Chimeric protein vaccine

Abstract

The present invention provides a chimeric or fusion protein for inducing an immune response to Porphyromonas pharyngolaris (P. guae), the protein comprising a first polypeptide and a second polypeptide, wherein: A) the first polypeptide comprises or consists of the following: an amino acid sequence of an active site of Arg gingival protease or Lys gingival protease of Porphyromonas pharyngolaris, or a sequence at least 80% identical to the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas pharyngolaris; and B) the second polypeptide comprises or consists of the following amino acid sequence of the DUF2436 domain of Porphyromonas pharyngolaris Arg gingival protease or Lys gingival protease, and the amino acid sequence of the adhesin domain of Porphyromonas pharyngolaris Arg gingival protease or Lys gingival protease.

Description

Technical Field

[0001] This invention relates to substances that can be used to induce the growth of Porphyromonas pharyngosum (Porphyromonas pharyngosum). P. gulae Chimeric polypeptides that provide immune responses to porphyria, compositions comprising such polypeptides, and their use in the prevention and treatment of symptoms and diseases associated with Porphyromonas pharynx.

[0002] Related applications

[0003] This application claims priority to Australian Provisional Application AU 2023902373, the entire contents of which are hereby incorporated by reference. Background Technology

[0004] If dental plaque accumulates around the teeth at the gingival margin, it can cause gingivitis (inflammation of the gums). Chronic gingivitis can lead to the growth of Porphyromonas aeruginosa at the base of the periodontal pockets. Porphyromonas sp. Periodontal pathogens cause chronic infection and the development of severe disease. This severe form of periodontitis is called periodontitis and can lead to tooth loss during the immune system's attempt to eliminate the infection.

[0005] Periodontitis is an inflammatory disease of the tooth-supporting tissues associated with disordered subgingival plaque, which causes destruction of these tissues and loss of tooth attachment in humans and companion animals. By age three, more than 80% of dogs show signs of periodontitis, and by the same age, 70% of cats do as well. Therefore, periodontitis poses a significant disease burden in companion animal populations.

[0006] The dominant periodontal pathogen in companion animals (specifically dogs) is *Porphyromonas pharyngiomas* (… P. gulae ).

[0007] There are currently no commercially approved treatments for the prevention or reduction of the incidence and / or severity of Porphyromonas pharyngiomatosis infection, or for the treatment of Porphyromonas pharyngiomatosis infection and disease in companion animals.

[0008] Therefore, there is a need for novel and / or improved methods for designing and preparing agents for treating, preventing, or reducing the severity of Porphyromonas pharyngoides infection.

[0009] References to any prior art in this specification are not an admission or implication that such prior art constitutes part of common common sense in any jurisdiction, nor are they an admission or implication that such prior art can be reasonably expected or understood by a person skilled in the art, or be regarded as relevant and / or combined with other prior art. Summary of the Invention

[0010] This invention provides a chimeric or fusion protein for inducing an immune response in *Porphyromonas pharyngoides*, the protein comprising a first polypeptide and a second polypeptide, wherein:

[0011] A) The first polypeptide comprises or consists of the following: an amino acid sequence of the active site of an Arg gingival protease or Lys gingival protease homolog of Porphyromonas pharyngoides; and B) The second polypeptide comprises or is composed of the following: The amino acid sequence of the DUF2436 domain of the Arg gingival protease or Lys gingival protease or hemagglutinin surface complex of *Porphyromonas pharyngioma*, preferably as shown in SEQ ID NO: 3 or 4, or at least 80% identical thereto; and The amino acid sequence of the adhesin domain, also found in the surface complex of the Arg gingival protease and Lys gingival protease homologs of *Porphyromonas pharyngioma*, preferably wherein the adhesin domain comprises at least the amino acid sequence of SEQ ID NO: 86 and / or SEQ ID NO: 85 (such as the sequence of SEQ ID NO: 88) or at least 80% identical thereto, more preferably wherein the adhesin domain comprises the amino acid sequence of SEQ ID NO: 20 or at least 80% identical thereto.

[0012] In another embodiment, the chimeric or fusion protein comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of a *Porphyromonas phlebotomyces* Arg gingivase or Lys gingivase homolog, or a sequence at least 80% identical thereto. The one or more additional polypeptides comprising or consisting of the active site of a *Porphyromonas phlebotomyces* Arg gingivase or Lys gingivase homolog may be located at the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, the N-terminus of the second polypeptide, or the C-terminus of the second polypeptide. In some embodiments, at least two additional polypeptides may be present, which comprise or consist of the amino acid sequence of the active site of a *Porphyromonas phlebotomyces* Arg gingivase or Lys gingivase homolog, or a sequence at least 80% identical thereto. In such embodiments, the two additional polypeptides may be located at the N-terminus of the second polypeptide, the C-terminus of the second polypeptide, or both the N-terminus and C-terminus of the second polypeptide.

[0013] The one or more additional polypeptides may be linked to the first polypeptide or the second polypeptide of the chimeric or fusion protein, preferably through a linker of no more than 50 amino acids, or directly linked to the first polypeptide or the second polypeptide.

[0014] In any embodiment, the first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 or 2, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it.

[0015] The one or more additional polypeptides preferably comprise or consist of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 1 or 2, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it.

[0016] In any embodiment, the first polypeptide and the other polypeptide comprising the amino acid sequence of the active site of an Arg gingivase or Lys gingivase homolog of *Porphyromonas pharyngioma*, or composed thereof, comprise or consist of the following: amino acid sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to each other, or wherein said amino acid sequences are identical to each other. The first polypeptide and the other polypeptide may be derived from the active site of a heterologous gingivase (e.g., gingivase from a different strain of *Porphyromonas pharyngioma*). The first polypeptide and the other polypeptides may have amino acid sequences derived from different gingival proteases (e.g., one of the polypeptides has an amino acid sequence from the active site of Kgp and the other polypeptide has an amino acid sequence from the active site of Rgp; or alternatively, one of the polypeptides has an amino acid sequence from the active site of RgpA and the other polypeptide has an amino acid sequence from the active site of RgpB).

[0017] In any embodiment, the first polypeptide and / or the other polypeptide comprises or consists of the amino acid sequence (R) of the active site of the Arg gingival protease homolog of *Porphyromonas pharyngoides*, as illustrated in SEQ ID NO: 2, said amino acid sequence may include a substitution of a cysteine ​​residue (located at position 5 in the sequence of SEQ ID NO: 2). Optionally, the amino acid substitution may be changed to a serine, valine, or alanine residue.

[0018] In any embodiment, the amino acid sequence of the adhesin domain (found in the surface complex of Arg gingival protease and Lys gingival protease homologs of Porphyromonas pharyngioma) further comprises one or more amino acid substitutions selected from the following: a) One or more cysteine ​​amino acids are substituted compared to the naturally occurring Porphyromonas pharyngosum sequence in the corresponding region; b) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of SEQ ID NO: 85 (equivalent to residues 68 to 71 of the sequence of SEQ ID NO: 20 or residues 68 to 71 of SEQ ID NO: 88) or located at positions equivalent to said residues; c) Substitution of motif NxFA to SxYQ in the sequence corresponding to residues 2 to 5 of SEQ ID NO: 85 (equivalent to residues 64 to 67 of the sequence of SEQ ID NO: 20 or residues 64-67 of SEQ ID NO: 88) or located at positions equivalent to said residues; d) Substitution of tyrosine residues corresponding to or located at positions 10 of SEQ ID NO: 86 and tryptophan residues corresponding to or located at positions 23 of SEQ ID NO: 85 with alanine residues (equivalent to tyrosine at position 10 of SEQ ID NO: 20 and tryptophan at position 85).

[0019] The substitution of cysteine ​​amino acid residues in the adhesin domain (e.g., the adhesin domain shown in SEQ ID NO: 88 or 20) may be a substitution with a serine residue or a valine residue. Preferably, the one or more cysteine ​​substitutions may comprise one or more substitutions that change to serine residues.

[0020] Optionally, only one cysteine ​​residue is substituted. In other embodiments, two cysteine ​​residues are substituted. In some embodiments, the cysteine ​​residues are substituted with a combination of valine and serine residues. In other embodiments, all substituted cysteine ​​residues are substituted with serine or all substituted cysteine ​​residues are substituted with valine.

[0021] Preferably, the adhesin domain comprises or consists of the sequence shown in any one of SEQ ID NO: 20, 22, 23 or 24, or a sequence that is at least 80% identical thereto, wherein one or more cysteine ​​residues are substituted with serine or valine residues.

[0022] In any embodiment, the adhesin domain may contain substitutions of proline residues and / or asparagine residues in the motif PxxN corresponding to positions 68 to 71 of the sequence in SEQ ID NO: 20 or located at positions equivalent to those positions.

[0023] The substitution of proline amino acids specifically involves replacing them with alanine residues.

[0024] The asparagine amino acid substitution can be a substitution of a proline residue or an alanine residue. Preferably, the asparagine residue is substituted with a proline residue. In other embodiments, the asparagine residue is not substituted.

[0025] In a particularly preferred embodiment, the motif PxxN in the adhesin domain is replaced with, for example, AxxP (e.g., AVQP, SEQ ID NO: 64), such that the adhesin domain contains an amino acid sequence as shown in SEQ ID NO: 21 or a sequence at least 80% identical thereto, wherein the sequence contains AxxP at positions 68 to 71 of the sequence equivalent to that in SEQ ID NO: 21.

[0026] In a particularly preferred embodiment, the amino acid sequence of the adhesin domain comprises one or both of the following: a) One or more cysteine ​​amino acids are substituted compared to the sequences of naturally occurring Porphyromonas pharyngoides Arg gingival protease or Lys gingival protease homologs in the corresponding region; b) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 68 to 71 of the sequence of SEQ ID NO: 20 or located at positions equivalent to said residues.

[0027] In a particularly preferred embodiment, the amino acid sequence of the adhesin domain comprises the following: a) The substitution of two cysteine ​​amino acid residues in the adhesin A domain compared to the A domain naturally present in the protein complex sequence of Arg gingival protease and Lys gingival protease found in the corresponding region. b) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 68 to 71 of the sequence of SEQ ID NO: 20 or located at positions equivalent to said residues.

[0028] Therefore, the adhesin domain may contain an amino acid sequence as shown in any of SEQ ID NO: 24 to 27 or a sequence that is at least 80% identical thereto, wherein the sequence contains AxxP at positions 68 to 71 of the sequence equivalent to SEQ ID NO: 21, and wherein one or more cysteine ​​residues are substituted with serine or valine residues.

[0029] As mentioned above, the second polypeptide preferably comprises the amino acid sequence of the DUF2436 domain of the Arg gingival protease and Lys gingival protease surface complex of Porphyromonas pharyngoides (as defined in SEQ ID NO: 3 and 4).

[0030] In any embodiment, the second polypeptide comprises the amino acid sequence of SEQ ID NO: 4 or at least 80% identical thereto.

[0031] In any embodiment, the second polypeptide comprises an amino acid sequence of the DUF2436 domain as shown in SEQ ID NO: 3, optionally, the amino acid sequence further comprising one or more amino acid substitutions. Preferably, the one or more amino acid substitutions are one or more substitutions of cysteine ​​residues in the domain.

[0032] In any embodiment, only one cysteine ​​residue is substituted. Alternatively, any two, three, or all four cysteine ​​residues in the DUF domain may be substituted.

[0033] The cysteine ​​residues in the DUF domain can be substituted with any suitable amino acid residue to reduce the likelihood of disulfide bond formation between other cysteine ​​residues in the chimeric or fusion protein. In a preferred embodiment, the substitution may be with a valine residue, a serine residue, or an alanine residue. Optionally, the one or more cysteine ​​residues are substituted with one or more serine residues. Optionally, the one or more cysteine ​​residues are substituted with one or more alanine residues.

[0034] Exemplary amino acid sequences comprising a DUF domain substituted with one or more cysteine ​​residues are shown in SEQ ID NO: 5 to 19. Therefore, in any embodiment, the chimeric or fusion protein of the present invention comprises a second polypeptide comprising the DUF2436 amino acid sequence as shown in any of SEQ ID NO: 5 to 19.

[0035] Based on the various embodiments disclosed above, it will be understood that the present invention provides a chimeric or fusion protein for generating an immune response against *Porphyromonas pharyngoides*. Furthermore, it will be understood that preferably the chimeric or fusion protein comprises a "core" structure, said structure comprising the following: -Active site domain (e.g., as shown in SEQ ID NO: 1 or 2, also referred to herein as the "K" domain or the "R" domain, respectively referring to derivatives of the active site of Lys gingival protease or Arg gingival protease from Porphyromonas pharyngoides). - As defined in this paper, the DUF2436 structural domain (D); and - As defined in this paper, the adhesin domain (A). The domain may include additional modifications as defined herein, and optionally further include one or more additional active site domains.

[0036] Therefore, in one embodiment, the chimeric or fusion protein comprises an amino acid sequence as shown in any of SEQ ID NO: 32 to 35 or a functional variant thereof having at least 80% identity with it.

[0037] Alternatively, the chimeric or fusion protein may comprise an amino acid sequence as shown in any of SEQ ID NO: 77 to 82 or a functional variant thereof having at least 80% identity with it.

[0038] In another embodiment, compared to the naturally occurring *Porphyromonas pharyngioma* Arg gingival protease or Lys gingival protease homolog sequence in the corresponding region, the chimeric or fusion protein (including chimeric or fusion proteins having the amino acid sequence of any one of SEQ ID NO: 32 to 35 or SEQ ID NO: 77 to 82) further comprises one or more amino acid substitutions of the adhesin domain, such as (e.g.) one or more cysteine ​​amino acid substitutions as shown herein; and / or b) substitutions of proline and / or asparagine residues in sequence PxxN corresponding to residues 68 to 71 of the sequence of SEQ ID NO: 20 or located at positions equivalent to said residues. Thus, in some embodiments, the chimeric or fusion protein comprises the DUF domain derived from SEQ ID NO: 4, and the chimeric or fusion protein comprises amino acids as shown in any one of SEQ ID NO: 36 to 45 or functional variants thereof having at least 80% identity with them. In yet another embodiment, the chimeric or fusion protein comprises a DUF domain derived from an amino acid sequence as shown in SEQ ID NO: 3, and the chimeric or fusion protein comprises an amino acid as shown in any of SEQ ID NO: 46 to 76 or a functional variant thereof having at least 80% identity with it.

[0039] In a particularly preferred embodiment, the chimeric or fusion protein comprises or consists of the amino acid sequence of SEQ ID NO: 28 or 29 or a functional variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and preferably wherein the functional variant comprises cysteine ​​substitution and AVQP (SEQ ID NO: 64) substitution in the adhesin domain as defined herein.

[0040] In any embodiment of the invention, the chimeric or fusion protein comprises, or is substantially comprised of, the sequences of the first polypeptide and the second polypeptide as defined herein. Therefore, it should be understood that the chimeric or fusion protein includes an arrangement or configuration of domains that differ from the configuration of those domains in the naturally occurring gingival protease polyprotein sequence. In other words, the first polypeptide and the second polypeptide and their domains have a spatial configuration different from that of the naturally occurring gingival protease polyprotein.

[0041] In any embodiment of any aspect of the invention, the first polypeptide and the second polypeptide are linked. The first polypeptide and the second polypeptide may be linked directly, via a linker, or via a polypeptide sequence of no more than 100, preferably no more than 50 amino acids. Preferably, the first polypeptide and the second polypeptide are directly linked, or linked via a sequence of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. Most preferably, the first polypeptide and the second polypeptide are directly linked.

[0042] In any embodiment, the C-terminal residue of the first polypeptide may be directly linked to the N-terminal residue of the second polypeptide, either via a linker or via a polypeptide sequence of no more than 50 amino acids. Alternatively, the N-terminal residue of the first polypeptide may be directly linked to the C-terminal residue of the second polypeptide, either via a linker or via a polypeptide sequence of no more than 50 amino acids.

[0043] In any embodiment, the DUF2436 domain and the adhesin domain, derived from Arg gingivase or Lys gingivase (or hemagglutinin), can be directly linked or conjugated via a linker or via a polypeptide sequence. Preferably, the DUF2436 domain and the adhesin domain are linked by a short linker sequence comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In a particularly preferred embodiment, the DUF2436 domain and the adhesin domain are linked by a short linker sequence not greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0044] In some embodiments, the DUF2436 domain and the adhesin domain (having representative amino acid sequences of SEQ ID NO: 3, 4 and 20, respectively) (or containing various amino acid substitutions as described herein) can be linked by peptide or polypeptide linkers, as described in other parts of this document.

[0045] In any embodiment, the additional polypeptide may bind directly to or via a linker to the chimeric or fusion protein comprising the first polypeptide and the second polypeptide. In embodiments where the additional polypeptide binds to the C-terminal region of the fusion protein via the second polypeptide, preferably the C-terminus of the second polypeptide binds directly to the N-terminus of the additional polypeptide. (For example, the C-terminus of the adhesin domain preferably binds directly to the N-terminus of the active site amino acid sequence). In cases comprising more than one additional polypeptide, copies of the additional polypeptide may bind directly to each other or via a linker sequence.

[0046] In any embodiment of any aspect of the invention, the linker region is an amino acid sequence of no more than 15 amino acids, and preferably more than 2 amino acids. Suitable linkers used in protein constructs (including those with minimal impact on solubility) are known in the art. Useful linkers include glycine-serine (GlySer) and glycine-threonine (GlyThr) linkers, which are well known in the art and comprise glycine and serine units in various combinations in various sequences. Examples include, but are not limited to, (GS), (GSGGS)n (SEQ ID NO: 102), (GGGS)n (SEQ ID NO: 103), and (GGGGS)n (SEQ ID NO: 104), where n is an integer of at least one, typically from 1 to 10, for example, from 1 to 8, from 1 to 6, or from 1 to 5. Other useful linkers include DSSG (SEQ ID NO: 105), DSSGAS (SEQ ID NO: 106), KLDSSG (SEQ ID NO: 107), or other linkers described herein. In some embodiments, the connector region may be derived from a natural gingival protease multiprotein sequence (such as sequence PNGT, SEQ ID NO: 101).

[0047] The present invention also provides a nucleic acid encoding any chimeric or fusion protein as defined herein.

[0048] Preferably, the nucleic acid has a nucleotide sequence encoding any one or more of the amino acid sequences defined in Table 1 herein.

[0049] Optionally, the nucleic acid has a nucleotide sequence as shown in Table 2 herein (e.g., a nucleic acid sequence comprising any of SEQ ID NO: 83, 84 and 89 to 96).

