination schedule in Figure 2E and Materials and Methods section). Right graphs: 0 to 81 days after vaccination via the intramuscular and intramammary routes with Formulation #2 with 15 days between prime and boost (see vaccination schedule in Figure 3A or Materials and Methods section). Subsequently, the production of IFNy and IL-17 was evaluated by ELISA. A stronger response was observed through the modification of 5. cerevisiae for the expression of the antigen on the surface and in the cytoplasm. This strategy allowed for an increased amount of OVA per vaccine dose.

[0114] Figure 6: In vitro evaluation of two yeast-based vaccine formulations using OmpA as reporter antigen. A. PBMC were collected from three cows 30 days after immunisation with recombinant OmpA. Cells were stimulated in vitro with the following stimuli: yeast strain #0 (EBY100 expressing OmpA in the cytoplasm), yeast strain #1.2 (EBY100 expressing OmpA on the surface), yeast strain #2.2 (EBY100 expressing OmpA in the cytoplasm and on the surface), EBY100 transformed with empty p425-GPD (EBY 1001) or EBY100 co-transformed with empty pYDl and empty p425-GPD (EBY 1002). Empty medium and recombinant OmpA were used as controls. After 72h of culture, supernatants were collected and the production IFNy and IL-17 was estimated by ELISA. B. Production of IFNy and IL-17 after stimulation. Figure 7: Analysis of OmpA expression by western blot. Expression levels were estimated taking 10 ng of OmpA as reference. Strain #0 corresponds to EBY100 transformed with p425-GPD-OmpA (intracellular OmpA). Yeast strain #1.2 corresponds to EBY100 transformed with PYDl-OmpA (surface OmpA). Strain #2.2 corresponds to EBY100 cotransformed with PYDl-OmpA and p425-GPD-OmpA (surface and intracellular OmpA). EBY100 transformed with empty vectors were used as controls. Numbers represent a ratio between tested conditions and reference (OmpA 10 ng) calculated as follows: tested condition band intensity level divided by reference band intensity level.

[0115] Figure 8: In vivo evaluation of vaccine formulation #3 (cellular response). Somatic cell count in the mammary gland of vaccinated animals before and after stimulation with vaccine antigens. UspA induced an increase of cell count in both vaccinated animals whereas PyrR induced this result only in one of the evaluated animals.

[0116] Figure 9: In vivo evaluation of vaccine formulation #3 (humoral response). Serum from vaccinated and control animals was tested for the presence of vaccine-specific antibodies. Anti-PyrR, coagulase and UspA antibodies were observed in the serum of vaccinated animals at different time points after vaccination.

[0117] DETAILED DISCLOSURE OF PARTICULAR EMBODIMENTS

[0118] MATERIALS AND METHODS

[0119] Whole-blood and mammary primary cells stimulation (see Figures 1A, B, C and D)

[0120] EDTA-treated blood samples were collected by jugular venepuncture from eight Holstein cows in lactation bred in the Unite Experimentale de Physiologie Animale (UEPAO INRAE, France). One hundred microliters of whole blood were mixed with 100 pl of WBA medium (RPMI 10% FBS, 2 mM glutamine, 1 mM sodium pyruvate, 100 nM non-essential amino acids, 50 pM p-mercaptoethanol, 10 mM HEPES) containing 106CFU of heat- inactivated EBY100 (56°C for 30 minutes), 5. aureus or E. coli (70°C for 30 minutes). Bovine primary mammary epithelial cells were prepared as described in

[0012] and stimulated with 105CFU of heat-inactivated EBY100, 5. aureus or E. coli. Cultures were kept for 48h at 38.5°C with 5% CO2 and supernatants were collected for cytokine measurement by ELISA. Ex vivo stimulation of mammary tissue (see Figures 1C and E)

[0121] Teats were collected from MGs with no signs of disease from four slaughtered dairy cows in a French commercial abattoir. Teat explants were processed and stimulated with 106CFU of heat-inactivated EBY1OO, 5. aureus or E. coli as previously described

[0013] . Supernatants were collected 24h after stimulation and cytokine production was measured by ELISA.

[0122] Production of S. cerevisiae-based vaccine formulations

[0123] In what follows, formulation #2 and yeast strains #2.1 and #2.2 are formulation and yeast strains according to the present invention while formulation #1, yeast strain #0, yeast strain #1.1 and yeast strain #1.2 are control formulation and yeast strains not part of the present invention.

[0124] Cloning strategy

[0125] Production ofDNA constructs for antigen expression on yeast surface: Gallus gallus ovalbumin (OVA) coding sequence (depleted of its first 50 aminoacids) or Escherichia coli outer membrane protein A (OmpA) coding sequence (depleted of its N-terminus peptide signal) were inserted into pYDl vector backbone (Invitrogen) in frame with Aga2p coding sequence to generate the DNA constructs pYDl-OVA and pYDl-OmpA, respectively. Recombinant DNA constructs were cloned in E. coli DH5a (New England Biolabs).

[0126] Production of DNA constructs for intracellular antigen expression on yeast surface: Gallus gallus ovalbumin (OVA) coding sequence (depleted of its first 50 aminoacids) or Escherichia coli outer membrane protein A (OmpA) coding sequence (depleted of its N- terminus peptide signal) were inserted into p425-GDP vector backbone

[0014] to generate DNA constructs p425GDP OVA and p425-GDP-OmpA. Recombinant DNA constructs were cloned in E. coli DH5a (New England Biolabs).

[0127] Yeast transformation

[0128] S. cerevisiae strain EBY100 (ATCC, MATa AGA1::GAL1-AGA1::URA3 ura3-52 trpl leu2-delta200 his3-delta200 pep4::HIS3 prbdl.6R canl GAL) was cultivated in YPD broth to ODeoo of 0.6 - 0.8 and routinely transformed using the LiAc / SS carrier DNA / PEG method [8]. Yeast strain #0: 6 - 8.107UFC of EBY100 transformed with 1 ng of p425-GPD- OmpA. Transformants were selected in minimal agar #3 plates (0.67% Yeast Nitrogen Base with ammonium sulfate, 2% glucose, dropout aminoacids mixture (76 mg / L each) without leucine, 2% bacto agar).

