Chimeric Polypeptides

Abstract

A chimeric or fusion protein for inducing an immune response against P. gingivalis, the protein comprising a first polypeptide linked to a second polypeptide, wherein A) the first polypeptide comprises or consists of the amino acid sequence of an active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto, and B) the second polypeptide comprises or consists of the amino acid sequence of an adhesin domain of Arg- or Lys-gingipain of P. gingivalis, , the sequence of one or more adhesin binding motifs (ABM), and the second polypeptide a) does not include a truncated adhesin domain (CAD) sequence or a portion thereof, b) comprises an amino acid sequence substantially corresponding to the entire length of the DUF2436 domain of an Arg- or Lys-gingipain, c) comprises one or more cysteine ​​amino acid substitutions in the adhesin domain compared to a naturally occurring Arg- or Lys-gingipain sequence in the corresponding region, and / or d) comprises one or more additional amino acid motif substitutions as defined herein.

Description

[Technical field]

[0001] The present invention relates to chimeric polypeptides that are useful for inducing an immune response to P. gingivalis, compositions comprising same, and their use for the prevention and treatment of P. gingivalis-associated conditions and diseases.

[0002] Related Applications This application claims priority from Australian Provisional Application No. 2022 / 900101, the contents of which are incorporated herein by reference. [Background technology]

[0003] If plaque is allowed to build up around the teeth at the gingival (gum) margin, it causes inflammation of the gums (gingivitis). Chronic gingivitis can lead to the development of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis) at the base of the periodontal pocket, resulting in chronic infection and the development of severe disease. This severe form of periodontal disease is called periodontitis, and the immune system's approach to eliminating the infection can result in tooth loss.

[0004] Chronic periodontitis is an inflammatory disease of the supporting tissues of the teeth, leading to alveolar bone resorption and eventual tooth loss. The disease is a major public health problem in all societies, affecting up to 30% of the adult population, with an estimated 12-15% of the adult population suffering from severe forms.

[0005] One in three adults has moderate to severe periodontitis. Epidemiological studies have linked periodontitis to an increased risk of inflammatory diseases, including cardiovascular disease, certain cancers, premature birth, rheumatoid arthritis, and dementia. More recent studies have linked chronic infection with P. gingivalis to dementia and rheumatoid arthritis. For example, in one study, 96% of Alzheimer's disease (AD) brain samples showed the presence of P. gingivalis. Another study shows that chronic oral infection of mice with P. gingivalis leads to brain plaques associated with AD in humans, and that P. gingivalis proteases can cleave amyloid precursor and tau proteins to form plaques and tangles associated with AD.

[0006] Several virulence factors have been reported that contribute to the pathogenicity of P. gingivalis, including LPS, fimbriae, hemagglutinins, hemolysins, and extracellular hydrolases (especially Arg-X and Lys-X specific proteinases), also known as "P. gingivalis gingipains."

[0007] The magnitude of the public health problem is such that a vaccine and a means to provide same is needed that provides a strong protective response against P. gingivalis infection.

[0008] One problem has been that it is unclear how to obtain a strong protective response against P. gingivalis infection with a plethora of virulence factors to select for.

[0009] Currently, there are no commercially approved vaccines for use in preventing or reducing the incidence and / or severity of P. gingivalis infection, or for treating P. gingivalis infection and disease in a subject.

[0010] Thus, there is a need for alternative and / or improved approaches for the design and manufacture of P. gingivalis vaccines, and alternative and / or improved vaccines produced from P. gingivalis.

[0011] The reference to any prior art in this specification is not an admission or suggestion that this prior art forms part of the common general knowledge in any jurisdiction, or that this prior art would be understood by, considered relevant, and / or could reasonably be expected to be combined with other prior art by a person skilled in the art. Summary of the Invention

[0012] The present invention provides a chimeric or fusion protein for inducing an immune response against P. gingivalis, the protein comprising a first polypeptide and a second polypeptide, A) the first polypeptide comprises or consists of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; B) the second polypeptide comprises or consists of the amino acid sequence of an adhesin domain of Arg- or Lys-gingipain of P. gingivalis; the second polypeptide comprises the sequence of one or more adhesin binding motifs (ABMs), preferably the ABMs correspond to part or all of the ABM between the DUF2436 domain and the truncated adhesin domain (CAD) of P. gingivalis gingipain; Preferably, one or more of the ABMs comprises a sequence as set forth in SEQ ID NO: 15 or 20, (ABM2), and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence at least 80% identical thereto; More preferably, one or more of the ABMs comprises a sequence as set forth in SEQ ID NO: 15 or 20 (ABM2), SEQ ID NO: 14 or 19 (ABM1), and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence at least 80% identical thereto; Most preferably, one or more ABMs comprise a sequence as set forth in any of SEQ ID NO: 16 or SEQ ID NO: 18 or 22 or 27, or a sequence at least 80% identical thereto; The second polypeptide is a) does not contain the sequence of a truncated adhesin domain (CAD) or a part thereof, preferably the sequence set forth in SEQ ID NO: 12 or 13, or the sequence of a CAD having at least 80% identity thereto, and / or b) comprising an amino acid sequence substantially corresponding to the entire length of the DUF2436 domain of Arg- or Lys-gingipain, preferably as set forth in SEQ ID NO: 23, or a sequence at least 80% identical thereto; Contains one or more amino acid substitutions selected from the following: c) one or more cysteine ​​amino acid substitutions in the adhesin domain compared to naturally occurring Arg- or Lys-gingipain sequences in the corresponding regions; and / or d) one or more amino acid motif substitutions selected from the following: i) substitution of proline and / or asparagine residues in the sequence PxxN at positions corresponding to or equivalent to residues 6 to 9 of SEQ ID NO: 14 or 19 (ABM1); ii) a substitution of the motif NxFA with SxYQ in the sequence at a position corresponding to or equivalent to residues 2 to 5 of SEQ ID NO: 14 or 19 (ABM1); iii) Substitution of a second tyrosine residue at a position corresponding to or equivalent to residue 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue at a position corresponding to or equivalent to residue 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue.

[0013] Preferably, the chimeric or fusion protein comprises one or more additional polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto. The one or more additional polypeptides comprising or consisting of the active site of Arg- or Lys-gingipain of P. gingivalis 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 certain embodiments, there may be at least two additional polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence that is 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 the N-terminus and C-terminus of the second polypeptide.

[0014] The one or more additional polypeptides may be linked to the first or second polypeptide of the chimeric or fusion protein via a linker, preferably of 50 amino acids or less, or may be linked directly to the first or second polypeptide.

[0015] In any embodiment, the first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0016] The one or more further polypeptides preferably comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0017] In any embodiment, the first and further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis comprise or consist of an amino acid 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 or have identical amino acid sequences. The first and further polypeptides may be derived from the active site of a heterologous gingipain (e.g., from a gingipain of a different strain of P. gingivalis). The first polypeptide and the further polypeptide may have amino acid sequences derived from different gingipains (e.g., one of the polypeptides has the active site amino acid sequence derived from Kgp and the other polypeptide has the active site amino acid sequence derived from Rgp, or alternatively, one of the polypeptides has the active site amino acid sequence derived from RgpA and the other polypeptide has the active site amino acid sequence derived from RgpB).

[0018] In a first aspect, the present invention provides a chimeric or fusion protein for inducing an immune response against P. gingivalis, the protein comprising a first polypeptide and a second polypeptide, A) the first polypeptide comprises or consists of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; B) the second polypeptide comprises or consists of the amino acid sequence of an adhesin domain of Arg- or Lys-gingipain of P. gingivalis; the second polypeptide comprises the sequence of one or more adhesin binding motifs (ABMs), preferably the ABMs correspond to part or all of the ABM between the DUF2436 domain and the truncated adhesin domain (CAD) of P. gingivalis gingipain; Preferably, one or more of the ABMs comprises a sequence as set forth in SEQ ID NO: 15 or 20 (ABM2), and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence at least 80% identical thereto; More preferably, one or more of the ABMs comprises a sequence as set forth in SEQ ID NO: 15 or 20 (ABM2), SEQ ID NO: 14 or 19 (ABM1), and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence at least 80% identical thereto; Most preferably, one or more ABMs comprise a sequence as set forth in any of SEQ ID NO: 16 or SEQ ID NO: 18 or 22 or 27, or a sequence at least 80% identical thereto; The second polypeptide is a) does not contain a truncated adhesin domain (CAD), preferably a CAD having the sequence set forth in SEQ ID NO: 12 or 13, or a sequence at least 80% identical thereto; Preferably, the chimeric fusion protein comprises one or more further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto.

[0019] The one or more further polypeptides comprising or consisting of the active site of Arg- or Lys-gingipain of P. gingivalis may be located at the N-terminus of the first polypeptide, at the C-terminus of the first polypeptide, at the N-terminus of the second polypeptide at the C-terminus of the second polypeptide. The one or more further polypeptides may be linked to the first or second polypeptide of the chimeric or fusion protein via a linker, preferably of 50 amino acids or less, or may be linked directly to the first or second polypeptide.

[0020] In any embodiment, the first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0021] The one or more further polypeptides preferably comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0022] In any embodiment of the first aspect of the invention, the first and further polypeptides comprising or consisting of an amino acid sequence of an active site of Arg- or Lys-gingipain of P. gingivalis comprise or consist of an amino acid 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 or are identical in amino acid sequence. The first and further polypeptides may be derived from the active site of a heterologous gingipain (e.g. from a gingipain of a different strain of P. gingivalis). The first polypeptide and the further polypeptide may have amino acid sequences derived from different gingipains (e.g., one of the polypeptides has the active site amino acid sequence derived from Kgp and the other polypeptide has the active site amino acid sequence derived from Rgp, or alternatively, one of the polypeptides has the active site amino acid sequence derived from RgpA and the other polypeptide has the active site amino acid sequence derived from RgpB).

[0023] In a second aspect, the present invention provides a chimeric or fusion protein for inducing an immune response against P. gingivalis, the protein comprising a first polypeptide linked to a second polypeptide, A) the first polypeptide comprises or consists of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; B) the second polypeptide comprises or consists of the amino acid sequence of an adhesin domain of Arg- or Lys-gingipain of P. gingivalis; the second polypeptide comprises the sequence of one or more adhesin binding motifs (ABMs), preferably the ABMs correspond to part or all of the ABM between the DUF2436 domain and the truncated adhesin domain (CAD) of P. gingivalis gingipain; Preferably, one or more of the ABMs comprises a sequence as set forth in SEQ ID NO: 15 or 20 (ABM2) and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence at least 80% identical thereto; More preferably, one or more of the ABMs comprises a sequence as set forth in SEQ ID NO: 15 or 20 (ABM2), SEQ ID NO: 14 or 19 (ABM1), and / or SEQ ID NO: 17 or 21 (ABM2), or a sequence at least 80% identical thereto; Most preferably, one or more ABMs comprise a sequence as set forth in SEQ ID NO: 16 or any of SEQ ID NOs: 18 or 22, or a sequence at least 80% identical thereto; The second polypeptide is a) does not contain a truncated adhesin domain (CAD), preferably a CAD having the sequence set forth in SEQ ID NO: 12 or 13 or a portion thereof, or a sequence at least 80% identical thereto; and b) comprises an amino acid sequence substantially corresponding to the entire length of the DUF2436 domain of Arg- or Lys-gingipain, preferably as set forth in SEQ ID NO: 23, or a sequence at least 80% identical thereto.

[0024] As used herein, an amino acid sequence substantially corresponding to the entire length of the DUF2436 domain, or a sequence at least 80% identical thereto, refers to 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% of the length of the DUF2436 domain of an Arg- or Lys-gingipain.

[0025] In a preferred embodiment, the amino acid sequence of the DUF2436 domain of Arg- or Lys-gingipain is the sequence set forth in SEQ ID NO: 23 or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0026] Most preferably, the second polypeptide comprises or consists of a sequence set forth in SEQ ID NO: 34, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In an alternative embodiment, the second polypeptide comprises a sequence set forth in SEQ ID NO: 76, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0027] Preferably, the chimeric or fusion protein comprises one or more further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis or a sequence at least 80% identical thereto. The one or more further polypeptides comprising or consisting of the active site of Arg- or Lys-gingipain of P. gingivalis may be located at the N-terminus of the first polypeptide, at the C-terminus of the first polypeptide, at the N-terminus of the second polypeptide at the C-terminus of the second polypeptide. The one or more further polypeptides may be linked to the first or second polypeptide of the chimeric or fusion protein via a linker, preferably of 50 amino acids or less, or directly to the first polypeptide.

[0028] In any embodiment, the first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0029] The one or more further polypeptides preferably comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0030] In any embodiment, the first and further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis comprise or consist of an amino acid 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 or have identical amino acid sequences. The first and further polypeptides may be derived from the active site of a heterologous gingipain (e.g., from a gingipain of a different strain of P. gingivalis). The first polypeptide and the further polypeptide may have amino acid sequences derived from different gingipains (e.g., one of the polypeptides has the active site amino acid sequence derived from Kgp and the other polypeptide has the active site amino acid sequence derived from Rgp, or alternatively, one of the polypeptides has the active site amino acid sequence derived from RgpA and the other polypeptide has the active site amino acid sequence derived from RgpB).

[0031] In particularly preferred embodiments of the second aspect of the invention, the chimeric or fusion protein comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 28, 50, 51, 52, 53 or 54, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0032] In a third aspect of the invention, the present invention provides a chimeric or fusion protein for inducing an immune response against P. gingivalis, the protein comprising a first polypeptide and a second polypeptide, A) the first polypeptide comprises or consists of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; B) the second polypeptide comprises or consists of the amino acid sequence of an adhesin domain of Arg- or Lys-gingipain of P. gingivalis; the second polypeptide comprises the sequence of one or more adhesin binding motifs (ABMs), preferably the ABMs correspond to part or all of the ABM between the DUF2436 domain and the truncated adhesin domain (CAD) of P. gingivalis gingipain; Preferably, one or more ABMs comprise a sequence as set forth in SEQ ID NO: 15 or 20 (ABM2), and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence at least 80% identical thereto; More preferably, one or more of the ABMs comprises a sequence as set forth in SEQ ID NO: 15 or 20 (ABM2), SEQ ID NO: 14 or 19 (ABM1), and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence at least 80% identical thereto; Most preferably, one or more ABMs comprise a sequence as set forth in any of SEQ ID NO: 16 or SEQ ID NO: 18 or 22 or SEQ ID NO: 27, or a sequence at least 80% identical thereto; The second polypeptide comprises one or more amino acid substitutions selected from: a) one or more cysteine ​​amino acid substitutions in the adhesin domain compared to naturally occurring Arg- or Lys-gingipain sequences in the corresponding regions; and / or b) one or more amino acid motif substitutions selected from the following: i) substitution of proline and / or asparagine residues in the sequence PxxN at positions corresponding to or equivalent to residues 6 to 9 of SEQ ID NO: 14 or 19 (ABM1); ii) a substitution of the motif NxFA with SxYQ in the sequence at a position corresponding to or equivalent to residues 2 to 5 of SEQ ID NO: 14 or 19 (ABM1); iii) Substitution of a second tyrosine residue at a position corresponding to or equivalent to residue 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue at a position corresponding to or equivalent to residue 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue.

[0033] The one or more cysteine ​​amino acid substitutions may be substitutions to serine or valine residues. Preferably, the one or more cysteine ​​substitutions may include one or more substitutions to serine residues.

[0034] In certain embodiments, only one cysteine ​​residue may be substituted. In other embodiments, two or three cysteine ​​residues may be substituted. In particularly preferred embodiments, 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.

[0035] In a particularly preferred embodiment, the adhesin domain comprises a DUF2436 domain or a portion thereof, and a cysteine ​​residue in the DUF2436 domain is substituted with serine or valine, preferably serine, In other embodiments, the adhesin domain comprises a DUF2436 domain, and a cysteine ​​residue in the DUF2436 domain is not substituted, and preferably one or more cysteine ​​residues in the remainder of the adhesin domain are substituted.

[0036] Optionally, the adhesin domain comprises or consists of the sequence set forth in SEQ ID NO:23, or a sequence at least 80% identical thereto, wherein the cysteine ​​residue at position 115 is substituted with a serine or valine residue.

[0037] Optionally, the adhesin domain comprises or consists of a sequence set forth in SEQ ID NO:25, or a sequence at least 80% identical thereto, wherein the cysteine ​​residue at position 77 is substituted with a serine or valine residue.

[0038] Preferably, the adhesin domain comprises or consists of a sequence as set forth in SEQ ID NO: 34 or SEQ ID NO: 76, or a sequence at least 80% identical thereto, wherein one or more cysteine ​​residues are substituted by a serine or valine residue.

[0039] In a particularly preferred embodiment of the third aspect of the invention there is provided a chimeric or fusion protein for inducing an immune response against P. gingivalis, the protein comprising a first polypeptide and a second polypeptide, A) the first polypeptide comprises or consists of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; B) the second polypeptide comprises or consists of the amino acid sequence of an adhesin domain of Arg- or Lys-gingipain of P. gingivalis; the second polypeptide comprises the sequence of one or more adhesin binding motifs (ABMs), preferably the ABMs correspond to part or all of the ABM between the DUF2436 domain and the truncated adhesin domain (CAD) of P. gingivalis gingipain; Preferably, one or more of the ABMs comprises a sequence as set forth in SEQ ID NO: 15 or 20 (ABM2), SEQ ID NO: 14 or 19 (ABM1), and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence at least 80% identical thereto; More preferably, one or more ABMs comprise a sequence as set forth in any of SEQ ID NO: 16 or SEQ ID NO: 18 or 22 or SEQ ID NO: 27, or a sequence at least 80% identical thereto; The second polypeptide is c) containing one or more cysteine ​​to serine amino acid substitutions compared to a naturally occurring adhesin domain sequence; d) comprising proline and / or asparagine substitutions in the sequence PxxN at positions corresponding or equivalent to residues 6 to 9 of SEQ ID NO: 14 or 19.

[0040] Preferably, the chimeric or fusion protein comprises one or more further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence at least 80% identical thereto. The one or more further polypeptides comprising or consisting of the active site of Arg- or Lys-gingipain of P. gingivalis may be located at the N-terminus of the first polypeptide, at the C-terminus of the first polypeptide, at the N-terminus of the second polypeptide at the C-terminus of the second polypeptide.

[0041] In a preferred embodiment, the second polypeptide comprises the amino acid sequence of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, the adhesin domain comprising a sequence as set forth in SEQ ID NO: 18 or 22 or a sequence at least 80% identical thereto, wherein one or both of the cysteine ​​residues in SEQ ID NO: 18 or 22 have been replaced by a serine residue, and a proline and / or asparagine residue has been replaced in the sequence PxxN at positions 63 to 66 in SEQ ID NO: 18 or SEQ ID NO: 22 (corresponding to positions 6 to 9 in SEQ ID NO: 14 or 19) or equivalent positions. Optionally, the proline residue has been replaced by an alanine residue and / or the asparagine residue has been replaced by a proline or alanine residue, preferably the proline has been replaced by an alanine and the asparagine has been replaced by a proline, such that the sequence at positions 63 to 66 in SEQ ID NO: 18 or SEQ ID NO: 22 is AxxP (e.g. AVQP).

[0042] In a particularly preferred embodiment, the second polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 34, or a sequence at least 80% identical thereto, in which one, two or three cysteine ​​residues are substituted with serine residues. Preferably, the cysteine ​​residue at residue 115 of SEQ ID NO: 34, or an equivalent position, is not substituted with either a serine or a valine residue. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 34, or an equivalent position, is not substituted with either a serine or a valine residue, and the cysteine ​​residues at positions 208 and 222 of SEQ ID NO: 34, or an equivalent position, are substituted with serine residues.

[0043] In a particularly preferred embodiment, the second polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 34, or a sequence at least 80% identical thereto, in which one, two or three cysteine ​​residues have been replaced by serine residues, and the proline and asparagine residues in the sequence PxxN at or equivalent positions from 235 to 238 have been replaced. Preferably, the cysteine ​​residue at residue 115 of SEQ ID NO: 34, or at a position equivalent thereto, is not replaced by either a serine or a valine residue. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 34, or at a position equivalent thereto, is not replaced by either a serine or a valine residue, the cysteine ​​residues at positions 208 and 222 of SEQ ID NO: 34, or at positions equivalent thereto, are replaced by serine residues, the proline residue at position 235 or at a position equivalent thereto is replaced by an alanine residue, and the asparagine residue at position 238 or at a position equivalent thereto is replaced by proline.

[0044] In particularly preferred embodiments, the second polypeptide comprises or consists of a sequence set forth in any one of SEQ ID NOs: 35-49, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0045] In any embodiment, the first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0046] The one or more further polypeptides preferably comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0047] In any embodiment, the first and further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis comprise or consist of an amino acid 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 or have identical amino acid sequences. The first and further polypeptides may be derived from the active site of a heterologous gingipain (e.g., from a gingipain of a different strain of P. gingivalis). The first polypeptide and the further polypeptide may have amino acid sequences derived from different gingipains (e.g., one of the polypeptides has the active site amino acid sequence derived from Kgp and the other polypeptide has the active site amino acid sequence derived from Rgp, or alternatively, one of the polypeptides has the active site amino acid sequence derived from RgpA and the other polypeptide has the active site amino acid sequence derived from RgpB).