[0050] In any embodiment, such nucleic acid is included in an expression construct wherein the nucleic acid is operatively linked to a promoter. Such expression constructs may be present in a vector (e.g., a plasmid) or a viral vector.

[0051] The present invention also provides a cell comprising nucleic acids or nucleic acid vectors as described herein. Examples of cells of the present invention include bacterial cells, yeast cells, insect cells, or mammalian cells. Preferably, the cells are isolated, substantially purified, or recombinant.

[0052] The present invention also provides a composition comprising a chimeric or fusion protein as described herein, wherein the composition is optionally combined with a pharmaceutically acceptable carrier.

[0053] The composition may also contain an adjuvant for enhancing the immune response to the chimeric or fusion protein.

[0054] Therefore, the present invention further provides a vaccine or immunostimulatory composition for inducing an immune response against *Porphyromonas pharyngoides* in a subject, said composition comprising: - Immunogens in the form of chimeric or fusion proteins as described in this article, and - An adjuvant used to enhance the immune response to the immunogen in the subject.

[0055] Preferably, the sole immunogen provided in the compositions, vaccines, or immunostimulatory compositions of the present invention is a chimeric or fusion protein as described herein.

[0056] The present invention also provides a method for inducing an immune response against Porphyromonas pharyngota in a subject, the method comprising administering to a subject in need a chimeric or fusion protein, vaccine, or immunostimulatory composition as described herein.

[0057] The present invention also provides a method for inducing a humoral immune response against Porphyromonas pharyngoides in a subject, the method comprising administering to the subject a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as defined herein.

[0058] Preferably, the induced immune response includes a switch from a Th1 to a Th2 immune response.

[0059] The present invention also provides a method for inducing an immune response against Porphyromonas pharyngoides in a subject, the method comprising administering to the subject a first primary dose of a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition as defined herein, and further comprising administering a second booster dose of a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition as defined herein.

[0060] It should be understood that, in any embodiment, the immune response induced by administration of the chimeric or fusion protein described herein or a vaccine or other composition containing said chimeric or fusion protein is preferably antigen-specific. Therefore, in preferred embodiments, the methods and compositions described herein, as well as the chimeric protein, are used to induce an immune response, preferably a protective immune response, against the *Porphyromonas pharyngoides* gingival protease antigen.

[0061] In any embodiment, the compositions, chimeric proteins, and methods of the present invention can be used to enhance the immune response (e.g., protective immune response) of a subject to Porphyromonas pharyngosum.

[0062] The present invention also provides a method for immunizing a subject against Porphyromonas pharyngoides infection, the method comprising administering to the subject a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition as defined herein.

[0063] In any embodiment, subjects who have received the protein or have been administered a composition or vaccine comprising the chimeric or fusion protein of the present invention have improved levels of protection against the severity of one or more symptoms of Porphyromonas pharyngoides infection or present Porphyromonas pharyngoides infection compared to subjects who have not received the chimeric or fusion protein, composition or vaccine of the present invention.

[0064] Furthermore, the present invention provides a method for treating a subject with Porphyromonas pharyngoides infection, the method comprising administering to a subject in need a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition as defined herein, thereby treating the subject's presence of Porphyromonas pharyngoides infection.

[0065] This invention provides a method for reducing or minimizing the severity of symptoms associated with Porphyromonas pharyngoides infection, the method comprising administering to an individual in need a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition as defined herein, wherein the symptoms are selected from the group consisting of: swollen or edematous gums, bleeding gums, receding gums, periodontal pockets around teeth, loss of tooth supporting tissues (periodontal ligaments, cementum, and / or alveolar bone), pus between the gums and teeth, and gingivitis.

[0066] In any embodiment, administration of the chimeric protein as described herein can reduce the load of Porphyromonas pharynx bacteria (i.e., the number of Porphyromonas pharynx cells) in the saliva of the subject who administered the chimeric protein.

[0067] In any embodiment, administration of the chimeric protein as described herein can reduce the load of Porphyromonas pharyngiomas (i.e., the number of Porphyromonas pharyngiomas cells) in the subgingival plaque of the subject who administered the chimeric protein.

[0068] The present invention also provides a method for treating Porphyromonas pharyngoides-associated disease in a subject, the method comprising administering to an individual in need a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition as defined herein. Preferably, the Porphyromonas pharyngoides-associated disease includes periodontal disease.

[0069] The present invention provides a treatment method comprising administering the chimeric or fusion protein, composition, vaccine or immunostimulatory composition of the present invention, and may further comprise administering one or more of the following: antimicrobial compound, anti-inflammatory agent.

[0070] It should also be understood that any method or use described herein may also include a method or use comprising administering one or more additional immunogens, therapeutic agents, or prophylactic agents. In any embodiment, the chimeric or fusion protein of the present invention may be a component of a combination vaccine or immunostimulatory composition.

[0071] The present invention also provides the use of a chimeric or fusion protein as defined herein for the preparation of a medicament for: - Induce an immune response (preferably a protective immune response) against Porphyromonas pharynx in subjects. -Immunize the subject against Porphyromonas pharyngoides infection; - Treating subjects with Porphyromonas pharynx infection; - To minimize or reduce the severity of one or more symptoms of Porphyromonas pharyngosum infection; or - To treat subjects with Porphyromonas pharynx-related diseases.

[0072] This invention also provides chimeric or fusion proteins, compositions, vaccines, or immunostimulatory compositions as defined herein for use in: - Induce an immune response (preferably a protective immune response) against Porphyromonas pharynx in subjects. -Immunize the subject against Porphyromonas pharyngoides infection; - Treating subjects with Porphyromonas pharynx infection; - To minimize or reduce the severity of one or more symptoms of Porphyromonas pharyngosum infection; or - To treat subjects with Porphyromonas pharynx-related diseases.

[0073] In any method, use, or protein, composition, or vaccine used according to the invention, the subject may be any subject who is already infected with or at risk of infection with *Porphyromonas pharyngiosum*. According to the invention, the subject is preferably a veterinary subject, such as a companion animal (e.g., a cat or dog) that is already infected with or at risk of infection with *Porphyromonas pharyngiosum*.

[0074] The present invention also provides a method for obtaining antibodies targeting *Porphyromonas pharyngiosum*, the method comprising administering the chimeric or fusion protein, composition, vaccine, or immunostimulatory composition of the present invention to a non-human animal, thereby generating antibodies targeting *Porphyromonas pharyngiosum* in the animal. Preferably, the method further comprises isolating the antibodies from the animal (e.g., from the animal's blood) or from its eggs (preferably chickens in the case of avian species).

[0075] The present invention also provides an antibody preparation comprising an antibody against Porphyromonas pharyngiosum, wherein the antibody preparation is obtained by administering the chimeric or fusion protein, composition, vaccine or immunostimulatory composition of the present invention to a non-human animal, thereby generating an antibody against Porphyromonas pharyngiosum in the animal, and isolating the antibody from the animal or its eggs.

[0076] Antibodies targeting Porphyromonas pharyngioma can be used therapeutically to eliminate or reduce Porphyromonas pharyngioma infection, or prophylactically to prevent or reduce the severity of Porphyromonas pharyngioma infection.

[0077] The present invention also provides a kit comprising a composition including a chimeric or fusion protein as defined herein, wherein optionally the kit comprises one or more cytokines and / or adjuvants in a sealed container.

[0078] Preferably, the kit includes a label or packaging insert indicating that the composition is intended for immunizing an individual, and optionally the label or packaging insert includes instructions for use.

[0079] Throughout this specification, unless the context otherwise requires, the terms “comprise,” “comprises,” and “comprising” will be understood to imply inclusion of the stated steps or elements or groups of steps or elements, but not to exclude any other steps or elements or groups of steps or elements. Thus, the use of the terms “comprise,” etc., indicates that the listed elements are necessary or mandatory, but other elements are optional and may or may not be present. “consisting of” means including and limited to anything following the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are necessary or mandatory, and other elements may be absent. “Substantially consisting of” means including any element listed following the phrase and is limited to other elements that do not interfere with or facilitate the activities or actions specified in this disclosure for the listed elements. Therefore, the phrase “consistent with…” indicates that the listed elements are required or mandatory, but other elements (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more additional amino acid residues at the N-terminus or C-terminus of the polypeptide sequence) are optional and may be present or absent, depending on whether they affect the activity or function of the listed elements.

[0080] As used herein, unless the context otherwise requires, the term “comprise” and variations thereof, such as “comprising,” “comprises,” and “comprised,” are not intended to exclude additional additions, components, wholes, or steps.

[0081] Further aspects of the invention and further embodiments of the aspects described in the foregoing paragraphs will become apparent from the following description, which is given by way of example and with reference to the accompanying drawings. Attached Figure Description

[0082] Figure 1 : Porphyromonas pharyngoides chimeric protein KD F AK-2S-AVQP and KD A Expression and solubility of AK-2S-AVQP. (A) Lysis fractions; (B) Small-scale purification test using a Ni-NTA spinning column: 1. Clarified lysis buffer before loading onto the column; 2. Flow-through buffer; 3. Column wash; 4. Eluted protein fractions. In A) and B), the inset on the left: KD F AK-2S-AVQP, and the small image on the right: KD A AK-2S-AVQP.

[0083] Figure 2 SDS-PAGE analysis of expressed *Porphyromonas pharyngoides* antigens and cell lysis fractions. (A) KD F AK-2S-AVQP and KD A AK-2S-AVQP expression persisted for 2–3 hours in LB and TB media. (B) Lysis under non-reducing conditions. (C) (i and ii) Attempts to dissolve the insoluble fractions of KD from non-reducing lysis using reducing buffers (5 mM and 100 mM DTT). F AK-2S-AVQP; (iii) the reaction of protein KD under reducing conditions (10 mM DTT) F AK-2S-AVQP was used for lysis. M: protein standard; TC: total cell lysate; Sup: clarified lysate.

[0084] Figure 3 Ni affinity chromatography. (A) KD under normal non-reducing conditions. A (A) Elution curve of AK-2S-AVQP and reduced SDS-PAGE of column fractions. (B) Elution curve of antigen-F and reduced SDS-PAGE of column fractions under reducing conditions (R). (C) KD under normal non-reducing conditions (NR). F Elution curve of AK-2S-AVQP and reduction SDS-PAGE of column fractions. TC: Total cell lysate; Sp: Clarified supernatant before loading onto the column; FT: Flow-through buffer; W: Column wash; M: Protein standard.

[0085] Figure 4 Purification was performed using anion exchange chromatography (AIEX). (A) KD under normal non-reducing conditions. A Elution curves of AK-2S-AVQP and reduction SDS-PAGE of column fractions. (B) KD under reduction conditions (R) FElution curves of AK-2S-AVQP and reduction SDS-PAGE of column fractions. (C) KD under normal non-reducing conditions (NR). F Elution curve of AK-2S-AVQP and reduction SDS-PAGE of column fractions. BL: before loading; FT: flow-through buffer; M: protein standard.

[0086] Figure 5 Size exclusion chromatography under non-reducing (NR) conditions. (A) Elution curve KD A AK-2S-AVQP and column fractionation of reduced SDS-PAGE. (B) KD purified under reducing conditions (R) prior to this step. F Elution curves of AK-2S-AVQP and reducing SDS-PAGE of the column fraction. (C) KD purified under non-reducing conditions (NR) prior to this step. F Elution curve of AK-2S-AVQP and reduction SDS-PAGE of column fractions. BL: before loading; M: protein standard.

[0087] Figure 6 PAGE analysis of the final products of *Porphyromonas pharyngoides* antigen. (A) SDS-PAGE; (B) On native PAGE or non-denaturing gel. Version A: KD A AK-2S-AVQP; FR: KD purified under reducing conditions F AK-2S-AVQP up to final size exclusion steps; F-NR: KD purified under non-reducing conditions F AK-2S-AVQP. R: Reducing; NR: Non-reducing; M: Protein standard.

[0088] Figure 7 Mouse periodontitis model: treatment with vaccination. Schematic diagram of experimental timeline.

[0089] Figure 8 Bone loss induced by *Porphyromonas pharyngoides*. Statistical analysis - one-way ANOVA and post-hoc Dunnett T3. # (p < 0.05 compared with the original control); ## (p < 0.05 compared with the infected control).

[0090] Figure 9 Antibody isotype response against *Porphyromonas pharyngioma*. Antibody titers against heat-inactivated whole cells of *Porphyromonas pharyngioma* in mouse serum (individual). (A) Total IgG titer; (B) IgG1 subtype titer; (C) IgG2a subtype titer.

[0091] Figure 10Antibody IgG response against *Porphyromonas pharyngoides* protease complex. Antibody titers against purified *Porphyromonas pharyngoides* RgpA / Kgp protease complex in mouse serum (individual). (A) Total IgG titer; (B) IgG1 subtype titer; (C) IgG2a subtype titer.

[0092] Figure 11 Antibody IgG anti-vaccine antigen immune response. Antibody titer in mouse serum (individual serum) against the vaccine antigen used to immunize the mouse.

[0093] Figure 12 : A schematic diagram of an experimental protocol used for canine serological studies.

[0094] Figure 13: Effects of immunoantigen (KD) A IgG response to AK-2S-AVQP-6His. A) Collected serum; b) Individual serum; c) Analysis of individual serum; d) Titration of individual serum.

[0095] Figure 14 Serological endpoint titer (2x baseline) relative to whole cells of Porphyromonas pharyngoides.

[0096] Figure 15 : Using KD A Comparison of titer responses to Porphyromonas pharyngoides KAS2 protein after immunization with AK-2S-AVQP-6His.

[0097] Figure 16 Midpoint titer at each serum collection time point. For 10 dogs in each group, the midpoint titer value at each serum collection time point was plotted. The mean and mean standard error (SEM) for each serum collection time point are shown. The ordinate represents the serum dilution required to reach the titration midpoint. Serum time point pairs showing statistically significant differences (p<0.05) between means are represented by the bars above the plot.

[0098] Figure 17 Serological endpoint titers at each serum collection time point. For 10 dogs in each group, endpoint titer values ​​were plotted at each serum collection time point. The mean and mean standard error (SEM) for each serum collection time point are shown. The ordinate represents the serum dilution required to reach the titration endpoint. Serum time point pairs showing statistically significant differences (p < 0.05) between means are represented by the bars above the plot.

[0099] Sequence information Table 1: Amino acid sequence information

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116] Table 2: Nucleic Acid Sequence Information

[0117]

[0118]

[0119]

[0120]

[0121]

[0122] Detailed Implementation

[0123] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or apparent in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0124] Reference will now be made to certain embodiments of the invention. Although the invention will be described in conjunction with embodiments, it should be understood that it is not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined in the claims.

[0125] Those skilled in the art will recognize that many methods and materials can be similar to or equivalent to those described herein, and can be used to practice this invention. This invention is by no means limited to the methods and materials described. It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or apparent in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0126] All patents and publications mentioned in this article are incorporated herein by reference in their entirety.

[0127] For the purposes of interpreting this specification, terms used in the singular will also include the plural form, and vice versa.

[0128] In the work that led to this invention, the inventors investigated various chimeric or fusion proteins for inducing an immune response against Porphyromonas pharyngoides, and methods for the large-scale production of such chimeras for use as vaccine candidates.

[0129] Recent studies in dogs and cats have shown that companion animals harbor similar periodontal pathogens, one of which, *Porphyromonas pharynx*, is closely associated with the key human pathogen *Porphyromonas gingivalis*, and is commonly found in cats and dogs with severe disease. Although *Porphyromonas pharynx* is a known causative agent of periodontitis in companion animals, there are currently no preventative or therapeutic treatments.

[0130] Although *Porphyromonas gingivalis* and *Porphyromonas pharynx* are different species, they possess similar virulence domains, and although encoded by different genes, their proteins are secreted and assembled on the cell surface in a similar manner. This has led the inventors to develop chimeric or fusion proteins containing these virulence genes for inducing an immune response against *Porphyromonas pharynx*, as well as methods and uses comprising said chimeric or fusion proteins.

[0131] Gingival protease The pathogenicity of *Porphyromonas gingivalis* is attributed to numerous surface-associated virulence factors, including cysteine ​​proteases (gingival protease homologs), pili, heme-binding proteins, and outer membrane transporters. Specifically, the extracellular Arg-specific and Lys-specific proteases of the associated pathogen *Porphyromonas gingivalis*, 'gingival protease homologs' (RgpA / B and Kgp), are considered key virulence factors, crucial for colonization, invasion of host tissues, dysregulation of the immune response, ecological imbalance, and disease.

[0132] Gingival proteases, particularly the Lys-specific protease Kgp, are crucial for the ability of *Porphyromonas gingivalis*, an associated pathogen, to induce alveolar bone resorption in a mouse model of periodontitis. High concentrations of gingival proteases have also been found in gingival tissue near subgingival plaque and in distal sites deep within the gingival tissue at sites of severe periodontitis. Lys-specific and Arg-specific proteases have been shown to degrade a variety of host proteins in vitro, such as fibrinogen, fibronectin, and laminin. Plasma host defense and regulatory protease inhibitors α-trypsin, α2-macroglobulin, antichymotrypsin, antithrombin III, and antifibrinolysin have also been degraded by Lys and Arg proteases from *Porphyromonas gingivalis*. This leads to the development of a compelling mechanism to explain the key role *Porphyromonas gingivalis* plays in the development of chronic periodontitis.

[0133] The RgpA, RgpB, and Kgp genes of *Porphyromonas gingivalis* all encode an N-terminal signal peptide of approximately 22 amino acids, an unusually long propeptide of approximately 200 amino acids, and a catalytic domain of approximately 480 amino acids. The C-terminus of the catalytic domain is a large hemagglutinin-adhesin (HA) domain, which contains a "domain of unknown function" (designated DUF2436, defined as a conserved Pfam domain of unknown function; IPR018832) and an adhesin domain (containing an adhesin-binding domain). The specific arrangement of the adhesin domain and DUF varies between Kgp and RgpA / B, and specifically between Kgp gingival proteases and Rgp gingival proteases from different *Porphyromonas* species.

[0134] In vivo, RgpA and Kgp precursor proteins are cleaved into multiple domains that remain non-covalently associated, forming a large outer membrane protein complex. Thus, Arg-specific and Lys-specific proteases have been found in cell-associated complexes of non-covalently associated proteases and adhesins in vivo. One such complex has been named the RgpA-Kgp protease-adhesin complex (previously known as the PrtR-PrtK protease-adhesin complex). This complex consists of a 45 kDa Arg-specific calcium-stabilized cysteine ​​protease and seven sequence-associated adhesin domains.

