Recombinant pyolysin protein

Abstract

The invention provides a recombinant pyolysin protein. Preferably a recombinant pyolysin protein is provided comprising at least 80% sequence identity to SEQ ID NO: 1, wherein the amino acid sequence comprises (i) an amino acid deletion at amino acid position 472, and / or (ii) an amino acid substitution at amino acid position 496 and / or at 497. Also provided by the invention are nucleic acid sequences encoding the recombinant pyolysin protein, and methods for producing of the same. The invention lastly provides for methods of amelioration and / or prevention of intrauterine disease, liver abscess and / or foot rot or vaccine compositions for use in prophylaxis of the same.

Description

[0001] RECOMBINANT PYOLYSIN PROTEIN

[0002] Field of the invention

[0003] The present invention relates to a recombinant pyolysin, a nucleic acid sequence encoding the recombinant pyolysin, a vaccine comprising the recombinant pyolysin, and a vaccine comprising the recombinant pyolysin for use in the prophylaxis of intrauterine disease.

[0004] Background

[0005] The bacterium Trueperella pyogenes (T. pyogenes), formerly also known as Arcanobacterium pyogenes, is a part of the biota of skin and mucous membranes of the upper respiratory, gastrointestinal, or urogenital tracts of animals, but also an opportunistic pathogen. It is found in farm animals such as cows, pigs and sheep, but also in wild animals. Infection with T. pyogenes can contribute to diseases including intrauterine disease, liver abscess and hoof rot. Due to a reduction of meat and / or milk yield, as well as decreased reproductive efficiency and sometimes the necessity to cull diseased animals, the diseases T. pyogenes is implicated in generate significant economic losses in livestock.

[0006] An infection with T. pyogenes can be treated with antibiotics. However, given the risk of antibiotic resistance, there is a need for prophylactic treatment against diseases associated with T. pyogenes infection.

[0007] Pyolysin is a hemolysin (i.e., pyolysin lyses red blood cells) that is secreted by T. pyogenes and contributes significantly to the pathogenicity of an infection by T. pyogenes. Pyolysin (PLO) is a member of a large group of toxins known as cholesterol-dependent cytolysins (CDCs). CDCs, including PLO, disrupt the cell membrane of cells and causing their lysis by a mechanism of pore formation: CDCs are secreted as water-soluble monomeric proteins. The toxins bind to the target membrane and oligomerize into a ring-like structure, generally referred to as the “pre-pore complex” for cholesterol-dependent cytolysins (see, Heuck, A.P., Moe, P.C., Johnson, B.B. (2010) Subcellular Biochemistry, vol 51)). A conformational change then leads to the insertion of transmembrane p-hairpins into the bilayer to form the aqueous pore.

[0008] WO 2014 / 084964 describes a vaccine formulation comprising whole cells of T. pyogenes or PLO protein. Preparing these vaccine formulations requires a step of treating T. pyogenes or PLO with formalin to inactivate so that the bacteria and / or bacterial components have less or no pathogenicity to the subject to which the formulation is administered.

[0009] The use of chemical agents in the inactivation of T. pyogenes or PLO is one with major drawbacks. One of such chemical agents used in the inactivation is formalin. Formalin is the aqueous solution of formaldehyde, and is known to be irritating, corrosive and highly toxic and carcinogenic. Moreover, formalin is readily adsorbed by the mammalian (human) body when in direct contact with the solution. Formalin, or formaldehyde, is commonly used in the inactivation or detoxification of bacterial toxins. Therefore, during the manufacture of the vaccine formulation comprising T. pyogenes or PLO the bacteria are exposed to a formalin solution, for example a 0.1% formalin solution, for a prolonged period of time, e.g., 12h, to inactivate T. pyogenes or PLO. In the manufacture of industrial batches of a vaccine, large quantities of formalin are required to inactivate sufficient T. pyogenes or PLO to produce such large batches. The using of such large quantities of the toxic and carcinogenic formalin in the manufacturing process poses a serious risk of exposure, and thus a serious health risk, to laboratory workers and vaccine production teams. Moreover, a certain amount of formalin typically remains present as constituent in the final vaccine composition, thereby affecting the vaccine composition such that administration potentially results in a harmful immune response (Moghaddam et al. Nat Med 12, 905-907 (2006)). Therefore, there is a need to dispense of the chemical inactivation step of pyolysin and / or of T. pyogenes.

[0010] Further there remains a constant need, for example considering the continuously increasing costs of veterinary healthcare, for improving vaccine compositions and the methods of producing the same.

[0011] It is an object of the present invention to at least meet the above-mentioned needs.

[0012] Summary of the invention

[0013] To meet the object of the invention there is provided, in a first aspect, for a recombinant pyolysin protein comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 , and comprising:

[0014] (i) an amino acid mutation at amino acid position 472, and

[0015] (ii) an amino acid mutation at amino acid position 496 and at amino acid position 497. In a second aspect there is provided for a nucleic acid sequence encoding the recombinant pyolysin protein according to the invention.

[0016] In a further aspect there is provided for a vector comprising a nucleic acid sequence encoding the recombinant pyolysin protein according to the invention.

[0017] In a further aspect there is provided for a host cell comprising a nucleic acid sequence encoding the recombinant pyolysin protein according to the invention. There further is provided for a cell culture of one or more of said host cells.

[0018] In a further aspect there is provided for a vaccine composition comprising the recombinant pyolysin protein according to the invention, or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

[0019] In a further aspect there is provided for a vaccine composition comprising the recombinant pyolysin according to the invention for use in the amelioration and / or prophylaxis of intrauterine disease, liver abscess and / or foot rot.

[0020] In a further aspect there is provided for a method of producing a vaccine composition comprising the recombinant pyolysin protein according to the invention.

[0021] In a further aspect there is provided for a method of improving the reproductive function of a ruminant.

[0022] In a further aspect there is provided for a method of ameliorating and / or preventing a diseased state in a ruminant.

[0023] Lastly, in one aspect there is provided for a kit of parts comprising the recombinant pyolysin protein according to the invention.

[0024] Figure descriptions

[0025] Fig. 1 : SDS PAGE (A) and Western blot (B) using anti His antibody (GenScript,

[0026] Cat.No.A00186)) analysis of p.l61A_D238R_ pET15b in E. coli expression in construct pET 15b. SDS PAGE (C) and Western blot (D) using anti His antibody (GenScript, Cat.No.A00186)) analysis of p.P472del_T496L_L497T_D238R_ pET15b in E. coli expression in construct pET 15b. SDS PAGE (E) and Western blot (F) using anti His antibody (GenScript, Cat.No.A00186)) analysis of p.T496L_L497T_P472del in E. coli expression in construct pET 15b.

[0027] (A), (C), (E): Lane Mi : Protein marker, Lane PCi: BSA (1 pg), Lane PC2: BSA (2 pg), Lane NC: Cell lysate without induction, Lane 1 : Cell lysate with induction for 16 h at 15°C, Lane 2: Cell lysate with induction for 4h at 37°C, Lane NC1: Supernatant of cell lysate without induction, Lane 3: Supernatant of cell lysate with induction for 16 h at 15°C, Lane 4: Supernatant of cell lysate with induction for 4h at 37°C, Lane NC2: Pellet of cell lysate without induction, Lane 5: Pellet of cell lysate with induction for 16h at 15°C, Lane 6 : Pellet of cell lysate with induction for 4h at 37°C.

[0028] (B), (D), (F): Lane M2: Western blot marker, Lane NC: Cell lysate without induction , Lane 1 : Cell lysate with induction for 16 h at 15°C, Lane 2: Cell lysate with induction for 4h at 37°C, Lane NC1: Supernatant of cell lysate without induction, Lane 3: Supernatant of cell lysate with induction for 16 h at 15°C, Lane 4: Supernatant of cell lysate with induction for 4h at 37°C, Lane NC2: Pellet of cell lysate without induction, Lane 5: Pellet of cell lysate with induction for

[0029] Fig. 2: (A) Hemolysis inhibition assay with tested dilutions: 1 :2, 1 :4, 1 :8, 1 :16, 1 :32, 1 :64, 1 :128, 1 :256, 1 :512, 1 :1024, 1 :2048, 1 :4096. T. pyogenes supernatant contains the recombinant pyolysin protein according to the invention. At dilutions 1 :1 , 1 :2, 1 :4, 1 :8, 1 :16, 1 :32 hemolytic activity was detected (pyolysin titer). At dilution 1 :64 the T. pyogenes supernatant comprising the recombinant pyolysin protein according to the invention lost hemolytic activity. A pyolysin sample with known hemolytic activity, namely a native PLO from T. pyogenes sup. was used as a positive control, and heat inactivated pyolysin was used as a negative control. The positive control showed hemolytic activity at a dilution of 1 :128. As negative control a heat-inactivated pyolysin sample was used.

[0030] (B) Reference samples for the hemolysis assay (see, Figure 20) wherein the positive control was a native PLO (pyolysin sample with known hemolytic activity), negative control was a heat-inactivated pyolysin sample. The negative control heat-inactivated pyolysin sample has a titer of <2. The positive control pyolysin sample with known hemolytic activity has a titer or 64.

[0031] (C) Hemolysis assay wherein truncated versions of pyolysin were tested for hemolytic activity. Of these truncated versions three samples were selected namely: Sample #2: pyolysin with amino acid mutations 161 A & D238R (cone. 0,97 mg / ml); Sample #3: pyolysin with amino acid mutations P472del, T496L, L497T & D238R (cone. 4,34 mg / ml); Sample #4: pyolysin with amino acid mutations T496L, L497T & P472del (cone. 3,98 mg / ml). Sample #3 and Sample #4 have lost hemolytic activity. Definitions

[0032] For purposes of the present invention, the following terms are defined below.

[0033] As used herein, the singular form terms “a”, “an” and “the” include the plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a cell” includes a combination of two or more cells, and the like.

[0034] As used herein, one amino acid sequence is 100% "identical" or has 100% “sequence identity” to a second amino acid sequence when the amino acid residues of both sequences, when aligned, are identical, when determined by using the computer program " BLAST®" by selecting sub-program: "Global Alignment” using standard settings, that can be found at https: / / blast.ncbi.nlm.nih.gov / Blast.cqi. Accordingly, an amino acid sequence (or nucleic acid sequence) can be 50% "identical" to a second amino acid sequence (or second nucleic acid sequence) when 50% of the amino acid residues of the two amino acid sequences (or nucleic acids of the two nucleic acid sequences) are identical when determined by using the computer program "BLAST®" by selecting sub-program: "Global Alignment” using standard settings. The sequence comparison is performed over a contiguous block of amino acid residues comprised by a given protein, e.g., a protein, or a portion of the polypeptide being compared. In a particular embodiment, selected substitutions, deletions, or insertions that either do or do not alter the correspondence between the two amino acid sequences are also encompassed. Similarly, a nucleic acid sequence is 100% "identical" or has 100% “sequence identity” to a second nucleic acid sequence when the nucleic acid residues of both sequences, when aligned, are identical, when determined by using the computer program "BLAST®" by selecting sub-program: "Global Alignment” using standard settings.