[0048] In a preferred embodiment of the third aspect of the invention, the chimeric or fusion protein comprises or consists of an amino acid sequence set forth in any of SEQ ID NOs: 55, 56 or 57, or a sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0049] In a fourth aspect, the present invention provides a chimeric or fusion protein for inducing an immune response against P. gingivalis, the protein comprising a first polypeptide and a second polypeptide, A) the first polypeptide comprises or consists of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; B) the second polypeptide comprises or consists of the amino acid sequence of an adhesin domain of Arg- or Lys-gingipain of P. gingivalis; the second polypeptide comprises the sequence of one or more adhesin binding motifs (ABMs), preferably the ABMs correspond to part or all of the ABM between the DUF2436 domain and the truncated adhesin domain (CAD) of P. gingivalis gingipain; Preferably, one or more of the ABMs comprises a sequence as set forth in SEQ ID NO: 15 or 20 (ABM2), SEQ ID NO: 14 or 19 (ABM1), and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence at least 80% identical thereto; More preferably, one or more ABMs comprise a sequence as set forth in SEQ ID NO: 16 or any of SEQ ID NOs: 18 or 22, or a sequence at least 80% identical thereto; The second polypeptide is a) comprising an amino acid sequence substantially corresponding to the entire length of the DUF2436 domain of Arg- or Lys-gingipain, preferably as set forth in SEQ ID NO: 23, or a sequence at least 80% identical thereto; One or more amino acid substitutions selected from the following: b) one or more cysteine ​​amino acid substitutions in the adhesin domain compared to naturally occurring Arg- or Lys-gingipain sequences in the corresponding regions; and / or d) one or more amino acid motif substitutions selected from the following: i) substitution of proline and / or asparagine residues in the sequence PxxN at positions corresponding to or equivalent to residues 6 to 9 of SEQ ID NO: 14 or 19 (ABM1); ii) a substitution of the motif NxFA with SxYQ in the sequence at a position corresponding to or equivalent to residues 2 to 5 of SEQ ID NO: 14 or 19 (ABM1); iii) Substitution of a second tyrosine residue at a position corresponding to or equivalent to residue 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue at a position corresponding to or equivalent to residue 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue.

[0050] The one or more cysteine ​​amino acid substitutions may be substitutions to serine or valine residues. Preferably, the one or more cysteine ​​substitutions may include one or more substitutions to serine residues.

[0051] In certain embodiments, only one cysteine ​​residue is replaced. In other embodiments, two or three cysteine ​​residues are replaced. In particularly preferred embodiments, the cysteine ​​residues are replaced with a combination of valine and serine residues. In other embodiments, all of the replaced cysteine ​​residues are replaced with serine, or all of the replaced cysteine ​​residues are replaced with valine.

[0052] In a particularly preferred embodiment, the adhesin domain comprises a DUF2436 domain, in which a cysteine ​​residue in the DUF2436 domain has been substituted with serine or valine, preferably serine, in another embodiment, the adhesin domain comprises a DUF2436 domain, in which a cysteine ​​residue in the DUF2436 domain has not been substituted, and preferably one or more cysteine ​​residues in the remainder of the adhesin domain have been substituted.

[0053] Preferably, the adhesin domain comprises or consists of a sequence as set forth in SEQ ID NO: 34 or SEQ ID NO: 76, or a sequence at least 80% identical thereto, wherein one or more cysteine ​​residues are substituted by a serine or valine residue.

[0054] According to this aspect of the invention, the sequence PxxN at a position corresponding or equivalent to residues 235-238 of SEQ ID NO: 34 comprises a substitution of a proline and an asparagine residue. Preferably, the substitution is of PxxN to AxxP.

[0055] In particularly preferred embodiments of this aspect of the invention, the second polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 34, or a sequence at least 80% identical thereto, in which one, two or three cysteine ​​residues have been substituted with serine residues. Preferably, the cysteine ​​residue at residue 115, or an equivalent position, of SEQ ID NO: 34 is not substituted with either a serine or a valine residue. Preferably, the cysteine ​​residue at position 115, or an equivalent position, of SEQ ID NO: 34 is not substituted with either a serine or a valine residue, and the cysteine ​​residues at positions 208 and 222, or equivalent positions, of SEQ ID NO: 34 are substituted with serine residues.

[0056] In a particularly preferred embodiment, the second polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 34, or a sequence at least 80% identical thereto, in which one, two or three cysteine ​​residues have been replaced by serine residues, and the proline and asparagine residues in the sequence PxxN at or equivalent positions from 235 to 238 have been replaced. Preferably, the cysteine ​​residue at residue 115 of SEQ ID NO: 34, or at a position equivalent thereto, is not replaced by either a serine or a valine residue. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 34, or at a position equivalent thereto, is not replaced by either a serine or a valine residue, the cysteine ​​residues at positions 208 and 222 of SEQ ID NO: 34, or at positions equivalent thereto, are replaced by serine residues, the proline residue at position 235 or at a position equivalent thereto is replaced by an alanine residue, and the asparagine residue at position 238 or at a position equivalent thereto is replaced by proline.

[0057] In particularly preferred embodiments, the second polypeptide comprises or consists of a sequence set forth in any one of SEQ ID NOs: 35-49, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0058] In particularly preferred embodiments, the chimeric or fusion protein comprises or consists of a sequence set forth in any one of SEQ ID NOs: 62-63, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0059] Preferably, the chimeric fusion protein comprises one or more further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence at least 80% identical thereto. The one or more further polypeptides comprising or consisting of the active site of Arg- or Lys-gingipain of P. gingivalis may be located at the N-terminus of the first polypeptide, at the C-terminus of the first polypeptide, at the N-terminus of the second polypeptide at the C-terminus of the second polypeptide.

[0060] In any embodiment, the first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0061] The one or more further polypeptides preferably comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0062] In any embodiment, the first and further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis comprise or consist of an amino acid 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 or have identical amino acid sequences. The first and further polypeptides may be derived from the active site of a heterologous gingipain (e.g., from a gingipain of a different strain of P. gingivalis). The first polypeptide and the further polypeptide may have amino acid sequences derived from different gingipains (e.g., one of the polypeptides has the active site amino acid sequence derived from Kgp and the other polypeptide has the active site amino acid sequence derived from Rgp, or alternatively, one of the polypeptides has the active site amino acid sequence derived from RgpA and the other polypeptide has the active site amino acid sequence derived from RgpB).

[0063] In a fifth aspect, the present invention provides a chimeric or fusion protein for inducing an immune response against P. gingivalis, the protein comprising a first polypeptide linked to a second polypeptide, A) the first polypeptide comprises or consists of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; B) the second polypeptide comprises or consists of the amino acid sequence of an adhesin domain of Arg- or Lys-gingipain of P. gingivalis; the second polypeptide comprises the sequence of one or more adhesin binding motifs (ABM), preferably the ABM corresponds to part or all of the ABM between the DUF2436 domain and the truncated adhesin domain (CAD) of P. gingivalis gingipain; Preferably, one or more of the ABMs comprises a sequence as set forth in SEQ ID NO: 15 or 20 (ABM2) and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence at least 80% identical thereto; More preferably, one or more of the ABMs comprises a sequence as set forth in SEQ ID NO: 15 or 20 (ABM2), SEQ ID NO: 14 or 19 (ABM1), and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence at least 80% identical thereto; Most preferably, one or more ABMs comprise a sequence as set forth in SEQ ID NO: 16 or any of SEQ ID NOs: 18 or 22, or a sequence at least 80% identical thereto; The second polypeptide is a) does not contain the sequence of a truncated adhesin domain (CAD) or a part thereof, preferably the sequence of a CAD having the amino acid sequence set forth in SEQ ID NO: 12 or 13, or a sequence at least 80% identical thereto; and b) comprises an amino acid sequence substantially corresponding to the entire length of the DUF2436 domain of Arg- or Lys-gingipain, preferably as set forth in SEQ ID NO: 23, or a sequence at least 80% identical thereto; and Contains one or more of the following amino acid substitutions: c) one or more cysteine ​​amino acid substitutions in the amino acid sequences of the DUF2436 domains and ABMs compared to naturally occurring Arg- or Lys-gingipain sequences in the corresponding regions, and / or (preferably, d) one or more amino acid motif substitutions selected from the following: i) substitution of proline and / or asparagine residues in the sequence PxxN at positions corresponding to or equivalent to residues 6 to 9 of SEQ ID NO: 14 or 19 (ABM1); ii) a substitution of the motif NxFA with SxYQ in the sequence at a position corresponding to or equivalent to residues 2 to 5 of SEQ ID NO: 14 or 19 (ABM1); iii) Substitution of a second tyrosine residue at a position corresponding to or equivalent to residue 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue at a position corresponding to or equivalent to residue 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue.

[0064] The one or more cysteine ​​amino acid substitutions may be substitutions to serine or valine residues. Preferably, the one or more cysteine ​​substitutions may include one or more substitutions to serine residues.

[0065] In certain embodiments, only one cysteine ​​residue is replaced. In other embodiments, two or three cysteine ​​residues are replaced. In particularly preferred embodiments, the cysteine ​​residues are replaced with a combination of valine and serine residues. In other embodiments, all of the replaced cysteine ​​residues are replaced with serine, or all of the replaced cysteine ​​residues are replaced with valine.

[0066] In a particularly preferred embodiment, the adhesin domain comprises a DUF2436 domain, in which a cysteine ​​residue in the DUF2436 domain has been substituted with serine or valine, preferably serine, in another embodiment, the adhesin domain comprises a DUF2436 domain, in which a cysteine ​​residue in the DUF2436 domain has not been substituted, and preferably one or more cysteine ​​residues in the remainder of the adhesin domain have been substituted.

[0067] Preferably, the adhesin domain comprises or consists of the sequence set forth in SEQ ID NO: 34, or a sequence at least 80% identical thereto, wherein one or more cysteine ​​residues are substituted by a serine or valine residue.

[0068] According to this aspect of the invention, the sequence PxxN at a position corresponding or equivalent to residues 235-238 of SEQ ID NO: 34 comprises a substitution of a proline and an asparagine residue. Preferably, the substitution is of PxxN to AxxP.

[0069] In particularly preferred embodiments of this aspect of the invention, the second polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 34, or a sequence at least 80% identical thereto, in which one, two or three cysteine ​​residues have been substituted with serine residues. Preferably, the cysteine ​​residue at residue 115, or an equivalent position, of SEQ ID NO: 34 is not substituted with either a serine or a valine residue. Preferably, the cysteine ​​residue at position 115, or an equivalent position, of SEQ ID NO: 34 is not substituted with either a serine or a valine residue, and the cysteine ​​residues at positions 208 and 222, or equivalent positions, of SEQ ID NO: 34 are substituted with serine residues.

[0070] In a particularly preferred embodiment, the second polypeptide comprises an amino acid sequence as set forth in SEQ ID NO: 34, or a sequence at least 80% identical thereto, in which one, two or three cysteine ​​residues have been replaced by serine residues, and the proline and asparagine residues in the sequence PxxN at or equivalent positions from 235 to 238 have been replaced. Preferably, the cysteine ​​residue at residue 115 of SEQ ID NO: 34, or at a position equivalent thereto, is not replaced by either a serine or a valine residue. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 34, or at a position equivalent thereto, is not replaced by either a serine or a valine residue, the cysteine ​​residues at positions 208 and 222 of SEQ ID NO: 34, or at positions equivalent thereto, are replaced by serine residues, the proline residue at position 235 or at a position equivalent thereto is replaced by an alanine residue, and the asparagine residue at position 238 or at a position equivalent thereto is replaced by proline.

[0071] In particularly preferred embodiments, the second polypeptide comprises or consists of a sequence set forth in any one of SEQ ID NOs: 35-49, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0072] In particularly preferred embodiments, the chimeric or fusion protein comprises or consists of a sequence set forth in any one of SEQ ID NOs: 58-61, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0073] Preferably, the chimeric or fusion protein comprises one or more further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence at least 80% identical thereto. The one or more further polypeptides comprising or consisting of the active site of Arg- or Lys-gingipain of P. gingivalis may be located at the N-terminus of the first polypeptide, at the C-terminus of the first polypeptide, at the N-terminus of the second polypeptide at the C-terminus of the second polypeptide.

[0074] In any embodiment, the first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0075] The one or more further polypeptides preferably comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0076] In any embodiment, the first and further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis comprise or consist of an amino acid 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 or have identical amino acid sequences. The first and further polypeptides may be derived from the active site of a heterologous gingipain (e.g., from a gingipain of a different strain of P. gingivalis). The first polypeptide and the further polypeptide may have amino acid sequences derived from different gingipains (e.g., one of the polypeptides has the active site amino acid sequence derived from Kgp and the other polypeptide has the active site amino acid sequence derived from Rgp, or alternatively, one of the polypeptides has the active site amino acid sequence derived from RgpA and the other polypeptide has the active site amino acid sequence derived from RgpB).

[0077] In a sixth aspect, the present invention provides a chimeric or fusion protein for inducing an immune response against P. gingivalis, the protein comprising a first polypeptide linked to a second polypeptide, A) the first polypeptide comprises or consists of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; B) the second polypeptide comprises or consists of the amino acid sequence of an adhesin domain of Arg- or Lys-gingipain of P. gingivalis; the second polypeptide comprises the sequence of one or more adhesin binding motifs (ABM), preferably the ABM corresponds to part or all of the ABM between the DUF2436 domain and the truncated adhesin domain (CAD) of P. gingivalis gingipain; Preferably, one or more of the ABMs comprises a sequence as set forth in SEQ ID NO: 15 or 20 (ABM2) and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence at least 80% identical thereto; More preferably, one or more of the ABMs comprises a sequence as set forth in SEQ ID NO: 15 or 20 (ABM2), SEQ ID NO: 14 or 19 (ABM1), and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence at least 80% identical thereto; Most preferably, one or more ABMs comprise a sequence as set forth in SEQ ID NO: 16 or any of SEQ ID NOs: 18 or 22, or a sequence at least 80% identical thereto; The second polypeptide is a) does not contain the sequence of a truncated adhesin domain (CAD) or a part thereof, preferably the sequence of a CAD having the amino acid sequence set forth in SEQ ID NO: 12 or 13, or a sequence at least 80% identical thereto; and and containing one or more amino acid substitutions selected from the following: b) one or more cysteine ​​amino acid substitutions in the amino acid sequence of the ABM compared to naturally occurring Arg- or Lys-gingipain sequences in the corresponding regions; and / or c) one or more amino acid motif substitutions selected from the following: i) substitution of proline and / or asparagine residues in the sequence PxxN (e.g., PVQN) at positions corresponding to or equivalent to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1); ii) a substitution of the motif NxFA to SxYQ (e.g., NEFA to SEYQ) in the sequence at a position corresponding to or equivalent to residues 2 to 5 of SEQ ID NO: 14 or 19 (ABM1); iii) Substitution of a second tyrosine residue at a position corresponding to or equivalent to residue 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue at a position corresponding to or equivalent to residue 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue.

[0078] In this aspect of the invention, it will be understood that, preferably, the chimeric or fusion protein does not contain an amino acid sequence corresponding to or derived from the DUF2436 domain of Arg- or Lys-gingipain as set forth in SEQ ID NO:23, or a sequence at least 80% identical thereto.

[0079] According to the sixth aspect of the invention, the one or more cysteine ​​amino acid substitutions may be substitutions to serine or valine residues. Preferably, the one or more cysteine ​​substitutions may include one or more substitutions to serine residues.

[0080] Optionally, only one cysteine ​​residue is replaced. In other embodiments, two cysteine ​​residues are replaced. In a particularly preferred embodiment, the cysteine ​​residues are replaced with a combination of valine and serine residues. In other embodiments, all of the replaced cysteine ​​residues are replaced with serine, or all of the replaced cysteine ​​residues are replaced with valine.

[0081] Preferably, the adhesin domain comprises or consists of a sequence as set forth in SEQ ID NO: 18 or 22, or a sequence at least 80% identical thereto, wherein one or more cysteine ​​residues are substituted by a serine or valine residue.

[0082] According to this aspect of the invention, the sequence PxxN (e.g., PVQN) at a position corresponding to or equivalent to residues 63-66 of SEQ ID NO: 18 comprises a substitution of a proline and an asparagine residue. Preferably, the substitution is of PxxN to AxxP, (e.g., AVQP).

[0083] In a particularly preferred embodiment of this sixth aspect of the invention, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 18, or a sequence at least 80% identical thereto, in which one or two cysteine ​​residues are replaced by serine residues.

[0084] In a particularly preferred embodiment, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 18, or a sequence at least 80% identical thereto, in which one or two cysteine ​​residues have been replaced by serine residues, and proline and asparagine residues have been replaced in the sequence PxxN at positions 63 to 66, or at equivalent positions.

[0085] In any embodiment of the sixth aspect of the present invention, the first and second polypeptides may be directly connected or connected via a linker. Preferably, the linker comprises a series of amino acid sequences, preferably from about 1 amino acid residue to about 20 amino acid residues, preferably about 5 amino acids or less, or about 10 amino acids or less, or about 15 amino acids or less in length.

[0086] In a preferred embodiment, a first and a second polypeptide (having the representative amino acid sequences of SEQ ID NOs: 1 and 18, respectively (i.e., the amino acid sequences without the amino acid substitutions described herein) can be linked via a peptide sequence having the amino acid sequence DMEVEDDSP.

[0087] In particularly preferred embodiments, the chimeric or fusion protein comprises or consists of a sequence set forth in any one of SEQ ID NOs: 80 or 81, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0088] Preferably, the chimeric or fusion protein comprises one or more further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence at least 80% identical thereto. The one or more further polypeptides comprising or consisting of the active site of Arg- or Lys-gingipain of P. gingivalis may be located at the N-terminus of the first polypeptide, at the C-terminus of the first polypeptide, at the N-terminus of the second polypeptide at the C-terminus of the second polypeptide.

[0089] In any embodiment, the first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0090] The one or more further polypeptides preferably comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0091] In any embodiment, the first and further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis comprise or consist of an amino acid 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 or have identical amino acid sequences. The first and further polypeptides may be derived from the active site of a heterologous gingipain (e.g., from a gingipain of a different strain of P. gingivalis). The first polypeptide and the further polypeptide may have amino acid sequences derived from different gingipains (e.g., one of the polypeptides has the active site amino acid sequence derived from Kgp and the other polypeptide has the active site amino acid sequence derived from Rgp, or alternatively, one of the polypeptides has the active site amino acid sequence derived from RgpA and the other polypeptide has the active site amino acid sequence derived from RgpB).

[0092] In any embodiment of any aspect of the invention, the chimeric or fusion protein consists or essentially consists of the sequences of the first and second polypeptides defined herein. It will therefore be understood that the chimeric or fusion protein comprises an arrangement or organization of domains that differs from the organization of those domains in the naturally occurring gingipain polyprotein sequence. In other words, the first and second polypeptides and the domains therein have a different spatial organization than the naturally occurring gingipain polyprotein.

[0093] In any embodiment of any aspect of the invention, the first and second polypeptides are linked. The first and second polypeptides may be linked directly, via a linker, or via a polypeptide sequence of 100 amino acids or less, preferably 50 amino acids or less. Preferably, the first and second polypeptides are directly linked or linked by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids or less. Most preferably, the first and second polypeptides are directly linked.

[0094] In any embodiment of any aspect of the invention, the C-terminal residue of a first polypeptide may be linked to the N-terminal residue of a second polypeptide directly, via a linker, or via a polypeptide sequence of 50 amino acids or less. Alternatively, the N-terminal residue of a first polypeptide may be linked to the C-terminal residue of a second polypeptide directly, via a linker, or via a polypeptide sequence of 50 amino acids or less.

[0095] In any of the second to fifth aspects of the present invention, the DUF2436 domain and the ABM domain derived from Arg- or Lys-gingipain can be directly linked or can be connected via a linker or via a polypeptide sequence. Preferably, the DUF2436 and ABM domains are linked via 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 DUF domain and the ABM domain are linked via a short linker sequence of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids or less.

[0096] In a preferred embodiment, DUF2436 and the ABM domain (having the representative amino acid sequences of SEQ ID NOs: 23 and 18, respectively (i.e., the amino acid sequences without the amino acid substitutions described herein) may be linked via a peptide sequence having the amino acid sequence EVEDDSP.

[0097] In any embodiment, the additional polypeptide may be connected directly or via a linker to a chimeric or fusion protein comprising the first and second polypeptides. In embodiments in which the additional polypeptide is connected to the C-terminal region of the fusion protein via a second polypeptide, preferably the C-terminus of the second polypeptide is directly connected to the N-terminus of the additional polypeptide. (For example, the C-terminus of the adhesin domain is preferably directly connected to the N-terminus of the active site amino acid sequence.)

[0098] When two or more additional polypeptides are included (e.g., as exemplified in SEQ ID NO:54, the copies of the additional polypeptides can be directly connected to each other or can be linked via a linker sequence.

[0099] In any embodiment of any aspect of the invention, the linker region is an amino acid sequence of 15 amino acids or less, preferably more than 2 amino acids. Suitable linkers for use in protein constructs, including those that have minimal impact on solubility, are known in the art. Useful linkers include glycine-serine (GlySer) linkers, which are well known in the art and contain glycine and serine units combined in various orders. Examples include, but are not limited to, (GS), (GSGGS)n, (GGGS)n, and (GGGGS)n, where n is an integer of at least 1, typically 1 to about 10, such as 1 to about 8, 1 to about 6, or 1 to about 5. Other useful linkers include DSSG, DSSGAS, KLDSSG, or others described herein. In certain embodiments, the linker region may be derived from a native ginigipain protein sequence.

[0100] The present invention also provides a nucleic acid encoding any of the chimeric or fusion proteins defined herein.

[0101] Preferably, the nucleic acid has a nucleotide sequence that encodes any one or more of the amino acid sequences defined in Table 1 herein.

[0102] In any embodiment, such a nucleic acid is included in an expression construct in which the nucleic acid is operably linked to a promoter. Such an expression construct can be in a vector, for example, a plasmid or a viral vector.

[0103] The present invention also provides a cell comprising a nucleic acid or a nucleic acid vector described herein. Exemplary cells of the present invention include bacterial cells, yeast cells, insect cells, or mammalian cells. Preferably, the cells are isolated, substantially purified, or recombinant.

[0104] The present invention also provides compositions comprising a chimeric or fusion protein described herein, optionally in combination with a pharma- ceutically acceptable carrier.

[0105] The composition can also include an adjuvant to enhance the immune response to the chimeric or fusion protein.

[0106] Accordingly, the present invention provides a vaccine or immunostimulatory composition for inducing an immune response against P. gingivalis in a subject, the composition comprising: i) an immunogen in the form of a chimeric or fusion protein as described herein; and ii) an adjuvant for enhancing the immune response to the immunogen in the subject.

[0107] Preferably, the only immunogen provided in the composition, vaccine, or immunostimulatory composition of the invention is a chimeric or fusion protein as described herein.

[0108] The present invention also provides a method for inducing an immune response against P. gingivalis in a subject, the method comprising administering to a subject in need thereof a chimeric or fusion protein, vaccine, or immunostimulatory composition described herein.