[0129] Yeast strain #1.1: 6 - 8.107UFC of EBY100 were transformed with 1 pg of pYDl- OVA. Transformants were selected in minimal agar #1 plates (0.67% Yeast Nitrogen Base with ammonium sulfate, 2% glucose, dropout aminoacids mixture (76 mg / l each) without tryptophan, 2% bacto agar).

[0130] Yeast strain #1.2: 6 - 8.107UFC of EBY100 were transformed with 1 pg of pYDl- OmpA. Transformants were selected in minimal agar #1 plates (0.67% Yeast Nitrogen Base with ammonium sulfate, 2% glucose, dropout aminoacids mixture (76 mg / l each) without tryptophan, 2% bacto agar).

[0131] Yeast strain #2.1: 6 - 8.107UFC of EBY100 transformed with pYDl-OVA (strain #1) were transformed with 1 pg of p425-GPD-OVA. Transformants were selected in minimal agar #2 plates (0.67% Yeast Nitrogen Base with ammonium sulfate, 2% glucose, dropout aminoacids mixture (76 mg / L each) without tryptophan and leucine, 2% bacto agar).

[0132] Yeast strain #2.2: 6 - 8.107UFC of EBY100 previously transformed with pYDl- OmpA were transformed with 1 pg of p425-GPD-OmpA. Transformants were selected in minimal agar #2 plates.

[0133] Induction of antigen expression

[0134] Yeast strains #1.1 and #1.2 were inoculated at a ODeoo of 0.2 in minimal broth #1 (0.67% Yeast Nitrogen Base with ammonium sulfate, 2% raffinose, dropout aminoacids mixture (76 mg / l each) without tryptophan). Yeast strains #2.1 and #2.2 were inoculated at a ODeoo of 0.2 in minimal broth #2 (0.67% Yeast Nitrogen Base with ammonium sulfate, 2% glucose, 2% raffinose, dropout aminoacids mixture (76 mg / l each) without tryptophan and leucine). After overnight incubation at 28°C with agitation (250 rpm), cultures were diluted to an ODeoo of 0.2 in minimal broth #1 or #2 and incubated at 28°C with agitation (250 rpm). At a ODeoo of 0.5, antigen expression on surface was induced with 2% galactose and cultures were incubated in agitation (250 rpm) for 7h. In order to obtain cultures with an ODeoo of 1 - 2, temperature was adjusted from 26 to 28°C. Preparation of vaccines

[0135] After galactose induction (yeast strain #1.1, yeast strain #2.1 and yeast strain #2.2) or overnight culture in minimal broth #3 (0.67% Yeast Nitrogen Base with ammonium sulfate, 2% glucose, dropout aminoacids mixture (76 mg / l each) without leucine) (yeast strain #0), cultures were concentrated to an ODeoo of 10 - 20 in DPBS without Ca+2and Mg+2and heat- inactivated at 56°C for 30 min. After 30 min at room temperature, yeast cells were centrifuged at 3500 rpm for 10 min at 10°C and washed twice with wash buffer (DPBS without Ca+2and Mg+2, 2% FBS, 2 mM EDTA). Dry yeast pellets were kept at -20°C until vaccination. For the preparation of vaccine doses, the required amount of vaccine yeast (refer to Table 1 below) was resuspended in 3 ml of DPBS without Ca+2and Mg+2.

[0136] A comparison of vaccine formulation #1 and formulation #2 is presented in the

[0137] Table 1 below

[0138] Table 1

[0139] Vaccination with formulation #1 (EBYIOO-OVA on the surface) and clinical monitoring (see Figure 2 and Figure 5) Six non-pregnant Holstein cows at the dry-off period were recruited for the immunisation protocol carried out at the Plateforme d'lnfectiologie Experimentale (PFIE INRAE, France). Selected animals showed low somatic cell counts in udder quarter milk (< 200 000 cells / ml) and absence of intramammary infection by major pathogens (Staphylococcus aureus, Escherichia coli or streptococci) before dry-off. For prime immunization, 2.109CFU of Formulation #1 were administered intramuscularly at the prescapular region. After 60 days, animals were administered with an intramammary booster as follows: rear quarters received 109CFU of EBYIOO-OVA, front right quarter received 108CFU of EBYIOO-OVA and front left quarter was used as control. After immunisations, the presence of systemic and local reactogenicity signs was evaluated and recorded by a single operator using a scoring grid. As a complement, the presence of subcutaneous edema at the MG cistern was monitored by ultrasound using an Esaote Piemedical MyLab30 ultrasound device equipped with a linear LA332 probe (Hospimedi France, Valdampierre, France). Vaccination with Formulation #2 (EBYIOO-OVA + EBYlOO-OmpA on the surface and intracellularly) and clinical monitoring (see Figure 3, Figure 4 and Figure 5)

[0140] Six non-pregnant Holstein cows at the dry-off period were recruited for the immunisation protocol carried out at the Plateforme d'lnfectiologie Experimentale (PFIE INRAE, France). Selected animals showed low somatic cell counts in udder quarter milk (< 200 000 cells / ml) and absence of intramammary infection by major pathogens (Staphylococcus aureus, Escherichia coli or streptococci) before dry-off. For prime immunization, 1010CFU of Formulation #2 were administered intramuscularly at the prescapular region. After 15 days, animals were administered with an intramammary booster as follows: right quarters received 2.109CFU of Formulation #2 and left quarters were used as control. After immunisations, the presence of systemic and local reactogenicity signs was evaluated and recorded by a single operator using a scoring grid. As a complement, the presence of subcutaneous edema at the MG cistern was monitored by ultrasound using an Esaote Piemedical MyLab30 ultrasound device equipped with a linear LA332 probe (Hospimedi France, Valdampierre, France).

[0141] Vaccination with recombinant OVA and OmpA (see Figure 2M and Figure 6)

[0142] OVA immunizations: Three Holstein cows in lactation were selected as previously described and immunised intramuscularly at the prescapular region with 50 pg of pyrogen- free ovalbumin (Calbiochem) dissolved in 0.8 mL phosphate-buffered saline and emulsified in 1.2 mL of Montanide™ ISA 61 VG (Seppic) (Figure 2M).