[0035] As used herein, the term “formalin” refers to an aqueous solution of formaldehyde.

[0036] As used herein, the term “host cell” refers to a cell used to express a recombinant insulin precursor. Typically, the host cell is a microorganism such as a yeast cell or a bacterium. A suitable host cell may be Escherichia coli (E. coli).

[0037] A “pharmaceutically acceptable carrier” refers to a biocompatible medium, viz. a medium that after administration does not induce significant adverse reactions in the treated subject, capable of presenting the antigen to the immune system of the animal after administration of the composition comprising the carrier. Such a pharmaceutically acceptable carrier may for example be a liquid containing water and / or any other biocompatible solvent or a solid carrier such as commonly used to obtain freeze-dried vaccines (based on sugars and / or proteins), optionally comprising an adjuvant. As used herein, the term “prophylaxis” refers to a measure that is taken to protect against a post-vaccination infection. Typically, a composition comprising a pharmaceutically active agent is administered by and / or to a subject for a prophylactic effect. Compositions suitable for prophylaxis of a disease I disorder may be referred to as a “vaccine”. Compositions suitable for prophylaxis of a disease I disorder may activate the immune system (e.g., immunization) of the subject to which a prophylactic composition is administered.

[0038] The terms “protection” or “protect” against a pathogenic infection with an infectious agent means arriving at protective immunity in an animal, i.e., aiding in preventing, ameliorating or curing (an) adverse effect(s) caused by the infection with that agent, for example, by reducing the virulence of a virulent factor that is known to contribute to the pathogenicity of an infection in an animal by a microorganism.

[0039] The term “protein” refers to a molecular chain of amino acids. Included within the definition of protein for the invention, are polypeptides, peptides and oligopeptides.

[0040] The term “recombinant protein” refers to a protein of which the amino acid sequence does not match with a protein occurring in nature. Such a protein thus has a molecular make-up that is artificial, and manmade.

[0041] As used herein, the term “vector” refers to a construct used to introduce DNA into a host cell. After the vector is introduced into the host cell, the DNA present on the vector can then be expressed by the host cell, resulting in the production of a protein encoded by the DNA. The vector can for example be a virus, but is typically a plasmid, e.g., a pET plasmid or a pGEX plasmid.

[0042] The term “vaccine” is herein used to refer to a composition suitable for administration to a mammal, comprising one or more antigens of an infectious agent in an immunologically effective amount, typically combined with a pharmaceutically acceptable carrier, which upon administration to the animal induces an immune response that protects the animal against a pathogenic infection with the infectious agent.

[0043] Detailed description of the invention

[0044] The invention is defined herein and in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention. It is contemplated that any product, method, use or composition described herein can be implemented with respect to any other product, method, use or composition described herein. Embodiments disclosed in the context of products, methods, uses or compositions of the invention may be employed with respect to any other product, method, use, or composition described herein. Thus, an embodiment pertaining to one product, method, use or composition may be applied to other products, methods, uses or compositions of the invention as well.

[0045] Surprisingly, the inventors found that by genetically inactivating a pyolysin protein the obtained recombinant pyolysin protein had desirable characteristics.

[0046] Amino acid sequences

[0047] The current invention provides for a recombinant pyolysin comprising an amino acid sequence at least 80% identical to SEQ ID NO: 1 and comprising:

[0048] (i) an amino acid deletion at position 472; and / or

[0049] (ii) an amino acid substitution at position 496 and at position 497.

[0050] It is preferred that the current invention provides for a recombinant pyolysin comprising an amino acid sequence at least 80% identical to SEQ ID NO: 1 and comprising:

[0051] (i) an amino acid deletion at position 472; and

[0052] (ii) an amino acid substitution at position 496 and at position 497.

[0053] It was found that the pyolysin protein having at least 80% sequence identity to SEQ ID NO: 1 and comprising an amino acid deletion at amino acid position 472 and an amino acid substitution at position 496 and at 497 has desirable feature characteristics, such as but not limited to, desirable yield (e.g., improved yield compared to other recombinant pyolysin constructs) of said protein by methods, e.g., conventional methods, of producing said protein. Moreover, it was surprisingly found that by way of the current invention a method step comprising the use of a chemical agent, e.g., formalin, for the inactivation (i.e., by reducing or removing the toxicity) of pyolysin can be avoided. It was, for example, surprisingly found that the recombinant pyolysin protein according to the invention is less toxic than a native pyolysin protein. Hence, it is understood herein that a recombinant pyolysin protein of the invention has a reduced hemolytic activity compared to a non-recombinant pyolysin protein, e.g., native (wild type) pyolysin protein. In other words, the recombinant pyolysin protein is genetically inactivated. Accordingly, the recombinant pyolysin protein of the invention may be used in a vaccine composition that is safe and able to induce an antibody without the need of having to inactivate the protein by use of chemical agents, e.g., formalin. Preferably, the resulting vaccine formulation is non-toxic.

[0054] Therefore, in a first aspect of the invention, there is provided for a recombinant pyolysin protein comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 , and comprising:

[0055] (i) an amino acid deletion at position 472, and

[0056] (ii) an amino acid substitution at position 496 and at position 497.

[0057] It is to be understood that the numbering of the residue positions (e.g., the amino acid positions) corresponds to the numbering of amino acid residue positions of the amino acid sequences of the PLO proteins disclosed herein (e.g., SEQ ID NO: 1 , 2, 4, 6 and 8, see Table 8) and / or nucleic acid residue positions of nucleic acid sequences encoding PLO proteins disclosed herein (e.g., SEQ ID NO: 3, 5, 7 and 9, see Table 8)). For example, when referring to position 472 in SEQ ID NO: 1 there is meant the amino acid residue on position 472 in SEQ ID NO: 1 , which in this case is a “Proline”.

[0058] It will be understood that the current invention also encompasses any amino acid sequences having at least 80% sequence identity to SEQ ID NO: 1 and at least comprising an amino acid deletion at amino acid position 472, and an amino acid substitution at position 496 and at 497. Accordingly, it is encompassed by the current invention that there is provided for a recombinant pyolysin protein in accordance with the invention (for example, an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 , and comprising an amino acid deletion at amino acid position 472, and an amino acid substitution at position 496 and at 497) wherein the amino acid sequence further comprises mutations that align with a codon optimized nucleic acid sequence, preferably a nucleic acid sequence that is colon-optimized for expression in E. coli, encoding the recombinant pyolysin protein. For example, it is contemplated that a nucleic acid sequence of native PLO as disclosed by Machado et al., PLoS ONE, 2014, 9:e91734 can be codon optimized, preferably for E. coli, to arrive at a nucleic acid sequence encoding an amino acid sequence having at least 80% sequence identity to SEQ ID NO:1. Codon optimization of nucleic acids, e.g., for expression in a host cell, is a method that is commonly known and used in the art and commonly is used for selecting synonymous codons that may improve protein expression in a host cell.

[0059] It will also be understood that, for the proteins encompassed herein, natural variations can exist between individual T. pyogenes strains. These variations may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions, or additions of (an) amino acid(s) in said sequence. Amino acid substitutions which do not essentially alter biological and immunological activities, have been described, e.g., by Neurath et al in "The Proteins" Academic Press New York (1979). Amino acid replacements between related amino acids or replacements which have occurred frequently in evolution are, inter alia, Ser / Ala, Ser / Gly, Asp / Gly, Asp / Asn, lle / Val (see, Dayhof, M.D., Atlas of protein sequence and structure, Nat. Biomed. Res. Found., Washington D.C., 1978, vol. 5, suppl. 3). Other amino acid substitutions include Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Thr / Phe, Ala / Pro, Lys / Arg, Leu / lle, Leu / Val and Ala / Glu. Based on this information, Lipman and Pearson developed a method for rapid and sensitive protein comparison (Science, 227, 1435-1441 , 1985) and determining the functional similarity between homologous proteins. Such amino acid substitutions of the exemplary embodiments of this invention, as well as variations having deletions and / or insertions are within the scope of the invention as long as the resulting proteins retain their immune reactivity.

[0060] This explains why recombinant pyolysin proteins according to the invention, when isolated from different field isolates, may have sequence identity levels of at least 80% sequence identity, while still representing the same protein with the same immunological characteristics. Those variations in the amino acid sequence of a certain protein according to the invention that still provide a protein capable of inducing an immune response against infection with T. pyogenes or at least against the clinical manifestations of the infection are considered as not essentially influencing the immunogenicity and are therefore also encompassed by the current invention.

[0061] It is preferred that the amino acid sequence has at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably at least 95% sequence identity to SEQ ID NO: 1. It is understood that the amino acid of the recombinant pyolysin protein according to the invention comprises at least, with increasing preference, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% sequence identity to the amino acid sequence according to SEQ ID NO: 1. Therefore, in some embodiments, the amino acid sequence of the recombinant pyolysin protein may comprise a further amino acid change, such as a mutation, insertion, deletion and / or substitution. It is preferred that the amino acid changes do not alter the protective immunogenicity of the recombinant pyolysin protein provided herein.

[0062] In one preferred embodiment the recombinant pyolysin protein comprises that the amino acid deleted by the amino acid deletion at position 472 of SEQ ID NO: 1 is a Proline (P). It was found that the proline residue is associated with a loss of hemolytic activity, thereby reducing the virulence of the pyolysin protein. In one preferred embodiment the recombinant pyolysin protein comprises that the amino acids substituted by the amino acid substitutions at position 496 and 497 of SEQ ID NO: 1 are: a) at position 496 a Threonine (T) is substituted for a Leucine (L); and b) at position 497 a Leucine (L) is substituted for a Threonine (T).

[0063] It was found that these substitutions, in the recombinant pyolysin protein according to the invention results in a reduced binding of the pyolysin protein to membrane cholesterol. It is contemplated that these amino acid substitutions in a recombinant pyolysin protein positively contribute, individually or in combination, to a reduced hemolytic activity of pyolysin. Hence, it may be that one of these substitutions is present in the recombinant pyolysin protein according to the invention. Therefore, in one optional embodiment the recombinant pyolysin protein comprises that the amino acids substituted by the amino acid substitutions at position 496 and 497 of SEQ ID NO: 1 are that at position 496 a Threonine (T) is substituted for a Leucine (L) or that at position 497 a Leucine (L) is substituted for a Threonine (T). It is preferred that both substitutions at position 496 and 497 of SEQ ID NO: 1 , wherein at position 496 a Threonine (T) is substituted for a Leucine (L) and at position 497 a Leucine (L) is substituted for a Threonine (T) are present in the recombinant pyolysin protein according to the invention.

[0064] Therefore, there is further herein provided for a recombinant pyolysin protein according to the invention comprising:

[0065] (i) an amino acid deletion at position 472 of SEQ ID NO: 1 ; and

[0066] (ii) an amino acid substitution at position 496 and at 497 of SEQ ID NO: 1 ; wherein the amino acid deleted by the amino acid deletion at position 472 is a Proline (P), and wherein the amino acids substituted by the amino acid substitutions at position 496 and position 497 are: a) at position 496 a Threonine (T) is substituted for a Leucine (L); and b) at position 497 a Leucine (L) is substituted for a Threonine (T).