[0109] The present invention also provides a method for inducing a humoral immune response against P. gingivalis in a subject, the method comprising administering to the subject a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition defined herein.

[0110] Preferably, the induced immune response comprises a switch from a Th1 to a Th2 immune response.

[0111] It will be appreciated that in any embodiment, the immune response elicited by administration of the chimeric or fusion proteins described herein, or vaccines or other compositions comprising same, is preferably antigen-specific. Thus, in preferred embodiments, the methods and compositions and chimeric proteins described herein are for inducing an immune response, preferably a protective immune response, against P. gingivalis gingipain antigens.

[0112] In any embodiment, the compositions, chimeric proteins, and methods of the present invention can be for enhancing a subject's immune response (eg, a protective immune response) against P. gingivalis.

[0113] The invention also provides a method of immunising a subject against P. gingivalis infection comprising administering to the subject a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as defined herein.

[0114] In any embodiment, a subject who has received or been administered a chimeric or fusion protein of the present invention, or a composition or vaccine comprising same, has a level of protection against infection with P. gingivalis, or an increased severity of one or more symptoms of P. gingivalis infection, compared to a subject who does not receive the protein, composition, or vaccine.

[0115] Still further, the present invention provides a method of treating a P. gingivalis infection in a subject, the method comprising administering to a subject in need thereof a chimeric or fusion protein, composition, vaccine or immunostimulatory composition defined herein, thereby treating the P. gingivalis infection in the subject.

[0116] The present invention provides a method for reducing or minimizing the severity of symptoms associated with infection with P. gingivalis comprising administering to an individual in need thereof a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition defined herein, wherein the symptoms are selected from the group consisting of swollen or puffy gums, gums that bleed easily, receding gums, periodontal pockets around the teeth, loss of supporting tissues for the teeth (periodontal ligament, cementum, and / or alveolar bone), pus between the gums and teeth, and gingivitis.

[0117] The present invention also provides a method for treating a P. gingivalis associated disease in a subject, the method comprising administering to an individual in need thereof a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition as defined herein. Preferably, the P. gingivalis associated disease comprises periodontal disease.

[0118] The invention provides that a method of treatment comprising administration of a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition of the invention may also include administration of one or more of an antibacterial compound, an anti-inflammatory agent.

[0119] The present invention also provides Induce an immune response (preferably a protective immune response) against P. gingivalis in a subject; - immunize subjects against P. gingivalis infection, - Treating a P. gingivalis infection in a subject; minimize or reduce the severity of one or more symptoms of P. gingivalis infection, or - The use of a chimeric or fusion protein as defined herein in the manufacture of a medicament for treating P. gingivalis associated disease in a subject is provided.

[0120] The present invention also provides Induce an immune response (preferably a protective immune response) against P. gingivalis in a subject; - immunize subjects against P. gingivalis infection, - treating a P. gingivalis infection in a subject; minimize or reduce the severity of one or more symptoms of P. gingivalis infection, or - There is provided a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as defined herein for use in treating P. gingivalis associated disease in a subject.

[0121] In any method, use, or protein, composition, or vaccine for use according to the present invention, the subject may be any subject infected with or at risk of infection with P. gingivalis. The subject may be a human. The subject may be a veterinary subject, such as a pet, infected with or at risk of infection with P. gingivalis.

[0122] The invention also provides a method for obtaining antibodies directed against P. gingivalis, the method comprising administering to a non-human animal a chimeric or fusion protein, composition, vaccine or immunostimulatory composition of the invention, thereby producing antibodies directed against P. gingivalis in the animal. Preferably, the method further comprises isolating the antibodies from the animal (e.g. extracting them from the blood of the animal) or from its eggs (if the animal is an avian species, preferably a chicken).

[0123] The invention also provides an antibody preparation comprising antibodies directed against P. gingivalis, the antibody preparation being obtained by administering to a non-human animal a chimeric or fusion protein, composition, vaccine or immunostimulatory composition of the invention, thereby producing antibodies directed against P. gingivalis in the animal, and isolating the antibodies from the animal or its eggs.

[0124] Antibodies directed against P. gingivalis may be used therapeutically to eliminate or reduce a P. gingivalis infection, or prophylactically to prevent or reduce the severity of a P. gingivalis infection.

[0125] The present invention also provides a kit comprising a composition comprising a chimeric or fusion protein as defined herein, optionally wherein the kit also comprises one or more cytokines and / or adjuvants in a sealed container.

[0126] Preferably, the kit includes a label or package insert indicating that the composition is used to immunize an individual, and optionally, the label or package insert includes instructions for use.

[0127] Throughout this specification, unless the context requires otherwise, the words "comprise", "comprises", and "comprising" will be understood to mean the inclusion of a recited step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. Thus, use of a term such as "comprise" indicates that the recited elements are required or essential, but other elements are optional and may or may not be present. "Consisting of" means inclusive and limited to everything following the phrase "consisting of". Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and other elements may or may not be present. "Consisting essentially of" means including any elements listed following the phrase, and limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure for the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are necessary or essential, but that 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 than 20 additional amino acid residues at the N-terminus or C-terminus of the polypeptide sequence) are optional and may or may not be present depending on whether they affect the activity or action of the recited elements.

[0128] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and referring to the accompanying drawings, in which: [Brief description of the drawings]

[0129] [Figure 1-1] A. Mouse periodontitis model - therapeutic vaccination B. Schematic diagram showing the domain structure of the Kgp gingipain polyprotein and components derived therefrom for generating chimeric or fusion proteins of the invention. [Figure 1-2] (As stated above.) [Diagram 2]A. SDS-PAGE of E. coli fractions expressing recombinant variants containing either full-length or truncated DUF domains with or without the K1 domain. Low temperature and low IPTG conditions were used. Full-length DUF is essential for solubility. Full-length K1 adversely affects solubility. B. Native PAGE of chimeric constructs KDA and KDAK1 in the presence and absence of DTT. Addition of the K1 domain to KDA reduces solubility but the protein multimerizes. [Figure 3-1] Elimination of multimerization A. Diagram of Kgp polyprotein showing the location of ABM1 and ABM2 domains. B-D. Native_PAGE analysis of purified recombinant protein variants subjected to electrophoresis in the presence or absence of DTT. B: Study of the "PVQN" mutant variant. C: Study of the "PVQN" mutant variant with an additional Cys>Ser mutation. D: Study of mutations in the loop preceding the "PVQN" motif (lanes 2-4) and of the highly conserved hydrophobic Tyr and Trp residues in ABM2 and ABM1, respectively (lanes 5-7). E. Native PAGE analysis of purified chimeric KDcAK1n and recombinant ABM21 with cysteine ​​and PVQP mutations. KDcAK1n was purified from inclusion bodies and recombinant ABM21 was purified as a soluble protein from an E. coli expression strain. F. Native PAGE analysis of small scale purified recombinant variants. G. (i) 4-12% SDS gels and (ii) 3-12% native gels of various vaccine candidates. Lanes 1 and 2: KDAK-3S-AVQP (1: non-reduced, 2: reduced), lanes 3 and 4: KDAK-1V-2S-AVQP (3: non-reduced, 4: reduced), lanes 5 and 6: KDAK-1V-AVQP (5: non-reduced, 6: reduced), lanes 7 and 8: KDAK-AVQP (7: non-reduced, 8: reduced), lanes 9 and 10: KDcAK1n (9: non-reduced, 10: reduced). Gels were stained with Coomassie blue. [Figure 3-2] (As stated above.) [Figure 3-3] (As stated above.) [Diagram 3-4] (As stated above.) [Figure 4]Bone Loss Study. Statistical Analysis-One-way ANOVA and post-hoc Dunnett's T3. #Significantly different from control loading group. [Diagram 5] Antibody isotype responses of antigens (individual sera) against heat-killed P. gingivalis whole cells. Naïve = control unchallenged. Gingivalis = control challenge with P. gingivalis. Chimeric = KDcAK1n antigen. Amox = amoxicillin. [Figure 6] Bone Loss Study. Statistical Analysis-One-way ANOVA and post-hoc Dunnett's T3. #Significantly different from control loading group. [Figure 7] Statistical analysis of bone loss study - One-way ANOVA and post-hoc Dunnett's T3. #p<0.01, ##p<0.05 (T-test) significantly different from the control loading group. [Figure 8] Bone loss study. S=soluble fraction, Urea=urea used in purification, AC=affinity column purification, IB=inclusion bodies, Batch=batch purification method. Statistical analysis: One-way ANOVA and post-hoc Dunnett's T3. #p<0.01 significantly different from control loading group. [Figure 9] Bone loss study following immunization of animals with various fusion protein constructs. Statistical analysis: One-way ANOVA and post hoc Dunnett's T3 significantly different from control challenged group #p<0.05##p<0.01###p<0.001####p<0.0001. [Figure 10] Bone loss study following immunization of animals with various fusion protein constructs. Statistical analysis: One-way ANOVA and post hoc Dunnett's T3. ###(p<0.05), ####(p<0.01), significantly different from control challenged group. [Figure 11] Bone loss study following immunization of animals with various fusion protein constructs. Statistical analysis: One-way ANOVA and post-hoc Dunnett's T3. #(p<0.05) significantly different from control challenged group. [Figure 12]Bone loss study after immunization of animals with various fusion protein constructs. Except for KDcAK1n, all antigens tested also contain the "AVQP" substitution, i.e., KDAK-3S=KDAK-3S-AVQP, KDAK-1V2S=KDAK-1V2S-AVQP, KDAK-3S-tag=KDAK-3S-AVQP-tag. Statistical analysis: One-way ANOVA and post-hoc Dunnett's T3. *(p<0.05) significantly different from naive (i.e., control unloaded group). [Figure 13-1] Final product purity of P. gingivalis antigens tested in Example 4. PAGE analysis of His-tagged antigens (A and B) and untagged KDAK-3S-AVQP (C). (A) SDS-PAGE, (B) on native PAGE or native gel. (C) on SDS-PAGE and native PAGE or native gel with loaded samples either heated (95° C.) or not heated. R: reduced gel load sample, NR: non-reduced gel load sample, M: protein standard. His-tagged KDAK-3S-AVQP was purified in parallel with other His-tagged proteins. [Figure 13-2] (As stated above.) [Figure 14] Bone loss study following immunization of animals with various fusion protein constructs. Statistical analysis-One-way ANOVA and post hoc Dunnett's T3. # (p<0.05 compared to naive controls (i.e. control unchallenged)), ## (p<0.05 compared to infected controls (i.e. control challenged)). [Figure 15] Antibody titers in individual mouse sera against heat-killed P. gingivalis whole cells. (A) Total IgG titer, (B) IgG1 subtype titer, (C) IgG2a subtype titer. [Figure 16] Dose response trends of exemplary fusion proteins of the invention with protection of periodontal bone loss by Kgpcat cross-reactive Ab titers.

[0130] Sequence information [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0131] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of which various combinations constitute various alternative aspects of the invention.

[0132] Reference will now be made in detail to certain specific embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that it is not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims.

[0133] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used to practice the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these various combinations constitute various alternative aspects of the invention.

[0134] All patents and publications referenced herein are incorporated by reference in their entirety.

[0135] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0136] In the work leading to the present invention, the inventors investigated a variety of chimeric or fusion proteins for use in inducing an immune response to P. gingivalis, and methods for the large-scale production of such chimeras for use as vaccine candidates.

[0137] The present inventors have identified various problems associated with large-scale manufacturing and production of chimeric or fusion proteins derived from components of the P. gingivalis gingipain. First, the chimeric or fusion proteins described in the prior art are difficult to produce as soluble proteins in sufficiently large quantities. More specifically, the chimeric proteins of the prior art are typically produced in inclusion bodies in E. coli, making it difficult to produce large amounts of soluble protein for downstream clinical product development.

[0138] The reduced solubility when produced in E. coli also contributes substantially to the time and scale of production, as well as the reduced stability of the protein once solubilized and refolded from the inclusion bodies.

[0139] Furthermore, gingipain-derived chimeric proteins described in the prior art also suffer from multimerization. The formation of beta-sheets between domains in chimeric proteins not only makes the evaluation of the final vaccine product difficult from a regulatory point of view, but also contributes to a reduction in the potential immune response when administered.

[0140] Thus, the present invention relates to improved design of chimeric or fusion proteins for use in inducing an immune response against P. gingivalis, and methods and uses comprising same.

[0141] In any embodiment, the chimeric or fusion proteins of the present invention have improved solubility and / or stability compared to chimeric or fusion proteins described in the prior art, which provides a major advantage for large-scale production of fusion proteins for use in a clinical environment.

[0142] In an alternative embodiment, the chimeric or fusion proteins of the present invention have a reduced tendency to aggregate and multimerize compared to prior art chimeric or fusion proteins. Aggregation and multimerization can hinder large-scale manufacturing and production of therapeutic / prophylactic proteins. As a result, the reduced tendency of the chimeric proteins of the present invention to aggregate and / or multimerize represents a significant improvement over prior art chimeric proteins for use in inducing an immune response against P. gingivalis.

[0143] Still further, in any embodiment, the chimeric or fusion proteins of the present invention have increased immunogenicity as compared to prior art chimeric or fusion proteins.

[0144] Thus, the inventors have identified a variety of new approaches to obtain chimeric or fusion proteins for inducing an immune response against P. gingivalis, which result in chimeric or fusion proteins that have one or more advantages over the prior art.

[0145] The inventors believe that one or more of the new approaches for generating chimeric or fusion proteins for inducing an immune response against P. gingivalis, as described herein, may provide advantages including increased ease of manufacture, improved shelf life, and / or improved immunogenicity of suitable immunogenic compositions for inducing an immune response against P. gingivalis.

[0146] Ginger Pine The pathogenicity of P. gingivalis is attributed to a number of surface-associated virulence factors, including cysteine ​​proteinases (gingipains), pili, heme-binding proteins, and outer membrane transport proteins, among others. In particular, the extracellular Arg- and Lys-specific proteinases "gingipains" (RgpA / B and Kgp) of P. gingivalis have been implicated as major virulence factors important for colonization, penetration into host tissues, dysregulation of immune responses, dysbiosis, and disease.

[0147] Gingipains, especially the Lys-specific proteinase Kgp, are essential for P. gingivalis to induce alveolar bone resorption in a murine periodontitis model. Gingipains have also been found in gingival tissue at sites of severe periodontitis, at high concentrations proximal to the subgingival plaque and at lower concentrations at deeper distal sites in the gingival tissue. Lys- and Arg-specific proteinases have been shown to degrade various host proteins in vitro, such as fibrinogen, fibronectin, and laminin. The plasma host defense and regulatory proteinase inhibitors α-trypsin, α2-macroglobulin, antichymotrypsin, antithrombin III, and antiplasmin are also degraded by Lys- and Arg-proteinases from P. gingivalis. This has led to the development of a plausible mechanism to explain the important role played by P. gingivalis in the development of chronic periodontitis.

[0148] The RgpA, RgpB, and Kgp genes all encode an N-terminal signal peptide of about 22 amino acids in length, an unusually long propeptide of about 200 amino acids in length, and a catalytic domain of about 480 amino acids. C-terminal to the catalytic domain is a large hemagglutinin adhesin (HA) domain composed of an adhesin binding domain (ABM, or 5 different sequences have been described), a "domain of unknown function" (designated DUF2436, defined as a conserved Pfam domain of unknown function, IPR018832), and a C-terminal adhesin domain or truncated adhesin domain (or CAD). The specific arrangement of the ABM, DUF, and CAD varies between Kgp and RgpA / B.

[0149] The domain structure in the Kgp polyprotein is shown in Figure 1 B. For example, Kgp contains (from N-terminus to C-terminus) a catalytic domain, a first ABM (ABM1), a domain containing DUF2436, ABM2, ABM1, ABM3, two CAD domains (designated K1 and K2), a further domain containing ABM2 and ABM1, a further CAD domain (designated K3), ABM2, and a C-terminal domain.

[0150] As used herein, references to ABM1, 2, and 3 will be understood to generally refer to the ABMs found in the order ABM2, ABM1, and ABM3 in the sequence immediately C-terminal to DUF2436 of Kgp, as shown in FIG. 1B.

[0151] The catalytic domains of RgpB and RgpA share a high degree of sequence homology. However, RgpB lacks an HA domain and is located in a monomeric form on the outer membrane. Some of the HA domains have alternatively been described as C-terminal adhesin domains or truncated adhesin domains (CADs), and some are DUF ("Domain of Unknown Function") 2436 domains (conserved Pfam domain of unknown function, IPR018832).

[0152] The RgpA and Kgp precursor proteins are cleaved into multiple domains that remain non-covalently associated to form a large outer membrane protein complex. Thus, in vivo, the Arg- and Lys-specific proteinases are found in cell-associated complexes of non-covalently associated proteinases and adhesins. One such complex has been designated the RgpA-Kgp proteinase-adhesin complex (previously called the PrtR-PrtK proteinase-adhesin complex). The complex is composed of a 45 kDa Arg-specific calcium-stabilized cysteine ​​proteinase and seven sequence-related adhesin domains.

[0153] As used herein, the Lys-gingipain catalytic domain may also be referred to as a KAS domain or a PAS domain. As used herein, the Arg-gingipain catalytic domain may also be referred to as a RAS domain or a PAS domain. Typically, the catalytic domain of Lys-gingipain or Arg-gingipain is located in the N-terminal approximately 480 amino acid region of the protein. The active site within the catalytic domain is typically located at amino acid residues 426-446 (for RgpA) and 432-453 (for Kgp). Exemplary active site peptides found within the catalytic domain are set forth in Table 1 as SEQ ID NOs: 1-11.

[0154] As used herein, an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis will be understood to refer to a region of Arg- or Lys-gingipain that is typically C-terminal to the catalytic or active site domain. The adhesin domain (also referred to as the HA domain) typically contains a domain of unknown function (DUF) domain (particularly DUF2436 conserved Pfam domain of unknown function, IPR018832), as well as several adhesin binding motif (ABM) domains and a truncated adhesin domain (CAD).

[0155] First Polypeptide The chimeric or fusion proteins of the invention comprise a first polypeptide comprising or consisting of the amino acid sequence of the active site of the Arg-X or Lys-X proteinase of P. gingivalis (also referred to herein as Arg- or Lys-gingipain, respectively), or a sequence that is at least 80% identical thereto.

[0156] In any embodiment, the first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0157] Improving immunogenicity Preferably, the chimeric or fusion protein comprises one or more further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis or a sequence at least 80% identical thereto. The one or more further polypeptides comprising or consisting of the active site of Arg- or Lys-gingipain of P. gingivalis 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 further polypeptides may be linked to the first or second polypeptide of the chimeric or fusion protein via a linker, preferably of 50 amino acids or less, or directly to the first polypeptide.

[0158] The one or more further polypeptides preferably comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-11, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

[0159] In any embodiment, the first polypeptide and the further polypeptide comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis comprise or consist of 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.

[0160] In a preferred embodiment, a chimeric or fusion protein of the invention comprises no more than two, or no more than three, or no more than four polypeptides comprising or consisting of the amino acid sequence of an active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto. Preferably, a chimeric or fusion protein of the invention has fewer than five, more preferably fewer than four, and most preferably fewer than three polypeptides comprising or consisting of the amino acid sequence of an active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto.

[0161] Second Polypeptide A chimeric or fusion protein of the invention comprises a second polypeptide comprising or consisting of the amino acid sequence of the adhesin domain of the ArgX or Lys-X proteinase of P. gingivalis, or a sequence that is at least 80% identical thereto.

[0162] It will be appreciated that the second polypeptide will typically comprise at least a sequence corresponding to one or more of the adhesin binding motifs (ABMs), which are domains / motifs recognised in the adhesin domains of the ArgX or Lys-X proteinases of P. gingivalis (such as Kgp and RgpA).

[0163] There are five ABMs defined for Kgp and RgpA (ABM1-5). Exemplary sequences of these ABMs are listed in Table 1.

[0164] Typically, the second polypeptide comprises two or more ABMs, preferably the second polypeptide comprises at least ABM1 and ABM2, or a sequence at least 80% identical to each of ABM1 or ABM2. The second polypeptide may also comprise the sequence of ABM3, or a sequence at least 80% identical thereto.

[0165] It will also be understood that the arrangement of the ABM peptides in the second polypeptide need not correspond to the arrangement of the ABM peptides found in a naturally occurring adhesin domain. For example, the ABM peptides may be arranged in the second polypeptide in the sequence (N-terminus to C-terminus) ABM1, ABM2, ABM3, etc. Alternatively, the ABM peptides may be arranged to reflect their arrangement in naturally occurring adhesin domains such as ABM2, ABM1, and ABM3, as present in adhesin domain 1 of Kgp.

[0166] Still further, the ABMs in the second polypeptide may be positioned adjacently or separated from one another by an amino acid sequence of 50 amino acids or less. One skilled in the art will appreciate that the spacing between the ABMs is not critical in designing a chimeric or fusion protein of the present invention.

[0167] In any embodiment, the second polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 16, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0168] In any embodiment, the second polypeptide comprises an amino acid sequence set forth in SEQ ID NO:18 or SEQ ID NO:27, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0169] In any embodiment, the second polypeptide comprises an amino acid sequence set forth in SEQ ID NO:22 or SEQ ID NO:27, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0170] Improved solubility The inventors have demonstrated that prior art methods involving simply selecting various domains found in naturally occurring gingipains for use in chimeric or fusion proteins typically result in the production of proteins that are poorly expressed in recombinant systems and have a tendency to be expressed as insoluble proteins (e.g., in inclusion bodies in E. coli).

[0171] With reference to the Examples herein, the inventors recognized that the sequence set forth in SEQ ID NO: 13 (i.e., a truncated form of the CAD domain) represents how the Kgp polyprotein is proteolytically processed and assembled on the cell surface. Thus, inclusion of such a sequence was thought to be important for inducing an immune response against P. gingivalis. However, the inventors surprisingly found that, when present, this sequence contributed to reduced solubility of the recombinant protein and formation of inclusion bodies when expressed in E. coli.

[0172] The inventors attempted to correct this problem by including the complete sequence of CAD in the fusion protein. Surprisingly, this did not improve solubility and actually contributed to lower solubility.