[0135] As used herein, the Lys gingival protease catalytic domain sequence surrounding the active site histidine may also be referred to as the KAS or K domain. Similarly, the Arg gingival protease catalytic domain sequence surrounding the active site histidine may also be referred to as the RAS or R domain. Typically, the catalytic domains of Lys or Arg gingival protease are located in the N-terminal region of the protein. Table 1 lists exemplary histidine active site peptides, such as SEQ ID NO: 1 and 2, as found within the catalytic domain.

[0136] As used herein, the HA domain of Arg gingivase or Lys gingivase in *Porphyromonas pharyngioma* is understood to generally refer to the region of an Arg gingivase or Lys gingivase homolog located at the C-terminus of a homologous HA domain sequence encoded by an independent multi-adhesin gene such as Hag. The HA domain typically contains a domain of unknown function (DUF) (particularly the conserved undefined Pfam domain of DUF 2436; IPR018832) and an adhesin domain containing an adhesin-binding domain (ABM). Once these domains are expressed and secreted onto the cell surface, they form non-covalent complexes with the DUF and ABM domains to create Arg-specific and Lys-specific protease catalytic domains, thus forming a virulence coat.

[0137] First polypeptide The chimeric or fusion protein of the present invention comprises a first polypeptide comprising or consisting of the amino acid sequence of the active site of a homolog of the Arg-X protease or Lys-X protease of Porphyromonas pharyngoides (also referred to herein as Arg gingival protease or Lys gingival protease, respectively).

[0138] In any embodiment, the first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or 2, or the same sequence as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0139] In embodiments where the first polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 2, the sequence may optionally include a substitution of the cysteine ​​residue at position 5. The substitution may be a serine, valine, or alanine residue, or any other residue suitable for reducing the likelihood of a cysteine ​​residue in a peptide sequence that may form a disulfide bond.

[0140] Preferably, the chimeric or fusion protein comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of a homolog of *Porphyromonas pharyngioma* Arg gingivase or Lys gingivase, or a sequence that is at least 80% identical thereto. The one or more additional polypeptides comprising or consisting of the active site of *Porphyromonas pharyngioma* Arg gingivase or Lys gingivase may be located at the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, the N-terminus of the second polypeptide, or the C-terminus of the second polypeptide. The one or more additional polypeptides may be linked to the first polypeptide or the second polypeptide of the chimeric or fusion protein, preferably through a linker of no more than 50 amino acids, or directly linked to the first polypeptide.

[0141] The one or more additional polypeptides preferably comprise or consist of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 1 or 2, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it.

[0142] In embodiments where one or more additional polypeptides comprise or consist of the amino acid sequence of SEQ ID NO: 2, the sequence may optionally include a substitution of the cysteine ​​residue at position 5. The substitution may be a serine, valine, or alanine residue, or any other residue suitable for reducing the likelihood of a cysteine ​​residue in a peptide sequence that may form a disulfide bond.

[0143] In any embodiment, the first polypeptide comprising or composed of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas pharyngioma, and the other polypeptide comprising or composed of the following: identical amino acid sequences or sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to each other.

[0144] In a preferred embodiment, the chimeric or fusion protein of the present invention comprises no more than two, three, or four polypeptides, said polypeptides comprising or consisting of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease from *Porphyromonas pharyngioma*, or a sequence that is at least 80% identical thereto. Preferably, the chimeric or fusion protein of the present invention has fewer than five polypeptides, more preferably fewer than four, and most preferably fewer than three, said polypeptides comprising or consisting of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease from *Porphyromonas pharyngioma*, or a sequence that is at least 80% identical thereto.

[0145] Second polypeptide The chimeric or fusion protein of the present invention comprises a second polypeptide, the second polypeptide comprising or consisting of the following: i) The amino acid sequence of the DUF2436 domain of the Arg gingival protease and Lys gingival protease surface complex of *Porphyromonas pharyngoides*, preferably wherein the amino acid sequence of the DUF2436 domain is as shown in SEQ ID NO: 3 or 4, or is at least 80% identical thereto; and ii) The amino acid sequence of the adhesin domain of the surface complex of the Arg gingival protease and Lys gingival protease homolog of *Porphyromonas pharyngioma*, preferably wherein the adhesin domain comprises at least the amino acid sequence of SEQ ID NO: 86 and / or SEQ ID NO: 85 (such as the sequence of SEQ ID NO: 88) or at least 80% identical thereto, more preferably wherein the adhesin domain comprises the amino acid sequence of SEQ ID NO: 20 or at least 80% identical thereto.

[0146] DUF structural domain In *Porphyromonas pharyngiomas*, there are two distinct forms of the DUF2436 domain: an "F" variant (e.g., listed in SEQ ID NO: 3) and an "A" variant (e.g., listed in SEQ ID NO: 4). The inventors have found that both variants can be used in the chimeric or fusion proteins of the present invention and for inducing an immune response against *Porphyromonas pharyngiomas*.

[0147] In some embodiments, the DUF2436 domain comprises an amino acid sequence of the “A” variant as shown in SEQ ID NO: 4 or a functional variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0148] In another embodiment, the inventors envision various substitutions for cysteine ​​residues found in the F variant of the DUF 2436 domain. The inventors believe that reducing the number of cysteine ​​residues in the domain will improve the solubility of the chimeric or fusion protein of the present invention and simplify its preparation.

[0149] Therefore, the inventors have conceived of chimeric or fusion proteins as defined herein, wherein the chimeric or fusion proteins comprise a second polypeptide, wherein the second polypeptide comprises a DUF2436 domain having one or more cysteine ​​substitutions.

[0150] One or more cysteine ​​residues may be replaced with any amino acid selected from the following: serine, alanine, and valine.

[0151] Preferably, the one or more cysteine ​​residues are replaced with one or more serine residues.

[0152] In a preferred embodiment, the sequence of the DUF2436 domain comprises an amino acid sequence of the “A” variant as shown in SEQ ID NO: 2, or a functional variant thereof, such as an amino acid sequence having the amino acid sequence shown in SEQ ID NO: 5 to 19, or a functional variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

[0153] Adhesin domain The inventors have also determined that the various domains in gingivase have a tendency to polymerize, which helps reduce the solubility of chimeric or fusion proteins and reduces immunogenicity upon polymerization or aggregation. Therefore, the formation of polymers has an impact on the ease of large-scale production of chimeric proteins derived from gingivase sequences and their use as vaccines, but may also affect the generation of protective responses.

[0154] Specifically, the inventors have identified two mechanisms for polymer formation: the formation of disulfide bridges between cysteine ​​residues and the β-chain exchange between ABM domains within the adhesin domain.

[0155] Therefore, in order to reduce the polymerization of the chimeric protein intended to be used according to the present invention, the inventors aim to target the cysteine ​​residues of the adhesin domain that may lead to β-sheet formation (and thus promote polymerization).

[0156] The inventors have identified one or more of the following modifications that help reduce polymerization of chimeric fusion proteins derived from Kgp and Rgp multiproteins: a) One or more cysteine ​​amino acids are substituted compared to the naturally occurring Arg gingival protease or Lys gingival protease sequence in the corresponding region; b) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of SEQ ID NO: 85 (equivalent to residues 68 to 71 of the sequence of SEQ ID NO: 20 or residues 68 to 71 of SEQ ID NO: 88) or located at positions equivalent to said residues; c) Substitution of motif NxFA to SxYQ in the sequence corresponding to residues 2 to 5 of SEQ ID NO: 85 (equivalent to residues 64 to 67 of the sequence of SEQ ID NO: 20 or residues 64-67 of SEQ ID NO: 88) or located at positions equivalent to said residues; d) Substitution of tyrosine residues corresponding to or located at positions 10 of SEQ ID NO: 86 and tryptophan residues corresponding to or located at positions 23 of SEQ ID NO: 85 with alanine residues (equivalent to tyrosine at position 10 of SEQ ID NO: 20 and tryptophan at position 85).

[0157] In a particularly preferred embodiment of the invention, the second polypeptide comprises one or more cysteine ​​amino acid substitutions compared to the naturally occurring adhesin domain sequence of the *Porphyromonas pharyngoides* gingival protease sequence.

[0158] Cysteine ​​amino acid substitutions can be substitutions that change to serine or valine residues. Preferably, one or more cysteine ​​substitutions comprise one or more substitutions that change to serine residues.

[0159] One or both cysteine ​​residues in the adhesin domain may be substituted with serine or valine residues, preferably serine residues. In a preferred embodiment of the invention, one or both cysteine ​​residues at positions 41 and 55, or equivalent to positions in SEQ ID NO: 20, are substituted, optionally with serine or valine residues, preferably with serine residues. Exemplary sequences containing one or two amino acid substitutions are shown in SEQ ID NO: 22 to 24.

[0160] Therefore, in a particularly preferred embodiment, the chimeric or fusion protein of the present invention comprises an amino acid sequence corresponding to or at least 80% identical to the sequence shown in any of SEQ ID NO: 22 to 24 and contains cysteine ​​substitutions.

[0161] The inventors further determined that mutations in the conserved motifs present in the adhesin domain can also reduce polymerization.

[0162] In one instance, the inventors considered that modification of motif PxxN (e.g., PVQN (SEQ ID NO: 108) in Porphyromonas pharyngoides Kgp, as shown in SEQ ID NO: 20) significantly contributes to reduced polymerization between ABM domains and reduced β-chain exchange.

[0163] Therefore, in a preferred embodiment, the chimeric or fusion protein of the present invention includes a PxxN motif modification in the region of the chimeric or fusion protein corresponding to the adhesin domain of *Porphyromonas pharyngoides* gingival protease. Thus, the second polypeptide preferably comprises proline and asparagine substitutions in the sequence PxxN corresponding to or at positions 68 to 71 of SEQ ID NO: 20.

[0164] The proline amino acid substitution is preferably a substitution of alanine residues.

[0165] The asparagine amino acid substitution can be a substitution of a proline residue or an alanine residue. Preferably, the asparagine residue is substituted with a proline residue. In other embodiments, the asparagine residue is not substituted.

[0166] In another example, the inventors considered the motif NEFA (SEQ ID NO: 109) in the sequence of the adhesin domain of the Kgp / Rgp polyprotein. This sequence is defined herein at residues 64 to 67 of SEQ ID NO: 20. The inventors believe that replacing the motif NEFA (SEQ ID NO: 109) with SEQY (SEQ ID NO: 110) by substituting the asparagine, phenylalanine, and alanine residues with serine, glutamine, and tyrosine, respectively, significantly reduces polymerization.

[0167] In another example, the inventors determined that the substitution of tyrosine residues corresponding to or at positions 10 of SEQ ID NO: 86 and tryptophan residues corresponding to or at positions 23 of SEQ ID NO: 85 with alanine residues also significantly reduced polymerization.

[0168] Finally, the inventors discovered that through cysteine ​​modification... and Combinations of one or more of the following substitutions almost eliminate polymerization: i) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 68 to 71 of the sequence of SEQ ID NO: 20 or located at positions equivalent to said residues; ii) A substitution of motif NxFA for SxYQ in the sequence corresponding to residues 64 to 67 of SEQ ID NO: 20 or located at positions equivalent to said residues; or iii) Substitution of a second tyrosine residue corresponding to or located at a position equivalent to the residue at position 10 of SEQ ID NO: 86 and a tryptophan residue corresponding to or located at a position equivalent to the residue at position 23 of SEQ ID NO: 85.

[0169] In a particularly preferred embodiment, the inventors found that a combination of one or more cysteine ​​modifications, preferably two cysteine ​​substitutions to serine, and modifications of ABM1 to the PXXN motif of AXXP, eliminated the polymerization of the resulting recombinant chimeric protein.

[0170] In summary, through cysteine ​​modification and PXXN motif modification, the present invention provides a chimeric or fusion protein as described herein, wherein the second polypeptide of the chimeric protein comprises an amino acid sequence corresponding to the region of the adhesin domain of *Porphyromonas pharyngoides* Arg gingival protease or Lys gingival protease, wherein the sequence is as shown in any of SEQ ID NO: 21 to 27, preferably comprising the sequence shown in SEQ ID NO: 27 or at least 80% identical thereto, wherein one or two cysteine ​​residues are replaced with serine residues, and / or wherein proline residues and asparagine residues in the sequence PXXN at positions 72 to 75 of SEQ ID NO: 23 or equivalent positions are replaced.

[0171] The linking of the first polypeptide and the second polypeptide In the chimeric or fusion proteins of the present invention, the C-terminal residues of the first peptide may be covalently linked to the N-terminal residues of the second peptide (corresponding to the adhesin domain peptide), or the N-terminal residues of the first peptide may be covalently linked to the C-terminal residues of the second peptide (corresponding to the adhesin domain peptide). In this arrangement, the first peptide and the adhesin domain peptide are referred to as "directly linked" or "adjacent".

[0172] In other embodiments, the chimeric or fusion protein includes a connector for linking the first peptide to the adhesin domain polypeptide. The connector can be any connector capable of binding the peptide to the polypeptide, including both amino acid connectors and non-amino acid connectors.

[0173] Preferably, the linker is non-immunogenic. Typically, the linker contains amino acids and can therefore be called a peptide linker.

[0174] Linkers are typically peptides of up to 20 amino acids in length, although they may be longer. The terms “linked with” or “fused with” refer to a covalent bond (e.g., a peptide bond) formed between two parts. Therefore, in the context of this invention, the length of a linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 or more amino acids. For example, the chimeric or fusion proteins provided herein may comprise a linker between a first polypeptide and a second polypeptide (e.g., between the N-terminus of the second polypeptide and the C-terminus of the first polypeptide), wherein the first polypeptide comprises or is composed of an amino acid sequence of or corresponds to the active domain of *Porphyromonas pharyngoplasmin* gingival protease, and the second polypeptide corresponds to the adhesin domain of *Porphyromonas pharyngoplasmin* gingival protease. An advantage of such linkers is that they make it more likely that the different polypeptides of the fusion protein will fold independently and exhibit the expected behavior. A suitable linker can be up to 50 amino acids long, although less than 20, 15, or 5 amino acids are preferred. The linker can serve to bring the first peptide and the adhesin domain peptide closer together in spatial arrangement than is typically observed in *Porphyromonas pharyngoides* trypsin-like enzymes. Alternatively, it can separate the first peptide from the second peptide (corresponding to the adhesin domain peptide).

[0175] Suitable linkers used in protein constructs (including linkers with minimal impact on solubility) are known in the art. Linkers can be any linker known to those skilled in the art and can be flexible linkers (e.g., linkers containing repeating sequences of glycine and serine residues), rigid linkers (e.g., linkers containing glutamic and lysine residues flanking alanine repeating sequences), and / or cleavable linkers (e.g., sequences susceptible to protease cleavage). Examples of such linkers are known to those skilled in the art and are described, for example, in Chen et al., (2013) *Advanced Drug Delivery Reviews*, 65: 1357-1369.

[0176] Useful linkers include glycine-serine (GlySer) linkers, which are well known in the art, and comprise glycine and serine units in various combinations in a sequence. Examples include, but are not limited to, (GS), (GSGGS)n (SEQ ID NO: 102), (GGGS)n (SEQ ID NO: 103), and (GGGGS)n (SEQ ID NO: 104), where n is an integer of at least one, typically from 1 to 10, such as from 1 to 8, from 1 to 6, or from 1 to 5.

[0177] In some embodiments, the peptide linker may comprise glycine and serine amino acids of various lengths and combinations. In some aspects, the peptide linker may comprise the sequences Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS, SEQ ID NO: 103), or Gly-Gly-Gly-Gly-Ser (GGGGS, SEQ ID NO: 104), as well as variants or repeating sequences thereof. In some aspects, the peptide linker may comprise the amino acid sequence GGGGS (a linker of 6 amino acids in length, SEQ ID NO: 104) or even longer. The linker may comprise a series of repeating glycine and serine residues (GS) of varying lengths, i.e., (GS)n, where n is any number from 1 to 15 or greater. For example, the linker may be (GS)3 (i.e., GSGSGS, SEQ ID NO: 111) or longer (GS)11 or longer. It should be understood that n may be any number including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or greater. Fusion proteins with linkers of this length are included within the scope of this invention. Similarly, the linker can be a series of repeating glycine residues separated by serine residues. For example, (GGGGS)3 (i.e., the linker can contain the amino acid sequence GGGGSGGGGSGGGGS, (G4S)3, SEQ ID NO: 112) and its variants.

[0178] In one embodiment, the peptide linker may comprise the amino acid sequence GGGGS (a linker of 6 amino acids in length, SEQ ID NO: 104) or even longer. The linker may be a series of repeating glycine and serine residues (GS) of varying lengths, i.e., (GS)n, where n is any number from 1 to 15 or greater. For example, the linker may be (GS)3 (i.e., GSGSGS, SEQ ID NO: 111) or longer (GS)11 or longer. It should be understood that n may be any number including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or greater.

[0179] Other useful adapters include DSSG (SEQ ID NO: 105), DSSGAS (SEQ ID NO: 106), KLDSSG (SEQ ID NO: 107), and their variants. Examples of other suitable adapters are described in Chen et al., (2013) Advanced Drug Delivery Review, 65: 1357-1369.

[0180] Chimeric or fusion proteins and recombinant proteins The chimeric or fusion proteins of the present invention can be prepared by any of a variety of conventional techniques, although recombinant techniques are typically used to prepare peptides.

[0181] For recombinant peptides, well-known molecular genetic techniques can be used to subclone the DNA fragment encoding the desired peptide into a suitable vector (see, for example, Maniatis et al., *Molecular Cloning: A Laboratory Manual*, 2nd edition (Cold Spring Harbor, 1982); Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd edition (Cold Spring Harbor, 1989). The fragment can be transcribed, and the peptide is subsequently translated in vitro. Commercially available kits can also be used (e.g., those prepared by Clontech, Palo Alto, California; Amersham Pharmacia Biotech Inc., Piscataway, NJ; InVitrogen, Carlsbad, California, etc.). Polymerase chain reaction can optionally be used to manipulate nucleic acids.

[0182] A “fragment” is a part of the polypeptide of the present invention that retains substantially similar functional activity or substantially the same biological function or activity as the polypeptide, which can be determined using the assays described herein.

[0183] The "percentage of amino acid sequence identity (%)" or "percentage of identical amino acids (%)" for a polypeptide sequence (i.e., the polypeptide of the present invention as defined herein) is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific polypeptide of the present invention, after sequence alignment and the introduction of vacancies (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of sequence identity. A variant mentioned herein that has "at least x% sequence identity" with the stated sequence means that the variant is at least x% identical to the stated sequence.