[0067] Accordingly, in one preferred embodiment, the amino acid sequence of the recombinant pyolysin protein according to the invention has 80% sequence identity to SEQ ID NO: 2, in a more preferred embodiment it is understood that the amino acid of the recombinant pyolysin protein according to the invention comprises at least, with increasing preference, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% of the amino acid sequence according to SEQ ID NO: 2. It will be understood that the synthetic amino acid sequence according to SEQ ID NO: 2 comprises a recombinant pyolysin protein in accordance with the invention, wherein said amino acid sequence according to SEQ ID NO: 2 is encoded by a nucleic acid sequence that is codon optimized for expression in a host cell, preferably E. coli. As provided herein said amino acid sequence according to SEQ ID NO: 2 comprises a recombinant genetically inactivated form of a pyolysin protein. Said amino acid sequence according to SEQ ID NO: 2 is 506 amino acid residues in length and comprises at position 495 a Leucine (L) and at position 496 a Threonine (T). Moreover, a Proline (P) has been deleted on position 472, e.g., compared to SEQ ID NO: 1 and / or SEQ ID NO: 8.

[0068] It will be understood that the recombinant pyolysin protein of the invention provided herein may comprise further mutations in the amino acid sequence of said protein. One of such mutations can comprise the substitution at position 238 of SEQ ID NO: 2, wherein the Asparagine (D) is replaced by a different amino acid, preferably by an Arginine (R). Therefore, in one preferred embodiment, the recombinant pyolysin according to the invention comprises at least, with increasing preference, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% of the amino acid sequence according to SEQ ID NO: 4 and / or SEQ ID NO: 6.

[0069] Mutations in amino acid sequences of a pyolysin protein can be obtained by using standard techniques known in the art, such as introducing the mutations in a plasmid comprising a PLO gene and using standard DNA manipulation procedures, e.g. by using primers, to obtain plasmids comprising nucleic acid sequences encoding the recombinant pyolysin proteins described herein. A skilled person will be able to introduce mutations in amino acid sequences to obtain mutant amino acid sequences. An exemplary method to which the current invention is not limited is described in Example 1.

[0070] Nucleic acid sequences

[0071] In a second aspect of the invention, there is provided for a nucleic acid sequence encoding the recombinant pyolysin protein according to the invention.

[0072] In one embodiment of the invention there is provided for a nucleic acid sequence encoding for at least 80% of the amino acid sequence according to SEQ ID NO: 1. Preferably, there is provided for a nucleic acid sequence encoding at least 80% of the amino acid sequence according to SEQ ID NO: 1 wherein said amino acid sequence at least comprises an amino acid deletion at position 472 of SEQ ID NO: 1 and an amino acid substitution at position 496 and at 497 of SEQ ID NO: 1 , wherein the amino acid deleted by the amino acid deletion at position 472 is a Proline (P), and wherein the amino acids substituted by the amino acid substitutions are that at position 496 a Threonine (T) is substituted for a Leucine (L) and that at position 497 a Leucine (L) is substituted for a Threonine (T). In a preferred embodiment of the invention there is provided for a nucleic acid sequence encoding for at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, most preferably 100% sequence identity of the amino acid sequence according to SEQ ID NO: 2.

[0073] Preferably, the nucleic acid sequence has at least 80% sequence identity to the PLO gene encoding native (wild type) pyolysin protein, such as the nucleic acid sequence of the PLO gene from T. pyogenes ATCC49698 disclosed by Machado et al., PLoS ONE, 2014, 9:e91734 and incorporated herein by reference. In certain aspects, the nucleic acid sequence encoding native pyolysin is codon optimized for expression in a host cell, e.g., a microorganism such as E. coli.

[0074] It is preferred that the nucleic acid sequence has at least 80%, preferably at least 85%, more preferably 90%, even more preferably at least 95%, even more preferably at least 99%, most preferably 100% sequence identity to SEQ ID NO: 3. It is understood that the nucleic acid encoding the recombinant pyolysin protein according to the invention comprises at least, with increasing preference, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% of the nucleic acid sequence according to SEQ ID NO: 3. Therefore, in some embodiments, the nucleic acid encoding the recombinant pyolysin protein may comprise one, or more than one nucleotide changes, such as a mutation, insertion, deletion and / or substitution. It is preferred that the changes in nucleotide do not alter the protective immunogenicity of the encoded recombinant pyolysin protein.

[0075] The nucleic acid sequences according to the invention can be encompassed in a vector. Therefore, in an aspect of the invention there is also provided for a vector comprising the nucleic acid molecule encoding a recombinant pyolysin protein as broadly disclosed herein. The nucleic acid sequence can be cloned in the vector using restriction enzymes recognizing restriction sites present in the vector, such as Xho\, BamHI, EcoRI, Nde\, Bgll, / 7 / ndlll. The vector preferably is a (live) bacterial vector, more preferably a bacterial vector plasmid. Transcription and translation of the nucleic acid molecule encoding the recombinant pyolysin protein that is comprised in the vector results in the antigen being expressed in cells infected with the vector. The nucleic acid molecule encompassed in the vector preferably comprises a full gene encoding the recombinant pyolysin protein or may be fragment thereof encoding a virulent section of the protein. It is preferred that the vector is a recombinant plasmid, such as pET-15b.

[0076] It will be understood herein that the invention comprises a vector, such as a plasmid molecule, that comprises a nucleic acid sequence that encodes for the recombinant pyolysin protein provided herein. In a preferred embodiment, the vector comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 3 and / or comprises a nucleic acid sequence encoding an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0077] Typically, the vector comprises a nucleic acid sequence encoding an amino acid sequence encoding the recombinant pyolysin according to the invention, wherein the nucleic acid sequence further comprises a startcodon (e.g., the nucleic acid sequence “ATG”) at the 5’ terminal and a stopcodon at the 3’ terminal (e.g., the nucleic acid sequence “TAATGA”). Also, the vector may further comprise a nucleic acid sequence encoding an amino acid sequence that comprises a His-tag (e.g., the amino acid sequence “HHHHHH”). Further, the vector may comprise a nucleic acid sequence encoding an amino acid sequence comprising a cleavage site for enzymes, e.g., thrombin, and / or nucleic acid sequences that can act as restriction site for restriction enzymes (e.g., “GGATCC” for BamHI and / or “CATATG” for Nde\). Typically, the amino acid sequence may further comprise a nucleic acid sequence encoding for a linker, such as a sequence encoding for a flexible linker, e.g., in the form of repeats of serine and / or glycine residues (Ser / Gly linkers). Generally, a skilled person is aware of methods suitable for cloning nucleic acids, e.g., nucleic acids encoding proteins of interest, in vectors and with suitable or desirable additional constructs, such as a startcodon, stopcodon, His-tag, linker, cleavage site and / or restriction site to incorporate in such methods. The amino acid sequences and / or nucleic acid sequences provided in the herein disclosed sequences in SEQ ID Nos 1 - 9 do not include the any one of the amino acid sequences of a flexible linker, His-tag, vector, cleavage site and / or restriction site and / or nucleic acid sequences encoding for a flexible linker, His-tag, vector, cleavage site and / or restriction site. A skilled person is able to select suitable / desirable amino acid sequences of a flexible linker, His-tag, vector, cleavage site and / or restriction site and / or nucleic acid sequences encoding for a linker, His-tag, vector, cleavage site and / or restriction site for the intended purpose, e.g., for the expression of recombinant pyolysin in an organism.

[0078] Any of the recombinant proteins described herein can typically be produced recombinantly using conventional techniques, such as obtaining from a suitable expression system the recombinant proteins. In a further aspect the invention provides for a host cell comprising a nucleic acid sequence encoding the recombinant pyolysin protein. The host cell preferably is transformed by using the vector, e.g., a recombinant plasmid, comprising the nucleic acid molecule encoding a recombinant pyolysin protein according to the invention and thereby the host cell can express the recombinant pyolysin protein. Typically, the host cell is a microorganism, such as a bacterium or a yeast cell. It is preferred that the host cell is an Escherichia coli (E. coli) cell. It is preferred that the nucleic acid sequence encoding the recombinant pyolysin protein according to the invention is codon optimized for the host cell. It is understood that codon optimization may be used for optimizing the nucleic acid sequences for expression in other microorganisms other than E. coli. Hence, it is understood that such optimized nucleic acid sequences are also encompassed by the current disclosure.

[0079] Typically, the recombinant pyolysin protein is expressed in a population of host cells in order to produce larger quantities of the recombinant protein. Therefore, in one aspect there is provided for a cell culture of one or more host cells. Further encompassed herein are methods for purification of the recombinant pyolysin protein from a (population of) host cells. A skilled person generally is familiar with methods suitable for purification of recombinant proteins, e.g., recombinant pyolysin, for example by using a nickel-column purification method.

[0080] Vaccine

[0081] In a further aspect the invention provides for a vaccine composition comprising the recombinant pyolysin protein according to the current invention, or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier. It was surprisingly found that vaccine compositions comprising the recombinant pyolysin protein according to the invention were able to induce an antibody inducing response in a bovine. Moreover, it was found that vaccine compositions comprising the recombinant pyolysin protein according to the invention were able to induce the antibody inducing response in bovine at a significantly lower dose compared to formalin inactivated pyolysin (see, Example 4). Also, it was found that the vaccines comprising the recombinant pyolysin protein according to the invention are safe. Typically, a vaccine composition may comprise any pharmaceutically acceptable carrier that is suitable for uses according to the invention, e.g., a liquid, a solid, a powder, etc. The liquid may be aqueous or oily; the solid may be frozen or freeze-dried. Also, a vaccine composition typically comprises one or more adjuvants. Adjuvants may stimulate the immune response of a target in a non-specific manner. Many different adjuvants are known in the art. Examples of adjuvants are: complete- or incomplete Freund’s adjuvant, vitamin E or alpha-tocopherol, non-ionic block polymers and polyamines such as dextran sulphate, Carbopol™, pyran, Saponin, such as: Quil A™, or Q-vac™. Saponin and vaccine components may be combined in an ISCOM™. Furthermore, peptides such as muramyl dipeptides, dimethylglycine, tuftsin, are often used as adjuvant, and mineral oil e.g. Bayol™, Drakeol™, Klearol™, or Marcol™, Montanide™ or light mineral (paraffin) oil; non-mineral oil such as squalene, squalane; vegetable oils or derivatives thereof, e.g. ethyl- oleate. Also, combination products such as ISA™ (Seppic), or DiluvacForte™ and Xsolve™ (both MSD Animal Health) can advantageously be used. A further option is the use of SVEA adjuvant (comprising squalane and vitamin E-acetate) as disclosed in WO 2018 / 115435. Adjuvants such as Emunade® (MSD Animal Health) can be advantageously used and are formulated as oil-in-water (O / W) emulsions. Also, adjuvants can be formulated as water-in-oil (W / O) emulsions. A handbook on adjuvants and their uses and effects is: “Vaccine adjuvants” (Methods in molecular medicine, vol. 42, D. O’Hagan ed., 2000, Humana press, NJ, ISBN: 0896037355). Typically, vaccine compositions may further comprise other pharmaceutically acceptable constituents, not limited to the examples of such constituents selected from preservatives and fillers.