[0173] Thus, the inventors have found that the inclusion of sequences corresponding to one or more truncated adhesin domains (CADs) or portions thereof contributes to reduced solubility of chimeric proteins and vaccine compositions derived from the proposed gingipain sequences.

[0174] Thus, in a particularly preferred embodiment of the invention, the second polypeptide does not comprise the sequence set forth in SEQ ID NO: 13 or a sequence at least 80% identical thereto.

[0175] Alternatively, the second polypeptide does not comprise the sequence set forth in SEQ ID NO: 12, or a sequence at least 80% identical thereto, or a portion thereof.

[0176] The inventors have also found that when a chimeric or fusion protein of the present invention contains only a portion of the DUF2436 domain (i.e., an N-terminally truncated DUF2436 domain), the solubility of the chimeric or fusion protein of the present invention is reduced.

[0177] With reference to the Examples herein, the inventors recognized that prior art approaches to P. gingivalis vaccine design include an N-terminally truncated form of the DUF2436 domain. Although the truncated DUF2436 domain represents how the Kgp polyprotein is proteolytically processed and assembled on the cell surface, the inventors found that providing the full length sequence of the DUF2436 domain (i.e., including the N-terminal portion of the DUF2436 domain) substantially improves production of soluble recombinant protein.

[0178] Thus, in a preferred embodiment, the second polypeptide in a chimeric or fusion protein of the invention comprises residues 1 to 37 of the DUF2436 domain, and preferably the chimeric protein comprises an amino acid sequence that substantially corresponds to the entire length of the DUF2436 domain of Arg- or Lys-gingipain, or a sequence that is at least 80% identical thereto.

[0179] As used herein, an amino acid sequence substantially corresponding to the entire length of the DUF2436 domain, or a sequence at least 80% identical thereto, refers to a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the DUF2436 domain of an Arg- or Lys-gingipain.

[0180] Preferably, the DUF2436 domain comprises the sequence set forth in SEQ ID NO:23, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0181] Reduced multimerization The inventors have also demonstrated that various domains in gingpains have a tendency to multimerize, which, when multimerized or aggregated, contributes to reduced solubility and reduced immunogenicity of chimeric or fusion proteins. Thus, multimer formation has implications for the ease of large-scale production and use as vaccines of chimeric proteins derived from gingpain sequences, but may also affect the generation of protective responses.

[0182] In particular, we identified two mechanisms for multimerization: formation of disulfide bridges between cysteine ​​residues and beta-strand exchange between ABM domains. Cross-referencing the schematic showing the various domains of Kgp in FIG. 1B, we hypothesize that ABM1 may interact with its next available neighbor ABM2 during folding of the Kgp polyprotein in vivo. Separately, we show that co-expression of recombinant ABM1 and ABM2 as separate proteins was able to form a stable beta-sheet complex structure.

[0183] Therefore, to reduce multimerization of the chimeric proteins proposed for use according to the invention, we targeted cysteine ​​residues in the DUF and ABM domains, in addition to specific motifs within ABM2 and 1 that may be involved in beta-sheet formation.

[0184] The inventors have identified that one or more of the following modifications contribute to reduced multimerization of chimeric fusion proteins derived from the Kgp and Rgp polyproteins: c) comprising one or more cysteine ​​amino acid substitutions in the amino acid sequences of the DUF2436 domains and ABMs compared to naturally occurring Arg- or Lys-gingipain sequences in the corresponding regions; and / or d) containing one or more amino acid motif substitutions selected from the following: i) substitution of proline and / or asparagine residues in the sequence PxxN at positions corresponding to or equivalent to residues 6 to 9 of SEQ ID NO: 14 or 19 (ABM1); ii) a substitution of the motif NxFA with SxYQ in the sequence at a position corresponding to or equivalent to residues 2 to 5 of SEQ ID NO: 14 or 19 (ABM1); iii) Substitution of a second tyrosine residue at a position corresponding to or equivalent to residue 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue at a position corresponding to or equivalent to residue 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue.

[0185] Thus, 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.

[0186] Cysteine ​​residues are found (at least) in the DUF2436 domain, ABM2, and the K1 CAD sequence in the P. gingivalis Kgp sequence. It will therefore be appreciated that the present invention contemplates chimeric or fusion proteins in which one or more, two or more, or three or more cysteine ​​residues have been substituted to reduce, minimize, or abolish the formation of disulfide bridges.

[0187] The cysteine ​​amino acid substitutions may be to serine or valine residues. Preferably, the one or more cysteine ​​substitutions include one or more to serine residues.

[0188] In a particularly preferred embodiment, the cysteine ​​residues in the DUF2436 domain are not substituted, but one or more cysteine ​​residues in the region of the adhesin domain between the C-terminus of the DUF2436 domain and the N-terminus of the K1 CAD domain may be substituted. An exemplary sequence of the region between the DUF domain and the CAD domain is set forth in SEQ ID NO: 18. In a preferred embodiment of the present invention, one or both of the cysteine ​​residues at positions 36 and 50 or equivalent are optionally substituted with serine or valine residues, preferably serine residues. An exemplary sequence of the DUF2436 domain+ABM2+1+3 region is set forth in SEQ ID NO: 34. In a preferred embodiment of the present invention, one or both of the cysteine ​​residues at positions 208 and 222 or equivalent are optionally substituted with serine or valine residues, preferably serine residues.

[0189] In an alternative embodiment, the cysteine ​​residue in the DUF2436 domain is replaced with a serine or valine residue, preferably a serine residue, and one or both cysteine ​​residues in the region of the adhesin domain between the C-terminus of the DUF2436 domain and the N-terminus of the K1CAD domain may be replaced. An exemplary sequence of the region between the DUF domain and the CAD domain is set forth in SEQ ID NO: 18. In a preferred embodiment of the invention, one or both of the cysteine ​​residues at positions 36 and 50 or equivalent are optionally replaced with a serine or valine residue, preferably a serine residue. An exemplary sequence of the DUF2436 domain+ABM2+1+3 region is set forth in SEQ ID NO: 34. In a preferred embodiment of the invention, one or both of the cysteine ​​residues at positions 115 and 208 and 222 or equivalent are optionally replaced with a serine or valine residue, preferably a serine residue.

[0190] Thus, in a particularly preferred embodiment, the chimeric or fusion protein of the invention comprises an amino acid sequence corresponding to any one of SEQ ID NOs: 35 to 49, or a sequence at least 80% identical thereto (excluding cysteine ​​substitutions therein).

[0191] The inventors further demonstrated that multimerization was also reduced by mutation of conserved motifs present in ABM domains 2, 1, and 3.

[0192] In one example, the inventors investigated domain modifications at the N-terminus of the adhesin domain ABM1. More specifically, it was found that substitution of the motif PxxN (e.g. PVQN in ABM1 of P. gingivalis Kgp as set forth in SEQ ID NO: 14) contributes substantially to reduced multimerization between ABM domains and reduced beta-strand exchange.

[0193] Thus, in a preferred embodiment, the chimeric or fusion protein of the invention comprises a modification of the ABM1 PxxN motif in a region of the chimeric or fusion protein which corresponds to the adhesin domain of P. gingivalis gingipain.The second polypeptide thus preferably comprises a proline substitution and an asparagine substitution in the sequence PxxN at positions corresponding to or equivalent to positions 6 to 9 of SEQ ID NO: 14 or 19 which define ABM1.

[0194] The proline amino acid substitution is preferably a substitution to an alanine residue.

[0195] The asparagine amino acid substitution may be with a proline or an alanine residue. Preferably, the asparagine residue is substituted with a proline residue. In other embodiments, the asparagine residue is not substituted.

[0196] In a further example, the inventors considered the motif NEFA in the sequence of ABM1 in the Kgp / Rgp polyprotein, which sequence is defined herein as residues 2-5 of SEQ ID NOs: 14 and 19. As further shown in the examples, modification of the motif NEFA to SEQ ID NO: 14 via replacement of asparagine, phenylalanine, and alanine residues with serine, glutamine, and tyrosine, respectively, significantly reduces multimerization.

[0197] In another example, the inventors determined that substitution of a tyrosine residue in ABM2 at a position corresponding to or equivalent to residue 5 of SEQ ID NO: 15 or 20, and a tryptophan residue in ABM1 at a position corresponding to or equivalent to residue 23 of SEQ ID NO: 14 or 19 with an alanine residue also significantly reduced multimerization.

[0198] Finally, the inventors found that multimerization was substantially eliminated through a combination of cysteine ​​modifications with one or more of the following substitutions: i) substitution of proline and / or asparagine residues in the sequence PxxN at positions corresponding to or equivalent to residues 6 to 9 of SEQ ID NO: 14 or 19 (ABM1); ii) a substitution of the motif NxFA with SxYQ in the sequence at a position corresponding to or equivalent to residues 2 to 5 of SEQ ID NO: 14 or 19 (ABM1); iii) Substitution of a second tyrosine residue at a position corresponding to or equivalent to residue 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue at a position corresponding to or equivalent to residue 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue.

[0199] In a particularly preferred embodiment, the inventors found that a combination of one or more cysteine ​​modifications, preferably at least two cysteine ​​substitutions to serine, and a modification of the PXXN motif of ABM1 to AXXP eliminated multimerization of the resulting recombinant chimeric protein.

[0200] Thus, taking the cysteine ​​modifications of the ABM1 domain and the modifications of the PXXN motif together, the invention provides a chimeric or fusion protein as described herein, wherein the second polypeptide of the chimeric protein corresponds to a region of the adhesin domain of P. gingivalis Arg or Lys gingipain and comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 34, or a sequence at least 80% identical thereto, in which one, two or three cysteine ​​residues have been replaced by serine residues, and / or the proline and asparagine residues in the sequence PXXN at positions 235 to 238 or equivalent thereto have been replaced.

[0201] Preferably, the cysteine ​​residue at residue 115 of SEQ ID NO:34, or an equivalent position, is not substituted with either a serine or a valine residue.

[0202] Preferably, the cysteine ​​residue at position 115 of SEQ ID NO:34, or an equivalent position, is not substituted with either a serine or a valine residue, the cysteine ​​residues at positions 208 and 222 of SEQ ID NO:34, or equivalent positions, are substituted with a serine residue, the proline residue at position 235, or an equivalent position, is substituted with an alanine residue, and the asparagine residue at position 238, or an equivalent position, is substituted with a proline residue.

[0203] Linking the First and Second Polypeptides In a chimeric or fusion protein of the invention, the C-terminal residue of a first polypeptide may be covalently linked to the N-terminal residue of a second polypeptide (corresponding to the adhesin domain polypeptide), or the N-terminal residue of a first peptide may be covalently linked to the C-terminal residue of a second polypeptide (corresponding to the adhesin domain polypeptide). In this arrangement, the first peptide and the adhesin domain polypeptide are said to be "directly linked" or "adjacent".

[0204] In other embodiments, the chimeric or fusion protein comprises a linker for linking the first peptide to the adhesin domain polypeptide. The linker can be any linker capable of connecting a peptide to a polypeptide, including both amino acid and non-amino acid linkers.

[0205] Preferably, the linker is non-immunogenic. Typically, the linker is composed of amino acids and may therefore be referred to as a peptide linker.

[0206] The linker is usually a peptide having a length of up to 20 amino acids, but may be longer. The term "linked to" or "fused to" refers to a covalent bond, e.g., a peptide bond, formed between two moieties. Thus, in the context of the present invention, the linker may have a length of 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, a chimeric or fusion protein provided herein may include a linker between a first polypeptide that includes or consists of an amino acid sequence of a P. gingivalis gingipain active domain and a second polypeptide that corresponds to an adhesin domain of P. gingivalis gingipain, such as between the N-terminus of the second polypeptide and the C-terminus of the first polypeptide. Such a linker has the advantage that it can increase the likelihood that the different polypeptides of the fusion protein will fold independently and act predictably. A suitable linker may be up to 50 amino acids in length, but is preferably less than 20, 15, or 5 amino acids. The linker may function to bring the first peptide and the adhesin domain polypeptide into closer spatial arrangement than is normally observed in P. gingivalis trypsin-like enzymes. Alternatively, it may space the first polypeptide and the second polypeptide (corresponding to the adhesin domain polypeptide).

[0207] Suitable linkers for use in protein constructs, including those that have minimal impact on solubility, are known in the art. The linker can be any linker known to those skilled in the art, and can be a flexible linker (such as one that includes repeats of glycine and serine residues), a rigid linker (such as one that includes repeats of glutamic acid and lysine residues, adjacent alanine), and / or a cleavable linker (such as a sequence that is 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.

[0208] Useful linkers include glycine-serine (GlySer) linkers, which are well known in the art and contain glycine and serine units arranged in various orders. Examples include, but are not limited to, (GS), (GSGGS)n, (GGGS)n, and (GGGGS)n, where n is an integer of at least 1, typically 1 to about 10, e.g., 1 to about 8, 1 to about 6, or 1 to about 5.

[0209] In some embodiments, the peptide linker may comprise the amino acids glycine and serine of various lengths and combinations. In some aspects, the peptide linker may comprise the sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS), or Gly-Gly-Gly-Ser (GGGGS) and variations or repeats thereof. In some aspects, the peptide linker may comprise the amino acid sequence GGGGS (a 6 amino acid long linker) or more. The linker is a series of repeats of glycine and serine residues (GS) of different lengths, i.e., (GS)n, where n can be any number from 1 to 15 or more. For example, the linker may be (GS)3 (i.e., GSGSGS) or longer (GS)11 or more. It will be understood that n can be any number, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more. Fusion proteins with linkers of such lengths are within the scope of the present invention. Similarly, the linker can be a series of repeated glycine residues separated by serine residues, for example, (GGGGS)3 (i.e., the linker can comprise the amino acid sequence GGGSGGGGSGGGGS(G4S)3) and variations thereof.

[0210] In one embodiment, the peptide linker may comprise the amino acid sequence GGGGS (a 6 amino acid long linker) or longer. The linker is a repeating series of glycine and serine residues (GS) of different lengths, i.e., (GS)n, where n can be any number from 1 to 15 or more. For example, the linker may be (GS)3 (i.e., GSGSGS) or longer (GS)11 or more. It will be understood that n can be any number including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more.

[0211] Other useful linkers include DSSG, DSSGAS, KLDSSG, and variations thereof. Examples of other suitable linkers are described in Chen et al., (2013) Advanced Drug Delivery Reviews, 65:1357-1369.

[0212] Chimeric or Fusion Proteins and Recombinant Proteins A chimeric or fusion protein of the invention can be prepared by any of a number of conventional techniques, although typically the polypeptide will be made using recombinant techniques.

[0213] For recombinant polypeptides, a DNA fragment encoding the desired peptide can be subcloned into an appropriate vector using well-known molecular genetic techniques (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1982); Sambrook et al., Molecular Cloning A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1989). The fragment can be transcribed in vitro and the polypeptide can then be translated. Commercially available kits can also be used (such as those manufactured by, e.g., Clontech, Palo Alto, Calif.; Amersham Pharmacia Biotech Inc., Piscataway, NJ; InVitrogen, Carlsbad, Calif., etc.). The polymerase chain reaction can optionally be used in the manipulation of nucleic acids.

[0214] A "fragment" is a portion of a polypeptide of the invention that retains substantially similar functional activity or substantially the same biological function or activity as the polypeptide, as can be determined using the assays described herein.

[0215] "Percent amino acid sequence identity" or "percent identity" with respect to a polypeptide sequence, i.e., a polypeptide of the invention as defined herein, is defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular polypeptide of the invention, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not taking into account any conservative substitutions as part of the sequence identity. Reference herein to a variant having "at least x% sequence identity" to a recited sequence means that the variant is at least x% identical to the recited sequence.

[0216] In various aspects and embodiments of the invention, defined polypeptides are described by reference to variants having at least 80% homology or more to a reference sequence. Percentage of homology (%) generally refers to a 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 particular polypeptide of the invention, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity.

[0217] The amino acids glycine, alanine, valine, leucine, and isoleucine can often be substituted for one another (amino acids with aliphatic side chains). Of these possible substitutions, glycine and alanine are preferably used to replace one another (because they have relatively short side chains), and valine, leucine, and isoleucine are preferably used to replace one another (because they have larger aliphatic side chains that are hydrophobic). Other amino acids that can often be substituted for one another 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).

[0218] Substitutions of this nature are often referred to as "conservative" or "semi-conservative" amino acid substitutions.

[0219] Amino acid deletions or insertions can also be made to the native sequence of P. gingivalis protein. Thus, for example, amino acids can be deleted that do not substantially affect the activity of the polypeptide, or at least do not eliminate such activity. Such deletions can be advantageous, especially for long polypeptides, since they can reduce the total length and molecular weight of the polypeptide while still retaining activity. This can reduce the amount of polypeptide required for a particular purpose, for example, reducing dosage levels.

[0220] Amino acid insertions into the sequence of a native polypeptide may also be made in order to alter the properties of the polypeptide for use in the present invention (e.g., to improve antigenicity).

[0221] The amino acid changes can be made using any suitable technique, for example, using site-directed mutagenesis or solid phase synthesis.

[0222] It is to be understood that amino acid substitutions or insertions within the scope of the present invention may be made using naturally occurring or non-naturally occurring amino acids. Whether natural or synthetic amino acids are used, it is preferred that only L-amino acids are present.

[0223] Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms (non-limiting examples of which are described below) necessary to achieve maximum alignment over the entire length of the sequences being compared. When amino acid sequences are aligned, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, it can be expressed as a given amino acid sequence A having or containing a certain percent amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as follows: percent amino acid sequence identity=X / Y100, where X is the number of amino acid residues scored as identical matches by the alignment of A and B of a sequence alignment program or algorithm, 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, the percent amino acid sequence identity of A to B is not equal to the percent amino acid sequence identity of B to A.

[0224] When calculating percent identity, typically exact matches are counted. Determining percent identity between two sequences can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm used to compare two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. MoI. Biol. 215:403. To obtain gapped alignment for comparison purposes, Gapped BLAST (in BLAST2.0) can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When using BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment can also be performed manually by inspection. Another non-limiting example of a mathematical algorithm used to compare sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW can compare sequences and align entire amino acid or DNA sequences, thus providing data on sequence conservation of 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 (Invitrogen Corporation, Carlsbad, CA). After alignment of amino acid sequences with ClustalW, percent amino acid identity can be assessed.A non-limiting example of a software program useful for analyzing ClustalW alignments is GENEDOC™ or JalView (http: / / www.jalview.org / ). GENEDOC™ allows the assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, version 10 (Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When utilizing the ALIGN program to compare amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0225] Polypeptides optionally contain amino and carboxyl termini. Polypeptides may contain D-amino acids, L-amino acids, or a mixture of D- and L-amino acids. However, the D-form of amino acids is particularly preferred, as polypeptides composed of D-amino acids are expected to retain their biological activity in vivo to a greater extent.

[0226] The term "conservative substitution" as used herein refers to the replacement of an amino acid present in a native sequence in a peptide with a naturally occurring or non-natural amino acid or peptidomimetic having similar steric properties. If the side chain of the substituted native amino acid is either polar or hydrophobic, then the conservative substitution should be a naturally occurring amino acid, non-natural amino acid, or peptidomimetic moiety that is also polar or hydrophobic (in addition to having the same steric properties as the side chain of the substituted amino acid).

[0227] Conservative amino acid substitution tables providing functionally similar amino acids are well known to those of skill in the art. The following six groups are examples of amino acids that may be considered conservative substitutions for one another: 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).

[0228] Naturally occurring amino acids are typically grouped according to their properties, so that conservative substitutions with naturally occurring amino acids can be determined, bearing in mind the fact that the substitution of a charged amino acid with a sterically similar uncharged amino acid is considered as a conservative substitution. It is also possible to use amino acid analogs (synthetic amino acids) well known in the art to create conservative substitutions with non-natural amino acids. Peptide mimetics of naturally occurring amino acids are well described in the literature known to those skilled in the art, and non-natural or unnatural amino acids are described further below. When affecting a conservative substitution, the substituted amino acid should have the same or similar functional group in the side chain as the original amino acid.

[0229] As used herein, the phrase "non-conservative substitution" or "non-conserved residue" refers to the replacement of an amino acid present in a parent sequence with another naturally occurring or non-natural amino acid having different electrochemical and / or steric properties. Thus, the side chain of the substituting amino acid may be significantly larger (or smaller) than the side chain of the natural amino acid being substituted and / or may have a functional group with significantly different electronic properties than the amino acid being substituted. Examples of this type of non-conservative substitution include the substitution of phenylalanine or cyclohexylmethylglycine for alanine, the substitution of isoleucine for glycine, or the substitution of -NH-CH[(-CH2)5-COOH]-CO- for aspartic acid. Non-conservative substitutions include any mutation that is not considered conservative.

[0230] Non-conservative amino acid substitutions can result from changes in: (a) the structure of the amino acid backbone in the area of ​​substitution, (b) the charge or hydrophobicity of the amino acid, or (c) the bulk of the amino acid side chain. In general, the substitutions expected to produce the greatest changes in protein properties are those in which: (a) a hydrophilic residue is substituted for (or by) a hydrophobic residue, (b) a proline is substituted for (or by) any other residue, (c) a residue with a bulky side chain, e.g., phenylalanine, is substituted for (or by) a residue with no side chain, e.g., glycine, or (d) a residue with a positively charged side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) a negatively charged residue, e.g., glutamyl or aspartyl.

[0231] Modification of the native amino acid sequence to produce a mutant polypeptide, such as by insertion, deletion, and / or substitution, can be performed by various means known to those skilled in the art. For example, site-specific mutations can be introduced by ligating synthetic oligonucleotides containing the modification 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 means for introducing mutations is the QuikChange Site-Directed Mutagenesis Kit (Stratagene, LaJolla, Calif.).

[0232] Any suitable expression vector (e.g., those described in Pouwels et al., Cloning Vectors: A Laboratory Manual (Elsevier, NY: 1985)) and corresponding suitable host may be used to produce recombinant polypeptides. Expression hosts include, but are not limited to, bacterial species within the genera Escherichia, Bacillus, Pseudomonas, Salmonella, mammalian or insect cell systems including baculovirus systems (e.g., those described by 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 recognize that the choice of expression host will have consequential effects on the type of polypeptide produced. For example, glycosylation of polypeptides produced in yeast cells or mammalian cells (e.g., COS-7 cells) will differ from glycosylation of polypeptides produced in bacterial cells such as Escherichia coli.