[0143] OmpA immunizations: Three Holstein cows in lactation were selected as previously described and immunised intramuscularly at the prescapular region with 20 pg of recombinant OmpA produced in E. coli dissolved in 0.8 mL phosphate-buffered saline and emulsified in 1.2 mL of Montanide™ ISA 61 VG (Seppic) (Figure 6).

[0144] In both protocols, PBMCs were isolated from blood samples 30 days after immunisations and stored in liquid nitrogen until analysis.

[0145] Evaluation of blood immune response to vaccination (see Figures 2H, I, J, N, O, Figure 3B, Figure 5 and Figure 6)

[0146] For the analysis of systemic immune response to immunization, whole blood, serum and PBMCs were routinely collected at the indicated time points. PBMC samples were kept in liquid nitrogen until the end of the experimental protocol and then defrosted and stimulated at the same time as follows: 3.105cells were mixed to 200 pl of WBA medium containing 106CFU of heat-inactivated recombinant yeast strains (detailed below) or 1 ng of recombinant protein (OVA or OmpA) in a 96 well round bottom plate (Nunc). Empty medium was used as control. For whole blood stimulation, 100 pl of EDTA-treated blood samples were mixed to 100 pl of WBA medium containing 106CFU of heat-inactivated recombinant yeast strains (detailed below) or 1 pg of recombinant protein (OVA or OmpA) in a 96 well round bottom plate (Nunc). Empty medium was used as control. Cultures were kept for48h at 38.5°C with 5% CO2 and supernatants were collected for cytokine measurement by ELISA.

[0147] The following recombinant yeast strains were tested when indicated: empty EBY100, EBY100 transformed with empty pYDl, EBY100 transformed with empty p425-GPD (EBY 1001), EBY100 co-transformed with empty pYDl and empty p425-GPD (EBY 1002), EBY100 transformed with p425-GPD-OmpA (yeast strain #0), EBY100 transformed with pYDl- OVA (yeast strain #1.1), EBY100 transformed with pYDl-OmpA (yeast strain #1.2), EBY100 cotransformed with pYDl-OmpA and p425-GDP-OmpA (yeast strain #2.2).

[0148] For the estimation of total antibody titers to OVA and EBY100 in blood serum, microtiter plates (Nunc Immunoplate Maxisorp) were coated by overnight incubation with 2 pg / ml of ovalbumin or EBY100 protein lysate (100 pl / well) in phosphate-buffered saline (PBS). Then, ELISA assays were carried out as described in

[0015] using an anti-bovine (H+L) antibody conjugated to horseradish peroxidase (AB_2337291, Jackson ImmunoResearch).

[0149] Intramammary stimulation, somatic cells count and analysis of cell populations by flow cytometry (see Figures 2K, L, and Figures 4A, B and C).

[0150] Figure 2 K and L: At day 100 (40 days after intramammary booster), control and boosted mammary quarters were administered with 10 pg of pyrogen-free ovalbumin (Calbiochem) dissolved in 3 ml of DPBS containing 1 mg of pyrogen-free bovine serum albumin (Sigma). Ten millilitres of mammary secretion were collected before and 24h after stimulation for the measurement of somatic cells count using an automated cell counter (Fossomatic model 90; Foss Food Technology, Hillerod, Denmark).

[0151] Figures 4A, B and C : At the indicated time point, control and boosted mammary quarters were stimulated with a following recombinant antigen solution: 10 pg of pyrogen- free ovalbumin (Calbiochem) mixed to 10 pg of pyrogen-free recombinant OmpA produced in E. coli, dissolved in 3 ml of DPBS containing 1 mg of pyrogen-free bovine serum albumin (Sigma).

[0152] To determine the response of mammary gland to local stimulation, 10 ml of mammary secretion were collected before and 24h after stimulation for the measurement of somatic cells count using an automated cell counter (Fossomatic model 90; Foss Food Technology, Hillerod, Denmark). For the analysis of cell populations by flow cytometry, 10 ml of mammary secretion were collected before and 24h after stimulation. Samples were mixed with 10 ml of FACS buffer (DPBS without Ca+2and Mg+2, 2 mM EDTA, 2% FBS) and centrifuged at 1400g for 10 min. Cell pellets were stained with the a mouse anti-bovine granulocytes antibody (CH138A, Biorad) and a rat anti-mouse secondary antibody conjugated to PE-Cy7 (115-126-075, Jackson ImmunoResearch) for the staining of neutrophils. For the analysis of IFNy and IL-17 producing T-lymphocytes, cells were separately stained with a mouse anti- bovine CD3 antibody (MM1A, Biorad) and an anti-mouse secondary antibody conjugated to BV711 (BDB565786, BD Biosciences). After fixation and permabilisation with BD Cytofix / Cytoperm™ (BD Biosciences) according to manufacturer's conditions, cells were stained in Perm / Wash Buffer (BD Biosciences) with an anti-bovine IFNy antibody conjugated to Alexa Fluor 488 (Clone CC302, Biorad) and an anti-human IL-17 antibody conjugated to PE- Cy7 (eBIO64DEC17, Ebiosciences). Dead cells were labeled using the Fixable Viability Dye eFluor450 (eBioscience). Samples were examined using a BD LSR Fortessa cytometer and data were analyzed with the Kaluza software (Beckman Coulter).

[0153] In vitro stimulation of mammary secretion cells (see Figure 4D)

[0154] Ten milliliters of mammary secretion were collected 90 days after intramammary booster, mixed with 10 ml of FACS buffer and centrifuged for 10 min at 1400g. After counting, 3.105cells were resuspensed in 200 pl of WBA medium containing the indicated amount of recombinant protein OVA or OmpA in a 96 well round bottom plate (Nunc). Empty medium was used as control. Cultures were kept for 72h at 38.5°C with 5% CO2 and supernatants were collected for cytokine measurement by ELISA.

[0155] Western blot (see Figure 2D and Figure 7)

[0156] Figure 2D: Two hundred microliters of EBY100 and EBY100-OVA cultivated in presence of galactose (ODeoo 1.0) were centrifuged at 3000 g and lysed with YeastBuster Protein Extraction Reagent (Merck) following manufacturer's condition. Ten micrograms of total protein were separated in a mPAGE™ 4-12% Bis-Tris Precast Gel (Merck) by electrophoresis, transferred onto 0.45 pm nitrocellulose membranes (GE) and blotted under standard conditions using a rabbit polyclonal antibody anti-OVA antibody (produced by our team, 0.5 pg / ml). A goat anti-rabbit IgG (H+L) antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch, 1 :5000) was used to generate immunocomplexes revealed with enhanced chemiluminescence (Thermo Scientific) and membranes were scanned in a Fusion FX imager (Vilber).