[0184] In various aspects and embodiments of the invention, the defined polypeptide is described by reference to a variant having at least 80% homology or higher with a reference sequence. Homology percentage (%) generally refers to the polypeptide of the invention as defined herein, defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific polypeptide of the invention, after sequence alignment and the introduction of vacancies (if necessary) to achieve the maximum sequence identity percentage, and without considering any conserved substitutions as part of sequence identity.

[0185] The amino acids glycine, alanine, valine, leucine, and isoleucine (amino acids with aliphatic side chains) can generally be substituted for each other. Among these possible substitutions, glycine and alanine are preferred to be substituted for each other (because of their relatively short side chains), and valine, leucine, and isoleucine are preferred to be substituted for each other (due to their larger hydrophobic aliphatic side chains). Other amino acids that can frequently be substituted for each other include: phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0186] This type of substitution is often referred to as "conservative" or "semi-conservative" amino acid substitution.

[0187] Amino acid deletions or insertions can also be made relative to the native sequence of *Porphyromonas pharyngoides* proteins. Thus, for example, amino acids that do not substantially affect the activity of the peptide, or at least do not eliminate such activity, can be deleted. Such deletions can be advantageous, especially for longer peptides, because the overall length and molecular weight of the peptide can be reduced while still maintaining its activity. This allows for a reduction in the amount of peptide required for a specific purpose, for example, by lowering the dosage level.

[0188] Amino acid insertions relative to the natural polypeptide sequence can also be performed. This can alter the properties of the polypeptide used in this invention (e.g., to enhance antigenicity).

[0189] Amino acid changes can be performed using any suitable technique, such as by using site-directed mutagenesis or solid-state synthesis.

[0190] It should be understood that amino acid substitutions or insertions within the scope of this invention can be made using naturally occurring or non-naturally occurring amino acids.

[0191] Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared (non-limiting examples described below). When aligning amino acid sequences, the percentage of amino acid sequence identity between a given amino acid sequence A and (and, or against) a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or containing a certain percentage of amino acid sequence identity with (and, or against) a given amino acid sequence B) can be calculated as: Amino acid sequence identity percentage = X / Y × 100, where X is the number of amino acid residues that are rated as identical matches by the alignment procedure or algorithm for A and B, and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the percentage of amino acid sequence identity between A and B will not be equal to the percentage of amino acid sequence identity between B and A.

[0192] When calculating the percentage of identity, exact matches are typically counted. The determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms. A non-restricted example of a mathematical algorithm for comparing two sequences is the following: Karlin and Altschul (1990) Proceedings of the National Academy of Sciences (PNAS) 87:2264, as modified in Karlin and Altschul (1993) PNAS 90:5873-5877. Such algorithms are incorporated into the following BLASTN and BLASTX procedures: Altschul et al. (1990) Journal of Molecular Biology 215:403. To obtain vacancy-free alignments for comparative purposes, vacancy-free BLAST (in BLAST 2.0) can be used, as described below: Altschul et al. (1997) Nucleic Acids Research. 25:3389. Alternatively, PSI-Blast can be used to perform iterative searches to detect distance relationships between molecules. See Altschul et al. (1997) Ibid. When using BLAST, vacancy-free BLAST, and PSI-Blast procedures, the default parameters of the respective procedures (e.g., BLASTX and BLASTN) can be used. Alignments can also be performed manually by inspection. Another non-limiting example of a mathematical algorithm for sequence comparison is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Research 22:4673-4680). ClustalW compares sequences and aligns the entire amino acid or DNA sequence, and thus can provide data on sequence conservation for the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA / amino acid analysis software packages, such as the ALIGNX module of the Vector NTI program suite (Ingenie, Carlsbad, California). After aligning amino acid sequences using ClustalW, the percentage of amino acid identity can be assessed. Non-limiting examples of software programs useful for analysis using ClustalW alignments are GENEDOC™ or JalView (http: / / www.jalview.org / ). GENEDOC™ allows for the assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller (1988), *Computers in the Biosciences (CABIOS)*, 4:11-17.This type of algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG WisconsinGenetics Software Package, version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When comparing amino acid sequences using the ALIGN program, the PAM 120 weighted residue table, vacancy length penalty 12, and vacancy penalty 4 can be used.

[0193] Peptides ideally contain an amino terminus and a carboxyl terminus. Peptides can contain D-amino acids, L-amino acids, or a mixture of D-amino acids and L-amino acids.

[0194] As used herein, the term "conservative substitution" refers to replacing an amino acid present in the natural sequence of a peptide with a naturally occurring or non-naturally occurring amino acid or a peptide mimic with similar stereochemical properties. If the side chain of the natural amino acid to be replaced is polar or hydrophobic, the conservative substitution should be the use of a naturally occurring amino acid, a non-naturally occurring amino acid, or a peptide mimic part that is also polar or hydrophobic (except for having the same stereochemical properties as the side chain of the replaced amino acid).

[0195] Conservative amino acid substitutions that provide functionally similar amino acids are well known to those skilled in the art. The following six groups are examples of amino acids that can be considered as conserved substitutions for each other: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0196] Since naturally occurring amino acids are typically grouped according to their properties, conservative substitutions of naturally occurring amino acids can be determined, taking into account the fact that replacing a charged amino acid with a spatially similar, uncharged amino acid is considered a conservative substitution. To generate conservative substitutions from non-natural amino acids, amino acid analogs (synthetic amino acids) well-known in the art can also be used. Peptide analogs of naturally occurring amino acids are well documented in the literature known to those skilled in the art, and non-natural or unnatural amino acids are further described below. When influencing conservative substitutions, the substituted amino acid should have the same or similar functional groups in its side chain as the original amino acid.

[0197] As used herein, the phrase “non-conservative substitution” or “non-conservative residue” refers to the replacement of an amino acid present in a parent sequence with another naturally occurring or non-naturally occurring amino acid that has different electrochemical and / or steric properties. Therefore, the side chain of the substituted amino acid can be significantly larger (or smaller) than the side chain of the substituted natural amino acid and / or can have functional groups with significantly different electronic properties than the substituted amino acid. Examples of such non-conservative substitutions include the substitution of alanine with phenylalanine or cyclohexylmethylglycine, the substitution of glycine with isoleucine, or the substitution of aspartic acid with -NH-CH[(-CH2)5-COOH]-CO-. Non-conservative substitutions include any mutation that is not considered conserved.

[0198] Non-conservative amino acid substitutions can result from changes in: (a) the structure of the amino acid backbone in the substituted region; (b) the charge or hydrophobicity of the amino acid; or (c) a large portion of the amino acid side chain. Substitutions that are generally expected to produce the greatest changes in protein properties are: (a) a hydrophilic residue replacing (or being replaced by) a hydrophobic residue; (b) proline replacing (or being replaced by) any other residue; (c) a residue with a large side chain (e.g., phenylalanine) replacing (or being replaced by) a residue without a side chain (e.g., glycine); or (d) a residue with a positively charged side chain (e.g., lysyl, arginyl, or histidine) replacing (or being replaced by) an electronegative residue (e.g., glutamyl or aspartic).

[0199] Alterations to the natural amino acid sequence to produce mutant peptides (e.g., through insertion, deletion, and / or substitution) can be performed in a variety of ways known to those skilled in the art. For example, site-specific mutations can be introduced by ligating a synthetic oligonucleotide containing the modified site into an expression vector. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used, such as those disclosed in Walder et al., *Gene* 42:133 (1986); Bauer et al., *Gene* 37:73 (1985); Craik, *Biotechniques*, 12-19 (January 1995); and U.S. Patent Nos. 4,518,584 and 4,737,462. A preferred method for introducing mutations is the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, California).

[0200] Any suitable expression vector (e.g., as described in Pouwels et al., *Cloning Vectors: A Laboratory Manual*, Elsevier, NY: 1985)) and a corresponding suitable host can be used to produce recombinant peptides. Expression hosts include, but are not limited to, *Escherichia coli* and *Bacillus*. Bacillus ), Pseudomonas spp. Pseudomonas Salmonella ( Salmonella Bacterial species, mammalian or insect host cell systems, including baculovirus systems (e.g., as described in Luckow et al., Bio / Technology 6:47 (1988)), and established cell lines such as COS-7, C127, 3T3, CHO, HeLa, and BHK cell lines. Those skilled in the art will understand that the choice of expression host influences the type of polypeptide produced. For example, the glycosylation of polypeptides produced in yeast or mammalian cells (e.g., COS-7 cells) will differ from that produced in bacterial cells (e.g., E. coli).

[0201] Alternatively, the polypeptides of the present invention can be synthesized using standard peptide synthesis techniques well known to those skilled in the art (e.g., summarized in Bodanszky, Principles of Peptide Synthesis (Springer-Verlag, Heidelberg: 1984)). Specifically, the polypeptides can be synthesized using solid-phase synthesis procedures (see, for example, Merrifield, *Journal of the American Chemical Society* 85: 2149-54 (1963); Barany et al., *International Journal of Peptide and Protein Research* 30: 705-739 (1987); and U.S. Patent No. 5,424,398). Automated peptide synthesizers can be used if desired. For example, the removal of the tert-butyloxycarbonyl (t-BOC) or 9-fluorenylmethoxycarbonyl (Fmoc) amino acid blocking groups and the separation of the polypeptide from the resin can be accomplished by acid treatment at low temperature. The polypeptide-containing mixture can then be extracted, for example, with dimethyl ether to remove non-peptide organic compounds, and the synthesized polypeptide can be extracted from the resin powder (e.g., with about 25% w / v acetic acid). Following polypeptide synthesis, further purification (e.g., using high-performance liquid chromatography (HPLC)) can optionally be performed to eliminate any incomplete polypeptides or free amino acids. The synthesized polypeptide can be subjected to amino acid and / or HPLC analysis to verify identity. For other applications according to the invention, it may be preferred to produce the polypeptide as part of a larger fusion protein (such as by the methods described herein or other genetic means) or as part of a larger conjugate (such as by physical or chemical conjugation, as known to those skilled in the art and described herein).

[0202] In any embodiment of the invention, the chimeric or fusion proteins of the invention may contain additional amino acid residues to facilitate expression in a recombinant expression system and / or to facilitate protein purification. Therefore, proteins as defined herein may include additional amino acids, such as one, two, three, four, or five amino acids in the N-terminal region. Typically, the additional amino acids will include N-terminal methionine to facilitate expression in a recombinant expression system, although it should be understood that such N-terminal residues are typically cleaved post-translationally. Therefore, it should be understood that for some proteins described herein (e.g., SEQ ID NO: 30 and 31), the N-terminal methionine will be treated by the expression organism, and the final protein product will not contain N-terminal methionine. In some embodiments, the N-terminal amino acid includes at least methionine and alanine residues.

[0203] Additionally, the chimeric or fusion proteins according to the invention may include additional amino acids, such as one, two, three, four, or five amino acids, in the N-terminal or C-terminal region, preferably to facilitate purification. It should be understood that such amino acid residues may facilitate the inclusion of purification tags (such as histidine tags) in the protein. Such residues may not be included when producing an untagged version of the protein.

[0204] The peptides of the present invention can also be modified, conjugated, or fused with another portion to facilitate purification, increase the in vivo half-life of the peptide, or be used in immunoassays using methods known in the art. For example, the peptides of the present invention can be modified by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or linkage with cellular ligands or other proteins.

[0205] Nucleic acid Nucleic acid molecules encoding any of the chimeric or fusion proteins or polypeptides of the present invention are also within the scope of the present invention. For example, nucleic acids can be used to prepare the polypeptides of the present invention and as therapeutic agents. The nucleic acids can be applied to cells in cultures or in vivo and can include secretion signals that direct or promote the secretion of the polypeptides of the present invention from cells. Expression vectors and host cells containing or including the nucleic acids of the present invention are also within the scope of the present invention (further described below). Although the nucleic acids of the present invention may be referred to as “isolated,” by definition, the polypeptides of the present invention are not wild-type polypeptides and therefore are not encoded by naturally occurring nucleic acids. Therefore, while the polypeptides and nucleic acids of the present invention may be “purified,” “substantially purified,” “isolated,” “recombinant,” or “synthetic,” this is not necessary to distinguish them from naturally occurring materials.

[0206] "Isolated" nucleic acid molecules are those identified and separated from at least one contaminant nucleic acid molecule typically associated with the natural source of nucleic acids. Isolated nucleic acid molecules differ from those found in nature or in their environment. Therefore, isolated nucleic acid molecules are distinct from those present in natural cells. However, isolated nucleic acid molecules include those typically found in cells expressing Kgp, wherein, for example, the nucleic acid molecule is located at a chromosomal location different from its position in natural cells.

[0207] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, namely deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotides of the present invention may be provided in isolated or purified form. The nucleic acid sequence “encoding” a selected polypeptide is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo. The boundaries of the coding sequence are defined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. For the purposes of this invention, such nucleic acid sequences may include, but are not limited to, cDNA from viral, prokaryotic, or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. The transcription termination sequence may be located at the 3' end of the coding sequence.

[0208] The polynucleotides of the present invention can be synthesized according to methods well known in the art, as described by way of example in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Press).

[0209] The polynucleotide molecules of the present invention can be provided in the form of expression cassettes, which include control sequences operatively linked to an insert sequence, thereby allowing the polypeptides of the present invention to be expressed in vivo in targeted subjects. In turn, these expression cassettes are typically provided within a vector (e.g., a plasmid or recombinant viral vector) suitable for use as a reagent for nucleic acid immunization. Such expression cassettes can be administered directly to a host subject. Alternatively, a vector containing the polynucleotides of the present invention can be administered to a host subject. Preferably, the polynucleotides are prepared and / or administered using a genetic vector. A suitable vector can be any vector capable of carrying a sufficient amount of genetic information and allowing expression of the polypeptides of the present invention.

[0210] Therefore, the present invention includes expression vectors comprising such polynucleotide sequences. Thus, the present invention provides a vector for the prevention or treatment of inflammatory diseases or symptoms, the vector comprising a polynucleotide sequence encoding a polypeptide of the present invention and optionally one or more additional polynucleotide sequences encoding different polypeptides as defined herein.

[0211] Furthermore, it should be understood that the compositions and products of the present invention may comprise a mixture of polypeptides and polynucleotides. Therefore, the present invention provides a composition or product as defined herein, wherein any one of the polypeptides is replaced with a polynucleotide capable of expressing the polypeptide.

[0212] Expression vectors are routinely constructed in the field of molecular biology and may involve, for example, the use of plasmid DNA and appropriate initiators, promoters, enhancers, and other elements, such as polyadenylation signals, which may be necessary and correctly oriented to allow expression of the peptides of the present invention. Other suitable vectors will be apparent to those skilled in the art. As another example in this regard, see Sambrook et al.

[0213] Therefore, the polypeptides of the present invention can be provided by delivering such vectors into cells and allowing transcription from the vectors. Preferably, the polynucleotides of the present invention, or those used in the vectors in the present invention, are operatively linked to a control sequence capable of providing expression of the coding sequence carried out by the host cell, i.e., the vector is an expression vector.

[0214] "Operationally linked" refers to an arrangement of elements in which the components described herein are configured to perform their usual functions. Therefore, in the presence of a suitable enzyme, a given regulatory sequence (such as a promoter) operably linked to a nucleic acid sequence can influence the expression of that sequence. The promoter need not be adjacent to the sequence, as long as it serves to guide its expression. Thus, for example, an intermediate untranslated but still transcribed sequence can exist between the promoter sequence and the nucleic acid sequence, and the promoter sequence and coding sequence can still be considered "operationally linked."

[0215] Various expression systems have been described in the art, each typically consisting of a vector containing a gene or nucleotide sequence of interest operatively linked to expression control sequences. These control sequences include transcription promoter sequences and transcription initiation and termination sequences. The vectors of the present invention can be, for example, plasmids, viral or phage vectors, provided with an origin of replication, a promoter optionally for the expression of the polynucleotide, and optionally a regulatory factor for the promoter. A “plasmid” is a vector in the form of an extrachromosomal genetic element. The vector may contain one or more optional marker genes, such as an ampicillin resistance gene in a bacterial plasmid or a resistance gene in a fungal vector. The vector can be used in vitro, for example, for the production of DNA or RNA, or for transfection or transformation of host cells, such as mammalian host cells. The vector can also be adapted for in vivo use, for example, to allow in vivo expression of polypeptides.

[0216] A promoter is a nucleotide sequence that initiates and regulates the transcription of a polynucleotide encoding a polypeptide. Promoters can include inducible promoters (where the expression of a polynucleotide sequence operatively linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressive promoters (where the expression of a polynucleotide sequence operatively linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. The term "promoter" or "control element" is intended to include both the full-length promoter region and the functional (e.g., controlling transcription or translation) segments of these regions.

[0217] The polynucleotides, expression cassettes, or vectors according to the invention may additionally include a signal peptide sequence. The signal peptide sequence is typically inserted in a manner operatively linked to a promoter, such that the signal peptide is expressed and promotes the secretion of a polypeptide encoded by a coding sequence, which is also operatively linked to a promoter.

[0218] Typically, signal peptide sequences encode 10 to 30 amino acids, such as peptides of 15 to 20 amino acids. These amino acids are usually predominantly hydrophobic. Under normal circumstances, the signal peptide targets the growth polypeptide chain carrying the signal peptide to the endoplasmic reticulum (ER) of the expressing cell. The signal peptide is cleaved in the ER, allowing the polypeptide to be secreted via the Golgi apparatus.

[0219] Immunogenicity and vaccine composition The present invention further provides compositions comprising chimeric or fusion proteins as defined herein, and the use of such chimeric or fusion proteins in immunogenic or vaccine compositions for the treatment or prevention of Porphyromonas pharyngoides infection.

[0220] As used herein, the term "vaccine composition" is defined as a composition intended to induce an immune response against an antigen (immunogen) within the composition to protect or treat an organism from disease.

[0221] As used herein, the terms “immunostimulatory composition,” “vaccine composition,” and “immunogenic composition” are generally used interchangeably.

[0222] In addition to one or more peptides of the present invention as therapeutic or prophylactic active ingredients, the immunostimulatory compositions or vaccines of the present invention may suitably include pharmaceutically acceptable carriers, excipients, diluents, adjuvants, mediators, buffers, or stabilizers. Such carriers include, but are not limited to, saline, buffered saline, dextran, liposomes, water, glycerol, polyethylene glycol, ethanol, and combinations thereof.