[0233] 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)). In particular, the polypeptides can be synthesized using solid-phase synthesis procedures (see, e.g., Merrifield, J. Am. Chem. Soc. 85: 2149-54 (1963); Barany et al., Int. J. Peptide Protein Res. 30: 705-739 (1987); and U.S. Pat. No. 5,424,398). If necessary, this can be done using an automated peptide synthesizer. Removal of t-butyloxycarbonyl (t-BOC) or 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking groups and separation of the polypeptide from the resin can be accomplished, for example, by acid treatment at low temperature. The polypeptide-containing mixture can then be extracted, for example, with dimethyl ether to remove non-peptidic organic compounds, and the synthesized polypeptide can be extracted from the resin powder (e.g., with about 25% w / v acetic acid). After synthesis of the polypeptide, further purification (e.g., using high performance liquid chromatography (HPLC)) can be optionally performed to eliminate any incomplete polypeptides or free amino acids. Amino acid and / or HPLC analysis can be performed on the synthesized polypeptide to verify its identity. For other uses according to the invention, it may be preferable to produce the polypeptide as part of a larger fusion protein, such as by methods described herein or other genetic means, or as part of a larger conjugate, such as via physical or chemical conjugation, known to those skilled in the art and described herein.

[0234] In any embodiment of the invention, the chimeric or fusion protein of the invention may comprise additional amino acid residues to facilitate expression in a recombinant expression system and / or to facilitate purification of the protein. Thus, the protein as defined herein may comprise additional amino acids, such as 1, 2, 3, 4, or 5 amino acids in the N-terminal region. Typically, the additional amino acids comprise an N-terminal methionine to facilitate expression in a recombinant expression system, although it will be understood that typically such N-terminal residues are cleaved after translation of the protein. In certain embodiments, the N-terminal amino acids comprise at least methionine and alanine residues.

[0235] Furthermore, chimeric or fusion proteins according to the invention may preferably comprise additional amino acids, such as 1, 2, 3, 4 or 5 amino acids in the N-terminal or C-terminal regions to facilitate purification. It will be appreciated that such amino acid residues may facilitate the inclusion of a purification tag (such as a histidine tag) in the protein. Such residues may not be included if an untagged version of the protein is produced.

[0236] The polypeptides of the invention can also be modified by conjugation or fusion to another moiety to facilitate purification or to increase the in vivo half-life of the polypeptide, or for use in immunoassays using methods known in the art. For example, the polypeptides of the invention can be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cellular ligands or other proteins, and the like.

[0237] nucleic acid Nucleic acid molecules encoding any of the chimeric or fusion proteins or polypeptides of the invention are also within the scope of the invention. Nucleic acids are useful, for example, in making the polypeptides of the invention and as therapeutic agents. They can be administered to cells in culture or in vivo and can include secretion signals that direct or promote secretion of the polypeptides of the invention from the cells. Expression vectors and host cells that contain or include the nucleic acids of the invention are also within the scope of the invention (described further below). The nucleic acids of the invention can be referred to as "isolated", but by definition the polypeptides of the invention will not be wild-type polypeptides and therefore will not be encoded by naturally occurring nucleic acids. Thus, the polypeptides and nucleic acids of the invention can be "purified", "substantially purified", "isolated", "recombinant", or "synthetic", but need not necessarily be such in order to be distinguished from naturally occurring substances.

[0238] An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Thus, an isolated nucleic acid molecule is distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes nucleic acid molecules that are ordinarily contained in cells that express Kgp, e.g., the nucleic acid molecule is in a chromosomal location different from that of natural cells.

[0239] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to polymeric forms of nucleotides of any length, either 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 invention may be provided in isolated or purified form. A nucleic acid sequence that "encodes" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For purposes of the present invention, such nucleic acid sequences include, but are not limited to, cDNA from viral, prokaryotic, or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, as well as synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0240] Polynucleotides of the invention can be synthesized according to methods well known in the art, for example as described in Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).

[0241] The polynucleotide molecules of the present invention can be provided in the form of expression cassettes that contain control sequences operably linked to the inserted sequence, thus allowing the in vivo expression of the polypeptides of the present invention in the target subject. These expression cassettes are then typically provided in vectors (e.g., plasmids or recombinant viral vectors) suitable for use as nucleic acid immunization reagents. Such expression cassettes can be administered directly to the host subject. Alternatively, vectors containing the polynucleotides of the present invention can be administered to the host subject. Preferably, the polynucleotides are prepared and / or administered using genetic vectors. A suitable vector can be any vector that can carry a sufficient amount of genetic information and allow the expression of the polypeptides of the present invention.

[0242] The present invention therefore includes expression vectors comprising such polynucleotide sequences.The present invention therefore provides a vector for use in the prevention or treatment of an inflammatory disease or condition comprising a polynucleotide sequence encoding a polypeptide of the present invention and, optionally, one or more further polynucleotide sequences encoding different polypeptides as defined herein.

[0243] It will further be understood that the compositions and products of the invention may comprise a mixture of polypeptides and polynucleotides. Thus, the invention provides a composition or product as defined herein which, in place of any one of the polypeptides, is a polynucleotide capable of expressing said polypeptide.

[0244] 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, for example, polyadenylation signals, which may be required and are positioned in the correct orientation, to allow expression of the peptides of the present invention. Other suitable vectors will be apparent to those skilled in the art. For further examples in this regard, see Sambrook et al.

[0245] Thus, a polypeptide of the invention can be provided by delivering such a vector to a cell and allowing transcription from the vector to occur. Preferably, a polynucleotide of the invention or for use in the invention in a vector is operably linked to a control sequence capable of providing for expression of the coding sequence by the host cell, i.e. the vector is an expression vector.

[0246] "Operably linked" refers to an arrangement of elements in which the components so described are configured to perform their normal functions. Thus, a given regulatory sequence, such as a promoter, operably linked to a nucleic acid sequence can effect expression of that sequence when the appropriate enzymes are present. The promoter need not be contiguous with the sequence, so long as it functions to direct its expression. Thus, for example, an intervening untranslated but transcribed sequence can be present between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0247] Many expression systems have been described in the art, each of which typically consists of a vector containing a gene or nucleotide sequence of interest operably linked to an expression control sequence. These control sequences include a transcription promoter sequence and transcription start and stop sequences. The vector of the invention can be, for example, a plasmid, virus or phage vector having an origin of replication, optionally a promoter for the expression of the polynucleotide, and optionally a regulator of the promoter. A "plasmid" is a vector in the form of an extrachromosomal genetic element. The vector can contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a resistance gene for a fungal vector. The vector can be used in vitro, for example for the production of DNA or RNA, or can be used to transfect or transform a host cell, for example a mammalian host cell. The vector can also be adapted to be used in vivo, for example to allow the in vivo expression of a polypeptide.

[0248] A "promoter" is a nucleotide sequence that initiates and regulates transcription of a polynucleotide encoding a polypeptide. Promoters can include inducible promoters (expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. The term "promoter" or "regulatory element" is intended to include full-length promoter regions as well as functional (e.g., transcription or translation controlling) segments of these regions.

[0249] The polynucleotide, expression cassette or vector according to the invention may further comprise a signal peptide sequence, which is generally inserted in operable linkage with a promoter such that the signal peptide is expressed and promotes secretion of a polypeptide encoded by the coding sequence also in operable linkage with the promoter.

[0250] Typically, a signal peptide sequence encodes a peptide of 10-30 amino acids, e.g., 15-20 amino acids. Often, the amino acids are predominantly hydrophobic. In a typical situation, the signal peptide targets the growing polypeptide chain bearing the signal peptide to the endoplasmic reticulum of the expressing cell. The signal peptide is cleaved off in the endoplasmic reticulum, allowing secretion of the polypeptide via the Golgi apparatus.

[0251] Immunogenic and Vaccine Compositions The present invention further provides compositions comprising chimeric or fusion proteins as defined herein, as well as the use of such chimeric or fusion proteins in immunogenic or vaccine compositions in the treatment or prevention of P. gingivalis infection.

[0252] The term "vaccine composition" as used herein is defined as a composition used to induce an immune response against an antigen (immunogen) within the composition in order to protect or treat an organism against disease.

[0253] As used herein, the terms "immunostimulatory composition," "vaccine composition," and "immunogenic composition" may generally be used interchangeably.

[0254] The immunostimulatory compositions or vaccines of the invention may suitably contain, in addition to one or more peptides of the invention as therapeutic or prophylactic active ingredients, a pharma- ceutically acceptable carrier, excipient, diluent, adjuvant, vehicle, buffer, or stabilizer, including, but not limited to, saline, buffered saline, dextrose, liposomes, water, glycerol, polyethylene glycol, ethanol, and combinations thereof.

[0255] The immunostimulatory or vaccine composition may be adapted for administration by any suitable route, for example, parenteral (including subcutaneous, intramuscular, intravenous or intradermal, or injection into the cerebrospinal fluid), oral (including buccal or sublingual), nasal, topical (including buccal, sublingual or transdermal), vaginal or rectal route. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the peptide with a carrier or excipient under sterile conditions. Typically, the vaccine composition is adapted for administration by subcutaneous, intramuscular, intravenous or intradermal route, typically by injection. Alternatively, the vaccine composition may be adapted for oral or nasal administration.

[0256] Immunostimulatory or vaccine compositions adapted for parenteral administration may be aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation substantially isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. Excipients that may be used for injectable solutions include, for example, water, alcohols, polyols, glycerin, and vegetable oils. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state requiring only the addition of a sterile liquid to be delivered, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0257] Immunostimulatory or vaccine compositions adapted for oral administration may be presented as discrete units such as capsules or tablets or lozenges, as a powder or granules, as a solution, syrup or suspension (in an aqueous or non-aqueous liquid, or as an edible foam or whip, or as an emulsion).

[0258] Suitable excipients for tablets or hard gelatin capsules include lactose, corn starch or its derivatives, stearic acid or its salts. Excipients suitable for use with soft gelatin capsules include, for example, vegetable oils, waxes, fats, semi-solid or liquid polyols, etc.

[0259] For the preparation of solutions and syrups, excipients which 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.

[0260] Immunostimulatory or vaccine compositions adapted for nasal administration where the carrier is a solid include a coarse powder having a particle size in the range, for example, 20 to 500 microns, administered in the manner of a nasal inhaler, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose. Suitable compositions where the carrier is a liquid for administration as a nasal spray or nasal drops may include aqueous or oily solutions of the active ingredient.

[0261] Compositions adapted for administration by inhalation include fine particle dusts or mists, which may be generated by means of various types of metered dose pressurized aerosols, nebulizers, or insufflators.

[0262] Immunostimulatory or vaccine compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis, as generally described in Pharmaceutical Research. 3(6):318 (1986).

[0263] Compositions adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions (e.g. mouthwash), pastes, gels, sprays, aerosols, or oils. In the case of infections of the eye or other external tissues, such as the mouth and skin, the compositions can be applied as topical ointments or creams. When formulated in an ointment, the active ingredient can be used with either a paraffinic or water-miscible ointment base. Alternatively, the active ingredient can be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye can include eye drops, in which the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth can include lozenges, pastes, or mouthwashes.

[0264] The immunostimulatory or vaccine composition may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts (the substances of the invention themselves may be provided in the form of pharma- ceutically acceptable salts), buffers, coating agents, or antioxidants.

[0265] The vaccine compositions of the invention may also contain, in addition to the chimeric or fusion protein as defined herein, one or more other prophylactically or therapeutically active agents.

[0266] Chimeric or fusion proteins for use in the vaccine compositions of the present invention may or may not be lyophilized.

[0267] The vaccine composition of the present invention may also comprise, in addition to the peptides defined herein, a pharma- ceutically acceptable adjuvant. Adjuvants are added to enhance the immunogenicity of the vaccine composition.

[0268] Adjuvants suitable for inclusion in the vaccine composition are known in the art and include incomplete Freund's adjuvant, complete Freund's adjuvant, Freund's adjuvant with MDP (muramyl dipeptide), alum (aluminum hydroxide), alum plus Bordatella pertussis and immune stimulating complexes (ISCOMs, typically a matrix of Quil A containing viral proteins), QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-arretin, MPC-026, Adjuvax, CpG ODN, Betafectin, and MF59.

[0269] The vaccine compositions of the invention may also comprise or be administered in conjunction with one or more costimulatory molecules.

[0270] The dosage of the vaccine composition of the present invention can vary within a wide range depending on the age and condition of the individual to be treated, and the doctor will ultimately determine the appropriate dosage to be used.

[0271] This dosage may be repeated as often as necessary, for example an initial dose of vaccine may be administered and then a booster dose may be administered at later dates.

[0272] For administration to mammals, particularly humans, the daily dosage of the active agent is expected to be between 1 μg / kg and 10 mg / kg body weight, typically about 10 μg / kg and 1 mg / kg body weight. In any event, the physician will determine the actual dosage most suitable for an individual, which will depend on factors including the individual's age, weight, sex, and response. The dosages listed above are illustrative of the average case. Of course, there may be cases where higher or lower dosages are required, and such cases are within the scope of the present invention.

[0273] The vaccine compositions of the invention may be administered by any convenient route as described herein, such as intramuscularly, intravenously, by inhalation, intraperitoneally or orally, or by injection into the cerebrospinal fluid.

[0274] The vaccine composition of the invention may be provided in a unit dosage form, generally in a sealed container, and may be provided as part of a kit. Such a kit will usually (but not necessarily) include instructions for use. It may contain a plurality of said unit dosage forms.

[0275] Thus, in a further aspect, the present invention provides a kit-of-parts comprising in a sealed container the vaccine composition of the invention and one or more cytokines and / or adjuvants.

[0276] Methods for immunizing a subject using a subject chimeric or fusion protein The present invention provides methods and compositions for treating or preventing infection or minimizing the likelihood of infection by P. gingivalis in an individual in need thereof, which methods comprise administering a fusion or chimeric protein of the invention.

[0277] The present invention further provides methods and compositions for inducing a humoral immune response against P. gingivalis in a subject. The humoral response may be for the purpose of obtaining protective / therapeutic anti-P. gingivalis antibodies directly in the individual in need of protection / treatment. Alternatively, the humoral response may be for the purpose of generating antibodies that are then isolated from the subject (or its eggs) so that they can be directly administered to the individual in need of treatment / protection with the antibodies.

[0278] Thus, the present invention includes methods and compositions for preventing infection with P. gingivalis, minimizing the likelihood of infection, and / or reducing the severity and duration of a P. gingivalis infection in a subject.

[0279] The invention also provides a method for obtaining antibodies directed against P. gingivalis, the method comprising administering to a non-human animal a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition of the invention, thereby producing antibodies directed against P. gingivalis in the animal. Preferably, the method further comprises isolating the antibodies from the animal (e.g., from the blood of the animal) or from the eggs of the animal (e.g., when producing IgY antibodies from chickens).

[0280] The invention also provides an antibody preparation comprising antibodies directed against P. gingivalis, the antibody preparation being obtained by administering to a non-human animal a chimeric or fusion protein, composition, vaccine or immunostimulatory composition of the invention, thereby producing antibodies directed against P. gingivalis in the animal, and isolating the antibodies from the animal or its eggs.

[0281] Antibodies directed against P. gingivalis may be used therapeutically to eliminate or reduce a P. gingivalis infection, or prophylactically to prevent or reduce the severity of a P. gingivalis infection.

[0282] As used herein, the term "treatment" or "treating" of a subject includes the application or administration of a composition of the invention to a subject (or the application or administration of a compound of the invention to a cell or tissue derived from a subject) for the purpose of delaying, slowing, stabilizing, curing, curing, palliating, alleviating, altering, remedying, reducing deterioration, ameliorating, improving, or affecting a disease or condition, symptoms of a disease or condition, or the risk (or susceptibility) of a disease or condition. The term "treating" refers to any indicator of successful treatment or amelioration of an injury, pathology, or condition, including objective or subjective parameters such as attenuation; remission; reduction in the rate of deterioration; reduction in the severity of a disease; stabilization, alleviation of symptoms, or making the injury, pathology, or condition more tolerable to the subject; slowing the rate of deterioration or decline; making the final point of deterioration less debilitating; or improving the physical or mental health of the subject.

[0283] As used herein, "preventing" or "prevention" is intended to refer to at least a reduction in the likelihood of the risk (or susceptibility) of acquiring a disease or disorder (i.e., preventing at least one of the clinical symptoms of the disease from developing in a subject who may be exposed to or has a predisposition to the disease but has not yet experienced or exhibited symptoms of the disease). Biological and physiological parameters for identifying such subjects are provided herein and are well known by physicians.

[0284] The vaccine compositions of the invention may be administered to subjects most likely to need it, for example, in the context of human patients, to children or the elderly or to individuals at risk of exposure to P. gingivalis. The vaccine compositions of the invention may also be administered to subjects suspected of or diagnosed with P. gingivalis infection.

[0285] The compositions and methods of the present invention extend equally to uses in both human and / or veterinary medicine, the generation of diagnostics, or the generation of other therapeutic reagents.

[0286] As used herein, the term "subject" is intended to mean any animal, including humans, e.g., mammals. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject may be a human. In a further example, the subject may be a veterinary subject, e.g., a pet (cat, dog, guinea pig, etc.).

[0287] As used herein, the terms "subject," "individual," and "patient" may be used interchangeably.

[0288] Those skilled in the art will be familiar with methods for determining successful vaccination / immunization with a chimeric or fusion protein or composition described herein. For example, those skilled in the art will be familiar with methods for quantifying the antibodies generated after immunization and / or the extent of a humoral (Th2) response induced after immunization.

[0289] kit In another embodiment, there is provided a kit or article of manufacture comprising one or more proteins, polypeptides or polynucleotides of the invention and / or immunogenic compositions as described above.

[0290] In yet another aspect, the present invention provides a kit-of-parts comprising a vaccine composition of the present invention and one or more adjuvants for separate, sequential or simultaneous administration to a subject.

[0291] In other embodiments, a kit for use in the above therapeutic or prophylactic applications is provided, the kit comprising: a container holding a protein, polypeptide, polynucleotide or immunogenic composition of the invention; and a label or package insert having instructions for use.

[0292] In any embodiment, the kit may contain one or more further active principles or active ingredients for inducing an immune response in a subject against P. gingivalis.

[0293] The kit or "article of manufacture" may include a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The containers may be formed from a variety of materials, such as glass or plastic. The container holds a therapeutic composition that is effective to treat a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the therapeutic composition is used to treat a selected condition. In one embodiment, the label or package insert includes instructions for use and indicates that the therapeutic or prophylactic composition may be used to treat an inflammatory disease or condition described herein.

[0294] The kit may include (a) a therapeutic or prophylactic composition and (b) a second container having a second active ingredient contained therein. The kit in this embodiment of the invention may further include a package insert indicating the composition, the other active ingredient may be used to treat disorders or prevent complications due to inflammatory diseases or conditions described herein. Alternatively, or in addition, the kit may further include a second (or third) container containing a pharma- ceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials that are desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0295] In any embodiment, the therapeutic composition may be provided in the form of a disposable or reusable device that includes a container for holding the therapeutic, prophylactic, or immunogenic composition. In one embodiment, the device is a syringe, autoinjector, or nanopatch. The device may hold 0.1-2 mL of the therapeutic or immunogenic composition. The therapeutic or prophylactic composition may be provided in the device ready for use or may require mixing, dissolving, or resuspension, or addition of further components.

[0296] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features mentioned or apparent from the text or drawings, all of which various combinations constitute various alternative aspects of the invention.

[0297] The following examples are presented to more fully illustrate some embodiments of the present invention. However, they should in no way be construed as limiting the broad scope of the present invention. Those skilled in the art can easily devise many variations and modifications of the principles disclosed herein without departing from the scope of the present invention. EXAMPLES

[0298] The following examples describe a series of in vitro and in vivo studies related to the chimeric or fusion proteins of the invention. Example 1 describes the materials and methods used in the studies described in Examples 2 and 3. Example 2 describes the results of the in vitro studies, and Example 3 describes the results of the in vivo studies.

[0299] Example 1: Materials and Methods 1.1 Materials and methods for in vitro studies Recombinant protein expression Recombinant proteins (i.e., the various chimeric or fusion proteins described herein) were expressed from pET28 vectors (or pDUET-1 for rABM1+rABM2 co-expression from one vector) by induction with isopropyl β-D-thiogalactosidase (IPTG) essentially as follows.

[0300] Nucleic acids encoding the various chimeras and components of the chimeric and fusion proteins (including active site and adhesin domains) were generated by standard PCR or DNA splicing via overlap extension ("SOEing") PCR using P. gingivalis W50 genomic DNA template and specifically designed oligonucleotide primers. PCR fragments or SOEn PCR fragments were purified, ligated into the cloning vectors pGEMTeasy or pBHA, and transformed into chemically competent E. coli α-Gold cells (Bioline, New South Wales, Australia).

[0301] DNA sequences encoding single additional KAS (i.e., active site) residues, either containing a "DSSG" amino acid linker region or without a linker, were added sequentially, one at a time, to recombinant chimeric and Kgp adhesin domains essentially as follows: restriction enzyme sites were introduced at the ends of DNA inserts in chimeric and adhesin variant mother clones by PCR using oligonucleotide primers containing nucleotides specific for the restriction enzyme. Synthetic DNA fragments encoding individual KAS sequences with corresponding restriction sequences were ligated to the insert ends of DNA constructs from the mother clones and recombinant clones were purified. Subsequently, if a second or third additional KAS was added, further restriction sequences were introduced at the ends of these cloned inserts and additional DNA fragments encoding KAS sequences with different restriction sites were ligated, one at a time. Mother clone constructs with single, double, or quadruple linear KAS coding sequences were subjected to specific restriction digestion and then subfragments could be exchanged via ligation to produce a large number of variants, including those exemplified in Table 1.

[0302] Residues in ABM1 and ABM2, as well as cysteine ​​residues, were mutated using the QuickChange II Site Directed Mutagenesis kit (Stratagene, La Jolla, Calif.) according to the manufacturer's instructions.