[0157] Figure 7: Yeast strain #0 (EBY100 transformed with p425-OmpA), yeast strain #1.2 (EBY100 transformed with pYDl-OmpA), yeast strain #2.2 (EBY100 transformed with p425- OmpA and pYDl-OmpA). EBY100 transformed empty p425-GPD, empty pYDl or both were used as control. Two hundred microliters of each culture at an ODeoo of 1.0 were centrifuged at 3000 g and lysed with CelLytic™ Y Cell Lysis Reagent (Sigma-Aldrich) following manufacturer's condition. Ten micrograms of total protein were separated in a mPAGE™ 4- 12% Bis-Tris Precast Gel (Merck) by electrophoresis, transferred onto 0.45 pm nitrocellulose membranes (GE) and blotted under standard conditions using a rabbit polyclonal antibody anti-OmpA antibody (produced by our team, 0.5 pg / ml). A goat anti-rabbit IgG (H+L) antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch, 1 :5000) was used to generate immunocomplexes revealed with enhanced chemiluminescence (Thermo Scientific) and membranes were scanned in a Fusion FX imager (Vilber).

[0158] Analysis of antigen expression by 5. cerevisiae using flow cytometry (see Figure 2C)

[0159] EBY100-OVA cultivated in presence of galactose or raffinose was heat-inactivated at 54°C or 56°C for 30 minutes. A non-inactivated galactose culture was used as control. One hundred microliters of yeast suspension (ODeoo 1.0) were centrifuged at 3000g and stained with a mouse monoclonal antibody anti-Xpress (AB_2556552 Invitrogen, 1:200) for 30 minutes in FACS buffer (DPBS without Ca+2and Mg+2, 2 mM EDTA, 2% FBS). A goat anti-mouse IgG (H+L) conjugated to Alexa Fluor 555 (AB_2535844, Thermo Fischer, 2 pg / ml) was used as secondary antibody. Stained yeast cells were examined using a BD LSR Fortessa cytometer and data were analyzed with the Kaluza software (Beckman Coulter). Cytokine measurement by ELISA

[0160] Cytokine levels were measured in culture supernatants with the following commercially available kits: IFNy (ELISA Flex: Bovine IFNy HRP, Mabtech, 3119-1H-20), IL-8 (ELISA Flex: Bovine IL-8 HRP, Mabtech, 3114-1H-6), TNFa (Bovine TNF alpha TNFSF2 Do-lt- Yourself ELISA, Kingfisher DIY0675B-003) and IL-6 (Bovine IL-6 Do-It-Yourself ELISA, Kingfisher DIY0670B-003). IL-17 was measured as described in

[0015] .

[0161] Statistical analyses

[0162] Data were analyzed and plotted using GraphPad Prism, version 6.0 (GraphPad Software Incorporation). Kruskal-Wallis test was used to compare groups and pairwise comparisons (treated versus control) were carried out using the Dunn's test. In time course analyses, data were analysed using Friedman's test and pairwise comparisons (indicated time point versus day 0) were carried out using the Dunn's test. Data shown represent the median and interquartile range. Black (treated versus control) and red (indicated time points versus day 0) asterisks denote statistically significant difference. *p < 0.05, **p < 0.01, ****p < 0.0001.

[0163] Test 1 and Test 2 (see Figure 5)

[0164] The inventors have implemented a comparison between two tests conducted in cattle. The same dairy cows were used for both tests, with an interval of 182 days. A summary of the differences between Test 1 and Test 2 is presented in below Table 2:

[0165] Table 2

[0166] RESULTS

[0167] Preliminary studies

[0168] The potential of yeasts as platforms for novel vaccines against bovine mastitis was evaluated. Initially, the capacity of Saccharomyces cerevisiae to stimulate bovine immune system was checked. The results showed that the 5. cerevisiae strain EBY100 (ATCC) is properly sensed by bovine blood cells and induces the production of inflammation and T-cell response markers, as mastitis causing bacteria (Figure 1A-B). Next, the suitability of 5. cerevisiae for the production of vaccine formulations to be administered via the intramammary (IMM) route was tested. High levels of proinflammatory cytokines have been reported to compromise the integrity of alveolar mammary epithelial cells (MECs), leading to a loss of milk production

[0016] . Thus, the inventors used primary cell cultures and a recently reported ex vivo model

[0013] to evaluate the inflammatory response of MECs and teat explants upon exposure to yeast (Figure 1C). As shown in the Figures 1D-E, 5. cerevisiae induced a lower release of pro-inflammatory cytokines than bacteria causing mastitis. It has been reported that receptors enabling the recognition of yeast cell wall sugars are mostly found on the surface of antigen presenting cells such as dendritic cells and macrophages

[0017] , whereas MECs are particularly armed to recognise bacteria-associated molecular patterns

[0018] . Therefore, antigen vectorisation by yeast might represent an efficient strategy to trigger adaptive immunity mechanisms in the MG circumventing an overproduction of inflammatory mediators by MECs.

[0169] Test 1: in vivo evaluation of Formulation #1

[0170] To test this hypothesis in vivo, the inventors set out a yeast-based vaccine and tested its immunogenicity in dairy cows. Ovalbumin (OVA) coding sequence was inserted into the cloning vector pYDl to construct pYDl-OVA (Figure 2A). EBY100 was transformed with pYDl-OVA to generate EBYIOO-OVA, which expresses the Aga2p-OVA fusion protein on its surface in presence of galactose (Figure 2B). Heat-inactivated EBYIOO-OVA was then resuspended in DPBS for the production of a model vaccine. The presence of OVA on EBY100 surface after heat-inactivation was validated by flow cytometry and western blot analysis, as shown in Figures 2C-D.