[0223] Immunostimulatory compositions or vaccine compositions may be adapted for administration via any suitable route, such as parenteral (including subcutaneous, intramuscular, intravenous, or intradermal, or by injection into cerebrospinal fluid), oral (including buccal or sublingual), nasal, local (including buccal, sublingual, or transdermal), vaginal, or rectal routes. In some embodiments, the vaccine or immunostimulatory composition may be delivered as a bolus injection or as a sustained-release composition. Such compositions may be prepared by any method known in the pharmaceutical field, such as by mixing peptides with a carrier or excipient under sterile conditions. Typically, vaccine compositions are adapted for administration via subcutaneous, intramuscular, intravenous, or intradermal routes, typically by injection. Alternatively, vaccine compositions may be adapted for oral or nasal administration.

[0224] Immunostimulatory compositions or vaccine compositions suitable for parenteral administration can be aqueous and non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickeners. Excipients that can be used in injectable solutions include, for example, water, alcohols, polyols, glycerol, and vegetable oils. The compositions can be present in single-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored under lyophilized (freeze-dried) conditions where a sterile liquid carrier, such as water for injection, is only required to be added immediately before use. Temporary injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0225] Immunostimulatory or vaccine compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, or lozenges; as powders or granules; as solutions, syrups, or suspensions (in aqueous or non-aqueous liquids; or as edible foams or pastes; or as emulsions).

[0226] Suitable excipients for use in tablets or hard gelatin capsules include lactose, corn starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients for use with soft gelatin capsules include, for example, vegetable oils, waxes, fats, semi-solid or liquid polyols.

[0227] For the preparation of solutions and syrups, excipients that can be used include, for example, water, polyols, and sugars. For the preparation of suspensions, oils (e.g., vegetable oils) can be used to provide oil-in-water or water-in-oil suspensions.

[0228] Where the carrier is a solid, suitable immunostimulatory or vaccine composition for nasal administration comprises coarse powder, for example, in the particle size range of 20 to 500 micrometers, which is administered by sniffing, i.e., rapidly inhaled through the nasal passages from a powder container near the nose. Where the carrier is a liquid, suitable compositions for administration as nasal sprays or drops may contain an aqueous or oil solution of the active ingredient.

[0229] Compositions suitable for inhalation administration include fine particulate dust or mist, which can be generated by various types of metered-dose pressurized aerosols, nebulizers, or blowers.

[0230] Immunostimulatory or vaccine compositions suitable for transdermal administration can be presented as discrete patches designed to maintain close contact with the recipient's epidermis for extended periods. For example, the active ingredient can be delivered from the patch via iontophoresis, as generally described in Pharmaceutical Research 3(6):318 (1986).

[0231] Compositions suitable for topical application can be formulated as ointments, creams, suspensions, lotions, powders, solutions (e.g., mouthwashes), pastes, gels, sprays, aerosols, or oils. For infections of the eyes or other external tissues (e.g., the mouth and skin), the composition can be applied as a topical ointment or cream. When formulated into an ointment, the active ingredient can be used with a paraffin-based ointment base or a water-miscible ointment base. Alternatively, the active ingredient can be formulated into a cream with an oil-in-water or water-in-oil base. Pharmaceutical compositions suitable for topical application to the eyes can comprise eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent. Pharmaceutical compositions suitable for topical application in the oral cavity can comprise lozenges, soft lozenges, or mouthwashes.

[0232] Immunostimulatory or vaccine compositions may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts (the substances of the present invention may be provided in the form of pharmaceutically acceptable salts), buffers, coating agents, or antioxidants.

[0233] In addition to chimeric or fusion proteins as defined herein, the vaccine compositions of the present invention may also contain one or more other prophylactic or therapeutic active agents.

[0234] The chimeric or fusion proteins used in the vaccine compositions of the present invention may or may not be lyophilized.

[0235] In addition to peptides as defined herein, the vaccine compositions of the present invention may also include pharmaceutically acceptable adjuvants. Adjuvants are added to enhance the immunogenicity of the vaccine composition.

[0236] Suitable adjuvants for inclusion in vaccine compositions are known in the art and include incomplete Freund's adjuvant, complete Freund's adjuvant, Freund's adjuvant with MDP (muramyldipeptide), alum (aluminum hydroxide), alum plus Bordatella pertussis and immunostimulatory complex (ISCOM, typically a matrix containing Quil A viral protein), QS-21, Detox-PC, MPL-SE, MoGM-CSF, TitreMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, alethine, MPC-026, Adjuvax, CpG ODN, betafectin, and MF59.

[0237] The vaccine composition of the present invention may also include one or more co-stimulatory molecules or be administered therewith.

[0238] The dosage of the vaccine composition of the present invention can vary within a range, depending on the age and condition of the individual to be treated, and the physician will ultimately determine the appropriate dosage to be used.

[0239] This dose can be repeated at an appropriate frequency. For example, an initial dose of vaccine can be administered, followed by a booster dose at a later date.

[0240] For administration to mammals (and particularly humans), the expected daily dose of the active agent will be from 1 μg / kg to 10 mg / kg body weight, typically from about 10 μg / kg to 1 mg / kg body weight. In any case, the physician will determine the most suitable actual dose for the individual based on factors including age, weight, sex, and response. The above doses are examples of general cases. Of course, there may be situations where higher or lower doses are justified, and such dose ranges are within the scope of this invention.

[0241] The vaccine compositions of the present invention can be administered by any convenient route as described herein, such as intramuscular, intravenous, inhalation, intraperitoneal, or oral routes, or by injection into the cerebrospinal fluid.

[0242] The vaccine compositions of the present invention may be provided in unit dosage forms, typically in sealed containers, and may be provided as part of a kit. Such kits typically (though not necessarily) include instructions for use. They may include various of the aforementioned unit dosage forms.

[0243] Therefore, in another aspect, the present invention provides a kit comprising the vaccine composition of the present invention and one or more cytokines and / or adjuvants in a sealed container.

[0244] Methods for immunizing subjects using thematic chimeric or fusion proteins The present invention provides methods and compositions for treating or preventing or minimizing the possibility of Porphyromonas pharyngoides infection in individuals in need, the methods comprising administering the fusion or chimeric protein of the present invention.

[0245] The present invention also provides methods and compositions for inducing a humoral immune response against *Porphyromonas pharyngoides* in a subject. The humoral response may be for the purpose of directly obtaining protective / therapeutic antibodies against *Porphyromonas pharyngoides* in an individual requiring protection / therapy. Alternatively, the humoral response may be for the purpose of generating antibodies, and then isolating said antibodies from the subject (or its eggs) so that the antibodies can then be directly administered to an individual requiring antibody treatment / protection.

[0246] Therefore, the present invention includes methods and compositions for preventing Porphyromonas pharyngoides infection in subjects, minimizing the likelihood of infection, and / or reducing the severity and duration of Porphyromonas pharyngoides infection.

[0247] The present invention also provides a method for obtaining antibodies targeting *Porphyromonas pharyngiosum*, the method comprising administering the chimeric or fusion protein, composition, vaccine, or immunostimulatory composition of the present invention to a non-human animal, thereby generating antibodies targeting *Porphyromonas pharyngiosum* in the animal. Preferably, the method further comprises isolating the antibodies from the animal (e.g., from the animal's blood) or from the animal's eggs (e.g., in the case of generating IgY antibodies from chickens).

[0248] The present invention also provides an antibody preparation comprising an antibody targeting Porphyromonas pharyngioma, wherein the antibody preparation is obtained by administering the chimeric or fusion protein, composition, vaccine or immunostimulatory composition of the present invention to a non-human animal, thereby generating an antibody targeting Porphyromonas pharyngioma in the animal, and isolating the antibody from the animal or its eggs.

[0249] Antibodies targeting Porphyromonas pharyngioma can be used therapeutically to eliminate or reduce Porphyromonas pharyngioma infection, or prophylactically to prevent or reduce the severity of Porphyromonas pharyngioma infection.

[0250] As used herein, the term "treatment" or "treating" refers to administering or applying the compositions of the present invention (or applying or applying compounds of the present invention to cells or tissues derived from the subject) to a subject with the aim of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, reducing deterioration, improving, enhancing, or influencing the disease or condition, symptoms of the disease or condition, or the risk (or susceptibility) to the disease or condition. The term "treatment" means any indicator of successful treatment or improvement of an injury, pathology, or condition, including any objective or subjective parameter such as reduction; relief; reduction of the rate of deterioration; reduction of the severity of the disease; stabilization, reduction of symptoms, or making the injury, pathology, or condition more tolerable for the subject; slowing the rate of deterioration or decline; making the late stages of deterioration less debilitating; or improving the physical or mental health of the subject.

[0251] As used herein, “preventing” or “prevention” is intended to refer to at least a reduction in the likelihood of acquiring a disease or condition (i.e., causing a subject who may be exposed to or susceptible to a disease but has not yet experienced or displayed symptoms of the disease to not develop at least one of the clinical symptoms of the disease). This document provides biological and physiological parameters for identifying such subjects, and these parameters are also well-known to physicians.

[0252] The vaccine composition of the present invention can be administered to subjects who need it most. The vaccine composition of the present invention can also be administered to subjects suspected of having or diagnosed with Porphyromonas pharyngoides infection.

[0253] The compositions and methods of the present invention are equally extended to use in both human medicine and / or veterinary medicine, in the production of diagnostic agents or other therapeutic agents.

[0254] As used herein, the term "subject" should be understood to mean any animal, preferably a non-human animal. Exemplary subjects include, but are not limited to, companion animals (cats, dogs, guinea pigs, etc.).

[0255] As used herein, the terms “subject,” “individual,” and “patient” are used interchangeably.

[0256] Those skilled in the art will be familiar with methods for determining successful vaccination / immunization with chimeric or fusion proteins or compositions as described herein. For example, those skilled in the art will be familiar with methods for quantifying antibodies produced after immunization and / or for quantifying the extent of humoral (Th2) responses induced after immunization.

[0257] Reagent test kit In another embodiment, a kit or article is provided comprising one or more proteins, peptides or polynucleotides of the present invention and / or immunogenic compositions as described above.

[0258] In another aspect, the present invention provides a kit comprising the vaccine composition of the present invention and one or more adjuvants for administration to a subject alone, subsequently or simultaneously.

[0259] In other embodiments, a kit is provided for use in the above-described therapeutic or preventative applications, the kit comprising: - A container for containing the protein, polypeptide, polynucleotide, or immunogenic composition of the present invention; - Includes a label or packaging insert with an instruction manual.

[0260] In any embodiment, the kit may contain one or more additional active principles or active ingredients for inducing an immune response against Porphyromonas pharyngosum in subjects.

[0261] The kit or "article" may include a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. The container may be formed from a variety of materials, such as glass or plastic. The container contains a therapeutic composition for the effective treatment of a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). The label or packaging insert indicates that the therapeutic composition is intended for the treatment of the selected condition. In one embodiment, the label or packaging insert includes instructions for use and indicates that the therapeutic or prophylactic composition may be used to treat the inflammatory disease or condition described herein.

[0262] The kit may comprise (a) a therapeutic or prophylactic composition; and (b) a second container containing a second active principle or active ingredient. The kit in this embodiment of the invention may further include a packaging insert indicating that the composition and other active ingredients can be used to treat a condition or prevent complications arising from the inflammatory disease or condition described herein. Alternatively or additionally, the kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0263] In any embodiment, the therapeutic composition may be provided in the form of a disposable or reusable device, including a container for containing a therapeutic, preventative, or immunogenic composition. In one embodiment, the device is a syringe, an autoinjector, or a nanopatch. The device may contain 0.1 to 2 mL of the therapeutic or immunogenic composition. The therapeutic or preventative composition may be provided in the device in a state ready for use or in a state requiring mixing, dissolution, resuspension, or the addition of additional components.

[0264] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or apparent in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0265] The following examples are described to more fully illustrate some embodiments of the invention. However, these examples should not in any way be construed as limiting the broad scope of the invention. Those skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0266] Example The following examples describe a series of in vitro and in vivo studies related to the chimeric or fusion proteins of this invention. Example 1 describes the in vitro materials and methods. Example 2 describes the results of the in vitro studies. Example 3 describes the materials and methods used in in vitro mouse studies, and Example 4 describes the results of these in vivo studies.

[0267] Example 1: In vitro materials and methods Cloning of Porphyromonas pharynx construct The encoding KD contains NcoI and XhoI restriction sequences at the 5' and 3' ends. F AK-2S-AVQP (DUF F variant; SEQ ID NO: 28) and KD A A DNA fragment of AK-2S-AVQP (DUF A variant, SEQ ID NO: 29) was synthesized by Bioneer Pacific (Australia) and ligated into the transfer vector pBHA to produce pBHA-gulA and pBHA-gulF. The cloning inserts were verified by DNA sequencing (Bioneer Pacific). Following the manufacturer's protocol, the pBHA-F and pBHA-A vectors were introduced into *E. coli* (…). E. coliRecombinant strains were selected in ABLE-K (Agilent Technologies) chemically competent cells on LB lamina containing Amp (100 μg / mL). Plasmid DNA was purified from positive clones, digested with NcoI and XhoI, and ligated into the NcoI / XhoI-digested pET28b plasmid vector (Novagen). The ligation product was introduced into *E. coli* ABLE-K chemically competent cells, and recombinant strains were selected on LB lamina containing kanamycin (30 μg / mL). Plasmid DNA was purified from positive clones, digested with NcoI / XhoI, and the plasmid digests were subjected to agarose gel electrophoresis to verify the presence of inserts of the correct size. The confirmed recombinant pET-gulA and pET-gulF variant constructs were then introduced into the *E. coli* expression host BL21(DE3) (Invitrogen) for recombinant protein expression. Selected positive clones were grown in LB-Kan (30 μg / mL) to approximately the intermediate logarithmic phase, and the glycerol stock solution (25% glycerol) was flash-frozen for storage at -70°C.

[0268] Small-scale expression testing of expression constructs To examine soluble expression levels, a small-scale expression trial was conducted as follows. Single bacterial colonies from freshly transformed LB-Kan plates were transferred to 5 mL of LB-Kan medium and grown overnight at 37°C with shaking at 200 rpm. These starting cultures were then used to inoculate fresh LB-Kan medium at a ratio of 1:100 (5 mL of culture) into 50 mL Falcon tubes. The cell cultures were allowed to grow at room temperature (approximately 24–26°C) until the cell density reached OD600 ≈ 0.6–0.8, and then induced with 0.5 mM IPTG at room temperature for 16 hours. 2 mL of the cell culture was centrifuged in an Eppendorf tube and resuspended in 0.5 mL of lysis buffer [20 mM sodium phosphate, 500 mM NaCl, 0.5% (v / v) Triton X-100, 20 mM imidazole, 1x protease inhibitor, pH 7.8], followed by brief sonication. Total cell lysates were collected for electrophoretic analysis, and the remainder was centrifuged at full speed using a benchtop Eppendorf centrifuge. Soluble fractions were collected, and insoluble fractions were resuspended in lysis buffer. SDS-PAGE was then performed on total cells, soluble cell fractions, and insoluble cell fractions equivalent to 4 μL of induced cell culture to assess expression and solubility.

[0269] Micro-purification of soluble recombinant proteins using a Ni-NTA spinning column The following small-scale purification of two recombinant *Porphyromonas pharyngioma* constructs was performed to evaluate His tag integrity / Nickel chromatography purification availability. Following the manufacturer's instructions, the soluble protein fraction (0.5 mL from the aforementioned 2 mL induced culture) was loaded onto a Ni-NTA rotating column, and the eluent representing crude nickel purification was collected, concentrations were measured, and approximately 3 to 5 μg of purified r-protein (along with flow-through and column wash fractions) was analyzed by SDS-PAGE.

[0270] Protein expression and cell lysis of His-tagged candidates As previously described, a His-tagged vaccine candidate for an animal model was expressed in *E. coli* BL21(DE3) as a recombinant C-terminal His-tagged fusion protein (SEQ ID NO: 30 and 31, His-tagged). Cells were grown at 37°C in LB medium or in Terrific broth supplemented with 50 μg / mL kanamycin. Protein expression was induced for 2–3 hours at 32°C with 0.2 mM IPTG at a culture OD600 of 1.0–1.2. Cells were harvested by centrifugation at 8000 g at 4°C and stored at -80°C for later use. After thawing, cells were lysed on ice by sonication at 35% power for 15 minutes and resuspended in lysis buffer [pH 7.5, 10 mM imidazole, 50 mM Tris, 300 mM NaCl, 1x EDTA without protease inhibitors (Sigma) (reducing lysis buffer for four Cys antigen-F: supplemented with 10 mM DTT)]. Cell lysates were clarified by centrifugation at 25,000 g for 40 minutes at 4°C.

[0271] Purification of His-tagged candidates from inclusion bodies After cell lysis, insoluble precipitates were washed twice with buffer PBS500 (20 mM NaPi, 500 mM NaCl, pH 7.4). Proteins expressed as inclusion bodies were dissolved in PBS500 with 8 M urea for 1 hour on a rolling platform at room temperature. The protein extract was centrifuged at 20,000 g. The supernatant was further filtered through a 0.22 μm filter unit and then mixed with nickel affinity resin (Thermofisher) and gently stirred in PBSU buffer (8 M urea, 20 mM NaPi, 500 mM NaCl, plus 20 mM imidazole, pH 7.8) for 2 hours. After thorough washing with PBSU (pH 7.8, then pH 6.5), the bound target proteins were eluted from the resin with 500 mM imidazole in the same buffer. The eluted proteins were progressively dialyzed into 6 M, 4 M, and then 2 M urea phosphate buffers (Fisher Biotec, Australia) in dialysis tubes with a molecular weight cutoff of 3.5 kDa. Prior to animal model experiments, the target proteins in the 2 M urea buffer were buffer-exchanged using a PD10 gel filter column to remove urea.

[0272] Purification of His-tagged candidates from soluble fractions Ni affinity chromatography This is the first chromatographic step in the purification of His-tagged proteins. The clarified cell lysate was filtered through a 0.22 μm filter unit and loaded onto a HisTrap or HisPrep Ni affinity column (GE Healthcare) in loading buffer TBS300 (50 mM Tris·Cl, 300 mM NaCl, pH 7.5) with 10 mM imidazole. To reduce purification, the cell lysate was diluted 4-fold with imidazole-containing TBS300 and then filtered before loading onto a column in reducing loading buffer containing 2 mM DTT. The column was thoroughly washed with appropriate loading buffer and then thoroughly washed with 20 mM imidazole in TBS300 or with 2 mM DTT for reducing conditions. Bound proteins were eluted using a 20–350 mM imidazole gradient in TBS300, and absorbance was monitored at 280 nm. Peak fractions were analyzed by SDS-PAGE. The eluted target protein was concentrated using an Amicon filter unit with a molecular weight cutoff of 10 kDa. The resulting protein solution was stored on ice for further purification.