[0303] The integrity of all inserts in the cloning vectors pGEMTeasy and pBH1 was confirmed by DNA sequencing (Applied Genetics and Diagnostics Facility, The University of Melbourne). Mutations were further verified by DNA sequencing of the entire insert. Verified constructs were then subjected to restriction digestion with selected enzymes and the inserts were cloned into the relevant pET expression vector in E. coli α-select chemically competent cells and subsequently cloned into the E. coli expression host, BL21-CodonPlus(DE3)RIPL (Stratagene, Australia).

[0304] Investigating chimeric features that affect recombinant protein solubility, small scale A single colony transformant was used to inoculate 2 mL of Luria Bertani (LB) broth containing 30 μg / ml kanamycin overnight at 37°C on an orbital shaker. This inoculum was then used to inoculate 2 ml of LB containing 30 μg / ml kanamycin (1:100). Cell cultures at OD600=0.6-1.0 were induced with 1 mM IPTG for 2 h at 37°C. The cell cultures were centrifuged and the pellets were resuspended in 250 μl of PBS, sonicated briefly using a CPX750 sonicator (Cole Parmer Instrument Company, USA), and centrifuged.

[0305] Total lysate, soluble (supernatant) and insoluble (pellet) fractions were analyzed by SDS-PAGE to assess recombinant protein solubility. Recombinant proteins that remained insoluble or showed low solubility under these initial pilot expression conditions were expressed under various conditions with lower temperatures (16°C-30°C) and lower IPTG (0.01-0.5 mM) to confirm optimal conditions for enhanced solubility. All recombinant protein inductions were scaled up (20-200 ml) under optimal conditions to test solubility at medium-large scale. Cultures (200 mL) were subjected to IPTG induction at various temperatures (e.g., 30°C for 5 h or 16°C for 16 h).

[0306] Cells were harvested and resuspended in lysis buffer (20 mM sodium phosphate, 500 mM NaCl, 0.5% v / v TritonX-100, 5 mg / ml DNAseI, 1× proteinase inhibitor cocktail, 1 mg / ml lysozyme, 10 mM imidazole, pH 8). The cell resuspension was incubated at 4° C. for 1 h, then the clarified lysate was centrifuged (8,000 g, 30 min, 4° C.) and the supernatant (soluble) and pellet (insoluble) fractions were collected and analyzed by SDS-PAGE and native-PAGE.

[0307] In-gel HIS staining of recombinant proteins The recombinant proteins subjected to SDS PAGE were stained using the Invision In Gel His Stain kit (Invitrogen) according to the manufacturer's instructions. This stain is highly specific for His-tagged proteins. This procedure was used to identify N-terminal degradation of the recombinant proteins since all candidates contain a C-terminal His tag.

[0308] Large-scale purification of chimeras for studies in animal models Purification conditions for each recombinant protein are detailed in the following sections.

[0309] Protein expression and cell lysis All recombinant proteins identified for use as vaccine candidates in animal models were expressed as recombinant C-terminal His-tagged fusion proteins or as "untagged" proteins in E. coli BL21(DE3) as previously described. Cells were grown at 37°C in LB medium, TB medium, or modified M9 minimal medium supplemented with 50 μg / mL kanamycin. Cultures at OD600=0.8-1.0 were induced with 0.4 mM IPTG for 2-4 h at 37°C or 34°C, 12 h at 25°C, or 16-20 h at 16°C. After harvesting by centrifugation at 8000 g at 4°C, cells were lysed in lysis buffer [0.35 mg / mL lysozyme, 40 μg / mL DNAse I in TBS150 (50 mM Tris·Cl, 150 mM NaCl, pH 7.5)] + 1% TritonX-100 and EDTA-free protease inhibitors (Complete ULTRA tablets, Sigma-Aldrich) for 1.5 h on ice unless otherwise indicated. Cell lysates were clarified by centrifugation at 23,500 g for 40 min at 4°C.

[0310] The initial Met residue (aa1) on all recombinant proteins shown in Table 1, beginning with the translated sequence MA, is removed in vivo by the E. coli N-terminal methionine processing enzyme, which cleaves off methionine if the penultimate residue is a small residue. This was confirmed by mass spectrometry of KDAK and KDAK variant recombinant proteins.

[0311] Purification of His-tagged proteins from the soluble fraction under non-denaturing conditions 1. Ni-affinity chromatography The cell lysate was clarified by centrifugation at 20,000 g at 4 °C to remove cell debris. After filtration through a 0.22 μm filter, the lysate was loaded onto a HisTrap Ni-affinity column (GE Healthcare) in loading buffer TBS300 (50 mM Tris·Cl, 300 mM NaCl, pH 7.5) + 10 mM imidazole and 1% TritonX-100. The column was washed extensively with loading buffer and then with 20 mM imidazole in TBS300. Bound proteins were eluted with a gradient of 20-350 mM imidazole in TBS300 while monitoring absorbance at 280 nm. Peak fractions were analyzed by SDS-PAGE and / or native PAGE. The eluted target protein was concentrated using Amicon filter units with a molecular weight cutoff of 3 kDa or 10 kDa depending on the protein size. The resulting protein solution was stored at -70°C for further purification.

[0312] 2. Anion Exchange Chromatography The isoelectric points (pI) of all candidate proteins were predicted to be in the acidic range using the online analytical ProtParam tool-ExPASy (https: / / web.expasy.org / protparam / ), so anion exchange chromatography was applied for further purification if necessary. Briefly, concentrated proteins from Ni-affinity purification were diluted into buffer A (50 mM Tris·Cl, 20 mM NaCl, pH 7.5) to reduce the ionic strength and then loaded onto an anion exchange HiTrap Q column (GE Healthcare) in buffer A. Proteins were eluted with a 20–400 mM NaCl gradient in Tris buffer while monitoring the absorbance at 280 nm. Target proteins in peak fractions were verified by SDS-PAGE and / or native PAGE and concentrated using Amicon filter units with appropriate molecular weight cutoff. Protein solutions were stored at -70 °C for further purification or buffer exchange using size exclusion chromatography or dialysis.

[0313] 3. Size Exclusion Chromatography Size-exclusion chromatography was performed on HiLoad Superdex 200 or HiLoad Superdex 75 columns (GE Healthcare) at 4 °C. Proteins from Ni-affinity purification or further steps of anion-exchange chromatography were loaded onto the appropriate column and eluted in a buffer of TBS150 or TBS100 (100 mM NaCl in 50 mM Tris·Cl, pH 7.5) while monitoring the absorbance at 280 nm. Target proteins in peak fractions were verified by SDS-PAGE and / or native PAGE. For isolation of single oligomeric species such as dimers, a second or further round of size-exclusion purification was performed. Fractions containing the best protein purity were pooled and concentrated. To prepare a mixture of protein homomultimers, the target protein fractions were simply pooled and concentrated. Protein identity was confirmed by determining intact protein molar mass using liquid chromatography-electrospray ionization spectroscopy (LC-MS, Agilent) or by sequence analysis using liquid chromatography-Orbitrap tandem mass spectrometry (LC-MS / MS, Agilent), as appropriate. Protein concentrations were quantified by determining absorbance at 280 nm using calculated extinction coefficients prior to submission to animal model experiments.

[0314] Purification of His-tagged proteins from inclusion bodies 1. Under oxidizing conditions After lysis of the cells (as described for purification from the soluble fraction), the insoluble pellet was washed twice with TBS150. Proteins expressed as inclusion bodies were solubilized with 8 M urea in TBS300 or 20 mM NaPi, 500 mM NaCl, pH 7.4 (PBS500) for 1 h on a rolling platform at room temperature. The protein extract was centrifuged at 20,000 g, filtered through a 0.22 μm filter unit and then loaded onto a HisTrap affinity column in urea containing buffer TBSU (8 M urea + 20 mM imidazole in TBS300) or PBSU (8 M urea, 20 mM NaPi pH 7.8, 500 mM NaCl + 20 mM imidazole). The column was washed extensively with the same 8 M urea buffer. Further washes with PBSU pH 6.5 were applied to the column with loaded protein in PBSU. For removal of urea, the target protein was eluted with a gradient of 20-500 mM imidazole in PBSU. When loose Ni-NTA resin (Thermofisher) was used, the urea extract was mixed with the resin for 2 h under gentle agitation in PBSU. After extensive washes with PBSU (pH 7.8, then pH 6.5), the bound protein was eluted from the resin with 500 mM imidazole in the same buffer. The eluted protein was dialyzed stepwise into 6M, 4M, then 2M urea phosphate buffer in dialysis tubing with a molecular weight cut-off of 3.5 kDa (Fisher Biotec, Australia). Protein in 2M urea buffer was further dialyzed into saline only and kept at 4°C before submission to animal model experiments, or else submitted in 2M urea buffer without further dialysis.

[0315] 2. Under reducing conditions Proteins were extracted from inclusion bodies using 6 M urea buffer in the presence of 5 mM TCEP and loaded onto a HisTrap column in the same buffer. After washing the column with 4 M urea buffer containing 2 M TCEP, the target protein was refolded on the column in the presence of 2 M TCEP with a decreasing gradient of urea from 4 to 0 M. The protein was then eluted in TBS300 + 2 mM TCEP with an increasing gradient of hemidazole from 20 to 350 mM. The eluted protein was then concentrated for size exclusion chromatography in reducing buffer (2 mM TCEP in TBS150) in the same way as described in the section for purifying protein dimers or multimers from the soluble fraction under non-denaturing conditions. After analysis using SDS-PAGE and native PAGE, mass spectrometry and quantification, the samples were stored at -70 °C in reducing buffer or submitted for animal model experiments.

[0316] Purification of His-tagged proteins from the soluble fraction under oxidative and denaturing conditions The cell lysate was clarified by centrifugation at 20,000g to remove debris and insoluble material, and then the lysate was immediately denatured by adding urea solution to a final concentration of 8M in PBS500 with stirring at room temperature for 1 hour. The resulting lysate was then centrifuged at 20,000g for 40 minutes at room temperature. The target protein was purified using HisTrap Ni-affinity column or loose Ni-NTA resin with the same approach as described in the section Purification from inclusion bodies under oxidizing conditions above. The purified antigen in saline was stored at 4°C before being submitted to animal model experiments.

[0317] Purification of untagged candidates from the soluble fraction Anion exchange chromatography This is the first chromatographic step to purify the untagged proteins. Similar to their His-tagged counterparts, the pI of the untagged candidates was also predicted to be in the acidic range using the online analytical ProtParam tool - ExPASy (https: / / web.expasy.org / protparam / ). Therefore, anion exchange chromatography was applied and two rounds were performed. In the first round, the clarified cell lysate was filtered through a 0.22 μm filter disk and loaded onto an anion exchange HiTrap Q column (GE Healthcare) in buffer AA (20 mM NaPi, pH 6.5 supplemented with 0.05x protease inhibitor cocktail). After washing with 10 column volumes of buffer AA, the bound proteins were eluted with a 0-100% NaCl gradient of buffer BA (1 M NaCl in 20 mM NaPi pH 6.5) and the absorbance at 280 nm was monitored. The target proteins in the peak fractions were verified by SDS-PAGE and / or native PAGE. In the second round, the best fractions from the first round of the anion exchange step were pooled, diluted 6-fold with buffer AA, and then reloaded onto an anion exchange HiTrap Q column (GE Healthcare) in the same buffer. Bound proteins were eluted with a 0-50% NaCl gradient of buffer BA and the absorbance at 280 nm was monitored. The target protein in the peak fractions was verified by SDS-PAGE and / or native PAGE, and the best fractions were concentrated using Amicon filter units with a molecular weight cutoff of 10 kDa. The protein solution was stored at -20 °C and further purified using size exclusion chromatography.

[0318] First round size-exclusion chromatography Size-exclusion chromatography was performed on a HiLoad Superdex200 column (GE Healthcare) at room temperature. Proteins from the anion exchange chromatography purification were loaded onto a size-exclusion column and eluted in AS buffer (1M (NH4)2SO4, 50 mM NaPi pH 7.0 supplemented with 0.05x protease inhibitor cocktail) while monitoring absorbance at 280 nm. Target proteins in peak fractions were verified by SDS-PAGE and / or native PAGE. Fractions with the best protein purity were pooled for further purification using hydrophobic interaction chromatography.

[0319] Hydrophobic interaction chromatography Pooled fractions of target protein in buffer AS from the first round of size exclusion chromatography were filtered through a 0.22 μm membrane and loaded onto a HiTrap Phenyl HP column pre-equilibrated in buffer AH (1 M (NH4)2SO4, 50 mM NaPi pH 7) at 4 °C. Bound protein was eluted with a decreasing ionic strength gradient of (NH4)2SO4 from 800 mM to 200 mM in buffer BH (50 mM NaPi, pH 7) while monitoring the absorbance at 280 nm. Target protein in peak fractions was verified by SDS-PAGE and / or native PAGE and the best fractions were pooled for the next purification or buffer exchange step of the second round of size exclusion chromatography and concentrated using an Amicon filter unit.

[0320] Second round size-exclusion chromatography The concentrated sample from the purification step involving hydrophobic interactions was loaded onto the size-exclusion column used in the first round of size-exclusion chromatography and eluted in a buffer of BTS (20 mM Bis-Tris, 150 mM NaCl, pH 6.5). Peak fractions were analyzed by SDS-PAGE and / or native PAGE. The highest purity fractions were pooled and concentrated. After identification by liquid chromatography-electrospray ionization mass spectrometry (LC-MS, Agilent) and quantification, the antigen was aliquoted and stored at -80°C for future use.

[0321] Protein Identification and Quantification of Untagged Candidates In addition to SDS-PAGE or native PAGE validation, protein identity was confirmed by determination of intact protein molar mass using LC-MS or by sequence analysis using liquid chromatography-Orbitrap tandem mass spectrometry (LC-MS / MS, Agilent) as appropriate. Protein concentration was quantified by determining absorbance at 280 nm using calculated extinction coefficients (Table 3). When buffer exchange was required prior to submission to animal model studies, Superdex 200 size exclusion columns (GE Healthcare) or Zeba™ spin desalting columns (ThermoFisher Scientific) were used.

[0322] 1.2 Materials and methods for in vivo studies Bacterial culture for a mouse model of periodontitis P. gingivalis strain, W50 (serotype C), was obtained from the culture collection at the Oral Health Cooperative Research Centre, The Melbourne Dental School, University of Melbourne, Australia. P. gingivalis W50 was grown on horse blood agar (HBA) (20 g / L HBA, Oxoid Ltd., Hampshire, UK) supplemented with 10% v / v lysed horse blood (37°C) in an anaerobic N2 atmosphere containing 5% CO2 in a MK3 anaerobic workstation (Don Whitley Scientific Ltd., Adelaide, Australia). Colonies were inoculated into 20 mL of starter medium composed of sterile brain heart infusion (37 g / L BHI, Oxoid Ltd., Hamsphire, UK) medium supplemented with 5 mg / L hemin and 0.5 mg / L cysteine ​​[McKee et al. (1986) Infect Immun 52:349-355] and incubated anaerobically (24 h, 37°C). The absorbance of the batch cultures was monitored at OD650 nm using a spectrophotometer (model 295E, Perkin-Elmer, Germany). Bacterial cells were harvested during late exponential growth by centrifugation (7,000 g, 20 min, 4°C). Bacterial purity was routinely confirmed by Gram staining [Slots (1982). In: Host-Parasite Interaction in Periodontal Disease, Genco, RJ and Merganhagan, SE (eds). Washington DC: American Society for Microbiology. pp. 27-45.].

[0323] Preparation of heat-killed bacteria P. gingivalis W50 cultures were harvested (6,500 g, 4° C.), washed once with phosphate-buffered saline (PBS) (0.01 M Na2HPO4, 1.5 mM KH2PO4, and 0.15 M NaCl, pH 7.4), and then pelleted by centrifugation (7,000 g, 20 min, 4° C.). The bacterial cells were resuspended in PBS and heated to 65° C. for 15 min. The suspension was centrifuged (7,000 g, 20 min, 4° C.) and resuspended in sterile PBS, which was repeated once. After the second wash, the supernatant was discarded and the cell pellet was resuspended in sterile PBS and diluted to 2×10 10 Cell densities in cells / mL were obtained and protein concentrations were determined using Biorad Protein Assay Dye Reagent Concentrate (Life Science, NSW, Australia).

[0324] Mouse periodontitis model Murine periodontitis experiments were performed as previously described by O'Brien-Simpson et al. (2005 J Immunol 175:3980-3989), modified from Baker et al.'s mode (1994). Arch Oral Biol 39:1035-1040). On day 0, mice (female BALB / c, 6-8 weeks old, 10 mice / group) were inoculated at 2-day intervals with four doses of P. gingivalis W50 [1 × 10 500 μl suspended in 20 μl PG buffer (50 mM Tris-HCL, 150 mM NaCl, 10 mM MgSO4, and 14.3 mM mercaptoethanol, pH 7.4) containing 2% w / v carboxymethylcellulose (CMC, Sigma, New South Wales, Australia)]. 10Mice were inoculated orally with P. gingivalis consisting of 10000 viable P. gingivalis W50 cells. The inoculum was prepared anaerobically and then immediately applied to the gingival margin of the maxillary molar. The viable count in each inoculum was verified by flow cytometry and CFU counting on blood agar. The groups of animals consisted of P. gingivalis W50 oral inoculation (infected control), non-bacterial inoculation control, and immunized group. For therapeutic vaccination, periodontitis model (Figure 1) mice were first orally inoculated with 50 μg of the vaccine candidate in saline / alum (Alhydrogel, 2% aluminum hydroxide wet gel suspension, Invivogen) and then immunized via intraperitoneal route on day 19. Mice received a second immunization (50 μg in saline / alum) via subcutaneous route on day 40. On day 62, mice were bled and killed by cardiac puncture. The maxilla was removed, halved across the midline, and divided into 10 sections to determine alveolar bone loss. Serum was used to determine antibody profiles using ELISA.

[0325] Measurement of alveolar bone loss in the mouse maxilla Maxillae to be examined for bone loss were boiled in deionized water (1 min), mechanically dissected, and immersed in 2% w / v potassium hydroxide (16 h, 25°C). Maxillae were washed twice in deionized water (25°C), dried (1 h, 37°C), and stained with 0.5% w / v aqueous methylene blue. Digital images of the buccal aspects of the maxillae were captured with an Olympus DP12 digital camera attached to a dissecting microscope using OLYSIA BioReport software version 3.2 (Olympus Australia Pty Ltd, New South Wales, Australia) to assess horizontal bone loss. The maxillae were positioned such that the cusps of the buccal and lingual molars were superimposed. Images were captured with a micrometer in the frame to allow standardization of measurements for each image. Horizontal bone loss was defined as loss occurring in a horizontal plane perpendicular to the alveolar bone crest, resulting in a reduction in crest height. The visible area from the cementum-enamel junction (CEJ) to the alveolar bone crest (ABC) of each molar was measured using OLYSIA BioReport software version 3.2 imaging software and expressed as mm 2 The total visible CEJ-ABC area in the gingivalis-induced alveolar bone loss (mm 2 ) was calculated by subtracting the total visible CEJ-ABC area in the non-vaccinated (NC) group from the total visible CEJ-ABC area in each experimental group. Alveolar bone loss measurements were determined in duplicate in a random and blinded protocol. Data are presented as mean + / - standard deviation (mm 2 ) and analyzed using one-way ANOVA and Dunnetts T3 post-hoc test.

[0326] Prescreening of vaccine candidates by enzyme-linked immunosorbent assay (ELISA) Several monoclonal antibodies against P. gingivalis were used to pre-screen vaccine candidates to determine whether the major domains and epitopes present in the vaccine construct were accessible to antibodies, thus allowing the generation of an antibody response in vivo. The antigens to be screened were coated on flat-bottom polyvinyl microtiter plates (Microtiter, Dynatech Laboratories, McLean, VA, US) in 0.1 M PBS (pH 7.4) for 16 hours at 4°C. The following antibody dilutions were used in these experiments: goat anti-mouse; 1 / 4000 dilution of IgG (M8642) antibody (Sigma, New South Wales, Australia). ELISA experiments were developed using 1 / 4000 dilution of horseradish peroxidase-conjugated swine anti-goat IgG antibody (M5420, Sigma, New South Wales, Australia).

[0327] Determination of subclass antibodies in serum using ELISA ELISAs were performed to assess antibodies to the domain subclasses using solutions of either HK W50 cells, domain subunits or epitopes (1 μg / mL) in 0.1 M PBS (pH 7.4) to coat (16 h, 4° C.) wells of flat-bottom polyvinyl microtiter plates (Microtiter, Dynatech Laboratories, McLean, VA, US) as described in Pathirana et al. (2007). Infect Immun 75:1436-1442). The following antibody dilutions were used in these experiments: 1 / 4000 dilution of goat anti-mouse, IgG (M8642), IgG1 (M8770), IgG2a (M4434) antibodies (Sigma, New South Wales, Australia). ELISA experiments were developed using a 1 / 4000 dilution of horseradish peroxidase-conjugated swine anti-goat IgG antibody (M5420, Sigma, New South Wales, Australia). For epitope ELISAs, biotinylated peptides were bound to preblocked streptavidin-coated flat-bottom plates (Pierce, Thermo-Fisher) at 10 μg / mL. After incubation with serum, ELISAs were developed with 1 / 4000 goat anti-mouse IgG and 1 / 4000 horseradish peroxidase-conjugated swine anti-goat IgG antibodies. All optical density measurements were performed at 405 nm on a Wallac VICTOR3 1420 Multilabel counter (Perkin Elmer).

[0328] Example 2: Results of in vitro studies Study 1: Chimeric constructs affecting the solubility of recombinant proteins One previously reported chimera (in WO2010 / 022463) consists of a KAS (K) peptide conjugated to an N-terminally truncated DUF2436 domain (Dc), an adhesin domain containing ABM213 (A), and a C-terminally truncated CAD domain (referred to as a truncated K1 domain, K1n). This chimera is referred to as "KDcAK1n" (SEQ ID NO: 64, which may also be referred to herein as the "original chimera" or "chimera" in comparison with the chimeras and fusion proteins of the present invention). The KDcAK1n protein is produced as an inclusion body in E. coli and is less soluble and less stable.

[0329] The truncated DUF and K1 domains of the A1 adhesin fragment represent how the Kgp polyprotein is naturally proteolytically processed and assembled on the P. gingivalis cell surface during infection, thus making them obvious candidates for inclusion in a vaccine to generate an immune response against gingipains.