[0171] Next, six dairy cows were immunized with EBYIOO-OVA by an intramuscular prime followed by an intramammary booster, as described in Figure 2E. During booster, each mammary quarter was administered with 0, 108or 109(two quarters) UFC of EBYIOO-OVA. None of the animals showed fever (Figure 2F) nor other reactogenicity events after prime. After booster, clinical examination showed only mild to moderate inflammation in the MG at 24 to 48 hours post immunisation (Figure 2G). To evaluate the systemic immune response to the vaccine vector and model antigen separately, PBMCs extracted ftom vaccinated cows, were stimulated in vitro with empty EBY100 or recombinant OVA and the production of I NFy and IL-17 was estimated. As shown in Figures 2H-I, vaccination lead to a significant increase of INFy and IL-17 responses against EBY100 but not OVA. In the same way, significant increase of serum antibody titters anti-EBYlOO but not anti-OVA was observed after vaccination (Figure 2J). We also evaluated the somatic cells count in mammary secretion before and after stimulation of the MG with recombinant OVA (Figures 2K-L) and results indicated that EBYIOO- OVA could not induce an OVA-specific neutrophilic inflammation in the MG, differently from our previous observations in cows immunized with OVA emulsified in an oil-in-water adjuvant

[0015] . To clarify these observations, immunization with adjuvanted OVA in three cows were repeated (Figure 2M) and analysed the response of their PBMCs to in vitro stimulation in comparison with PBMCs from EBYIOO-OVA immunized cows. Interestingly, cells from OVA- immunised cows showed INFy and IL-17 responses to EBYIOO-OVA and OVA (Figure 2N), whereas cells from EBYlOO-OVA-immunised animals responded only to EBY100 and EBYIOO- OVA (Figure 20).

[0172] Altogether, these results indicate that 5. cerevisiae antigens are dominant over OVA and similar observations have been reported upon vaccination with other immunogenic antigen vectors. Listeria monocytogenes-based vaccines failed to prime immune responses to cancer antigens due to the immunodominance its T-cell epitopes

[0016] . Similarly, immunogenitcity of adenovirus vaccine vectors has been associated to limited T-cell response to transgenic antigens

[0020] ,

[0173] Safety, affordability and adjuvant capacity of yeasts represent a window of opportunities to develop novel vaccines for mastitis. Nevertheless, this study indicates that strategies to counterbalance the immunodominance of 5. cerevisiae antigens are mandatory to assure the success of this organism as vaccine platforms.

[0174] Test 2: in vivo evaluation of Formulation #2

[0175] Following the initial in vivo protocol demonstrating the safety of their platform for bovine vaccination, the inventors constructed 5. cerevisiae EBY100 strains expressing two model antigens (ovalbumin - OVA or the OmpA protein from the bacterium Escherichia coli) on their surface and in the cytoplasm (Formulation #2). Six dried dairy cows were immunized via the intramuscular (IM) and intramammary (IMM) routes, according to the scheme presented in Figure 3A. In this second protocol, the inventors chose to shorten the time between the prime vaccination and the booster dose to 15 days. Only the right mammary quarters received the booster dose (vaccinated).

[0176] The two left quarters were used as controls. Clinical monitoring of the animals showed no signs of systemic or local reactogenicity following the immunizations. The inventors first assessed the animals' response to vaccination by ex vivo stimulation of blood samples with OVA or OmpA before and after vaccination (Figure 3B) .

[0177] For the analysis of the local response, the udder of immunized animals (all four quarters) was stimulated by the administration of the vaccine antigens (OVA and OmpA) via the IMM route 58 days after the IM prime (which corresponds to 43 days after booster) (Figure 3A) and cell populations of interest (neutrophils, T-cells producing IFNy or I L17) were analysed by flow cytometry. As shown in the Figure 4A, an increase of neutrophils in the mammary secretion was observed 24h after local stimulation with the vaccine antigens. Local stimulation also led to an increase of T-cells IFNy+(Figure 4B) and I L17+(Figure 4C) . Of note, these changes in cellularity were found out mammary quarters boosted and not boosted locally.

[0178] During the in vivo immunogenicity test, the local response of vaccinated animals to each of the two vaccine antigens (OVA and OmpA) could not be verified separately. To assess this, samples of mammary secretion were collected 90 days after local booster from all mammary quarters (whether locally boosted or not) and stimulated in vitro with OVA or OmpA, separately. After 3 days of stimulation, the release of IFNy and IL-17 was assessed by ELISA. Figure 4D shows that the mammary gland cells responded to stimulation with both antigens. Regarding the response to OVA, this proved to be more variable and less remarkable in comparison to OmpA, even at a higher dose (5 pg). In summary, these results show that immunization with a vaccine based on the yeast 5. cerevisiae induces antigen-specific IL-17 and I FNy responses in the blood and that vary from individual to individual. The analysis of the response in the mammary gland highlighted that despite differences at the blood level, this immunization strategy induced the migration of neutrophils and IL-17 and IFNy producing lymphocytes in an antigen-specific manner into the udder of all experimental animals. Various studies conducted by the inventors' team have shown that this mechanism plays a major role in the defense of the udder against mastitis, thereby reinforcing the interest in the vaccination strategy according to the invention for the prevention of mastitis.

[0179] Importantly, these results also show that partial vaccination of the udder (2 out of 4 quarters) is sufficient for the induction of defense mechanisms throughout the entire udder. This was demonstrated by the statistically significant increase of cells of interest even in the control quarters (which did not receive the booster dose) upon stimulation with the vaccine antigens. This observation could be justified by the anatomical peculiarities of the ruminant udder, which consists of four independent quarters that are drained by a common lymph node, where the mammary adaptive immune response is organized. From an industrial standpoint, this finding would have major implications for immunization strategies targeting the intramammary route, as a single booster dose instead of four would suffice for the protection of the udder.

[0180] Comparison of Formulation #1 and Formulation #2 (Test 1 and Test 2)

[0181] During both tests, blood samples were collected and stimulated in vitro with the vaccine antigen (recombinant OVA). Figure 5 shows that the vaccine formulation composed of S. cerevisiae carrying the antigen only on its surface induced a low production of IFNy and IL-17 by blood cells. However, a stronger cytokine response was observed during Test 2, when the animals were vaccinated with 5. cerevisiae carrying OVA on the surface and in the cytoplasm. This result and the preliminary estimation of the amount of antigen present in the two tested vaccine formulations suggest that the in vivo cellular response may be improved by modifications allowing 5. cerevisiae to carry more antigens. Figure 5 shows a better antigen-specific blood cell response following vaccination with Formulation #2, (in vitro stimulation with OVA without OmpA), suggesting a more effective delivery of OVA antigen in Formulation #2 by its presence in the cytoplasm and on the surface of EBY100. Additional analyses also indicated a higher presence of OVA in Formulation #2, as compared to Formulation #1.