[0082] The vaccine compositions according to the invention may comprise further antigenic constituents, e.g., antigenic fragments and / or inactivated whole cells. It is preferred that the further antigenic constituents are related to the bacterial causes of any one of intrauterine disease, bovine liver abscess (BLA) and / or bovine foot rot (BFR). Aside from T. pyogenes certain bacterial strains of Fusobacterium necrophorum (F. necrophorum) and Escherichia coli (E. coli) have been described to have been associated with intrauterine disease, bovine liver abscess (BLA) and / or bovine foot rot (BFR) (see, Machado et al., PLoS ONE, 2014, 9:e91734). Therefore, in a preferred embodiment of the invention a vaccine composition is provided comprising, as a first vaccine component, the recombinant pyolysin protein according to the invention and comprising, as further vaccine components, associated virulence components of F. necrophorum, preferably F. necrophorum leukotoxin (LKT), and / or virulence components of E. coli, preferably native and / or recombinant type 1 fimbrial adhesin (FimH). A further E. coli antigen that may be considered suitable in a vaccine composition is a native and / or recombinant pilus antigen. Preferably, at least one of the further vaccine components F. necrophorum leukotoxin (LKT) and / or recombinant type 1 fimbrial adhesin (FimH) is provided as an inactivated vaccine component. In some embodiments the F. necrophorum leukotoxin (LKT) is inactivated by using a chemical agent for inactivating the hemogenic activity of LKT, such as the chemical agent formalin. A preferred vaccine composition according to the invention at least comprises native F. necrophorum leukotoxin (nLKT), recombinant pyolysin protein according to the invention and recombinant E. coli type 1 fimbrial adhesin (rFimH), wherein rFimH may be as described by WO2014084964A1 , herein incorporated by reference in its entirety.

[0083] It was found that vaccine compositions comprising native F. necrophorum leukotoxin (nLKT), recombinant pyolysin protein according to the invention and recombinant E. coli type 1 fimbrial adhesin (rFimH) were able to induce an antibody inducing response for each one of the antigens contained in said vaccine composition in a bovine and are safe (see, Example 4).

[0084] Medical uses

[0085] In another aspect the invention provides for a vaccine composition comprising the recombinant pyolysin protein of the invention for use in the prophylaxis of diseases in mammals. The vaccine composition provided herein is particularly suitable in veterinary medicine. Therefore, the vaccine composition preferably is for use in the prophylaxis of diseases in ruminants, more preferably cattle (e.g., but not limited to, cows, pigs and sheep). In one embodiment the vaccine composition is for use in preventing diseases in mammals of the genus Bos, such as cows, oxen, yaks, buffalo etc. In preferred embodiments the ruminant is a cow. The vaccine composition of the invention is particularly suitable for use in the prophylaxis of intrauterine disease, bovine liver abscess (BLA) and / or bovine foot rot (BFR). It is, for example, contemplated that the vaccine composition of the current invention is an effective prophylactic for bacterial infection and / or contamination, such as intrauterine bacterial infection, bacterial hoof infection and / or infection of the liver. Further, the vaccine composition can be for use in the prophylaxis of metritis and / or endometritis that can occur as a result of bacterial infection in a ruminant. As is known in the art, metritis generally involves inflammation of the wall of the uterus, while endometritis generally involves inflammation of the endometrium.

[0086] As is shown in the Examples, the vaccine composition according to the invention was found to be safe and was found to be efficacious in the induction of antibody responses to a recombinant pyolysin protein, and further to one or more antigens selected from a F. necrophorum leukotoxin (nLKT) and a E. coli type 1 fimbrial adhesin. Therefore, in one embodiment there is provided for a method of inducing an immune response in a ruminant comprising the administration of a vaccine composition comprising the recombinant pyolysin protein according to the invention. The immune response can comprise a humoral and / or cell mediated response. The humoral response can comprise an increase in immunoglobulins specific for any protein administered in the vaccine composition. The immune response can provide a prophylactic effect against a disease, preferably a disease as described herein.

[0087] In further embodiments, the invention provides for method of treating (e.g., ameliorating and / or preventing) a disease in a ruminant, wherein said diseased state is treated, ameliorated and / or prevented by the subcutaneous administration to the ruminant of a vaccine composition as broadly encompassed herein. The disease that is treated, ameliorated and / or prevented by said administration of the vaccine composition comprises intrauterine disease, e.g., metritis, bovine liver abscess (BLA) and / or bovine foot rot (BFR).

[0088] In one embodiment, the vaccine composition is suitable for use in the prophyaxis of intrauterine disease, bovine liver abscess (BLA) and / or bovine foot rot (BFR). Preferably, the vaccine composition is administered subcutaneously. In one preferred embodiment the vaccine composition is administered to healthy ruminants so that a diseased state such as intrauterine disease, bovine liver abscess (BLA) and / or bovine foot rot (BFR) may be prevented in the ruminant to which the vaccine composition has been administered. Moreover, since metritis is most common in cattle such as ruminants after parturition, the vaccine composition can be considered suitable for administration to a pregnant ruminant, wherein the vaccine is administered subcutaneously to the pregnant ruminant at least once prior to calving. Therefore, in some aspects, the compositions are administered to a ruminant prior to parturition. It is contemplated that by administration of the vaccine bacterial infection and / or inflammation such as metritis as a result of infection may be prevented. The prevention of bacterial infection and / or inflammation such as metritis as a result of infection is considered to be beneficial for the reproductive function of a ruminant. Therefore, in further aspects, the invention provides for methods of improving the reproductive function of a ruminant, wherein the method comprises administering, preferably subcutaneously, to the ruminant a vaccine composition at least comprising the recombinant pyolysin protein according to the invention. An improvement in the reproductive function can comprise a reduction in a calving-to-conception interval relative to the calving-to-conception interval in a ruminant to which the vaccine composition has not been administered.

[0089] It is understood that the administration of the vaccine composition provided herein results in the lowering of the incidence of intrauterine disease, bovine liver abscess (BLA) and / or bovine foot rot (BFR), for example of puerperal metritis, in a group of animals, preferably ruminants. Therefore, in an embodiment there is provided for a method for the lowering of the incidence of intrauterine disease, bovine liver abscess (BLA) and / or bovine foot rot (BFR) in a group of ruminants, comprising administering to members of said group of animals a vaccine composition according to the invention, such that the incidence of intrauterine disease, bovine liver abscess (BLA) and / or bovine foot rot (BFR) in the group of animals of which members have been administered with a vaccine composition according to the invention is lower than when members of the group of animals had not been administered with the vaccine composition according to the invention.

[0090] Further it is envisioned that the invention provides for a use of a recombinant pyolysin protein according to the invention in the manufacture of a medicament, e.g., a vaccine composition, preferably to aid in the prophylaxis of intrauterine disease, bovine liver abscess (BLA) and / or bovine foot rot (BFR) in a ruminant.

[0091] It is preferred that vaccine compositions encompassed by the invention are administered in effective amounts, e.g., amounts I dosages sufficient to exhibit the desirable and prophylactic effects disclosed herein. Hence, it is envisioned that the invention provides for administration of an effective amount of recombinant pyolysin protein disclosed herein. Those skilled in the art will be able to determine, given the benefit of the recombinant pyolysin protein of the invention, when and how frequent to administer a vaccine compositions. Those skilled in the art will also be able to determine how much of the immunogenic agent may be included in a vaccine composition. In general, factors that go into this determination include, but are not limited to, type, size, age and overall health of the ruminant. In some aspects, the vaccine composition is administered to a ruminant in one dose, preferably in at least two separate doses. In some other aspects, when the vaccine composition is administered in two separate doses it is preferred that the administration of said doses is separated by at least one day, one week or at least two weeks or three weeks.

[0092] Methods & kits

[0093] In another aspect there is provided for a method for producing a vaccine composition comprising the recombinant pyolysin protein according to the invention. The method comprises the subsequent steps of: a. expressing the recombinant pyolysin protein through growth of a microorganism; b. lysing the microorganism to obtain the recombinant pyolysin protein. The microorganism inherently is a recombinant microorganism having a gene that is capable of expressing the recombinant pyolysin according to the invention. Preferably, microorganism growth is under optimal growth conditions for the used microorganism. Preferably, the method for producing the vaccine composition does not rely on the inactivation of whole cells of T. pyogenes and / or pyolysin. It is understood that the omission of the step of inactivation of T. pyogenes and / or pyolysin benefits the process of producing a vaccine composition. It is for example contemplated that at every (additional) step during the process of producing a vaccine there is a risk of denaturization of proteins included in the vaccine composition, hence any additional step can potentially result in a loss of efficacy of the produced vaccine product. Therefore, in preferred embodiments, the method does not comprise the step of inactivating the recombinant pyolysin protein by using a chemical agent suitable for inactivating pyolysin, for example formalin. Further, the method disclosed herein may comprise steps that are generally used in the production processes for vaccines, such as the further steps of:

[0094] • combining the recombinant protein with a pharmaceutically acceptable carrier;

[0095] • combining the recombinant protein with an adjuvant;

[0096] • combining the recombinant protein with a further immunogenic agent, preferably with further virulence components.

[0097] In preferred embodiments the method further comprises the steps of:

[0098] • combining the recombinant pyolysin protein with a pharmaceutically acceptable carrier;

[0099] • optionally, combining the recombinant pyolysin protein and pharmaceutically acceptable carrier with any one or more selected from the group consisting of an adjuvant, native F. necrophorum leukotoxin and E. coli FimH.

[0100] In certain aspects there is provided for a kit of parts, wherein the kit comprises at least one container, wherein the container comprises a veterinary composition comprising the recombinant pyolysin protein, F. necrophorum leukotoxin (LKT) and E. coli type 1 fimbrial adhesin (FimH). Preferably the container comprises an amount of each vaccine component sufficient for one dose. It is contemplated that the veterinary composition is a freeze-dried composition, such as a powder or in the form of flakes, granules, or pellets, that when suspended in a pharmaceutically acceptable carrier such as oily substance and / or aqueous substance, forms a suspension comprising the vaccine composition. The kit of parts optionally comprises a container of a pharmaceutically acceptable carrier.

[0101] Alternatively, there is provided for a kit of parts comprising a set of two, preferably three, containers at least comprising a first container comprises the recombinant pyolysin protein according to the invention and a pharmaceutically acceptable carrier and a second container comprising F. necrophorum leukotoxin (LKT) and a pharmaceutically acceptable carrier, and optionally a third container comprising E. coli type 1 fimbrial adhesin (FimH) and a pharmaceutically acceptable carrier. Preferably, the containers provided herein are sealed.

[0102] The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and / or adapt for various applications, such as specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

[0103] All references cited herein, including journal articles or abstract, published, or corresponding patent applications, patents, or any other references, are incorporated by reference herein in its entirety, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by reference.