[0330] Extensive attempts to produce soluble KDcAK1n were made. Variations in growth medium, growth conditions, IPTG concentration, E. coli expression strain, induction temperature, cell growth stage at induction, addition of growth stabilizers, extensive testing of lysis buffer and protein storage buffer were systematically investigated. Enhancement of soluble recombinant protein expression was observed in small-scale expression at low temperature and under IPTG induction. At small scale, KDcAK1n produced approximately 30-50% of the total recombinant protein expression as soluble protein. Nevertheless, the soluble recombinant could not be scaled to cultures larger than 10 ml, and attempts at scale-up resulted in inclusion body formation.

[0331] Effect of the K1 domain on solubility All intact DUF, ABM213, ABM21 and DUF-ABM213 domains of the Kgp polyprotein were expressed as highly soluble and stable recombinant proteins, however, the intact K1 domain alone was insoluble and expressed only as inclusion bodies (IBs).

[0332] Expression of a KAS motif conjugated to the ABM213 domain (i.e., "KA") or a KAS motif conjugated to both the DUF and ABM213 domains (i.e., "KDA") produced highly soluble and stable recombinant proteins.

[0333] However, as shown in Figure 2, addition of a C-terminally truncated K1 domain (i.e., the K1n fragment) or the complete K1 domain to recombinant KA or KDA (KAK1 or KDAK1) resulted in a loss of solubility.

[0334] Both KDAK1n (SEQ ID NO:65) and KDAK1 (SEQ ID NO:66) proteins (i.e., containing the K domain, DUF2436, and the adhesin domain including ABM, and truncated or full-length CAD domain) require more stringent growth conditions for soluble expression at significantly lower levels than KDA (i.e., the same protein excluding truncated or full-length CAD).

[0335] Taking the results of K1 domain expression together, it is clear that the K1 (CAD) domain contributes to the poor solubility of chimera-based recombinants.

[0336] Effect of DUF domains on protein solubility Truncation of the DUF domain in the chimeric protein construct resulted in the elimination of soluble protein expression, indicating that an intact DUF domain containing the extra 38 N-terminal residues is desirable for optimizing soluble expression and efficient stabilization of recombinant proteins.

[0337] Therefore, a construct was generated expressing a chimera variant with an extended Dc domain (chimera with Dc extended by 38 N-terminal residues). This recombinant was designated KDAK1n, and results show that the solubility of the chimera is significantly enhanced with an intact DUF domain. Large-scale expression of KDAK1n at 30 °C with 0.2-0.5 mM IPTG resulted in the expression of good yields of soluble recombinant protein.

[0338] Effect of K1 and full-length DUF domains on solubility Despite this soluble protein expression at this scale, the inclusion of K1n / K1 in KDAK1n / KDAK1 appears to make these K1-containing variants more problematic to prepare than KDA variants lacking K1n / K1, such that these variants may be less desirable for scale-up and commercial development.

[0339] To summarise: • A full-length DUF domain is desirable for solubility of recombinant variants (Figure 2A). • The K1 domain (whether full-length or truncated) was primarily responsible for the aggregation and insolubility of the variants. • In the absence of the full-length DUF domain (i.e., DUFc), the K1 and K1n-containing variants were completely insoluble (Figure 2A). In the presence of the full-length DUF domain, very low levels of solubility of K1 and K1n-containing variants were evident under very mild induction conditions, nevertheless this solubility was very low, with approximately 80–90% of all recombinant proteins expressed as inclusion bodies. Native PAGE gel analysis of mid-scale purification of soluble KDAK1 recombinants revealed disorganized aggregation of the recombinants (i.e., no neat, uniform ladder for the KDA variants). Small-scale purification of these variants (at low protein concentrations) using mini-nickel spin columns did not result in similar aggregation; however, small-scale purification of the truncated DUF variants also revealed that the characteristic ladder of proteins on native PAGE was heterogeneous, suggesting disorganized aggregation. The K1 and K1n domain-containing variants had an increased tendency for aggregation at higher concentrations and after freeze-thawing of purified samples. The K1 domain was responsible for this instability. However, all variants lacking K1 or K1n were highly stable upon freeze-thawing and at higher concentrations (e.g., concentrations above 30 mg / ml).

[0340] Variants containing full-length or truncated K1 domains are highly insoluble, and the low levels of soluble recombinant protein produced are unstable and prone to aggregation after purification. Efficient scale-up of soluble K1 containing variants has not been possible.

[0341] Therefore, extension of the DUF to its full length and / or removal of the K1 (or K1n) domain, preferably both, is important for the solubility and stability of the recombinant protein.

[0342] Study 2: Factors affecting multimerization Native PAGE analysis of purified soluble DUF-ABM213 and ABM213 and ABM21 recombinant proteins results in extensive and uniform laddering indicative of multimerization. Multimerization is also evident in the prior art KDcAK1n chimera through the formation of intermolecular disulfide bonds between the denatured domains. This disulfide bond multimerization was most evident in the KDcAK1n chimera purified from inclusion bodies.

[0343] During the course of studies on the recombinant chimeras and Kgp adhesins, it became apparent that multimerization of the recombinant proteins occurred via interactions between the ABM1 and ABM2 motifs. (See schematic diagram in Figure 3A) The recombinant proteins containing the ABM2(1), ABM2(2), and ABM3 domains could readily form multimers. Subsequent expression of a smaller recombinant protein (designated rABM213) encoding ABM2(1), ABM1(2), and ABM3, as well as a recombinant protein (designated rABM21) encoding ABM2(1) and ABM1(2), showed that both recombinants were readily able to multimerize. This multimerization was evident by the presence of an ordered ladder of bands on native PAGE.

[0344] It was proposed that during folding of the Kgp multidomain polyprotein, the ABM1 domain interacts with the ABM2 domain to form a stable FnIII-like beta-sheet complex structure. We hypothesized that ABM1(1) interacts with its next available neighbor ABM2(1), ABM1(2) interacts with ABM2(2), etc. during folding of the Kgp polyprotein (as shown diagrammatically in Figure 3A).

[0345] In a separate study, the inventors showed that co-expression of rABM1 and rABM2 as separate proteins could interact to form a stable beta-sheet complex structure. rABM2(1) could form a stable structure with either rABM1(1) or rABM1(2). More specifically, NMR spectroscopic analysis of the folded ABM domain shows that the domain multimerizes via beta-strand exchange involving the ABM1 and ABM2 motifs. These results provide evidence that chimeras and variants containing the ABM domain multimerize via interactions between the ABM1 and ABM2 motifs of a properly folded ABM domain.

[0346] Next, we aimed to mutate specific residues of ABM1 or ABM2 in the recombinant ABM21 (rABM21) protein, which encompasses residues 878-968 on the Kgp W50 polyprotein.

[0347] BLAST analysis of Kgp ABM1 and ABM2 sequences in all available bacterial genomes revealed that submotifs within the ABM1 and ABM2 motifs are very highly conserved across all phyla. Subsequent alignment of the ABM1 and ABM2 sequences to all ORFs in the W83 genome revealed that these residues are similarly highly conserved in P. gingivalis genes. The "PVQN" motif with a conserved proline residue was highly conserved across Phyla, including P. gingivalis.

[0348] Molecular modeling of the proposed tertiary fold using an online program revealed that this "PVQN" sequence is located at the start of a beta-sheet structure, and the sequence immediately N-terminal to this motif ("NEFA") was found to be most likely an unstructured "loop".

[0349] We mutated residues in the PVQN motif of rABM21, as well as in the loop immediately preceding PVQN. We targeted residues in the loop because we believed that the size and shape of the loop preceding the beta-sheet structure could affect the strength of adjacent beta-sheet folding. The number and position of proline residues in such "loop" structures have been reported to be related to adjacent beta-sheet interactions.

[0350] In addition, we mutated the highly conserved hydrophobic residues Y(878) and W(968) present in ABM2 and ABM1, respectively. We also introduced a substitution in the sequence of the motif NxFA in ABM1 to SxYQ (see the SSEYQ variant in Table 2 below).

[0351] Mutated rABM21 variants were purified and evaluated for their ability to multimerize. We sought to identify mutations that would result in stable, soluble recombinant molecules that could be purified as stable monomers. [Table 2]

[0352] All recombinant proteins (WT rABM213, rABM21, and rABM21 mutant variants) resulted in very high levels of expression of soluble recombinant proteins under stringent induction conditions (summarized in Table 2).

[0353] Purified recombinant proteins subjected to native PAGE analysis (Figure 3B-E) showed that rABM21 could form multimers via two mechanisms: (1) multimerization via the ABM1 motif “PVQN” and (2) multimerization via disulfide bonds.

[0354] Mutation of the highly conserved "PVQN" motif in rABM21 to AVQN or AVQA did not abolish multimerization.

[0355] Mutation of "PVQN" to AVQP resulted in a significant decrease in multimerization in the presence of DTT and a slight decrease in multimerization in the absence of DTT, suggesting that the change from PVQN to AVQP alone has some effect on reducing multimerization.

[0356] The AVQA mutation in combination with substitution of one or both cysteine ​​residues in the ABM domain with serine resulted in a slight decrease in multimerization. Mutation of "PVQN" to AVQP, together with mutation of one or both cysteine ​​residues in the ABM domain with serine, resulted in near or complete elimination of multimerization of rABM21.

[0357] Manipulation and mutation of residues in the modeled "loop" region immediately preceding the ABM1 "PVQN" motif in the modeled "beta sheet region" was also able to reduce multimerization, but not completely eliminate it. More specifically, "SSEYQ" substitutions in ABM1 (e.g., modification of the sequence SNEFA to SSEYQ immediately N-terminal to the highly conserved PVQN motif) resulted in a significant reduction in multimerization.

[0358] Mutation of Tyr-889 and Trp-964 residues (in ABM2 and 1, respectively) also resulted in the abrogation of multimerization of rABM21.

[0359] Engineering of the "PVQN" motif into AVQP together with the substitution of cysteine ​​residues within the ABM domain allowed for the expression and purification of a highly soluble monomeric protein using the E. coli pET expression system.

[0360] All chimera candidates constructed with the original sequence with the two Cys residues in the ABM21 domain intact showed extensive ladder multimerization (Figure 3B and C). However, although the substitution of one Cys residue in combination with the AVQP substitution resulted in a marked reduction in multimerization, the substitution of these two Cys residues did not result in a complete elimination of ladder multimerization (Figure 3C lanes 15 and 16). Complete elimination of ladder multimerization was only achieved by the substitution of the two ABM cysteine ​​residues plus the mutation of the motif PVQN to AVQP.

[0361] Interestingly, KDAK-1V-AVQP (SEQ ID NO: 70) and KDAK-AVQP (SEQ ID NO: 71), with or without mutation of the only DUF domain cysteine ​​residue, with the two ABM cysteine ​​residues remaining, exhibited significantly reduced multimerization under reducing conditions. The two proteins shared essentially the same profile on native gels, either reduced or not reduced (Figure 3G). Apparently, the DUF cysteine ​​residues did not contribute to ladder multimerization, but the presence of the two ABM cysteine ​​residues was important for the formation of ladder multimers in these two variants. This suggests that the DUF domain cysteine ​​residues are not significant contributors to multimerization.

[0362] Although ladder multimerization was abolished by mutation of cysteine ​​residues and the PVQN motif, an equilibrium between monomeric and oligomeric states still existed under certain conditions. This type of equilibrium was found to be temperature, pH, and concentration dependent from analysis with size exclusion chromatography. Increasing the temperature, decreasing the pH, and decreasing the concentration were found to favor the monomeric state. The presence of a His tag did not play a role in the equilibrium of the protein states. SEC-MALS analysis showed the overwhelming presence of monomers in solution at concentrations of 2 mg / mL or lower with high stability for the lead candidates.

[0363] Study 3: Active site (KAS) motif Additional KAS motifs were engineered into the recombinant chimeric variants to determine whether this would affect immunogenicity. A single KAS or two consecutive KAS were added to the variants at their N-terminus or both termini. In addition, a linear sequence of four KAS residues containing a DSSG linker sequence between each KAS motif was also added to the N-terminus of selected variants.

[0364] Recombinant purification profiles and His gel staining analysis revealed that the additional KAS residues caused some instability and degradation of the recombinant protein. Degradation and instability were greatest for the 4x linear KAS variant, suggesting that proteolytic processing was occurring within the additional KAS sequence. Subsequent removal of the DSSG linkers between adjacent KAS sequences did not improve stability.

[0365] We therefore concluded that these variants were prone to proteolytic processing when multiple consecutive KAS sequences were expressed in a linear array. Soluble variants with a single KAS at one or both termini were relatively stable and less susceptible to degradation and loss of purification yields.

[0366] Study 4: Large-scale protein yield and stability Most recombinant proteins expressed as soluble proteins and purified under non-denaturing conditions had relatively high yields of their final products (>10 mg / L culture). Some of them reached yields of more than 20 mg / L culture. In particular, the His-tagged KDAK-1V2S-AVQP protein (SEQ ID NO: 59, excluding the His-tag) had a yield of 45 mg / L culture when expressed in 2YT rich medium. The most remarkable yields were seen for the purified final products of untagged KDAK-1V2S-AVQP (SEQ ID NO: 59) and KDAK-3S-AVQP (SEQ ID NO: 69), which were expressed in Terrific Broth (TB) and had production of more than 80 and 60 mg / L culture, respectively, compared to cells grown in LB or 2YT medium. This remarkable improvement in target protein production was due to the high growth rate of the cells and the high cell density when the cells were grown in TB.

[0367] Yields of these same recombinant proteins were much lower (2.1-3.6 mg / L culture) when purified from inclusion bodies or soluble fractions under conditions that intentionally denatured them (e.g., urea). We noted that the denatured proteins had low binding affinity for Ni-affinity resins under denaturing conditions and low solubility when finally equilibrated in non-denaturing buffers. In addition, variants purified from denatured forms were unstable in unbuffered saline, regardless of whether the proteins were initially expressed as soluble proteins or insoluble forms and whether they were purified using pre-packed columns or using loose Ni-NTA resins. These results suggest that when denaturants are not required, it may be desirable to reduce or avoid their use.

[0368] A low expression temperature of 16°C proved to be effective in improving the soluble expression of poorly soluble proteins such as KDAK1 (SEQ ID NO: 66). Nevertheless, on-column concentration in low ionic strength buffers during anion exchange purification significantly impaired the yield due to on-column aggregation or precipitation, even at temperatures as low as 4°C. For these proteins, purification bypassing the anion exchange step resulted in higher yields, e.g., the yield of KDAK1 was doubled when produced in this way. Also, KDAK1n (SEQ ID NO: 65) was expressed as a soluble protein, but did not appear to be stable in solution during the subsequent purification process, with a yield of only 2.3 mg / L culture.

[0369] Considering that K1 is insoluble and addition of K1 or K1n to KDA or DA reduces the solubility of the recombinant variants in large-scale purification, the K1 region may be involved in the instability of K1-containing chimeric variants, such that, as described above, omission of K1 is preferred when the domain is not required for protection.

[0370] His staining of the gels demonstrated degradation within the region containing multiple KAS (K) residues, but no negative impact on yield was observed with Ni-affinity chromatography. For example, three protective antigens, KKDAK1nKK (SEQ ID NO: 78), KKDAKK (SEQ ID NO: 54), and KDAK1nK-4S-AVQP (SEQ ID NO: 63), had yields ranging from 12 to 21 mg / L culture with over 90% of the expected full-length protein species in the final product.

[0371] Reducing conditions were found to be favorable for extracting insoluble KDcAK1n from inclusion bodies using urea. Under non-reducing conditions, a higher concentration of urea was required than under reducing conditions. Apparently, the formation of disulfide bonds under denaturing conditions had a negative effect on the solubility of the protein. Interestingly, native PAGE gel analysis of KDcAK1n chimera versus a chimera in which all four cysteine ​​residues were mutated to serine (SEQ ID NO: 56) showed that the elimination of disulfide bonds in the mutated KDcAK1n chimera resulted in a more distinct ladder formation on native PAGE, consistent with only beta-strand exchange multimerization occurring. Furthermore, the chimeric mutant KDcAK1n PVQN>AVQP / 4Cys>Ser variant (SEQ ID NO: 57) showed higher solubility than KDcAK1n under identical non-reducing denaturing conditions in urea.

[0372] The concentration of some soluble proteins, such as His-tagged KDAK-1V2S-AVQP, could reach at least 40 mg / mL. The two leading untagged candidates, SEQ ID NO:59 and SEQ ID NO:69 (KDAK-1V2S-AVQP and KDAK-3S-AVQP), were concentrated up to over 30 mg / mL and 16 mg / mL, respectively, in the well buffer solution at pH 6.5, which is still not the maximum.

[0373] In contrast, the C-terminal His-tagged original chimera (KDcAK1n) and its variant KDcAK1n-4S purified from inclusion bodies were least stable in unbuffered saline. The highest achievable concentrations of KDcAK1n and KDcAK1n-4S in saline were 1.35 mg / mL and 2.3 mg / mL, respectively. Apparently, the formation of nonspecific disulfide bonds was one of the factors responsible for the significant protein aggregation and low solubility of the chimera. Removal of Cys residues in KDcAK1n-4S abolished the formation of intermolecular disulfide bonds and improved the solubility of the protein.

[0374] When the proteins were expressed under the same conditions, the C-terminal His tag was found to have no significant effect on protein production and solubility.

[0375] In the purification of the untagged candidates, anion exchange chromatography enriched the target protein, but this step could not efficiently separate the target protein from that of the E. coli host by tight binding affinity to the resin, because most E. coli proteins are acidic in the pI range of 4-7, and many of them have pIs close to that of the target protein. Interestingly, however, size-exclusion chromatography at room temperature with a buffer of 1 M (NH4)2SO4 appeared to be a critical step, providing a highly efficient separation of the target protein from that of the E. coli. After further purification with a second round of hydrophobic interaction and size-exclusion chromatography, both primary untagged candidates were purified to 99% purity with high homogeneity, which was evidenced by SDS-PAGE / native PAGE and SEC-MALS analysis. [Table 3-1] [Table 3-2]

[0376] Study 5: Evaluation of the importance of DUF and KAS domains In this study, we tested various constructs (antigens) to determine the importance of the DUF domain in the chimeric fusion protein and compare it to the solubility / stability of constructs containing active site sequences derived from arginine-dependent gingipains. The constructs produced were: KDAK-3S-AVQP, KDAK-2S-AVQP (His tag), KAK-2S-AVQP (His tag), and KDAR-3S-AVQP (His tag).

[0377] Protein production and yield All antigens were expressed well. The one remaining cysteine ​​residue in the DUF domain in the KDAK-2S-AVQP construct did not appear to affect protein solubility.

[0378] The RAS containing candidate KDAR-3S-AVQP was less soluble than other antigens, indicating interference of the RAS sequence with protein structure, but could still be used in subsequent mouse model experiments and could be purified under non-reducing conditions from the soluble fraction.

[0379] All antigens were purified from the soluble fraction under non-reducing conditions. The three His-tagged proteins were purified via Ni affinity, anion exchange, and size exclusion chromatography. KDAK-3S-AVQP (untagged) was purified via anion exchange, hydrophobic interaction, and size exclusion chromatography. The yield of purified antigens was high. Among the three His-tagged proteins, KDAK-2S-AVQP-His6 had the highest yield (>160 mg / L culture, 20 mg / g wet cells).

[0380] When the proteins were expressed under the same conditions, the C-terminal His tag did not appear to affect protein production and solubility, suggesting that the presence of the His tag did not interfere with the protein core structure.

[0381] In contrast, the KDAR-3S-AVQP-His6 antigen had a lower yield (55 mg / L culture, 7.2 mg / g wet cells) due to its low solubility. The antigen with the lowest molecular weight, KAK-2S-AVQP-His6, had a yield of 106 mg / L culture (14 mg / g wet cells). The identity of all antigens was confirmed by MS spectroscopy of the intact protein, and the first Met residue was missing.

[0382] As for the Hi-tagged protein, the higher solubility of the KAS-only antigen compared to the antigen-containing RAS was reflected from the peak intensity of the elution profile of the His-tagged protein in the Ni-affinity purification step. Nevertheless, when the RAS antigen is pure, it is also highly soluble and can be concentrated up to at least 15 mg / mL.

[0383] These proteins have so far been concentrated to the range of 15-33 mg / mL, but their high solubility may enable them to be concentrated to even higher concentrations.

[0384] Oligomerization Several minor bands were seen below each major full-length target protein on SDS gels of anion exchange fractions of KAK-2S-AVQP-His6 and KDAR-3S-AVQP-His6, suggesting the occurrence of minor degradation in these two proteins. Most of the degradation products were essentially removed by size exclusion chromatography, resulting in high-quality final products for each protein (Figure 13). Nevertheless, the construct not containing the DUF domain (KAK-2S-AVQP-His6) had more severe degradation that was persistent in its final product (Figure 13), suggesting that the DUF domain may provide some level of protection from degradation.

[0385] As seen from previous Ni-affinity chromatography, the two species in KAK-2S-AVQP-His6 and the ladder molecular weight species below the monomeric form in KDAR-3S-AVQP-His6 co-eluted in one predominant peak in AIEX.

[0386] The slightly asymmetric main peaks of both KAK-2S-AVQP-His6 and KDAR-3S-AVQP-His6 in the size-exclusion tailing profiles may also be indicative of the presence of degradants.

[0387] Such degradation was not evident for KDAK-2S-AVQP-His6 (e.g., FIG. 13). The presence of RAS destabilized the protein, and the DUF domain appeared to make the protein more stable.

[0388] Although minor degradation may have occurred, all final products except KAK-2S-AVQP-His6 had high purity and homogeneity, which was also supported by SEC-MALS analysis, which showed the predominant presence of monomers in 2 mg / mL solution for all these proteins (data not shown). KDAK-3S-AVQP and KDAK-2S-AVQP-His6 appeared to have the higher quality of the four antigens, and KAK-2S-AVQP-His6 appeared to have the least homogeneity by SDS-PAGE analysis, although minor species were not resolved by SEC-MALS analysis (e.g., FIG. 13).