[0182] Tests conducted on cattle indicated a better in vivo response in the mammary gland following vaccination with the second vaccine formulation (Formulation #2) based on 5. cerevisiae, which is composed of antigens expressed both in the cytoplasm and on the surface of the yeast.

[0183] To complete data shown in Figures 3-5, the inventors tested the immunogenicity of yeast strain #0 (antigen expressed only intracellularly), yeast strain #1.2 (antigen on the surface) and yeast strain #2.2 (antigen on the surface and intracellularly) in vitro using OmpA as a reporter antigen. For this, the inventors took advantage of blood cells (PBMC) collected from three dairy cows immunised with an experimental vaccine based on recombinant OmpA emulsified with an water in oil adjuvant (Montanide™ ISA 61G, Seppic, France). These cells were used as sources of OmpA-specifc immune cells, which were stimulated with the yeast strains expressing OmpA (yeast strain #0, yeast strain #1.2 and yeast strain #2.2) or recombinant OmpA, as described in below Table 3:

[0184] Table 3

[0185] All vaccine formulations induced higher cytokine responses than the empty yeast control (EBY100), which confirms that the evaluated responses are mostly against OmpA. Data also indicate that OmpA expression by 5. cerevisiae on the surface combined with intracellular expression (yeast strain #2.2, used in Formulation #2) induces higher release of I FNy and IL-17 (Figure 6). Interestingly, higher increase was observed for the production of IL-17 when compared to IFNy and this result suggests that double antigen expression by 5. cerevisiae (on the surface and in the cytoplasm) could be used as a way to favour IL-17-based immune mechanisms in bovine.

[0186] Analysis of antigen expression in different 5. cerevisiae-strains

[0187] Total protein extracted from OmpA vaccine yeast strain #0 (EBY100 expressing OmpA intracellularly), yeast strain#1.2 (EBY100 expressing OmpA on surface), yeast strain #2.2 (EBY100 expressing OmpA on surface and intracellularly) was analysed by western blot using an anti-OmpA antibody. EBY100 transformed with empty p425GPD or co-transformed with empty p425GPD and PYD1 were also analysed as controls. Expression levels were compared to 10 ng of recombinant OmpA. Results presented in Figure 7 show that yeast strain #2.2 expresses 2.5 more OmpA than yeast strain #1.2 and that yeast strain #2.2 expresses 18 more OmpA than yeast strain #0.

[0188] ADDITIONAL RESULTS

[0189] Differently from previous formulations, this prototype is based on a 5. cerevisiae cell wall mutant strain depleted of the gene Kre6, coding for a glucosyl hydrolase required for beta-1, 6-glucan biosynthesis (strain Y05574, Euroscarf). Besides, both plasmids used for the production this recombinant strain enable the production of 5. aureus antigens intracellularly (PyrR, Lipase-Coagulase fusion, Lipase-Enolase fusion and Lipase-EloTu fusion) and on the surface (UspA-PyrR fusion, UspA-Coagulase fusion, UspA-Enolase fusion, UspA-EloTu fusion) under a constitutive promoter. This modification facilitates bacterial antigen expression and reduces vaccine production costs (yeast vaccine strain is cultivated in glucose, circumventing galactose antigen induction). Additionally, yeast inactivation was performed chemically to facilitate vaccine production and reduce production costs.

[0190] Vaccine yeast production

[0191] 1 / Cloning strategy

[0192] 1.1 / Production of pPCl vector for antigen expression on yeast surface under a constitutive promoter: The Aga2p cassette was amplified from the PYD1 vector backbone (Invitrogen) and inserted into p423-TEF vector backbone

[0014] to generate the pPCl vector. 1.2 / Production of a DNA construct for antigen expression on yeast surface:

[0193] UspA-PyrR construct: A fragment of Staphylococcus aureus Universal stress protein (UspA, GenBank protein ID QKE58197.1) coding sequence was fused to a fragment of 5. aureus Bifunctional pyr operon transcriptional regulator / uracil phosphoribosyltransferase (PyrR, GenBank ID BCD43304.1) coding sequence and inserted into the pPCl vector in frame with Aga2p coding sequence to generate the DNA construct pPCl-UspA-PyrR.

[0194] UspA-Coagulase construct: A fragment of Staphylococcus aureus Universal stress protein (UspA, GenBank protein ID QKE58197.1) coding sequence was fused to a fragment of 5. aureus Staphylocoagulase (Coagulase, GenBank ID WPA42669.1) coding sequence and inserted into the pPCl vector in frame with Aga2p coding sequence to generate the DNA construct pPCl-UspA-Coagulase.

[0195] UspA-Enolase construct: A fragment of Staphylococcus aureus Universal stress protein (UspA, GenBank protein ID QKE58197.1) coding sequence was fused to a fragment of Staphylococcus aureus Enolase (Enolase, GenBank protein ID VDZ34627.1) coding sequence and inserted into the pPCl vector in frame with Aga2p coding sequence to generate the DNA construct pPCl-UspA-Enolase.

[0196] UspA-EloTu construct: A fragment of Staphylococcus aureus Universal stress protein (UspA, GenBank protein ID QKE58197.1) coding sequence was fused to a fragment of Staphylococcus aureus Elongation Factor Tu (EloTu, GenBank protein ID BCD42744.1) coding sequence and inserted into the pPCl vector in frame with Aga2p coding sequence to generate the DNA construct pPCl-UspA-EloTu.

[0197] Recombinant DNA construct was cloned in E. coli DH5a (New England Biolabs).

[0198] 1.3 / Production of a DNA construct for intracellular antigen expression:

[0199] PyrR construct: A fragment of 5. aureus Bifunctional pyr operon transcriptional regulator / uracil phosphoribosyltransferase (PyrR, GenBank ID BCD43304.1) coding sequence was inserted into the GDP-p425 vector backbone

[0014] to generate the DNA construct p425- GDP-PyrR.