[0104] It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

[0105] Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration and are not intended to be limiting to the present invention. Further aspects and embodiments will be apparent to those skilled in the art.

[0106] Examples

[0107] Example 1: Cloning and purification of recombinant pyolysin

[0108] The PLO gene lacking the coding region for the predicted signal sequence was amplified from T. pyogenes ATCC 49598 genomic DNA and cloned into a pTrcHisB plasmid as described in (Machado et al., PLoS ONE, 2014, 9:e91734). When expressed in E. coli, the expression of His-tagged pyolysin can be induced by isopropyl p-d-1 -thiogalactopyranoside (IPTG). The ORF region in each plasmid was built by de novo synthesis and subcloned into pET15b between Ndel and BamHI. Mutations were introduced in the resulting plasmid using standard DNA manipulation procedures. Example 2: Expression levels of recombinant pyolysin.

[0109] For the determining of protein expression levels E. coli BL21 (DE3) competent cells were transformed with the recombinant plasmid according to T able 1 . A single colony was inoculated into LB medium containing ampicillin; cultures were incubated in 37 °C at 200 rpm. Once cell density reached to OD=0.6-0.8 at 600 nm, 0.5 mM IPTG was introduced for induction of PLO expression. SDS-PAGE and blastwestern blot (using anti-His antibody (GenScript, Cat.No.A00186)) analysis of were used to monitor the expression. Results are shown in Table 2 and Figure 1 (A - H) for four different recombinant pyolysin constructs.

[0110] Table 1: Experimental protocol for Protein Expression Evaluation * Comprises ATG, linker, His tag, thrombin cleavage site and Nde\ restriction site Table 2: Results of SDS-PAGE and Western blot of recombinant pyolysin proteins

[0111] As will be apparent from the foregoing results presented in this Example, the p.T496L_L497T_P472del has the highest protein expression level (in mg / L) compared to other pyolysin constructs, which will be understood as resulting in an improved yield compared to different recombinant pyolysin constructs.

[0112] Example 3: Hemolysis inhibition and hemolytic activity of recombinant pyolysin

[0113] The plasmids encoding recombinant pyolysin were transformed into E. coli bacteria, and expression of recombinant pyolysin was induced by IPTG. 5 hours after induction, samples were collected from the supernatant of the respective E. coli cultures.

[0114] In a hemolysis inhibition assay (see, Figure 2A), a pyolysin sample with known toxic hemolytic activity, native PLO, was used as a positive control, and heat inactivated pyolysin was used as a negative control for a sample of supernatant of T. pyogenes comprising a recombinant pyolysin according to the invention. Positive Control, Negative control, PBS or supernatant samples were two-fold serial diluted with PBS. 0.5% sheep red blood cell (SRBC) suspension in PBS was added to each well in a ratio of SRBC suspension : sample = 1 :2. The plate was incubated at 36 ± 2°C for 120 ± 5 minutes, and the plate was observed for complete hemolysis of SRBC. The reciprocal of the highest dilution in which there is complete hemolysis was considered the pyolysin titer (see, Figure 2A).

[0115] In this hemolysis inhibition assay (see Fig. 2A) it was found that the supernatant of T. pyogenes comprising a recombinant pyolysin according to the invention lost hemolytic activity at a 1 :64 dilution, compared to a 1 :128 dilution of the native PLO (positive control). Further, it was found that even at high concentrations the recombinant pyolysin according to the invention (see Figure 2B and 2C) had lost hemolytic activity (i.e., lysis of erythrocytes), whereas native PLO maintained hemolytic activity and Sample #2 maintained toxic hemolytic activity, even at a concentration that was lower compared to a recombinant pyolysin according to the invention, e.g., such as shown in Sample #3 or #4.

[0116] It will be apparent from the foregoing results presented in the Example that a recombinant pyolysin construct according to the current invention loses hemolytic activity and therefore loses its toxicity compared to native PLO or different recombinant pyolysin constructs that maintain hemolytic activity and therefore remain toxic.

[0117] Example 4: Serological Comparison of Antigens and Dose Level of Metritis Vaccine

[0118] A vaccine composition comprising virulence factors from three of the most common bacteria associated with metritis, including the formalin inactivated native form of Fusobacterium necrophorum leukotoxin (LKT) or the recombinant domain 1 of LKT, the formalin inactivated Trueperella pyogenes recombinant pyolysin (PLO) toxoid or the genetically mutated (inactivated) PLO, and FimH antigen from Escherichia coli, were tested for their ability to induce antibody responses and safety.

[0119] This study compared serological responses to different dose levels of the new antigen preparations. Adjuvants used in the preparation of the vaccine compositions were Emunade® (herein: Emunade) and Seppic Montanide™ ISA 206 VG (herein: Seppic).

[0120] Calves were vaccinated subcutaneously (SQ) twice, 3 weeks apart, and were bled prior to each vaccination, and at various timepoints following the 2nd vaccination (study days -1 , 20, 35, 49, 77). Calves were palpated for the presence of injection site reactions at various time points following each vaccination. Serological responses to vaccination were evaluated using in-house serology ELISAs for LKT and FimH, and a hemolysis neutralization assay for PLO. The serological responses to LKT, PLO and FimH were compared between each treatment group. Table 3: Treatment groups (n=native, r=recombinant, gi=genetically inactivated)

[0121] Table 4: Antigen fractions of the various tested vaccine formulations

[0122] Biological Products used in experiment • Treatment Group 1 : 3-Way Recombinant Protein Vaccine Lot # I KC738-051 was formulated with native LKT at 40 pg / 2 ml dose, recombinant PLO (Cornell plasmid) at 200 pg / dose, and recombinant FimH (Cornell plasmid) at 100 pg / dose administered as a 2 mL dose for this study. • Treatment Group 2: 3-Way Vaccine Lot # 5014133-0127C-4 was formulated with native LKT at 5.2% final concentration (~ 30.9 pg / dose), recombinant genetically inactivated PLO at 20 pg / dose, and recombinant FimH160 (purified, refolded) at 20 pg / dose with Emunade adjuvant in a 2 mL dose.

[0123] • Treatment Group 3: 3-Way Vaccine Lot # 5014133-0127D-5 was formulated with native LKT at 2.6% final concentration (~ 15.4 pg / dose), recombinant genetically inactivated PLO at 10 pg / dose, and recombinant FimH160 (purified, refolded) at 5 pg / dose with Emunade adjuvant in a 2 mL dose.

[0124] • T reatment Group 4: 3- Way Vaccine Lot # 5014133-0127E-6 was formulated with native LKT at 2.6% final concentration (~ 15.4 pg / dose), recombinant genetically inactivated PLO at 10 pg / dose, and recombinant FimH160 (purified, refolded) at 5 pg / dose with Seppic Montanide ISA 206 VG adjuvant in a 2 mL dose.

[0125] • Treatment Group 5: Placebo Vaccine Lot # 5014133-0127J-9 was formulated with Emunade adjuvant.

[0126] • Treatment Group 6: Placebo Vaccine Lot # 5014133-0127K-10 was formulated with Seppic Montanide ISA 206 VG adjuvant.

[0127] Antigen Descriptions and Source

[0128] • F. necrophorum native LKT antigen lots # IVKC738-054 and ELN 0410544-0012H were produced and inactivated with 0.2% formalin in DeSoto, KS, R&D, according to the method used for the Bovine Liver Abscess (BLA) project. The recombinant domain 1 LKT lot 5014133-0127P was prepared by GenScript.

[0129] • T. pyogenes recombinant PLO lot 738-098 (Cornell) was produced in DeSoto, KS, R&D and lots 5014133-0147 (not genetically inactivated) and U028DEJ200-2 / P1 EJ001 (genetically inactivated) were produced by GenScript. PLO lots 738-098 and 5014133- 0147 were inactivated with 0.1 % formalin. All antigen lots were nickel-column purified.

[0130] • E. coli strain native pilus lot 5014133-0127L was produced in DeSoto, KS, R&D, using a method similar to the Scourmune product. The soluble recombinant FimH antigen lot 738-102 was produced in the DeSoto, KS, R&D department. The soluble recombinant FimH antigen lot U682SEK250-1 / P3EL001 was produced by GenScript. The insoluble recombinant FimH lot U2120EI230-2 / P3EJ002 was also produced by GenScript, but was extracted in DeSoto, KS R&D department to prepare Lot 5014133-0136d.

[0131] The genetically inactivated T. pyogenes recombinant PLO lot U028DEJ200-2 / P1 EJ001 comprises the recombinant PLO protein according to the current invention and comprises a pyolysin protein that is at least, with increasing preference, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identical (based on sequence identity) to the amino acid sequence according to SEQ ID NO: 2.

[0132] Experimental Procedures

[0133] 1.1. Pre-Vaccination Monitoring

[0134] 1.1.1. Physical Examination: Calves were examined prior to vaccination, and all calves appeared healthy.

[0135] 1.1.2. Blood samples: Blood samples (10 mL to 15 mL) for serum were collected from all calves in SST tubes on study day -1 for serological testing. All blood samples were individually labeled with animal number, study number, and the date of blood collection. The blood was allowed to clot at 37 ± 2°C for up to two hours, and then centrifuged to separate the serum. The serum was stored at -15°C or colder until tested for antibody titers /

[0136] 1.2. Vaccination

[0137] 1.2.1. Preparation of the Biological Products: All vaccines are in liquid, ready to use format. Administration of the Biological Product: The calves were vaccinated SQ with a 2 mL dose of vaccine administered in the left neck for the first vaccination and the right neck for the second vaccination. The first vaccination was administered on Day 0, the second vaccination was administered on Day 21. The vaccines were delivered using a 20 gauge x 1” needle. Following vaccination, any remaining vaccine was destroyed by autoclaving.

[0138] 1.2.2. Confirmation of Dose: Final product potency assays are not yet available for these antigens, so testing of the antigens was done prior to blending of the vaccines. Potency of the antigens was determined prior to vaccine blending using currently available SDS-PAGE and densitometry.

[0139] 1 .3. Post-Vaccination Procedures

[0140] 1.3.1. General Observations: Calves were observed daily for general health. No adverse events due to vaccination were observed following vaccination. 1 .3.2. Palpation for Injection Site Reactions: Calves were palpated for injection site reactions at the site of vaccination on days 4 (post-second vaccination only), 7, 14, 21 and 28 following each vaccination.

[0141] 1.3.3 Samples & sample collection: Blood samples for serum (10 mL to 15 mL) were collected from all calves in SST tubes on study days 20, 35, 49 and 77 for serological testing. All blood samples were individually labeled with animal number, study number, and the date of blood collection. The blood was allowed to clot at 37 ± 2°C for up to two hours, and then centrifuged to separate the serum. The serum was stored at -15°C or colder until tested for antibody titers.