[0273] Anion exchange chromatography The online ExPASy ProtParam tool (https: / / web.expasy.org / protparam / ) predicted the acid isoelectric point (pI) of the antigens: 5.07 for antigen-A and 4.87 for antigen-F. Therefore, anion exchange chromatography was applied after the Ni affinity chromatography step. Briefly, the concentrated protein purified from Ni affinity was diluted 10-fold to buffer A (50 mM Tris.Cl, 20 mM NaCl; 2 mm DTT for reduction buffer at pH 7.5) to reduce ionic strength, and then loaded onto an anion exchange HiTrap Q column (GE Healthcare) in buffer A. The protein was eluted with a 50–350 mM NaCl gradient, followed by elution with a 350–700 mM gradient. Absorbance was monitored at 280 nm during the process. The target protein in the peak fraction was validated by SDS-PAGE, and concentration was achieved using an Amicon filter unit. The protein solution was stored on ice for further purification and buffer exchange using size exclusion chromatography.

[0274] Size exclusion chromatography Size exclusion chromatography was performed on a HiLoad Superdex 200 column (GE Healthcare). The target protein solution purified by anion exchange was concentrated and loaded onto the size exclusion column, eluted in TBS150 buffer (50 mM Tris, 150 mM NaCl, pH 7.5), and the absorbance at 280 nm was monitored. The target protein in the peak fraction was validated by SDS-PAGE. Fractions with optimal protein purity were pooled and concentrated. After quantification by determining the absorbance at 280 nm using a Cary UV-vis spectrophotometer and calculating the theoretical extinction coefficient, the target protein was stored in aliquots at -80°C for future use.

[0275] ESI-LC-MS complete protein analysis Complete protein mass spectrometry analysis was performed using electrospray ionization time-of-flight mass spectrometry (ESI-TOF) combined with liquid chromatography. Protein samples were automated and passed through a C4 HPLC column (Phenomenex) with 0.1% formic acid aqueous solution as buffer A and acetonitrile + 0.1% formic acid as buffer B. The acetonitrile gradient for elution was set to 5-60%, followed by 60-95% buffer B, for a total of 15 minutes. MS data from 400 to 3200 m / z were collected on an Agilent 6520 QTOF mass spectrometer operated in positive mode, using in-situ internal standard mass references. Mass spectra were deconvoluted using Agilent Mass Hunter qualitative analysis software (B.05) with a maximum entropy algorithm to obtain the complete protein molar mass.

[0276] SEC-MALS SEC-MALS was run at room temperature on a Wyatt Technology system in buffer TBS150. Species were resolved on an HPLC size exclusion column (Shim-Pack Bio Diol-300) and transferred to a Wyatt 18-angle light scattering detector and a Wyatt refractive index monitor. The antigen concentration in the sample was 2 mg / mL, and the concentration of the control protein BSA used to establish the method was 5 mg / mL. Measurements were started following the step-by-step instructions on the control station screen, with the autosampler volume set to 10 μL per run. Data were processed using Wyatt Technology ASTRA software to obtain information on sample homogeneity in solution, protein aggregation, and the molar mass of the species.

[0277] Determine endotoxins Protein samples were diluted to 0.5 mg / mL using freshly prepared storage buffer TBS150. Endotoxin levels were determined using a commercial Pierce Chromogenic Endotoxin Quantification Kit (Thermo Scientific) following the manufacturer's instructions.

[0278] Assessment of nucleic acid contamination The A260 / A280 ratio was determined on a NanoDrop lite UV spectrophotometer (Thermo Scientific) to assess nucleic acid contamination.

[0279] Bacterial culture in a mouse model of periodontitis *Porphyromonas pharyngiosum* was obtained from the Oral Health Cooperative Research Centre, The Melbourne Dental School, University of Melbourne, Australia. *Porphyromonas pharyngiosum* was cultured in an anaerobic chamber (Whitley MG500 anaerobic workstation) at 37°C in brain-heart infusion (BHI) broth (BD Bacto Laboratories, USA) supplemented with cysteine ​​(1 g / L; Sigma-Aldrich, Australia), tryptone soybean broth (5% w / v, BD Bacto Laboratories, USA), heme (5 μg / mL, Calbiochem, Netherlands), and vitamin K (10 μg / mL, Sigma-Aldrich, USA) (5% CO2, 10% H2, and 85% N2).

[0280] The absorbance of batch cultures at OD 650 nm was monitored using a spectrophotometer (Model 295E, Perkin-Elmer, Germany). Bacterial cells were collected during the late exponential growth phase by centrifugation (7,000 g, 20 min, 4 °C). Bacterial purity was typically confirmed by Gram staining (Slots 1982).

[0281] Preparation of heat-inactivated bacteria Collect *Porphyromonas pharyngioma* culture (6,500 g, 4°C), wash once with phosphate-buffered saline (PBS) (pH 7.4: 0.01 M Na₂HPO₄, 1.5 mM KH₂PO₄, and 0.15 M NaCl), and then precipitate by centrifugation (7,000 g, 20 min, 4°C). Resuspend the bacterial cells in PBS and heat to 65°C for 15 min. Centrifuge the suspension (7,000 g, 20 min, 4°C) and resuspend in sterile PBS, repeating this process once. After the second wash, discard the supernatant and resuspend the cell pellet in sterile PBS to obtain 2 x 10⁻⁶ cells / mL. 10 Cell density was determined by cells / mL, and protein concentration was determined using Biorad protein assay dye reagent concentrate (Life Science, NSW, Australia).

[0282] Animal ethics All animal testing procedures were conducted in strict accordance with the recommendations in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

[0283] Example 2: Results of in vitro studies Antigen expression and solubility - small scale Small-scale expression assays of recombinant proteins containing DUF variants A or F induced at room temperature with 0.5 mM IPTG showed that both variants expressed relatively high levels of soluble recombinant protein. Figure 1 The results showed that the expected-sized recombinant protein in the soluble fraction was very prominent relative to the background of soluble E. coli proteins, indicating a high level of soluble expression. At room temperature, the chimeric protein containing DUF variant A exhibited very high levels of overall solubility, while recombinant protein was almost absent or absent in the insoluble fraction representing inclusion bodies. In contrast, the chimeric protein containing DUF variant F showed high levels of total recombinant protein expression in the insoluble fraction. However, despite the presence of relatively high levels of KD as insoluble inclusion bodies... F The AK-2S-AVQP recombinant protein, however, still exhibits relatively high levels of soluble protein expressed in the soluble fraction due to the extremely high overall level of total recombinant protein expressed by this strain under the tested conditions.

[0284] Antigen expression and solubility - large-scale KD F AK-2S-AVQP and KD A Both AK-2S-AVQP proteins were expressed at similar levels in LB or TB media. Figure 4 Under non-reducing conditions, antigen-A exhibits high solubility, as confirmed by SDS-PAGE analysis of its lysis fractions. Figure 2 In comparison, KD F The solubility of AK-2S-AVQP protein is much lower; after cleavage under the same non-reducing conditions, most of it exists as a precipitate. Figure 2 Furthermore, neither reducing buffers containing low (5 mM) nor high (100 mM) DTT could extract antigen-F from the precipitate fraction. Figure 2 However, when cells lyse under reducing conditions, KD... F AK-2S-AVQP is highly soluble, with most of the protein present in the soluble fraction. Figure 2 KD F The low solubility of AK-2S-AVQP is likely due to the presence of disulfide bonds formed by mispaired Cys residues, resulting in a misfolded protein structure. Once this aberrant structure forms, it is irreversible, and the disulfide bonds cannot be approached by reducing agents without the aid of unfolding forces such as SDS and heating. Therefore, although low-temperature expression may initially improve the soluble expression level of this protein, as seen in small-scale experiments, the purification of KD from the soluble fraction under non-reducing conditions remains challenging. F AK-2S-AVQP is not an ideal method. Finally, samples of this protein for animal model experiments were prepared by lysis and purification under reducing conditions until the final step to remove the included reducing agent DTT.

[0285] Purification of His-tagged control antigen from inclusion bodies This procedure was applied only to the purification of a prior art *Porphyromonas gingivalis* chimera (referred to as KDcAK1n), which is expressed in inclusion bodies as previously described (O'Brien-Simpson et al. 2016, *NPJ Vaccines* 1:16022). KDcAK1n was stable in 2 M urea after stepwise dialyzing to 2 M urea-phosphate buffer and was therefore stored at -80°C for further use after quantification. No precipitation was observed after buffer exchange via gel filtration prior to animal model experiments.

[0286] Purification of Porphyromonas pharynx antigen For the animal model experiments described in Examples 3 and 4, KD A AK-2S-AVQP was purified under non-reducing conditions, and KD F AK-2S-AVQP was purified under reducing conditions until the final buffer exchange and size exclusion purification steps. Figures 3 to 5 We also attempted to modify KD under non-reducing conditions. F AK-2S-AVQP was purified. KD was expressed in LB medium. A AK-2S-AVQP was used for purification under non-reducing conditions. KD expressed in TB medium... F AK-2S-AVQP was used for purification under reducing conditions, and proteins expressed in LB were used for non-reducing purification. KD A AK-2S-AVQP yielded the highest amount (78 mg / L culture, 18.6 mg / g wet cells) (Table 1). KD FWhen AK-2S-AVQP was purified from the lysate supernatant under reducing conditions, the yield of the final product was high (34 mg / L culture, 7.6 mg / g wet cells), but the yield was much lower when purified under non-reducing conditions (3.5 mg / L culture, 1.1 mg / g wet cells) (Table 1). The identities of the two proteins lacking the first Met residue were confirmed by intact protein MS spectroscopy (Table 3).

[0287] Table 3: Final products of *Porphyromonas pharyngioma* antigen purified from 1 L culture in LB or TB broth.

[0288]

[0289] KD respectively F AK-2S-AVQP and KD A AK-2S-AVQP was concentrated to 20 and 18 mg / mL in TBS150 buffer; however, these concentrations are not the maximum achievable concentrations (Table 1). Most of the expressed KD... F AK-2S-AVQP precipitates into an insoluble fraction via non-reducing cleavage. After purification to high quality under reducing conditions, the solution remains stable without redeposition even after removal of the reducing agent. This solution stability also resists freeze-thaw cycles in air. Therefore, sufficient material for animal models can be prepared in a single purification cycle.

[0290] Remove DnaK The host molecular chaperone protein DnaK, approximately 70 kDa, appeared to contaminate the purification of *Porphyromonas pharyngoides* antigen. Due to the interaction of DnaK with the target protein, it was eluted into the fraction with the greatest overlap with the target protein fraction during the Ni affinity purification step. This contaminant (along with other impurities) was well separated from the target protein by anion exchange chromatography. Figure 4 The interaction between DnaK and target proteins appears to be independent of reducing conditions. However, under reducing conditions, KD... F AK-2S-AVQP eluted to two peaks, possibly due to the presence of two forms ( Figure 4 Essentially, almost all fractions in the second peak were contaminated with DnaK. This is likely due to the stronger interaction between a small subset of misfolded antigen-F and reduced DnaK. Therefore, the second peak was excluded, and the optimal fraction from the earlier main peak was used for further purification using size exclusion chromatography. Figure 5 ).

[0291] Degradation and site prediction On anion-exchange fractionated SDS gels, smaller bands were visible beneath each major full-length target protein, including KD. F The presence of double bands in some fractions of AK-2S-AVQP indicates degradation. Figure 6 Size exclusion chromatography effectively removed most of the degradation products. Figure 5 ), and thus obtained high-quality final products for each protein, despite the presence of some mildly degraded species ( Figure 6 SEC-MALS analysis also confirmed the high purity and homogeneity of the final product, indicating that the two proteins exist mainly as monomers in solution at 2 mg / mL and have high stability (Table 4).

[0292] Table 4: Peak area percentage in SEC-MALS chromatograms of Porphyromonas pharyngosum antigen

[0293] Intramolecular disulfide bonds in antigen-F F-version antigen (KD) in response to reduction conditions F The SDS-PAGE spectrum of AK-2S-AVQP showed a slight band shift, indicating the presence of intramolecular disulfide bonds ( ). Figure 9 A). Even for purified KD involving a reduction purification process. F AK-2S-AVQP (FR) exhibits a band shift similar to that of proteins purified under non-reducing conditions (F-NR). During size exclusion purification, after the reducing agent is removed, FR also forms intramolecular disulfide bonds. These intramolecular disulfide bridges may play a role in maintaining protein structure and making the protein more compact, thus allowing for faster migration in SDS gels under non-reducing denaturing conditions. Figure 6 A). KD A The AK-2S-AVQP did not exhibit this type of strip displacement. Figure 6 A). KD F The slight reduction-dependent band shift of AK-2S-AVQP was indistinguishable in the native PAGE of unheated samples. Figure 6 B). Contrary to the results from SDS-PAGE, for heated samples on non-denaturing gels without SDS, the reduced KD... F The negative charge on the thiol ions of AK-2S-AVQP appears to affect protein migration, causing proteins to move faster than in unreduced samples. Figure 6 B).

[0294] Regardless of whether the sample was heated, the band displacement of F-NR on the SDS gel appeared to be slightly larger than that of FR. Figure 6A). It is speculated that intramolecular disulfide bonds form in F-NR during the cleavage phase, while disulfide bonds form in FR only after the reducing agent is removed during the final purification phase. This difference may have subtle effects on protein structure. However, FR purified from the reducing agent was found to have a similar overall structure to F-NR, only with slightly different tightness.

[0295] oligopoly In the SDS gel swimming lanes of heated non-reduced FR and F-NR samples, a weak band of approximately 90 kDa appeared, indicating the presence of a small amount of dimer due to the formation of intermolecular disulfides. Figure 6 A). This could be an artifact produced during the heat denaturation process, intended to temporarily expose Cys residues to form intermolecular disulfides, as this was not observed in the unheated sample. Figure 6 A).

[0296] Since the reduction conditions affect KD in non-denaturing gels regardless of whether the sample is heated. F The PAGE spectra of AK-2S-AVQP were unaffected. Figure 6 (B) Therefore, the possibility that the contained protein gradually forms polymers bridged by intermolecular disulfide bonds is ruled out.

[0297] SEC-MALS data showed some slight or minimal signs of polymerization in both antigens. Species with an estimated molar mass of 71.2 kDa for antigen-A and 84.7 kDa for FR (purified under reducing conditions) can be classified as dimer forms of the two proteins. As for F-NR (purified under non-reducing conditions), the peak of 394.2 kDa is likely due to the presence of a small amount of decameric form of this protein, although this species was not detected by SDS-PAGE. F The Cys residues in AK-2S-AVQP may make this protein more prone to forming higher levels of polymerization under non-reducing conditions during the cleavage phase. Therefore, trace amounts of decadals may be linked via intermolecular disulfides. Importantly, as estimated by integration of SEC-MALS liquid chromatography, these polymeric species comprise less than 1% of the total protein mass (Table 4).

[0298] Assessment of endotoxin and nucleic acid contamination Endotoxins were found to be at similarly low levels in both antigens. At a protein concentration of 0.5 mg / mL, the endotoxin contamination levels for the two proteins were 2.24 and 2.36 EU / mL, respectively (Table 5). Furthermore, the A260 / A280 values ​​for both antigens were below 0.6, indicating minimal nucleic acid contamination (Table 5).

[0299] Table 5: Assessment of endotoxin and nucleic acid contamination in Porphyromonas pharyngoides antigen.

[0300]

[0301] Example 3: Materials and Methods in In Vivo Studies in Mice Mouse periodontitis model On day 0, mice (female BALB / c; 6–8 weeks old, 10 mice / group) were orally inoculated with *Porphyromonas pharyngiosum*, which consisted of four doses of *Porphyromonas pharyngiosum* suspended in 20 μL of PG buffer (pH 7.4, 50 mM Tris-HCl, 150 mM NaCl, 10 mM MgSO4) containing 2% w / v carboxymethyl cellulose (CMC, Sigma, New South Wales, Australia). 4 Each dose of 1 x 10 (and 14.3 mM mercaptoethanol) 10 The inoculum consisted of [number of live *Porphyromonas pharyngoides* cells], administered at two-day intervals. The inoculum was prepared anaerobically and then immediately applied to the gingival margin of the maxillary molars. The number of live bacteria in each inoculum was verified by flow cytometry and CFU counting on blood agar. The animal groups consisted of: an orally administered *Porphyromonas pharyngoides* (infected control) group, a non-bacterial inoculation control group, and an immunization group. For the therapeutic vaccination periodontitis model, 21 mice were immunized on day 19 following an initial oral administration of 100 μg of the vaccine candidate in saline / alum (Alhydrogel; 2% aluminum hydroxide wet gel suspension; Invivogen, Inc.) via an intraperitoneal route. Mice received a second immunization (100 μg in saline / alum) subcutaneously on day 40. On day 62, mice were euthanized by exsanguination via cardiac puncture. The maxilla was removed and bisected along the midline, with 10 halves used to determine alveolar bone loss. Serum was used to determine the antibody curve using ELISA.

[0302] Measurement results of alveolar bone loss in the maxilla of mice The maxillae with bone loss to be examined were boiled in deionized water (1 minute), mechanically demineralized, and immersed in 2% w / v potassium hydroxide (16 hours, 25°C). The maxillae were washed twice with deionized water (25°C), dried (1 hour, 37°C), and stained with 0.5% w / v methylene blue aqueous solution. Coded digital images of the buccal side of the maxilla were taken using an Olympus DP12 digital camera mounted on a dissecting microscope to assess horizontal bone loss using ImageJ imaging analysis software (https: / / imagej.nih.gov / ij / index.html). The maxillae were oriented so that the buccal and lingual molar cusps overlapped. Images were taken in micrometers per frame to allow for normalization of measurements for each image. Horizontal bone loss was defined as loss occurring in a horizontal plane perpendicular to the alveolar ridge, resulting in a decrease in ridge height. The visible area from the cementum-enamel junction (CEJ) to the alveolar ridge (ABC) of each molar was measured using ImageJ version 1.3k imaging software, yielding results in mm. 2 Total visible CEJ-ABC area in mm². *Porphyromonas pharyngoides*-induced alveolar bone loss in mm² was calculated by subtracting the total visible CEJ-ABC area of ​​the uninoculated (NC) group from the total visible CEJ-ABC area of ​​each experimental group. Alveolar bone loss measurements were determined twice in a randomized and blinded protocol. Data are presented as mean + / - standard deviation (in mm²). 2 The results were expressed in units of 1, and the analysis was performed using one-way ANOVA and Dunnett's T3 post-hoc test.