[0200] Lipase-Coagulase construct: A fragment of Staphylococcus aureus Alpha / beta hydrolase (Lipase, GenBank ID WPA54171.1) was fused to a fragment of 5. aureus Staphylocoagulase (Coagulase, GenBank ID WPA42669.1) coding sequence and inserted into the GDP-p425 vector backbone

[0014] to generate the DNA construct p425-GDP-Lipase- Coagulase.

[0201] Lipase-Enolase construct: A fragment of Staphylococcus aureus Alpha / beta hydrolase (Lipase, GenBank ID WPA54171.1) was fused to a fragment of Enolase (Enolase, GenBank protein ID VDZ34627.1) coding sequence and inserted into the GDP-p425 vector backbone

[0014] to generate the DNA construct p425-GDP-Lipase-Enolase.

[0202] Lipase-EloTu construct: A fragment of Staphylococcus aureus Alpha / beta hydrolase (Lipase, GenBank ID WPA54171.1) was fused to a fragment of Elongation Factor Tu (EloTu, GenBank protein ID BCD42744.1) coding sequence and inserted into the GDP-p425 vector backbone

[0014] to generate the DNA construct p425-GDP-Lipase-EloTu.

[0203] Recombinant DNA construct was cloned in E. coli DH5a (New England Biolabs).

[0204] 2 / Yeast transformation

[0205] The Saccharomyces cerevisiae strain Y05574 (Euroscarf, BY4741; MATa; his3- deltal; leu2-delta0; metl5-delta0; ura3-delta0; YPR159w::kanMX4), depleted of the gene kre6 (for « Killer toxin Resistant 6 »), was cultivated in YPD broth to ODeoo of 0.6 - 0.8 and routinely transformed using the LiAc / SS carrier DNA / PEG method [8] as follows:

[0206] Formulation #3, yeast strain #3.1: 6 - 8.107UFC of Y05574 were transformed with 1 pg of pPCl-UspA-PyrR and 1 ug of p425-GPD-PyrR. Transformants were selected in minimal agar #3 plates (0.67% Yeast Nitrogen Base with ammonium sulfate, 2% glucose, dropout aminoacids mixture (76 mg / l each) without leucine, uracyl and histidine, 2% bacto agar).

[0207] Formulation #3, yeast strain #3.2: 6 - 8.107UFC of Y05574 were transformed with 1 pg of pPCl-UspA-Coagulase and 1 ug of p425-GPD-Lipase-Coagulase. Transformants were selected in minimal agar #3 plates (0.67% Yeast Nitrogen Base with ammonium sulfate, 2% glucose, dropout aminoacids mixture (76 mg / l each) without leucine, uracyl and histidine, 2% bacto agar).

[0208] Formulation #3, yeast strain #3.3: 6 - 8.107UFC of Y05574 were transformed with 1 pg of pPCl-UspA-Enolase and 1 pg of p425-GPD-Lipase-Enolase. Transformants were selected in minimal agar #3 plates (0.67% Yeast Nitrogen Base with ammonium sulfate, 2% glucose, dropout aminoacids mixture (76 mg / l each) without leucine, uracyl and histidine, 2% bacto agar). Formulation #3, yeast strain #3.4: 6 - 8.107UFC of Y05574 were transformed with 1 ng of pPCl-UspA-EloTu and 1 pg of p425-GPD-Lipase-EloTu. Transformants were selected in minimal agar #3 plates (0.67% Yeast Nitrogen Base with ammonium sulfate, 2% glucose, dropout aminoacids mixture (76 mg / l each) without leucine, uracyl and histidine, 2% bacto agar).

[0209] 3 / Production of yeast expressing S. aureus antigens

[0210] Strains #3.1, #3.2, #3.3 and #3.4 were inoculated at a ODeoo of 0.2 in minimal broth #3 (0.67% Yeast Nitrogen Base with ammonium sulfate, 2% glucose, dropout aminoacids mixture (76 mg / l each) without leucine, uracyl and histidine). Cultures were incubated overnight at 26-28°C with agitation (250 rpm) in order to obtain cultures with an ODeoo of 4 - 5.

[0211] 4 / Preparation of vaccines

[0212] Cultures were concentrated to an ODeoo of 10 - 20 in DPBS without Ca+2and Mg+2and inactivated with 4% Paraformaldehyde for 20 min at room temperature. Yeast cells were centrifuged at 3500 rpm for 10 min at 10°C and washed twice with wash buffer (DPBS without Ca+2and Mg+2, 2% FBS, 2 mM EDTA). Dry yeast pellets were kept at -20°C until vaccination. For the preparation of vaccine doses, the required amount of vaccine yeast (refer to table below) was resuspended in 3 ml of DPBS without Ca+2and Mg+2.

[0213] Vaccine formulation #3 in vivo evaluation (cellular response)

[0214] Two dairy cows were vaccinated with formulation #3 as follows: intramuscular prime followed of intramammary booster 15 days later (see Table 4 for vaccine composition). Forty-two days after booster, vaccinated cows were stimulated with an intramammary injection of 30 ug of recombinant UspA or PyrR resuspended in sterile DPBS in the front right and rear right mammary quarters, respectively. Mammary secretion volume and cell count were estimated before, 24h and 48h after stimulation with recombinant antigens. As shown in Figure 8, stimulation with UspA induced an increase of cell counts in both animals. Stimulation with PyrR resulted in an increase of cell counts in 1 of the animals.

[0215] Vaccine formulation #3 in vivo evaluation (humoral response) Five dairy cows were vaccinated with formulation #3 as follows: intramuscular prime followed of intramammary booster 15 days later (refer to Table 4 for vaccine composition). Five non vaccinated dairy cows were used as control. Serum from all experimental animals was collected at 0, 14, 28 and 48 days after prime vaccination to verify the presence of anti PyrR, Coagulase and UspA specific antibodies by ELISA (Figure 9).

[0216] Conclusion

[0217] - A vaccine formulation based in a cell wall mutant strain of 5. cerevisiae could be produced (Formulation #3);

[0218] - 5. aureus antigens could be produced by 5. cerevisiae under a constitute promoter, as a simplified and cost-efficient alternative to galactose-induced production;

[0219] - Vaccine formulation #3 could induce UspA and PyrR specific mammary cellular immune response. As this mechanism is described as the first barrier to bacterial infection in the mammary gland, these results suggest that Formulation #3 could be used to protect the bovine mammary gland against infections by 5. aureus.