[0142] 1.4. Analytical Methods

[0143] 1.4.1. Serology Assay (ELISA) for F. necrophorum LKT: Antibody responses to LKT antigen were evaluated using ELISA. Briefly, LKT antigen was directly bound to the ELISA plates. The antigen-coated plates were washed and then blocked with casein blocking buffer, then incubated with serial dilutions of the sera. Plates were washed, then incubated with anti-bovine peroxidase conjugate. Plates were washed again, then TMB was added, and the reaction was stopped with sulfuric acid. Plates were read at 450 nm.

[0144] 1 .4.2. Serology Assay (Hemolysis Inhibition or Neutralization) for T. pyogenes PLO: Antibody responses to PLO antigen were evaluated using a hemolysis neutralization assay. Briefly, twofold serial dilutions of the serum samples were made in 96-well V-bottom plates. Pyolysin toxin was added to all wells of the plates and the plates were incubated at 34-38°C for 1 hour. A 0.5% suspension of sheep red blood cells (SRBC) was added to all wells of the plate and the plates were incubated an additional 3 hours at 34-38°C. The plates were examined for the presence or absence of a SRBC pellet. The presence of a pellet was considered neutralization of hemolysis, and the reciprocal of the highest serum dilution with a pellet present was considered the endpoint.

[0145] 1 .4.3. Serology Assay (ELISA) for E. coli FimH: Antibody responses to E. coli antigens were evaluated using ELISA. Briefly, purified recombinant FimH protein was directly bound to the ELISA plates. The antigen-coated plates were washed and then blocked with casein blocking buffer, then incubated with serial dilutions of the sera. Plates were washed, then incubated with anti-bovine peroxidase conjugate, lates were washed again, then TMB was added, and the reaction was stopped with sulfuric acid. Plates were read at 450 nm.

[0146] 1.5. Antigen Potency Determination

[0147] 1.5.1. F. necrophorum LKT: For native F. necrophorum LKT, SDS-PAGE and densitometry of all formalin inactivated protein bands from approximately 120 kDa to the top of the gel were used to quantitate LKT. This method most likely measures other proteins in addition to LKT, but it is currently the only available method of quantitation. Measurement of domain 1 LKT was done by SDS-PAGE and densitometry. Testing done at a later date using the preliminary capture potency ELISA assay provided an estimate of the pg / dose in each vaccine.

[0148] 1.5.2. T. pyogenes PLO: The potency of the purified rPLO antigens were determined by GenScript using SDS-PAGE and densitometry and by BCA total protein analysis.

[0149] 1.5.3. E. coli FimH: The potency of native pilus antigen and the recombinant FimH were determined using SDS-PAGE and densitometry.

[0150] 1.6. Injection Site Reaction Measurements

[0151] 1.6.1. Animals were palpated for injection site reactions at the site of vaccination on day 4 following the second vaccination, and weekly up to day 28 following each vaccination. If an injection site reaction was present the reactions were measured for width, height and depth in centimeters using a caliper. The total injection site area (cm3) was determined by multiplying the 3 measurements.

[0152] 1.7 Data analysis

[0153] 1 .7.1 Outcome Variable: The primary outcome variable was the antibody responses to LKT, PLO, and FimH / pili following vaccination. Vaccine safety (injection site reaction scores) following each vaccination was a supporting variable.

[0154] 1.7.2. Experimental Unit: Individual calf.

[0155] 1 .7.3 Number of Replicates per T reatment: 8-11 calves in each vaccinated group

[0156] 1.7.4 Statistical Analysis: Serology data were not statistically analyzed. All calves were seropositive to the antigens prior to vaccination, so the change in antibody titers after vaccination were compared to the pre-vaccination titers for each group. In addition, geometric mean antibody titers (GMT) to each antigen were determined and compared between groups.

[0157] Results

[0158] 1 .8 Serological Antibody Responses to Vaccination

[0159] 1.8.1. F. necrophorum LKT Antibody Responses: Antibody responses to LKT were measured by serology ELISA. Calves in groups #1 through 4 vaccinated with 10 to 50 pg of native formalin inactivated LKT per dose all had similar antibody responses to LKT. Based on these results it appears that the minimum protective dose for native LKT could be 10 pg / dose or less. See Table 5 for the mean LKT ELISA titer results. Table 5: F. necrophorum LKT Geometric Mean Antibody ELISA Titers

[0160] 1 .8.2. T. pyogenes PLO Antibody Responses: Antibody responses to PLO were measured by hemolysis-neutralization assay. The highest neutralizing antibody titers were observed in the group 3 and 4 calves vaccinated with 10 g per dose of genetically inactivated recombinant PLO and the group 1 calves vaccinated with 200 pg of formalin inactivated recombinant PLO. See Table 6 for the mean PLO neutralizing antibody titer results. Table 6: T. Pyogenes PLO Geometric Mean Antibody ELISA Titers

[0161] NT = Not Tested

[0162] 1.8.3. E. coli anti-FimH ELISA Antibody Responses: Antibody responses to E. coli FimH were measured by serology ELISA. The highest anti-FimH antibody titers by far were observed in the group 1 calves vaccinated with approximately 100 g per dose of recombinant soluble

[0163] FimH (Cornell His-FimH) in a 2 mL dose. The calves in group 2, vaccinated with 20 pg of soluble recombinant FimH 160 had the next highest antibody responses, but the antibody titers were much lower than the titers in the group 1 calves. See Table 7 for the mean FimH ELISA titer results.

[0164] Table 7: E. coli anti-FimH Geometric Mean ELISA Antibody Titers 1 .8.4. Injection Site Reaction and Temperature Results: Very small injection site reactions were observed in less than 20% of the calves following the first vaccination, and only about 1 / 3rd of the calves had reactions following the second vaccination. All reactions were resolved by day 28 post-second vaccination, except in 3 calves. The vaccines comprising the recombinant genetically inactivated pyolysine appear to be acceptable in terms of safety.

[0165] Discussion

[0166] For the F. necrophorum native LKT antigen, calves vaccinated with 10 to 50 g of native formalin inactivated LKT per dose all had similar antibody responses to LKT. Based on these results it appears that the minimum protective dose for native LKT could be < 10 pg / dose. The recombinant LKT domain 1 at 6 pg / dose did not induce any antibody responses to LKT.

[0167] For the T. pyogenes PLO antigen, the highest hemolysis-neutralizing antibody titers were observed in calves vaccinated with lowest level (10 pg per dose) of genetically inactivated recombinant PLO. Hence, the genetically inactivated recombinant PLO is effective in inducing antibody response post-vaccination.

[0168] For E. coli, the calves responded best to recombinant soluble FimH antigen (Cornell His- FimH) at approximately 100 pg per dose, and calves vaccinated with 20 pg of recombinant soluble FimH 160 had detectable antibody responses to FimH.

[0169] Calves in groups 3 and 4 were administered vaccines formulated with the same antigen preparations and dose levels but with two different adjuvants, Emunade and Seppic, in order to compare responses to each adjuvant. The calves had similar antibody responses and injection site reaction scores were similar for both adjuvants. The LKT and FimH responses were slightly higher with Emunade compared to Seppic, and the PLO responses were slightly higher with the Seppic compared to Emunade adjuvant.

[0170] The vaccines, including the vaccines comprising the recombinant genetically inactivated recombinant pyolysin, appear to be acceptable in terms of safety. Very few calves (< 20%) had small injection site reactions following the first vaccination, and only about 1 / 3rd of the calves had reactions following the second vaccination.

[0171] Therefore, as will be apparent from the foregoing results presented in the Example, the vaccine composition comprising recombinant T. pyogenes pyolysin in accordance with the invention is safe and efficacious in inducing an antibody response. Further it is apparent that such a vaccine composition, when further comprising different FimH antigens and / or inactivated F. necrophorum native LKT antigen, is also safe to use and efficacious in inducing antibody responses to the LKT and / or FimH antigens.

[0172] Table 8: Amino acid and nucleic acid sequences

[0173] SEQ ID NO: 1 (native pyolysin protein)

[0174] AGLGNSSGLTDGLSAPRVSISPMDKVDLKSAQETDETSVDKYIRGLEYDPSGVLAVKGESIE

[0175] NVPVTKDQLKDGTYTVFKHERKSFNNLRSDISAFDANNAHVYPGALVLANKDLAKGSPTSIG

[0176] IARAPQTVSVDLPGLVDGKSKWINNPTKSSVTQGMNGLLDGWIQRNSKYPDHAAKIFYDET

[0177] MVTSKRQLEAKFGLGFEKVSAKLNVDFDAIHKRERQVAIASFKQIYYTASVDTPTSPHSVFG

[0178] PNVTAQDLKDRGVNNKNPLGYISSVSYGRQIFVKLETTSTSNDVQAAFSGLFKAKFGNLSTE

[0179] FKAKYADILNKTRATVYAVGGSARGGVEVATGNIDALKKIIKEESTYSTKVPAVPVSYSVNFL

[0180] KDNQLAAVRSSGDYIETTATTYKSGEITFRHGGGYVAKFGLKWDEISYDPQGKEIRTPKTWS

[0181] GNWVGRTLGFRETIQLPANARNIHVEAGEATGLAWDPWWTVINKKNLPLVPHREIVLKGTTL NPWVEENVKS

[0182] SEQ ID NO: 2 (recombinant PLO protein p.T496L_L497T_P472del)

[0183] AGLGNSSGLTDGLSAPRASISPTDKVDLKSAQETDETGVDKYIRGLKYDPSGVLAVKGESIE

[0184] NVPVTKDQLKDGTYTVFKHERKSFNNLRSDISAFDANNAHVYPGALVLANKDLAKGSPTSIG

[0185] IARAPQTVSVDLPGLVDGKNKVVINNPTKSSVTQGLNGLLDGWIQRNSKYPDHAAKISYDET

[0186] MVTSKRQLEAKLGLGFEKVSAKLNVDFDAIHKRERQVAIASFKQIYYTASVDTPTSPHSVFG

[0187] PNVTAQDLKDRGVNNKNPLGYISSVSYGRQIFVKLETTSTSNDVQAAFSGLFKAKFGNLSTE

[0188] FKAKYADILNKTRATVYAVGGSARGGVEVATGNIDALKKIIKEESTYSTKVPAVPVSYAVNFL

[0189] KDNQLAAVRSSGDYIETTATTYKSGEITFRHGGGYVAKFRLKWDEISYDPQGKEIRTPKTWS

[0190] GNWAARTLGFRETIQLPANARNIHVEAGEATGLAWDWWTVINKKNLPLVPHREIVLKGTLTN PWVEDNVKS

[0191] SEQ ID NO: 3 (nucleic acid encoding recombinant PLO protein p.T496L_L497T_P472del)