[0303] Enzyme-linked immunosorbent assay (ELISA) ELISA was performed to evaluate subclass antibodies in serum using a solution (1 μg / mL) of heat-inactivated (HK) Porphyromonas pharyngosum cells, recombinant domain subunits, or biotinylated peptides expressed in 0.1 M PBS (pH 7.4) to coat the wells of a flat-bottomed polyethylene microtiter (Dynatech Laboratories, McLean, VA, US) (16 h, 4 °C).

[0304] In these experiments, the following antibody dilutions were used: a 1 / 4000 dilution of goat anti-mouse antibody; IgG (M8642), IgG1 (M8770), and IgG2a (M4434) antibodies (Sigma, NSW, Australia). A 1 / 4000 dilution of horseradish peroxidase-conjugated porcine anti-goat IgG antibody (M5420; Sigma, NSW, Australia) was used to develop ELISA assays. For epitope ELISA, biotinylated peptides were conjugated to pre-blocked streptavidin-coated flat plates (Pierce; Thermo Fisher Scientific) at 10 μg / mL. After incubation with serum, ELISAs were developed using 1 / 4000 goat anti-mouse IgG and 1 / 4000 horseradish peroxidase-conjugated porcine anti-goat IgG antibodies. All densitometric measurements were performed at 405 nm on a Wallac VICTOR3 1420 multi-tag counter (PerkinElmer).

[0305] Example 4: Results of in vivo studies in mice Alveolar bone loss in the maxilla of mice induced by Porphyromonas pharynx.

[0306] The animal model used was a therapeutic vaccine mouse periodontitis model developed by O'Brien-Simpson et al. (2016, above). Figure 7 Compared with the original control, the infected control animals showed a significant level (P<0.001) of alveolar bone loss. Figure 8 The initial *Porphyromonas gingivalis* chimera (KDcAK1n) was used as a positive vaccine control in this study. Compared to infected control animals, the novel vaccine KDcAK1n based on the virulence domain sequence of *Porphyromonas pharyngiosum* showed improved efficacy of the present invention. F AK-2S-AVQP and KD A The AK-2S-AVQP provides a significant level of protection (P<0.01). Figure 8 ).

[0307] Antibody response Serum antibody subclass responses in immunized mice in a periodontitis model were detected by ELISA. Antiserum was used to detect heat-inactivated *Porphyromonas pharyngoides* as an adsorbed antigen. Figure 9 Antibody response was expressed as the obtained ELISA titer minus twice the background level, where each titer represents the mean ± standard deviation of 10 individual mice. All tested antigens elicited an IgG response to *Porphyromonas pharyngoides*, with the strongest response observed in the *Porphyromonas gingivalis* KDcAK1n vaccine. Compared to the negative control, both *Porphyromonas pharyngoides* vaccines KD... F AK-2S-AVQP and KD AAK-2S-AVQP produced significant levels of antibodies against the entire cell line. Figure 9 ).

[0308] The antibody titer against the purified RgpA / Kgp protease complex of Porphyromonas pharyngota was also measured. Figure 10 ).

[0309] KD F AK-2S-AVQP and KD A Both AK-2S-AVQP proteins produce significant antibodies against the RgpA / Kgp protease complex, with KD being the most effective. F AK-2S-AVQP produced higher titers against the RgpA / Kgp protease complex. Ultimately, Figure 11 The results showed that both Porphyromonas pharyngoides vaccines produced high-titer antibody responses against themselves.

[0310] In summary, the inventors generated a *Porphyromonas pharyngioma* vaccine using a *Porphyromonas pharyngioma*-specific sequence derived from a complex multi-protein gingival protease. Both tested proteins provided protection as a vaccine in mouse animal models.

[0311] Example 5: Materials and Methods for In Vivo Studies in Dogs A series of experiments were planned to determine the ability of the chimeric fusion protein to elicit an immune response in dogs.

[0312] The 9 dogs were divided into 3 groups as follows: -Group 1- Low dose: (Jake 3962, Phoebe 0346 and Rachael 8562) with low dose (100 μg) of Porphyromonas pharyngosum KD A Immunization was performed using the recombinant AK-2S-avqp-6His protein.

[0313] -Group 2-Medium dose: (Forest 5635, Milan 3711 and Moo 5498) with a medium dose (200 μg) of Porphyromonas pharyngosum KD A Immunization was performed using the recombinant AK-2S-avqp-6His protein.

[0314] -Group 3- High dose: (Bruno 8086, Kale 5636 and Lachie 5634) with high dose (400 μg) of Porphyromonas pharyngosum KD A Immunization was performed using the recombinant AK-2S-avqp-6His protein.

[0315] according to Figure 12The protocol involves two immunizations for the dogs, the first on day 0 (“primary”) and the second on day 21 (“boost”). Serum samples were collected from each dog at the following times: - Day 0 (= pre-immune serum, also known as pre-treatment serum, i.e., immediately before receiving the initial immunization dose).

[0316] - Day 21 (=primer serum, also known as pre-second treatment, i.e., immediately following the primer dose and before the booster).

[0317] - Day 35 (= booster serum, i.e., 14 days after receiving the booster).

[0318] Optimize ELISA Purified Porphyromonas pharynx KD A AK-2S-avqp-6His recombinant protein The ELISA plate wells were filled with 100 μL / well of *Porphyromonas pharyngoides* KD at concentrations of 10, 2.5, 1, 0.5, and 0.1 μg / mL. A AK-2S-avqp-6His recombinant protein was coated (diluted in 1×PBS). A PBS-only control was also included. After overnight incubation at 4°C, the recombinant protein solution was discarded, the wells were washed twice with distilled water, and the free protein binding sites were blocked by adding 200 μL / well of blocking solution. After incubation at 4°C for 6 hours, the blocking solution was discarded, the wells were rinsed twice with washing solution, and serum dilution was applied as described below.

[0319] Biotinylated peptides Spread 100 μL / well of 10 μg / mL streptavidin solution (diluted in 1×PBS) onto the ELISA plate wells. After incubation overnight at 4°C, discard the streptavidin solution, wash the wells twice with distilled water, and block the free protein binding sites by adding 200 μL / well of blocking solution (10% (w / v) skim milk powder in 1×PBS). After incubation at 4°C for 6 hours, the blocking solution was discarded, and the wells were washed twice with washing solution. Aliquots of 100 μL of biotinylated synthetic peptides prepared at concentrations of 10, 2.5, 1, 0.5, and 0.1 μg / mL in dilution buffer (Porphyromonas pharyngophyllum_KAS2 (SEQ ID NO: 1) and Porphyromonas pharyngophyllum_KAS2_random:biotin-KYKGWTNNSSTVLLQTNATLGVETFTHSPDSASDAK, SEQ ID NO: 113) were added to the wells coated with the blocked streptavidin. A dilution buffer-only control was also included. After overnight incubation at 4°C, the peptide solution was discarded, and the wells were washed six times with washing solution, followed by the application of serum dilution as described below.

[0320] intact heat-inactivated Porphyromonas pharyngoides cells The ELISA plate wells were coated with 100 μL / well of intact, heat-inactivated *Porphyromonas pharyngioma* cells at concentrations of 10, 2.5, 1, 0.5, and 0.1 μg / mL (diluted in 1×PBS). A PBS-only control was also included. After overnight incubation at 4°C, the *Porphyromonas pharyngioma* cells were discarded, the wells were washed twice with distilled water, and free protein binding sites were blocked by adding 200 μL / well of blocking solution. After incubation at 4°C for 6 hours, the blocking solution was discarded, the wells were rinsed twice with washing solution, and serum dilution was applied as described below.

[0321] Optimize ELISA: Serum application Aliquots of each Day 0 serum sample were pooled and diluted 1 / 10 in dilution buffer. Similarly, Day 35 serum samples were pooled and diluted 1 / 10 in dilution buffer. Aliquots (100 μL) of the 1 / 10 diluted serum samples were added to a blocking ELISA plate, and a 4-fold dilution series of 1 / 10 serum samples was constructed by sequentially transferring 25 μL to 75 μL of dilution buffer. Control wells that only accepted dilution buffer (i.e., serum-free) were also included. The plates were incubated overnight at 4°C, and the detection antibody was applied as described below.

[0322] Antibody detection After overnight incubation, discard the serum samples and wash the wells six times with wash solution. Add 100 μL of horseradish peroxidase (HRP)-conjugated goat anti-dog IgG (Fc-specific) antiserum (10 mg / mL) diluted 1 / 5,000 in dilution buffer to each well. After incubation at room temperature (22°C) for 2 hours, discard the labeled antibody, wash the wells six times with wash solution, and add the HRP substrate as described below.

[0323] HRP substrate TMB substrate buffer solution (90 μL) was added to the washed ELISA plate. To optimize the ELISA, color development was monitored spectrophotometrically at 370 nm in kinetic mode using a SpectraMax iD5 spectrophotometer. For titration of a single serum sample, color development was allowed to proceed for 20 minutes, and the reaction was terminated by adding 50 μL of 1 M H2SO4 to each well. The plate was read in terminated ELISA mode (450 nm) on the SpectraMax iD5 spectrophotometer. Analyzed data are described below.

[0324] ELISA for titration of a single dog serum Purified Porphyromonas pharynx KDA AK-2S-avqp-6His recombinant protein The ELISA plate wells were filled with 100 μL / well of 0.1 μg / mL Porphyromonas pharyngoides KD. A AK-2S-avqp-6His recombinant protein was coated (diluted in 1×PBS). After overnight incubation at 4°C, the recombinant protein solution was discarded, and the wells were washed twice with distilled water. Free protein binding sites were blocked by adding 200 μL / well of blocking solution. After incubation at 4°C for 6 hours, the blocking solution was discarded, and the wells were rinsed twice with washing solution. Aliquots (100 μL) of pre-immunization, primiparous, and booster sera from each dog were diluted 1 / 10, 1 / 10, and 1 / 500, respectively, in dilution buffer and added to the ELISA plate. A 1 / 5 dilution series was constructed by sequentially transferring 20 μL to 80 μL of dilution buffer. Control wells (i.e., serum-free) were also included. After overnight incubation at 4°C, detection antibodies were applied as described above.

[0325] Biotinylated Porphyromonas pharyngoides KAS2 peptide ELISA plate wells were coated with 100 μL / well of 10 μg / mL streptavidin solution (diluted in 1×PBS). After overnight incubation at 4°C, the streptavidin solution was discarded, and the wells were washed twice with distilled water. Free protein binding sites were then blocked by adding 200 μL / well of blocking solution. After incubation at 4°C for 6 hours, the blocking solution was discarded, and the wells were washed twice with wash solution. A 100 μL aliquot of 0.1 μg / mL biotinylated Porphyromonas pharyngoides_KAS2 peptide (diluted in dilution buffer) was added to each well of the streptavidin-coated plate. After overnight incubation at 4°C, the peptide solution was discarded, and the wells were washed six times with wash solution. Aliquots (100 μL) of pre-immunization, primiparous, and booster sera from each dog were diluted 1 / 10 in dilution buffer and added to the ELISA plate. A 1 / 5 dilution series was constructed by sequentially transferring 20 μL to 80 μL of dilution buffer. It also includes control wells with only dilution buffer (i.e., serum-free). After incubation overnight at 4°C, the detection antibody is applied as described above.

[0326] intact heat-inactivated Porphyromonas pharyngoides cells Spread 100 μL / well of 1 μg / mL intact heat-inactivated *Porphyromonas pharyngioma* cells (diluted in 1×PBS) onto the wells of the ELISA plate. After overnight incubation at 4°C, discard the *Porphyromonas pharyngioma* cells, wash the wells twice with distilled water, and block the free protein binding sites by adding 200 μL / well of blocking solution. After incubation at 4°C for 6 hours, discard the blocking solution and rinse the wells twice with washing solution. Aliquots (100 μL) of pre-immunization, primiparous, and booster sera from each dog were diluted 1 / 10 in dilution buffer and added to the ELISA plate, and a 1 / 5 dilution series was constructed by sequentially transferring 20 μL to 80 μL of dilution buffer. Control wells (i.e., serum-free) were also included. After overnight incubation at 4°C, the detection antibody was applied as described above.

[0327] Data Analysis: Determination of Antibody Titer ELISA assays based on the peroxidase-TMB system can be monitored in two ways: continuously recording absorbance at 370 nm; or terminating the reaction with acid after a set time and measuring absorbance at 450 nm. Continuous assays are often more accurate and have a wider dynamic range than terminated assays, but become impractical for multi-plate systems. Both assay types were used in this experiment.

[0328] All analyses were performed using scripts written for R-4.2.1 (R Foundation). The initial slope of consecutively read ELISA assays was calculated using quadratic regression of the absorbance response to interpret the curvature. The linear parameter of the quadratic equation corresponds to the slope at time = 0 (i.e., the initial slope). Dose-response curves for consecutive and acid-termination assays were calculated using R's drc function library. All samples were analyzed using a 4-parameter logarithmic logic equation (LL.4) (Equation 1 below). To address samples with low responses that did not fully cover the range, maximum and minimum parameters were determined at the plate level, the slope parameter was set to 1.0, and the inflection point parameter was unique for each serum sample.

[0329] Two antibody titer values ​​were calculated and reported: the midpoint titer is equivalent to the inflection point parameter in the LL.4 model. The endpoint titer was calculated as the predicted point on the LL.4 curve corresponding to twice the minimum response.

[0330] Equation 1:

[0331] Where: x = serum concentration; f(x) = absorbance response; a = maximum response; b = minimum response; c = slope (Hill coefficient); d = inflection point (midpoint). Example 6: Preliminary results of canine serological studies Figures 13 to 15 show preliminary results from the experimental protocol outlined in Example 5. In short, following immunization, serum was collected at specified time points, and ELISA was used to determine the resistance to the immunogen (KD). A Immune responses to AK-2S-avqp-6His, Porphyromonas pharyngoides KAS2 peptide, or whole cells of Porphyromonas pharyngoides.

[0332] As shown in Figure 13, all dogs showed a strong immune response to the antigen after primary and booster immunizations.

[0333] Figure 14 The results shown indicate that the response to whole cells of Porphyromonas pharyngoides is closely related to the response to immune antigens.

[0334] Figure 15 The results shown indicate that the response to the Porphyromonas pharyngoides KAS2 peptide is closely related to the response to immune antigens.

[0335] In summary, the results indicate that the animals tolerated the vaccine well and produced a strong immune response in the canine study.

[0336] Example 7: Proof-of-Concept Study in Dog Research A further canine study similar to those discussed in Examples 5 and 6 was conducted. Briefly, 20 dogs were organized into either a “treatment” or “control” group (10 dogs in each group). Dogs in the treatment group were given the chimeric protein antigen KD as described herein. A AK-2S-avqp was administered three times, starting on day 0 (primary immunization), followed by a booster immunization on day 21 (booster 1), and a final booster immunization on day 42 (booster 2). The control group received no treatment.

[0337] Serum was collected at the following time points: ●Day 0 = Before the immune response (immediately before receiving the initial immunization dose) ●Day 21 = Initial Immunization Response (immediately following the initial immunization dose and before the booster dose) ●Day 42 = Boost-1 response (immediately following 21 days after receiving Boost-1 and before receiving Boost-2) ● Day 56 = Enhanced-2 response (i.e., 14 days after receiving the enhanced-2 dose).

[0338] Serum IgG response to the Porphyromonas pharyngoides KAS2 peptide (SEQ ID NO: 1) was measured using an ELISA method similar to that described in Example 5.

[0339] Overview Figure 16 and 17The results showed that a strong immune response was generated after the immunization regimen.

[0340] In summary, when the mean midpoint titer or mean endpoint titer was used as a measure of the immune response to the KAS2 peptide, serum collected from dogs in the “control” group on days 21, 42, or 56 showed an increase of less than 1.3-fold relative to the mean pre-immunization (day 0) titer. This finding is consistent with this group, which included untreated control animals.

[0341] Conversely, at most serum dilutions, all dogs in the treatment groups developed titratable, enhanced (days 42 and 56) peptide-specific IgG responses greater than their pre-immunization (day 0) response. Side-by-side comparisons of midpoint and endpoint titers across all treatment groups showed a gradual increase in titers following continuous dose administration, appearing to peak in serum samples at day 42.

[0342] In a meta-analysis of peptide-specific serum IgG responses from all ten dogs in the treatment group, serum samples collected on days 21, 42, and 56 showed statistically significant (p<0.05) higher mean midpoint and mean endpoint titers compared to the mean values ​​of pre-immunization (day 0) serum samples. Furthermore, titers measured on day 42 and 56 were statistically significantly higher than those measured on day 21 (p<0.05). When using the mean midpoint titer as a measure of response, the titers of serum samples on days 21, 42, and 56 were 5, 94, and 96 times higher than the pre-immunization titers, respectively.

[0343] Example 8: Reducing bacterial load Simultaneously with the experiment described in Example 7, saliva and subgingival plaque samples were collected from canine subjects at the beginning and end of the study. The oral pharyngeal porphyria load of the samples was assessed using the qRT-PCR method described in the following literature: Maruyama et al., (2018) Polish Journal of Veterinary Sciences, 21: 127-132.

[0344] The results will show that, compared with the control, inoculation with protein antigen KD A The oral pharyngeal porphyria load was reduced in the saliva of subjects treated with AK-2S-avqp.

[0345] The results will show that, compared with the control, inoculation with protein antigen KD A The subjects who received AK-2S-avqp had a reduced load of oral pharyngeal porphyrinoma in subgingival plaque.

[0346] In addition, plasma samples were obtained from the subjects, and the levels of inflammatory cytokines present in them were assessed. The results showed that, compared to the control, inoculation with the protein antigen KD... A The presence of inflammatory cytokines in animals receiving AK-2S-avqp was reduced. The cytokines measured were one or more of the following: GM-CSF, IFN-γ, IL-2, IL-6, IL-7, IL-8, IL-10, IL-15, IL-18, IP-10, KC-like MCP-1, and TNF-α. Milliplex canine cytokine / chemokine magnetic bead plates were used to measure all of these cytokines.