[0220] - Vaccine formulation #3 could induce anti UspA, Coagulase and PyrR specific antibodies in the blood serum. These results suggest that Formulation #3 could be used to protect the bovine mammary gland against infections by 5. aureus.

[0221] Formulation #3 composition and in vivo evaluation-related information are summarized in the Table 4 below:

[0222] Table 4

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Claims

CLAIMS1) A recombinant yeast cell genetically modified to express (i) at least one first bacterial antigen or an immunogenic fragment thereof intracellularly and (ii) at least one second bacterial antigen or an immunogenic fragment thereof on its extracellular surface, the at least one first bacterial antigen or an immunogenic fragment thereof being identical to or different from the at least one second bacterial antigen or an immunogenic fragment thereof.2) The recombinant yeast cell according to claim 1, wherein said yeast is Saccharomyces cerevisiae.3) The recombinant yeast cell according to claim 1 or 2, wherein it comprises:(i') a first expression vector comprising a first nucleotide sequence encoding at least one first bacterial antigen or an immunogenic fragment thereof operably linked to at least one first sequence controlling the intracellular expression of the at least one first bacterial antigen or an immunogenic fragment thereof and(ii') a second expression vector comprising a nucleotide sequence encoding at least one second bacterial antigen or an immunogenic fragment thereof operably linked to at least one second sequence controlling the expression of the at least one second bacterial antigen or an immunogenic fragment thereof on the extracellular surface of this yeast cell.4) The recombinant yeast cell according to claims 3, wherein, in said second expression vector, the nucleotide sequence encoding at least one second bacterial antigen or an immunogenic fragment thereof is a nucleotide sequence encoding a fusion protein comprising at least one second bacterial antigen or an immunogenic fragment thereof and a yeast extracellular surface protein.5) The recombinant yeast cell according to any one of claims 1 to 4, wherein said recombinant yeast cell is inactivated.6) The recombinant yeast cell according to any one of claims 1 to 5, wherein the expression of at least one gene involved in the synthesis or assembly of cell wall sugar components is altered.7)°The recombinant yeast cell according to claim 6, wherein said at least one gene involved in the synthesis or assembly of cell wall sugar components the expression of which is altered is selected from the group consisting of genes gasl, krel, Ias21, mnn2, chs3, kre6, exgl,fksl and ccwl2.8) The recombinant yeast cell according to any one of claims 1 to 7, wherein said at least one first bacterial antigen and said at least one second bacterial antigen are independently selected from the group consisting of antigens from Escherichia coli, antigens from Klebsiella spp., antigens from Staphylococcus aureus, antigens from coagulase negative Staphylococcus sp., antigens from Streptococcus uberis, antigens from Streptococcus agalactiae, antigens from Mycobacterium bovis, antigens from Mycobacterium avium subspecies paratuberculosis (MAP), antigens from Trueperella pyogenes, antigens from Prevotella sp., antigens from Treponema sp. and antigens from Fusobacterium sp..9) The recombinant yeast cell according to any one of claims 1 to 8, wherein said at least one first bacterial antigen and the at least one second bacterial antigen are independently selected from the group consisting of Enterobacteriaceae cell division protein FtsA, Enterobacteriaceae rod shape-determining protein MreB, Enterobacteriaceae phosphoporin PhoE, Enterobacteriaceae cysteine synthase A, Enterobacteriaceae porin OmpF, E. coli Outer membrane protein A (OmpA), 5. aureus Enolase, 5. aureus Elongation FactorTu, 5. aureus 50S ribosomal protein L6, 5. aureus Bifunctional pyr operon transcriptional regulator / uracil phosphoribosyltransferase PyrR, 5. aureus Universal stress protein UspA, 5. aureus Ferritin-like protein 2, 5. aureus Galactose-6-phosphate isomerase, LacB subunit, 5. aureus Phosphoribosylformylglycinamide synthase, 5. aureus Immunodominant staphylococcal antigen IsaB, 5. aureus Asp23 / Gls24 family envelope stress response, 5. aureus Alpha / beta hydrolase, 5. aureus Staphylocoagulase, 5. uberis staphylokinase domaincontaining protein, 5. uberis surface-displayed alpha-enolase, 5. uberis sortase, 5. uberis cell division protein FtsZ, 5. uberis cell division protein FtsA, 5. uberis cell division protein FtsL, 5.uberis septation ring formation regulator EzrA, 5. uberis ubericin A precursor peptide, 5. uberis ubericin A immunity protein, 5. uberis circular bacteriocin uberolysin, M. bovis ESAT-6 like proteins, M. bovis CFP-10, Mycobacterium avium subsp. Paratuberculosis (MAP) antigen complex 85B, MAP antigen complex 85C, MAP superoxide dismutase, MAP polyprotein 74F and homologues thereof.10) A vaccine comprising as immunogen the at least one recombinant yeast cell according to any one of claims 1 to 9.11) A vaccine composition comprising at least one recombinant yeast cell according to any one of claims 1 to 9 and a pharmaceutically acceptable excipient.12) The recombinant yeast cell according to any one of claims 1 to 9, a vaccine according to claim 10 or a vaccine composition according to claim 11 for use in protecting a mammal and in particular a bovine, an ovine, a caprine, a swine and an equine from a disease caused by bacteria.13) The recombinant yeast cell, a vaccine or a vaccine composition for use according to claim 12, wherein said disease is selected from the group consisting of mastitis, bovine / ovine / caprine paratuberculosis, bovine tuberculosis, metritis and lameness.14) The recombinant yeast cell, a vaccine or a vaccine composition for use according to claim 12 or 13, wherein said disease is mastitis.15) The recombinant yeast cell, a vaccine or a vaccine composition for use according to claim 14, wherein said recombinant yeast cell, said vaccine or said vaccine composition is administered in at least two doses and advantageously in (i) at least two doses administered intramuscularly, (ii) at least two doses administered subcutaneously in the mammary gland, (iii) at least two doses administered subcutaneously in an area different from the mammary gland or (iv) the first dose administered intramuscularly and the second one subcutaneously in the mammary gland or via the intramammary route.

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