[0192] GCGGGTCTGGGTAACAGCAGCGGTCTGACCGACGGCCTGAGCGCGCCGCGTGCGAG

[0193] CATTAGCCCGACCGATAAGGTGGATCTGAAGAGCGCGCAGGAAACCGACGAAACCGGC

[0194] GTGGATAAGTACATCCGTGGTCTGAAATATGACCCGAGCGGCGTGCTGGCGGTTAAGG

[0195] GCGAGAGCATTGAAAACGTGCCGGTTACCAAGGACCAACTGAAAGATGGTACCTACAC

[0196] CGTTTTTAAGCACGAGCGTAAAAGCTTCAACAACCTGCGTAGCGACATCAGCGCGTTTG

[0197] ATGCGAACAACGCGCATGTGTACCCGGGTGCGCTGGTTCTGGCGAACAAGGACCTGGC

[0198] GAAAGGCAGCCCGACCAGCATCGGTATTGCGCGTGCGCCGCAGACCGTGAGCGTTGA CCTGCCGGGCCTGGTGGATGGCAAGAACAAAGTGGTTATTAACAACCCGACCAAAAGC AGCGTTACCCAGGGCCTGAACGGTCTGCTGGATGGTTGGATTCAGCGTAACAGCAAGT

[0199] ACCCGGACCACGCGGCGAAAATTAGCTATGATGAAACCATGGTGACCAGCAAGCGTCA

[0200] GCTGGAGGCGAAACTGGGCCTGGGTTTTGAAAAGGTGAGCGCGAAACTGAACGTTGAC

[0201] TTCGATGCGATCCACAAGCGTGAACGTCAGGTGGCGATCGCGAGCTTTAAACAAATTTA

[0202] CTATACCGCGAGCGTTGATACCCCGACCAGCCCGCACAGCGTGTTTGGTCCGAACGTT

[0203] ACCGCGCAAGACCTGAAGGATCGTGGCGTGAACAACAAAAACCCGCTGGGTTACATCA

[0204] GCAGCGTGAGCTATGGCCGTCAGATTTTTGTTAAGCTGGAAACCACCAGCACCAGCAAC

[0205] GACGTTCAAGCGGCGTTCAGCGGCCTGTTTAAGGCGAAATTCGGTAACCTGAGCACCG

[0206] AATTCAAGGCGAAATACGCGGATATCCTGAACAAAACCCGTGCGACCGTGTATGCGGTT

[0207] GGTGGCAGCGCGCGTGGTGGTGTTGAGGTGGCGACCGGTAACATTGACGCGCTGAAG

[0208] AAAATCATTAAAGAGGAGAGCACCTACAGCACCAAAGTGCCGGCGGTGCCGGTTAGCT

[0209] ATGCGGTTAACTTTCTGAAGGACAACCAACTGGCGGCGGTGCGTAGCAGCGGTGATTA

[0210] CATCGAAACCACCGCGACCACCTATAAAAGCGGCGAAATTACCTTTCGTCACGGTGGC

[0211] GGTTACGTTGCGAAGTTCCGTCTGAAATGGGACGAGATCAGCTATGATCCGCAGGGCA

[0212] AGGAAATTCGTACCCCGAAAACCTGGAGCGGTAACTGGGCGGCGCGTACCCTGGGTTT

[0213] CCGTGAAACCATCCAACTGCCGGCGAACGCGCGTAACATTCATGTTGAAGCGGGTGAA

[0214] GCGACCGGTCTGGCGTGGGATTGGTGGACCGTGATCAACAAGAAAAACCTGCCGCTGG

[0215] TTCCGCACCGTGAAATCGTTCTGAAAGGCACCCTGACCAATCCGTGGGTTGAAGATAAT GTGAAAAGC SEQ ID NO: 4 (recombinant PLO protein p.P472del_T496L_L497T_D238R)

[0216] AGLGNSSGLTDGLSAPRASISPTDKVDLKSAQETDETGVDKYIRGLKYDPSGVLAVKGESIE

[0217] NVPVTKDQLKDGTYTVFKHERKSFNNLRSDISAFDANNAHVYPGALVLANKDLAKGSPTSIG

[0218] IARAPQTVSVDLPGLVDGKNKVVINNPTKSSVTQGLNGLLDGWIQRNSKYPDHAAKISYDET

[0219] MVTSKRQLEAKLGLGFEKVSAKLNVDFDAIHKRERQVAIASFKQIYYTASVRTPTSPHSVFG

[0220] PNVTAQDLKDRGVNNKNPLGYISSVSYGRQIFVKLETTSTSNDVQAAFSGLFKAKFGNLSTE

[0221] FKAKYADILNKTRATVYAVGGSARGGVEVATGNIDALKKIIKEESTYSTKVPAVPVSYAVNFL

[0222] KDNQLAAVRSSGDYIETTATTYKSGEITFRHGGGYVAKFRLKWDEISYDPQGKEIRTPKTWS

[0223] GNWAARTLGFRETIQLPANARNIHVEAGEATGLAWDWWTVINKKNLPLVPHREIVLKGTLTN PWVEDNVKS

[0224] SEQ ID NO: 5 (nucleic acid encoding recombinant PLO protein p.P472del_T496L_L497T_D238R)

[0225] GCGGGTCTGGGTAACAGCAGCGGTCTGACCGACGGCCTGAGCGCGCCGCGTGCGAG

[0226] CATTAGCCCGACCGATAAGGTGGATCTGAAGAGCGCGCAGGAAACCGACGAAACCGGC

[0227] GTGGATAAGTACATCCGTGGTCTGAAATATGACCCGAGCGGCGTGCTGGCGGTTAAGG

[0228] GCGAGAGCATTGAAAACGTGCCGGTTACCAAGGACCAACTGAAAGATGGTACCTACAC

[0229] CGTTTTTAAGCACGAGCGTAAAAGCTTCAACAACCTGCGTAGCGACATCAGCGCGTTTG

[0230] ATGCGAACAACGCGCATGTGTACCCGGGTGCGCTGGTTCTGGCGAACAAGGACCTGGC

[0231] GAAAGGCAGCCCGACCAGCATCGGTATTGCGCGTGCGCCGCAGACCGTGAGCGTTGA

[0232] CCTGCCGGGCCTGGTGGATGGCAAGAACAAAGTGGTTATTAACAACCCGACCAAAAGC

[0233] AGCGTTACCCAGGGCCTGAACGGTCTGCTGGATGGTTGGATTCAGCGTAACAGCAAGT

[0234] ACCCGGACCACGCGGCGAAAATTAGCTATGATGAAACCATGGTGACCAGCAAGCGTCA

[0235] GCTGGAGGCGAAACTGGGCCTGGGTTTTGAAAAGGTGAGCGCGAAACTGAACGTTGAC

[0236] TTCGATGCGATCCACAAGCGTGAACGTCAGGTGGCGATCGCGAGCTTTAAACAAATTTA

[0237] CTATACCGCGAGCGTTCGTACCCCGACCAGCCCGCACAGCGTGTTTGGTCCGAACGTT

[0238] ACCGCGCAAGACCTGAAGGATCGTGGCGTGAACAACAAAAACCCGCTGGGTTACATCA

[0239] GCAGCGTGAGCTATGGCCGTCAGATTTTTGTTAAGCTGGAAACCACCAGCACCAGCAAC

[0240] GACGTTCAAGCGGCGTTCAGCGGCCTGTTTAAGGCGAAATTCGGTAACCTGAGCACCG

[0241] AATTCAAGGCGAAATACGCGGATATCCTGAACAAAACCCGTGCGACCGTGTATGCGGTT

[0242] GGTGGCAGCGCGCGTGGTGGTGTTGAGGTGGCGACCGGTAACATTGACGCGCTGAAG

[0243] AAAATCATTAAAGAGGAGAGCACCTACAGCACCAAAGTGCCGGCGGTGCCGGTTAGCT

[0244] ATGCGGTTAACTTTCTGAAGGACAACCAACTGGCGGCGGTGCGTAGCAGCGGTGATTA

[0245] CATCGAAACCACCGCGACCACCTATAAAAGCGGCGAAATTACCTTTCGTCACGGTGGC

[0246] GGTTACGTTGCGAAGTTCCGTCTGAAATGGGACGAGATCAGCTATGATCCGCAGGGCA

[0247] AGGAAATTCGTACCCCGAAAACCTGGAGCGGTAACTGGGCGGCGCGTACCCTGGGTTT

[0248] CCGTGAAACCATCCAACTGCCGGCGAACGCGCGTAACATTCATGTTGAAGCGGGTGAA

[0249] GCGACCGGTCTGGCGTGGGATTGGTGGACCGTGATCAACAAGAAAAACCTGCCGCTGG

[0250] TTCCGCACCGTGAAATCGTTCTGAAAGGCACCCTGACCAATCCGTGGGTTGAAGATAAT GTGAAAAGC

[0251] SEQ ID NO: 6 (recombinant PLO protein p.D238R_P472del)

[0252] AGLGNSSGLTDGLSAPRASISPTDKVDLKSAQETDETGVDKYIRGLKYDPSGVLAVKGESIE

[0253] NVPVTKDQLKDGTYTVFKHERKSFNNLRSDISAFDANNAHVYPGALVLANKDLAKGSPTSIG

[0254] IARAPQTVSVDLPGLVDGKNKVVINNPTKSSVTQGLNGLLDGWIQRNSKYPDHAAKISYDET

[0255] MVTSKRQLEAKLGLGFEKVSAKLNVDFDAIHKRERQVAIASFKQIYYTASVRTPTSPHSVFG

[0256] PNVTAQDLKDRGVNNKNPLGYISSVSYGRQIFVKLETTSTSNDVQAAFSGLFKAKFGNLSTE

[0257] FKAKYADILNKTRATVYAVGGSARGGVEVATGNIDALKKIIKEESTYSTKVPAVPVSYAVNFL

[0258] KDNQLAAVRSSGDYIETTATTYKSGEITFRHGGGYVAKFRLKWDEISYDPQGKEIRTPKTWS

[0259] GNWAARTLGFRETIQLPANARNIHVEAGEATGLAWDWWTVINKKNLPLVPHREIVLKGTTLN PWVEDNVKS SEQ ID NO: 7 (nucleic acid encoding recombinant PLO protein p.D238R_P472del)