[0347] Example 9: Evaluation of Alternative Protein Antigens A set of experiments similar to those described in Examples 7 and 8 were performed using one of the following alternative protein antigens: -KD F AK-2S-AVQP (e.g., SEQ ID NO: 28) -RD A AK-2S-AVQP (e.g., SEQ ID NO: 114) -R(S)D A AK-2S-AVQP (cysteine ​​in the R domain is replaced with serine) (e.g., SEQ ID NO:115) -KD F (S)AK-2S-AVQP (all four cysteine ​​residues in the D domain are replaced with serine residues) (e.g., SEQ ID NO: 76) -KD A AK-1S-AVQP (e.g., SEQ ID NO: 40) -KD A AK-2S (e.g., SEQ ID NO: 116) -KD A A-2S-AVQP (e.g., SEQ ID NO: 117) -KD A AK (e.g., SEQ ID NO: 31) -KD A A (e.g., SEQ ID NO: 32) Chimeras without amino acid substitutions in the D or A domains are expected to be less soluble than those with such substitutions. Nevertheless, the results show that all chimeras tested were able to induce a strong immune response, similar to the results shown in Example 7.

[0348] Chimeras containing only a single "K" domain are also expected to induce a slightly less pronounced immune response compared to chimeras with two copies of either the K or R domain. Nevertheless, the results show that all tested chimeras were able to induce a strong immune response, similar to the results shown in Example 7.

[0349] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or apparent in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

Claims

1. A method for inducing the activity of Porphyromonas pharyngoides (Porphyromonas pharyngoides) P. gulae A chimeric or fusion protein of an immune response, wherein the protein comprises a first polypeptide and a second polypeptide, wherein: A) The first polypeptide comprises or consists of the following: an amino acid sequence of the active site of an Arg gingival protease or Lys gingival protease homolog of Porphyromonas pharyngoides; and B) The second polypeptide comprises or is composed of the following: The amino acid sequence of the DUF2436 domain of the Arg gingival protease or Lys gingival protease surface complex of *Porphyromonas pharyngoides*; and The amino acid sequence of the adhesin domain of the surface complex of the Arg gingival protease and Lys gingival protease homologs of *Porphyromonas pharyngioma*, preferably wherein the adhesin domain comprises at least the amino acid sequence of SEQ ID NO: 86 and / or SEQ ID NO: 85 or at least 80% identical thereto.

2. The chimeric or fusion protein according to claim 1, wherein the amino acid sequence of the DUF2436 domain of the gingival protease homolog surface complex comprises or consists of the following: Amino acid sequences as shown in SEQ ID NO: 3 or 4, or sequences that are at least 80% identical to them.

3. The chimeric or fusion protein according to claim 1, wherein the amino acid sequence of the DUF2436 domain of the gingival protease homolog surface complex comprises or consists of the following: The amino acid sequence shown in SEQ ID NO: 4 or at least 80% identical to it.

4. The chimeric or fusion protein according to any one of claims 1 to 3, wherein the amino acid sequence of the adhesin domain of the gingival protease homolog surface complex comprises or is composed of the following: The amino acid sequence of SEQ ID NO: 88 or 20 or a sequence that is at least 80% identical thereto.

5. The chimeric or fusion protein according to any one of claims 1 to 4, wherein the chimeric or fusion protein comprises one or more additional polypeptides, said one or more additional polypeptides comprising or consisting of the following: The amino acid sequence of the active site of Arg gingival protease or Lys gingival protease homolog of Porphyromonas pharyngioma, or a sequence that is at least 80% identical to it.

6. The chimeric or fusion protein of claim 5, wherein the active site of an Arg gingival protease or Lys gingival protease homolog of Porphyromonas pharyngoides or one or more other polypeptides thereof is located at the N-terminus of the first polypeptide.

7. The chimeric or fusion protein of claim 5, wherein the active site of an Arg gingival protease or Lys gingival protease homolog of Porphyromonas pharyngoides or one or more other polypeptides thereof is located at the C-terminus of the first polypeptide.

8. The chimeric or fusion protein of claim 5, wherein the active site of an Arg gingival protease or Lys gingival protease homolog of Porphyromonas pharyngoides or one or more other polypeptides thereof is located at the N-terminus of the second polypeptide.

9. The chimeric or fusion protein of claim 5, wherein the active site of an Arg gingival protease or Lys gingival protease homolog of Porphyromonas pharyngoides or one or more other polypeptides thereof is located at the C-terminus of the second polypeptide.

10. The chimeric or fusion protein according to any one of claims 1 to 9, wherein the protein comprises at least two additional polypeptides that may be present, the at least two additional polypeptides comprising or consisting of the following: The amino acid sequence of the active site of Arg gingival protease or Lys gingival protease homolog of Porphyromonas pharyngioma, or a sequence that is at least 80% identical to it.

11. The chimeric or fusion protein of claim 10, wherein the active site of an Arg gingival protease or Lys gingival protease homolog of Porphyromonas pharyngoides or two or more other polypeptides thereof are located at the N-terminus of the second polypeptide, the C-terminus of the second polypeptide, or both the N-terminus and C-terminus of the second polypeptide.

12. The chimeric or fusion protein according to any one of claims 5 to 11, wherein the one or more additional polypeptides are linked to the first polypeptide or the second polypeptide of the chimeric or fusion protein via a linker of no more than 50 amino acids, or are directly linked to the first polypeptide or the second polypeptide.

13. The chimeric or fusion protein according to any one of the preceding claims, wherein the first polypeptide comprises or is composed of an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 or 2, is the same as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sequence.

14. The chimeric or fusion protein according to any one of claims 5 to 13, wherein the one or more additional polypeptides comprises or consists of an amino acid sequence selected from the group consisting of: SEQ ID NO: 1 or 2, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it.

15. The chimeric or fusion protein according to any one of claims 5 to 14, wherein the first polypeptide and the one or more other polypeptides comprise or are composed of the following: They have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences.

16. The chimeric or fusion protein according to any one of claims 5 to 14, wherein the first polypeptide and the one or more other polypeptides comprise or are composed of the same amino acid sequence.

17. The chimeric or fusion protein according to any one of claims 5 to 15, wherein the first polypeptide comprises or is composed of an amino acid sequence of the active site of a Kgp gingival protease homolog (e.g., as shown in SEQ ID NO: 1), and the other polypeptide comprises or is composed of an amino acid sequence of the active site of an Rgp gingival protease homolog (e.g., as shown in SEQ ID NO: 2).

18. The chimeric or fusion protein according to any one of claims 5 to 15, wherein the first polypeptide comprises or is composed of an amino acid sequence of the active site of an Rgp gingival protease homolog (e.g., as shown in SEQ ID NO: 2), and the other polypeptide comprises or is composed of an amino acid sequence of the active site of a Kgp gingival protease homolog (e.g., as shown in SEQ ID NO: 1).

19. The chimeric or fusion protein according to any one of claims 5 to 15, wherein the first polypeptide and the other polypeptide comprise or consist of an amino acid sequence of the active site of a Kgp gingival protease homolog (e.g., as shown in SEQ ID NO: 1).

20. The chimeric or fusion protein according to any one of claims 5 to 15, wherein the first polypeptide and the other polypeptide comprise or consist of an amino acid sequence of the active site of an Rgp gingival protease homolog (e.g., as shown in SEQ ID NO: 2).

21. The chimeric or fusion protein according to any one of the preceding claims, wherein the amino acid sequence of the adhesin domain of the *Porphyromonas pharyngoides* surface complex further comprises one or more amino acid substitutions selected from: a) One or more cysteine ​​amino acids are substituted compared to the naturally occurring Porphyromonas pharyngosum sequence in the corresponding region; b) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of SEQ ID NO: 85 (equivalent to residues 68 to 71 of the sequence of SEQ ID NO: 20 or 88) or located at positions equivalent to said residues; c) Substitution of motif NxFA to SxYQ in the sequence corresponding to residues 2 to 5 of SEQ ID NO: 85 (equivalent to residues 64 to 67 of the sequence of SEQ ID NO: 20 or residues 64-67 of SEQ ID NO: 88) or located at positions equivalent to said residues; d) Substitution of tyrosine residues corresponding to or located at positions 10 of SEQ ID NO: 86 and tryptophan residues corresponding to or located at positions 23 of SEQ ID NO: 85 with alanine residues (equivalent to tyrosine at position 10 of SEQ ID NO: 20 and tryptophan at position 85).

22. The chimeric or fusion protein of claim 21, wherein the amino acid sequence of the adhesin domain further comprises: a) One or more cysteine ​​amino acids are substituted compared to the naturally occurring Porphyromonas pharynx sequence in the corresponding region.

23. The chimeric or fusion protein of claim 21, wherein the amino acid sequence of the adhesin domain further comprises: b) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 68 to 71 of the sequence of SEQ ID NO: 20 or located at positions equivalent to said residues.

24. The chimeric or fusion protein of claim 21, wherein the amino acid sequence of the adhesin domain further comprises: a) One or more cysteine ​​amino acids are substituted compared to the naturally occurring Arg or Lys gingival protease sequences of *Porphyromonas pharyngioma* in the corresponding region; and b) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 68 to 71 of the sequence of SEQ ID NO: 20 or located at positions equivalent to said residues.

25. The chimeric or fusion protein according to any one of claims 21, 22 and 24, wherein the substitution of cysteine ​​amino acid residues in the adhesin domain is a substitution of serine or valine residues.

26. The chimeric or fusion protein of claim 25, wherein the one or more cysteine ​​substitutions comprise one or more substitutions that become serine residues.

27. The chimeric or fusion protein according to any one of claims 21, 22 and 24 to 26, wherein only one cysteine ​​residue in the adhesin domain is substituted.

28. The chimeric or fusion protein according to any one of claims 21, 22 and 24 to 26, wherein two cysteine ​​residues are substituted.

29. The chimeric or fusion protein according to any one of claims 21, 22, and 24 to 27, wherein the adhesin domain comprises or is composed of the following: The sequence shown in either SEQ ID NO: 20 or 23, or at least 80% identical thereto, wherein one or more cysteine ​​residues are substituted with serine or valine residues.

30. The chimeric or fusion protein according to any one of claims 21, 22, and 24 to 27, wherein the adhesin domain comprises or is composed of the following: The sequence shown in any of SEQ ID NO: 23 or at least 80% identical thereto, wherein two cysteine ​​residues are replaced with serine or valine residues.

31. The chimeric or fusion protein according to any one of claims 24 to 30, wherein the adhesin domain comprises a substitution of a proline residue and / or an asparagine residue in the motif PxxN corresponding to or located at a position equivalent to said position in the sequence of SEQ ID NO:

20.

32. The chimeric or fusion protein according to claim 31, wherein the proline amino acid substitution is a substitution of an alanine residue.

33. The chimeric or fusion protein according to claim 31, wherein the asparagine amino acid substitution is changed to a proline residue or an alanine residue, preferably a proline residue substitution.

34. The chimeric or fusion protein of claim 31, wherein the motif PxxN in the adhesin domain is replaced with AxxP (e.g., AVQP, SEQ ID NO: 64).

35. The chimeric or fusion protein of claim 23, wherein the adhesin domain comprises or is composed of the following: The amino acid sequence shown in SEQ ID NO: 21 or at least 80% identical thereto, wherein the sequence contains AxxP at positions 68 to 71 of the sequence equivalent to SEQ ID NO:

21.

36. The chimeric or fusion protein according to any one of claims 34 to 35, wherein the adhesin domain comprises or is composed of the following: The amino acid sequence shown in any of SEQ ID NO: 24 to 27 or at least 80% identical thereto, wherein the sequence contains AxxP at positions 68 to 71 of the sequence equivalent to SEQ ID NO: 21, and wherein one or more cysteine ​​residues are substituted with serine or valine residues.

37. The chimeric or fusion protein according to any one of claims 4 to 36, wherein the second polypeptide comprises an amino acid sequence of the DUF2436 domain as shown in SEQ ID NO: 3, the amino acid sequence further comprising one or more amino acid substitutions.

38. The chimeric or fusion protein of claim 37, wherein the one or more amino acid substitutions are one or more substitutions of cysteine ​​residues in the domain.

39. The chimeric or fusion protein of claim 38, wherein only one cysteine ​​residue is substituted.

40. The chimeric or fusion protein of claim 38, wherein any two, three, or all four cysteine ​​residues of the DUF domain are substituted.

41. The chimeric or fusion protein according to any one of claims 38 to 40, wherein the cysteine ​​residue in the DUF domain is replaced with a valine residue, a serine residue, or an alanine residue.

42. The chimeric or fusion protein of claim 41, wherein one or more cysteine ​​residues are replaced with one or more serine residues.

43. The chimeric or fusion protein according to any one of claims, wherein the DUF domain comprising one or more cysteine ​​residues substituted comprises an amino acid sequence as shown in any one of SEQ ID NO: 5 to 19.

44. The chimeric or fusion protein according to any one of claims 1 to 43, wherein the chimeric or fusion protein comprises or is composed of the following: an amino acid sequence as shown in any one of SEQ ID NO: 32 to 35, 97 or 98 or a functional variant thereof having at least 80% identity with it.

45. The chimeric or fusion protein according to any one of claims 1 to 43, wherein the chimeric or fusion protein comprises or is composed of the following: an amino acid sequence as shown in any one of SEQ ID NO: 77 to 82 or a functional variant thereof having at least 80% identity with it.

46. ​​The chimeric or fusion protein according to any one of claims 1 to 43, wherein the chimeric or fusion protein comprises or is composed of the following: an amino acid as shown in any one of SEQ ID NO: 36 to 45 or 99 or a functional variant thereof having at least 80% identity with it.

47. The chimeric or fusion protein according to any one of claims 1 to 43, wherein the chimeric or fusion protein comprises or is composed of the following: an amino acid as shown in any one of SEQ ID NO: 46 to 76 or a functional variant thereof having at least 80% identity with it.

48. The chimeric or fusion protein according to any one of claims 1 to 43, wherein the chimeric or fusion protein comprises or is composed of the following: The amino acid sequence of SEQ ID NO: 28, 29 or 100 or a functional variant thereof having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and preferably said functional variant comprising a cysteine ​​substitution in the adhesin domain and an AVQP (SEQ ID NO: 64) substitution changing to the motif PVQN (SEQ ID NO: 108).

49. The chimeric or fusion protein according to any one of claims 1 to 43, wherein the chimeric or fusion protein comprises or is composed of the following: The amino acid sequence of SEQ ID NO: 29 or its functional variants having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it.

50. A nucleic acid that encodes any chimeric or fusion protein according to any of the preceding claims, optionally wherein the nucleic acid has a nucleotide sequence encoding any one or more of the amino acid sequences defined in Table 1 herein.

51. The nucleic acid of claim 50, wherein the nucleic acid has a nucleotide sequence as shown in Table 2 herein.

52. A vector comprising the nucleic acid according to claim 50 or 51.

53. A cell comprising the vector according to claim 52 or the nucleic acid according to claim 50 or 51.

54. A composition comprising a chimeric or fusion protein according to any one of claims 1 to 49, wherein the composition is optionally combined with a pharmaceutically acceptable carrier.

55. The composition of claim 54, further comprising an adjuvant for enhancing the immune response to the chimeric or fusion protein.

56. A vaccine or immunostimulatory composition for inducing an immune response against *Porphyromonas pharyngoides* in a subject, said composition comprising: - An immunogen in the form of a chimeric or fusion protein according to any one of claims 1 to 49, and - An adjuvant used to enhance the immune response to the immunogen in the subject.

57. The vaccine of claim 56, wherein the chimeric or fusion protein is a single immunogen in the composition.

58. A method for inducing an immune response against Porphyromonas pharyngoides in a subject, the method comprising administering to a subject in need a chimeric or fusion protein according to any one of claims 1 to 49, a composition according to claim 54 or 55, or a vaccine or immunostimulatory composition according to claim 56.

59. A method for inducing a humoral immune response against Porphyromonas pharyngoides in a subject, the method comprising administering to the subject a chimeric or fusion protein according to any one of claims 1 to 49, a composition according to claim 54 or 55, or a vaccine or immunostimulatory composition according to claim 56.

60. The method according to claim 58 or 59, wherein the method is used to enhance the subject's immune response to Porphyromonas pharyngosum (e.g., a protective immune response).

61. A method for immunizing a subject against Porphyromonas pharyngoides infection, the method comprising administering to the subject a chimeric or fusion protein according to any one of claims 1 to 49, a composition according to claim 54 or 55, or a vaccine or immunostimulatory composition according to claim 56.

62. The method according to any one of claims 58 to 61, wherein subjects who have received or been administered the chimeric or fusion protein, composition, or vaccine have an improved level of protection against the severity of one or more symptoms of Porphyromonas pharyngoides infection compared to subjects who have not received the chimeric or fusion protein, composition, or vaccine.

63. A method for treating a subject with Porphyromonas pharyngoides infection, the method comprising administering to a subject in need a chimeric or fusion protein according to any one of claims 1 to 49, a composition according to claim 54 or 55, or a vaccine or immunostimulatory composition according to claim 56, thereby treating the subject with the Porphyromonas pharyngoides infection.

64. A method for reducing or minimizing the severity of symptoms associated with Porphyromonas pharyngoides infection, the method comprising administering to an individual in need a chimeric or fusion protein according to any one of claims 1 to 49, a composition according to claim 54 or 55, or a vaccine or immunostimulatory composition according to claim 56, optionally wherein the symptoms are selected from the group consisting of: swollen or edematous gums, bleeding gums, receding gums, periodontal pockets around teeth, loss of tooth supporting tissues (periodontal ligaments, cementum and / or alveolar bone), pus between the gums and teeth, and gingivitis.

65. A method for treating Porphyromonas pharynx-associated disease in a subject, the method comprising administering to an individual in need a chimeric or fusion protein according to any one of claims 1 to 49, a composition according to claim 544 or 55, or a vaccine or immunostimulatory composition according to claim 56.

66. The method of claim 65, wherein the Porphyromonas pharyngoides-associated disease includes periodontal disease.

67. The method according to any one of claims 58 to 66, further comprising administering an antimicrobial compound and / or an anti-inflammatory agent, or further comprising administering one or more additional immunogenic or stimulating compositions.

68. Use of a chimeric or fusion protein according to any one of claims 1 to 49, for the preparation of a medicament for: - Induce an immune response (preferably a protective immune response) against Porphyromonas pharynx in subjects. -Immunize the subject against Porphyromonas pharyngoides infection; - Treating subjects with Porphyromonas pharynx infection; - To minimize or reduce the severity of one or more symptoms of Porphyromonas pharyngosum infection; or Treatment of subjects with Porphyromonas pharynx-related diseases.

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