[0260] GCGGGTCTGGGTAACAGCAGCGGTCTGACCGACGGCCTGAGCGCGCCGCGTGCGAG

[0261] CATTAGCCCGACCGATAAGGTGGATCTGAAGAGCGCGCAGGAAACCGACGAAACCGGC

[0262] GTGGATAAGTACATCCGTGGTCTGAAATATGACCCGAGCGGCGTGCTGGCGGTTAAGG

[0263] GCGAGAGCATTGAAAACGTGCCGGTTACCAAGGACCAACTGAAAGATGGTACCTACAC

[0264] CGTTTTTAAGCACGAGCGTAAAAGCTTCAACAACCTGCGTAGCGACATCAGCGCGTTTG

[0265] ATGCGAACAACGCGCATGTGTACCCGGGTGCGCTGGTTCTGGCGAACAAGGACCTGGC

[0266] GAAAGGCAGCCCGACCAGCATCGGTATTGCGCGTGCGCCGCAGACCGTGAGCGTTGA

[0267] CCTGCCGGGCCTGGTGGATGGCAAGAACAAAGTGGTTATTAACAACCCGACCAAAAGC

[0268] AGCGTTACCCAGGGCCTGAACGGTCTGCTGGATGGTTGGATTCAGCGTAACAGCAAGT

[0269] ACCCGGACCACGCGGCGAAAATTAGCTATGATGAAACCATGGTGACCAGCAAGCGTCA

[0270] GCTGGAGGCGAAACTGGGCCTGGGTTTTGAAAAGGTGAGCGCGAAACTGAACGTTGAC

[0271] TTCGATGCGATCCACAAGCGTGAACGTCAGGTGGCGATCGCGAGCTTTAAACAAATTTA

[0272] CTATACCGCGAGCGTTCGTACCCCGACCAGCCCGCACAGCGTGTTTGGTCCGAACGTT

[0273] ACCGCGCAAGACCTGAAGGATCGTGGCGTGAACAACAAAAACCCGCTGGGTTACATCA

[0274] GCAGCGTGAGCTATGGCCGTCAGATTTTTGTTAAGCTGGAAACCACCAGCACCAGCAAC

[0275] GACGTTCAAGCGGCGTTCAGCGGCCTGTTTAAGGCGAAATTCGGTAACCTGAGCACCG

[0276] AATTCAAGGCGAAATACGCGGATATCCTGAACAAAACCCGTGCGACCGTGTATGCGGTT

[0277] GGTGGCAGCGCGCGTGGTGGTGTTGAGGTGGCGACCGGTAACATTGACGCGCTGAAG

[0278] AAAATCATTAAAGAGGAGAGCACCTACAGCACCAAAGTGCCGGCGGTGCCGGTTAGCT

[0279] ATGCGGTTAACTTTCTGAAGGACAACCAACTGGCGGCGGTGCGTAGCAGCGGTGATTA

[0280] CATCGAAACCACCGCGACCACCTATAAAAGCGGCGAAATTACCTTTCGTCACGGTGGC

[0281] GGTTACGTTGCGAAGTTCCGTCTGAAATGGGACGAGATCAGCTATGATCCGCAGGGCA

[0282] AGGAAATTCGTACCCCGAAAACCTGGAGCGGTAACTGGGCGGCGCGTACCCTGGGTTT

[0283] CCGTGAAACCATCCAACTGCCGGCGAACGCGCGTAACATTCATGTTGAAGCGGGTGAA

[0284] GCGACCGGTCTGGCGTGGGATTGGTGGACCGTGATCAACAAGAAAAACCTGCCGCTGG TTCCGCACCGTGAAATCGTTCTGAAAGGCACCACCCTGAATCCGTGGGTTGAAGATAAT GTGAAAAGC

[0285] SEQ ID NO: 8 (recombinant PLO protein p.l61A_D238R)

[0286] AGLGNSSGLTDGLSAPRASISPTDKVDLKSAQETDETGVDKYIRGLKYDPSGVLAVKGESAE

[0287] NVPVTKDQLKDGTYTVFKHERKSFNNLRSDISAFDANNAHVYPGALVLANKDLAKGSPTSIG

[0288] IARAPQTVSVDLPGLVDGKNKVVINNPTKSSVTQGLNGLLDGWIQRNSKYPDHAAKISYDET

[0289] MVTSKRQLEAKLGLGFEKVSAKLNVDFDAIHKRERQVAIASFKQIYYTASVRTPTSPHSVFG

[0290] PNVTAQDLKDRGVNNKNPLGYISSVSYGRQIFVKLETTSTSNDVQAAFSGLFKAKFGNLSTE

[0291] FKAKYADILNKTRATVYAVGGSARGGVEVATGNIDALKKIIKEESTYSTKVPAVPVSYAVNFL

[0292] KDNQLAAVRSSGDYIETTATTYKSGEITFRHGGGYVAKFRLKWDEISYDPQGKEIRTPKTWS

[0293] GNWAARTLGFRETIQLPANARNIHVEAGEATGLAWDPWWTVINKKNLPLVPHREIVLKGTTL NPWVEDNVKS

[0294] SEQ ID NO: 9 (nucleic acid encoding recombinant PLO protein p.l61A_D238R)

[0295] GCGGGTCTGGGTAACAGCAGCGGTCTGACCGACGGCCTGAGCGCGCCGCGTGCGAG

[0296] CATTAGCCCGACCGATAAGGTGGATCTGAAGAGCGCGCAGGAAACCGACGAAACCGGC

[0297] GTGGATAAGTACATCCGTGGTCTGAAATATGACCCGAGCGGCGTGCTGGCGGTTAAGG

[0298] GCGAGAGCGCGGAAAACGTGCCGGTTACCAAGGACCAACTGAAAGATGGTACCTACAC

[0299] CGTTTTTAAGCACGAGCGTAAAAGCTTCAACAACCTGCGTAGCGACATCAGCGCGTTTG

[0300] ATGCGAACAACGCGCATGTGTACCCGGGTGCGCTGGTTCTGGCGAACAAGGACCTGGC

[0301] GAAAGGCAGCCCGACCAGCATCGGTATTGCGCGTGCGCCGCAGACCGTGAGCGTTGA

[0302] CCTGCCGGGCCTGGTGGATGGCAAGAACAAAGTGGTTATTAACAACCCGACCAAAAGC

[0303] AGCGTTACCCAGGGCCTGAACGGTCTGCTGGATGGTTGGATTCAGCGTAACAGCAAGT

[0304] ACCCGGACCACGCGGCGAAAATTAGCTATGATGAAACCATGGTGACCAGCAAGCGTCA

[0305] GCTGGAGGCGAAACTGGGCCTGGGTTTTGAAAAGGTGAGCGCGAAACTGAACGTTGAC

[0306] TTCGATGCGATCCACAAGCGTGAACGTCAGGTGGCGATCGCGAGCTTTAAACAAATTTA

[0307] CTATACCGCGAGCGTTCGTACCCCGACCAGCCCGCACAGCGTGTTTGGTCCGAACGTT

[0308] ACCGCGCAAGACCTGAAGGATCGTGGCGTGAACAACAAAAACCCGCTGGGTTACATCA

[0309] GCAGCGTGAGCTATGGCCGTCAGATTTTTGTTAAGCTGGAAACCACCAGCACCAGCAAC

[0310] GACGTTCAAGCGGCGTTCAGCGGCCTGTTTAAGGCGAAATTCGGTAACCTGAGCACCG

[0311] AATTCAAGGCGAAATACGCGGATATCCTGAACAAAACCCGTGCGACCGTGTATGCGGTT

[0312] GGTGGCAGCGCGCGTGGTGGTGTTGAGGTGGCGACCGGTAACATTGACGCGCTGAAG

[0313] AAAATCATTAAAGAGGAGAGCACCTACAGCACCAAAGTGCCGGCGGTGCCGGTTAGCT

[0314] ATGCGGTTAACTTTCTGAAGGACAACCAACTGGCGGCGGTGCGTAGCAGCGGTGATTA

[0315] CATCGAAACCACCGCGACCACCTATAAAAGCGGCGAAATTACCTTTCGTCACGGTGGC

[0316] GGTTACGTTGCGAAGTTCCGTCTGAAATGGGACGAGATCAGCTATGATCCGCAGGGCA

[0317] AGGAAATTCGTACCCCGAAAACCTGGAGCGGTAACTGGGCGGCGCGTACCCTGGGTTT

[0318] CCGTGAAACCATCCAACTGCCGGCGAACGCGCGTAACATTCATGTTGAAGCGGGTGAA

[0319] GCGACCGGTCTGGCGTGGGATCCGTGGTGGACCGTGATCAACAAGAAAAACCTGCCG

[0320] CTGGTTCCGCACCGTGAAATCGTTCTGAAAGGCACCACCCTGAATCCGTGGGTTGAAG

[0321] ATAATGTGAAAAGC

Claims

Claims1. A recombinant pyolysin protein comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 1 , wherein the amino acid sequence comprises:(i) an amino acid mutation at amino acid position 472, and(ii) an amino acid mutation at amino acid position 496 and at amino acid position 497.

2. The recombinant pyolysin protein according to claim 1 , wherein the amino acid sequence has at least 85%, preferably at least 90%, more preferably at least 95% to SEQ ID NO: 1.

3. The recombinant pyolysin protein according to any one of the previous claims, wherein the amino acid mutation at amino acid position 472 is an amino acid deletion and / or wherein the amino acid mutation at amino acid position 496 and amino acid position 497 is an amino acid substitution.

4. The recombinant pyolysin protein according to any one of the previous claims, wherein at amino acid position 496 a Threonine is substituted for a Leucine and / or wherein at amino acid position 497 a Leucine is substituted for a Threonine.

5. The recombinant pyolysin protein according to any one of the previous claims, wherein the amino acid sequence has at least 85%, preferably at least 90%, more preferably at least 95%, more preferably 99% sequence identity to SEQ ID NO: 2.

6. A nucleic acid sequence encoding the recombinant pyolysin protein according to any one of the previous claims.

7. A vector comprising a nucleic acid sequence encoding the recombinant pyolysin protein according to any one of the claims 1 - 5.

8. A host cell comprising a nucleic acid sequence encoding the recombinant pyolysin protein according to any one of the claims 1 - 5, preferably wherein the nucleic acid sequence is codon optimized for the host cell, preferably wherein the host cell is an E. coli cell.

9. A vaccine composition comprising the recombinant pyolysin protein according to any one of the claims 1 - 5, or an immunogenic fragment thereof, and a pharmaceutically acceptable carrier.

10. The vaccine composition according to claim 9, further comprising any one or more antigens, preferably wherein the antigen is a F.necrophorum antigen and / or an E coli antigen, more preferably wherein the antigen is F. necrophorum leukotoxin (LKT) and / or E. coli type 1 fimbrial adhesin (FimH).

11. A vaccine composition for use in the prophylaxis of a disease and / or disorder in mammal, wherein the vaccine composition comprises the recombinant pyolysin according to any one of claims 1 - 5, and wherein the vaccine composition is administered to a mammal, preferably wherein the mammal is a ruminant, more preferably a cow.

12. The vaccine composition for use according to claim 11, wherein the disease and / or disorder is selected from the group consisting of intrauterine disease, liver abscess and / or foot rot.

13. The vaccine composition for use according to claim 11 - 12, wherein the vaccine is administered subcutaneously.

14. Method of improving the reproductive function of a ruminant comprising the subcutaneous administering to the ruminant a vaccine composition according to claim 9 or 10.

15. Method of ameliorating and / or preventing a diseased state in a ruminant comprising the subcutaneous administering to the ruminant a vaccine composition according to claim 9 or 10, wherein the diseased state comprises intrauterine disease, bovine liver abscess (BLA) and / or bovine foot rot (BFR).

16. A method for producing a vaccine composition according to claim 9 or 10, the method comprising the subsequent steps of: a. expressing the recombinant pyolysin protein according to any one of the claims 1 - 5 through growth of a microorganism; b. lysing the microorganism to obtain the recombinant pyolysin protein.

17. A kit of parts comprising at least one container, the container comprising a veterinary composition comprising the recombinant pyolysin protein according to any one of the claims 1 - 5, F. necrophorum leukotoxin (LKT) and E. coli type 1 fimbrial adhesin (FimH).

Images