[0389] Example 3: Animal Studies Prevention of P. gingivalis bone loss in a mouse model of periodontitis 1. Experiment 1 Antigens tested: KDcAK1n 50 μg, KDcAK1n 0.5 μg, KDA 50 μg, KDA 0.5 μg, KDAK1 50 μg, KDAK1 0.5 μg. All antigens were absorbed onto alum in PBS (pH 7.4).

[0390] KDcAK1n: was batch purified from inclusion bodies on Ni-NTA resin followed by dialysis into 2M urea-PBS under non-reducing conditions.

[0391] KDA and KDAK1: The soluble fraction was subjected to anion exchange chromatography with Ni-affinity (pre-packed column) and gradient elution, followed by dialysis into PBS under non-reducing conditions.

[0392] P. gingivalis-induced alveolar bone loss in the mouse maxilla KDcAK1n and KDA protected against bone loss in animal models. KDcAK1n protected against bone loss at both concentrations tested, whereas KDA only protected at 50 μg (FIG. 4). Data for KDAK1 are not shown.

[0393] Antibody response Serum antibody subclass responses of immunized mice in the periodontitis model were investigated by ELISA. Antisera were used to probe heat-killed P. gingivalis strain W50 as absorbed antigen. Antibody responses are expressed as the resulting ELISA titers minus 3-fold background levels, with each titer representing the mean ± sd of 10 individual mice (Figure 5). KDcAK1n (referred to as "chimera" in Figure 5) and KDA induced the strongest total IgG and IgG1 responses against P. gingivalis whole cells, followed by KDAK1. There were no significant differences in IgG and IgG1 antibody responses at 50 μg between the antigens tested.

[0394] 2. Experiment 2 Antigens tested: KDcAK1n 50 μg, KDcAK1n 0.5 μg, KDA 50 μg, KDA 0.5 μg, KDAK1 50 μg, KDAK1 0.5 μg. All antigens were absorbed onto alum in saline (pH 7.4).

[0395] The antigen was purified in the same manner as in experiment 1.

[0396] P. gingivalis-induced alveolar bone loss in the mouse maxilla In this experiment, the same antigens were investigated as in experiment 1, but they were absorbed into alum using saline rather than PBS. The bone loss results are mirrored in experiment 1 and Figure 4 (data not shown), showing that only KDcAK1n and KDA showed protection, and the use of PBS or saline in the alum preparation did not affect the experimental results.

[0397] Antibody response All antigens tested induced similar total IgG and IgG1 responses against whole cell P. gingivalis. KDA and KDAK1 induced stronger IgG2a responses compared to KDcAK1n (not shown).

[0398] 3. Experiment 3 Antigens tested: All antigens except KDcAK1n were fractionated as described: KDcAK1n (dimer), KDcAK1n (multimer), KDcAK1n (chimera, original), KDcAK1n-4S-AVQP, KDA (dimer), KDA (multimer).

[0399] KDcAK1n-4S-AVQP, KDcAK1n dimer and multimer: Urea extraction from inclusion bodies and Ni-affinity column purification followed by gel filtration of the two KDcAK1n samples under reducing conditions.

[0400] KDA dimers and multimers: From the soluble fraction, Ni-affinity column purification is performed followed by anion exchange and gel filtration under non-reducing conditions.

[0401] KDcAK1n: purified as in Experiment 1.

[0402] Pre-screening of vaccine candidates with mAbs against P. gingivalis epitopes In an attempt to develop a pre-screening assay to determine antigen suitability for animal models, mAbs against KAS2, ABM2, ABM3, and EP1 were used to screen the antigens by ELISA. All vaccine candidates were able to bind to KAS2, ABM3, and EP1 mAbs, as seen by the large titration curves compared to the negative control muBM4 mAb. KDcAK1n and KDcAK1n-4S-AVQP bound to ABM2 mAb, but KDcAK1n dimer, KDcAK1n multimer, KDA dimer, and KDA multimer did not bind - indicating that the epitope recognized by this mAb is not accessible in these constructs (not shown).

[0403] P. gingivalis-induced alveolar bone loss in the mouse maxilla Apart from KDcAK1n with a 2×His tag, none of the fraction antigens containing KDcAK1n with a single His tag protected against P. gingivalis -induced bone loss (Fig. 6 ), although there was a trend (not significant) for protection by the purified dimeric species of the single His tag chimera (KDcAK1n).

[0404] Antibody response Serum antibody subclass responses of immunized mice in a periodontitis model were investigated by ELISA. Antisera were used to probe heat-killed P. gingivalis strain W50 as the adsorbed antigen. All antigens tested generated varying degrees of IgG responses. KDcAK1n and KDA multimers generated strong IgG1 isotype responses.

[0405] Pooled serum samples were used to probe P. gingivalis domains adsorbed to ELISA plates (not shown). Total IgG responses to ABM21 (multimers and dimers) and ABM213 (multimers and dimers) were generated by all antigens except KDcAK1n-4S-AVQP and the KDA dimer.

[0406] IgG responses to DUF2436 were evident only in KDA (dimer and multimer), the only antigen tested in the model with an intact DUF domain. Pooled serum samples were used to probe for P. gingivalis epitope peptides (not shown). No clear pattern was observed between protective and non-protective sera.

[0407] Analysis of non-protective purified dimeric and multimeric antigens Analysis of the single KDA dimeric and multimeric species purified by anion exchange and gel filtration, which did not provide protection in experiment 3, using reducing and non-reducing native PAGE and SDS PAGE revealed that the single species purified in both cases was a disulfide-bridged denatured domain locked into a stable species, explaining why these molecular weight species are stable and can be purified by anion exchange and gel filtration chromatography.

[0408] 4. Experiment 4 Antigens tested: KKDA1nKK, KDAK1n, KDAK1nK-4S-AVQP, KKDAKK.

[0409] All antigens were purified from the soluble fraction under non-reducing conditions using Ni-affinity columns, anion exchange, and gel filtration chromatography, except for KDAK1nK-4S-AVQP, which did not use anion exchange chromatography.

[0410] Alum Absorption The ability of Alhydrogel 2% to absorb antigens was tested by incubating the antigens with alum at 4°C for 60 minutes with gentle mixing. The alum was then pelleted and Bradford protein assays were performed before and after alum adsorption of the antigens. All antigens bound to the alum flies at percentages ranging from 91.7% to 99.3%. SDS-PAGE gels were also run on samples before and after alum absorption and were consistent with the Bradford assay protein determinations.

[0411] Pre-screening of vaccine candidates with mAbs against P. gingivalis epitopes All vaccine candidates were able to bind to KAS2, ABM3, and EP1 mAbs, as seen by the large titration curves compared to the negative control muBM4 mAb. KDAK1nK-4S-AVQP bound to ABM2 mAb, but KKDA1nKK, KDAK1n, and KKDAKK did not - indicating that the epitope recognized by this mAb is not accessible in these constructs (not shown).

[0412] P. gingivalis-induced alveolar bone loss in the mouse maxilla KKDAK1nKK, KDAK1nK-4S-AVQP, and KKDAKK protected against P. gingivalis-induced bone loss (FIG. 7).

[0413] Antibody response Serum antibody subclass responses of immunized mice in a periodontitis model were investigated by ELISA. Antisera were used to probe heat-killed P. gingivalis strain W50 as the adsorbed antigen. All antigens tested generated IgG responses of various degrees. Protective KKDAK1nKK and KKDAKK induced robust total IgG and IgG1 responses against heat-killed P. gingivalis. Interestingly, KDAK1nK-4S-AVQP, which protected against bone loss, elicited lower antibody responses against whole-cell P. gingivalis than some of the less protective antigens (KDAK1n).

[0414] Pooled serum samples were used to probe P. gingivalis domains adsorbed to ELISA plates. Total IgG responses to ABM21 (multimers and dimers) and ABM213 (multimers and dimers) were generated by all antigens to various degrees. IgG responses to DUF2436 were evident only in antigens containing the complete DUF domain (KKDAK1nKK, KDAK1n, KDAK1nK-4S-AVQP, and KKDAKK). Pooled serum samples were also used to probe P. gingivalis epitope peptides, and although there was a clear trend for higher KAS titers in the protective antisera, again no clear pattern between protective and non-protective was observed.

[0415] 5. Experiment 5 Antigens tested All antigens were purified under non-reducing conditions using Ni-affinity chromatography followed by dialysis into saline.

[0416] From KDcAK1n-inclusion bodies, purified using urea and an affinity column (IB, urea, AC); from KDA-soluble fraction, purified using an affinity column (S, AC); from KDA-soluble fraction, purified using urea and an affinity column (S, urea, AC); from KDA-soluble fraction, purified using urea and a batch purification method (S, urea, batch); from KDAK1-soluble fraction, purified using urea and an affinity column (S, urea, AC); from KDAK1-inclusion bodies, purified using urea and an affinity column (IB, urea, AC); from KDAK1-inclusion bodies, urea and a batch purification method was used (IB, urea, batch).

[0417] Pre-screening of vaccine candidates with mAbs against P. gingivalis epitopes. All candidates were able to bind to KAS2, ABM3, and EP1 mAbs as seen by the large titration curves compared to the negative control muBM4 mAb. None of the antigens bound to ABM2 mAb, indicating that the epitopes recognized by this mAb are not accessible in these constructs (not shown).

[0418] P. gingivalis-induced alveolar bone loss in the mouse maxilla KDcAK1n (IB, urea, AC), KDA(S, AC), KDAK1(S, urea, AC), KDAK1(IB, urea, AC), KDAK1(IB, urea, batch), and KDA(S, urea, batch) showed significant protection against bone loss with KDcAK1n (IB, urea, AC), KDA(S, AC), and KDAK1(IB, urea, batch) providing the best protection. Note that the significant protection observed with KDA(S, AC) was lost upon denaturation with urea, e.g., KDA(S, urea, AC) (Figure 8).

[0419] It is noteworthy that unfractionated affinity purified antigen provided protection against bone loss, with particularly good protection observed with affinity purified soluble KDA, but this protection was partially lost upon treatment with urea under oxidizing conditions that would promote disulfide cross-linking of the denatured D and A domains. Again, these results suggest that disulfide cross-linking of the denatured domains may destroy antigen-induced protection, such that urea should be avoided and preparation of soluble folded domains is preferred.

[0420] 6. Experiment 6 Antigens tested All antigens were purified under non-reducing conditions using Ni-affinity chromatography followed by dialysis into saline. Antigens tested were: KDcAK1n-purified from inclusion bodies using urea and affinity columns (IB, urea, AC); KDAK-3S-AVQP, KDAK1nK, KDAK1n-4S-AVQP, KDAK, KKDAKK, KKDAK1nKK.

[0421] P. gingivalis-induced alveolar bone loss in the mouse maxilla All antigens tested protected against P. gingivalis-induced bone loss (Figure 9).

[0422] 7. Experiment 7 Antigens tested In this experiment, the inventors tested several new constructs based on the 4S, 3S, 4S-AVQP and 3S-AVQP mutations: KDcAK1n (prior art chimera), KDcAK1n-4S, KDAK, KDAK-3S, KDAK-3S-AVQP, KDAK1nK-4S-AVQP.

[0423] All antigens had a His tag and were purified using affinity chromatography. Antigens were absorbed onto alum by adding an equal volume of alum to the protein on a weight-to-weight ratio. The alum:protein compound was adjusted to the appropriate concentration (0.5 mg / mL) for IP or SC injection and then incubated at 4° C. with constant mixing. Mice were then injected IP (vaccination 1) or SC (vaccination 2). All antigens were purified under non-reducing conditions using Ni-affinity chromatography followed by dialysis into saline.

[0424] P. gingivalis-induced alveolar bone loss in the mouse maxilla Compared to naive (unloaded) controls, animals injected with KDcAK1n, KDAK-3S-AVQP, and KDAK did not develop significant bone loss (Figure 10). KDAK1nK-4S-AVQP provided partial protection. Infected control mice developed significant levels of alveolar bone loss (Figure 10).

[0425] Results were statistically different from control loading (###p<0.05, ###p<0.01, ###), one-way ANOVA and post hoc Dunnett's T3) (Figure 10).

[0426] 8. Experiment 8 Antigens tested In this experiment, the inventors tested several new constructs based on different permutations of the 1V, 1V-2S, 3S, and AVQP mutations: KDcAK1n (prior art chimera), KDAK-3S-AVQP, KDAK-1V-2S-AVQP, KDAK-1V-AVQP, and KDAK-AVQP.

[0427] All antigens had a His tag and were purified using affinity chromatography. Antigens were absorbed onto alum by adding an equal volume of alum to the protein on a weight-to-weight ratio. The alum:protein compound was adjusted to the appropriate concentration (0.5 mg / mL) for IP or SC injection and then incubated at 4° C. with constant mixing. Mice were then injected IP (vaccination 1) or SC (vaccination 2).

[0428] P. gingivalis-induced alveolar bone loss in the mouse maxilla Mice immunized with chimera (KDcAK1n), KDAK-3S-AVQP, KDAK-1V-2S-AVQP, KDAK-1V-AVQP, and KDAK-AVQP developed significantly less bone loss than infected control mice (Figure 11). Infected control mice developed significant levels of alveolar bone loss compared to naive mice. (Statistical analysis was performed by one-way ANOVA and post hoc Dunnett's T3. *=p<0.05 compared to control challenge group).

[0429] 9. Experiment 9 Antigens tested In this experiment, we tested several constructs carrying different permutations of the 1V-2S, 3S, and AVQP mutations at various concentrations and incorporating antigen purified without a His tag.

[0430] Groups were also included to determine the effect of alum on protein antigenicity. All antigens with His-tags and purified using affinity chromatography, and antigens without His-Tag were purified using a combination of anion exchange, size exclusion, and hydrophobic interaction chromatography (Figure 12). When appropriate, a constant concentration of alum (100 μg / dose) was used to absorb antigens onto alum, regardless of protein concentration (100 μg, 50 μg, and 25 μg). Alum:protein compounds were adjusted to appropriate antigen concentrations (1.0 mg / mL, 0.5 mg / mL, or 0.25 mg / mL) for IP and SC injections, and then incubated at 4°C with constant mixing. Mice were then injected IP (vaccination 1) and SC (vaccination 2).

[0431] Antigens tested were: KDcAK1n-His tagged, purified from inclusion bodies using urea and affinity columns (IB, urea, AC), KDAK-3S-AVQP untagged (100 μg, 50 μg, 25 μg doses), KDAK-1V-2S-AVQP untagged (100 μg, 50 μg, 25 μg doses), KDAK-3S-AVQP His tagged (100 μg dose), KDAK-3S-AVQP untagged, no alum.

[0432] P. gingivalis-induced alveolar bone loss in the mouse maxilla As shown in Figure 12, mice treated with 100 μg of the prior art chimera (KDcAK1n) did not develop significant bone loss compared to naive controls. Furthermore, mice treated with all concentrations of KDAK-3S-AVQP (100 μg, 50 μg, and 25 μg) also did not develop significant levels of alveolar bone loss compared to naive control mice.

[0433] KDAK-1V-2S-AVQP vaccinated mice were protected from alveolar bone loss at the highest dose of 100 μg, but developed significant bone loss when vaccinated with 50 μg and 25 μg or protein.

[0434] Removal of the His-tag from KDAK-3S-AVQP did not significantly affect the efficacy of protection, however removal of alum from the vaccination resulted in higher levels of bone loss for both vaccines.

[0435] Antibody response Serum antibody subclass responses of immunized mice in a periodontitis model were investigated by ELISA. Antisera were used to probe heat-killed P. gingivalis strain W50 as the adsorbed antigen. All antigens tested generated total IgG responses against P. gingivalis whole cells with KDA (S, urea, AC) followed by KDAK1 (IB, urea, batch) and KDcAK1n (IB, urea, AC) having strong IgG1 responses against P. gingivalis whole cells. Only KDAK1 (IB, urea, AC) induced a strong IgG2a response against P. gingivalis whole cells.

[0436] Pooled serum samples were used to probe P. gingivalis domains adsorbed to ELISA plates. Total IgG responses to ABM21 (multimers and dimers) and ABM213 (multimers and dimers) were generated to various degrees by all antigens, with KDA(S, urea, AC), KDcAK1n-1His(IB, urea, AC), and KDAK1(IB, urea, batch) consistently inducing the strongest responses, whereas KDA(S, AC) induced the weakest responses to ABM21 or ABM213 (multimers and dimers). Interestingly, KDA(S, urea, AC), KDA(S, AC), and KDAK1(IB, urea, AC) induced the strongest responses to the DUF2436 domain.

[0437] Pooled serum samples were used to probe for P. gingivalis epitope peptides. KDA(S, urea, AC), KDAK1(IB, urea, batch), and KDAK1(IB, urea, AC) induced strong IgG responses to the KAS and ABM3 epitopes. KDA(S, urea, AC) also induced IgG antibodies to the EP1 and ABM2a epitopes. All antigen groups except KDAK1(S, urea, AC) induced responses to the EP1 epitope. None of the protective antigens generated antisera that bound to the K1 epitope ABM5.

[0438] 10. Experiment 10 Antigens tested In this experiment, we further tested the in vivo activity of the constructs previously tested in Examples 6, 7, 8, and 9, and compared their activity to constructs that do not contain a DUF domain and to constructs that contain active site sequences derived from arginine-dependent gingipains.

[0439] The constructs tested were: KDAK-3S-AVQP (previously tested in experiments 6, 7, 8, and 9), KDAK-2S-AVQP (His tag), KAK-2S-AVQP (His tag), and KDAR-3S-AVQP (His tag).

[0440] P. gingivalis-induced alveolar bone loss in the mouse maxilla In this experiment, the antigen KDAK-3S-AVQP was used at two concentrations: 100 μg / mouse as previously tested, and a two-fold increase to 200 μg / mouse. There was a dose-dependent increase in protection from alveolar bone loss, with mice receiving the 200 μg dose losing less bone than the 100 μg group (Figure 14).

[0441] Restoration of one cysteine, KDAK-3S-AVQP versus KDAK-2S-AVQP, did not result in a significant difference in alveolar bone loss between these two variants (Figure 14).

[0442] Similarly, removal of the D domain, the difference between KDAK-2S-AVQP and KAK-2S-AVQP, did not significantly affect the observed alveolar bone loss (Figure 14), however the mean value for the KAK-2S-AVQP variant was higher and may have reached significance with greater power in the study (e.g., more animals).

[0443] When the C-terminal KAS was changed to the RAS group, the difference between KDAK-3S-AVQP and KDAR-3S-AVQP significantly increased the average alveolar bone loss even further when compared to other variants at equivalent vaccine doses (Figure 14). This highlights the importance of the KAS sequence to provide protection from bone loss in this model. Nevertheless, the KDAR-3S-AVQP antigen still produced a lower average bone loss than the infected control animals, thus still providing evidence that the RAS domain can be utilized in the context of the chimeric fusion protein antigens described herein.

[0444] Antibody response Serum antibody subclass responses of immunized mice in a periodontitis model were investigated by ELISA against several different antigens, including whole P. gingivalis, purified RgpA / Kgp complex, purified catalytic domain Kgpcat, and several different protective epitopes including KAS, and protective epitopes of the ABM domains, ABM3, and ABM2. (RgpA / Kgp complex and purified catalytic domain Kgpcat were purified from P. gingivalis and confirmed to be folded and enzymatically active in the assay.)

[0445] First, antisera were used to probe heat-killed P. gingivalis as an adsorbed antigen. Antibody responses are expressed as the resulting ELISA titers minus two-fold background levels, with each titer representing the mean ± sd of 10 individual mice. All antigens tested generated IgG responses against P. gingivalis, with the strongest responses observed after vaccination with 200 μg vaccination of KDAR-3S-AVQP and KDAK-3S-AVQP. The induced immune responses were predominantly IgG1 immune responses, which are associated with protective immune responses (Figure 15).

[0446] Antibody titers against purified RgpA / Kgp protease complex of P. gingivalis (data not shown) and recombinant Kgpcat from P. gingivalis (data not shown) were also measured. In both cases, the KDAK-3S-AVQP vaccine induced a strong, dose-dependent, and again predominantly IgG1 immune response. The KDAK-2S-AVQP vaccine induced higher IgG1 antibodies against RgpA / Kgp protease complex and recombinant Kgpcat compared to the other two vaccines tested, KAK-2S-AVQP and KDAR-3S-AVQP.

[0447] To further define reactivity against protective epitopes, serum samples were pooled and used to probe P. gingivalis specific biotinylated peptides bound to streptodavidin plates. For all antigens tested, very high titers were observed against the KAS2 peptide. However, KDAK-3S-AVQP induced the highest anti-KAS2 antibodies in a dose-dependent, IgG1-dominant manner. Of the three previously untested vaccines, vaccination with KDAK-2S-AVQP induced the greatest amount of anti-KAS2 antibodies, similar to the Kgpcat and RgpA / Kgp complex responses.

[0448] Compared to anti-KAS2 antibodies, lower levels of anti-ABM2 antibodies were induced after vaccination with all proteins, however the immune responses were still strong and predominantly IgG1. Vaccination with KDAK-3S-AVQP induced a dose-dependent IgG1-dominant immune response, whereas KAK-2S-AVQP induced the strongest immune response observed against ABM2.

[0449] Finally, dose-response data from experiments 9 and 10 on KDAK-3S-AVQP protection of P. gingivalis-induced bone loss correlated closely with antibody titers cross-reactive with purified native Kgpcat protease (FIG. 16).

[0450] Summary and discussion of the results presented in Examples 2 and 3 The studies described in this report compared the production and efficacy of various vaccine candidates to identify chimeric vaccine components that promote ease of production (such as components that contribute to solubility, stability, and reduced tendency to multimerize) and are most effective in eliciting an immune response to P. gingivalis and / or reducing P. gingivalis-induced alveolar bone loss.

[0451] Although effective in preventing periodontal bone loss in animal periodontitis models, the prior art vaccine, KDcAK1n, is expressed as inclusion bodies by E. coli and exhibits variable solubility and stability.

[0452] KDcAK1n was based on a fusion between the active site sequence (KAS or K) of the Lys-specific gingipain Kgp and the processed adhesin fragment (A1) of the Kgp polyprotein. From structural analysis of the Kgp polyprotein domain, it is now clear that the A1 adhesin fragment found on the cell surface contains three distinct structural domains: the DUF domain (D), the ABM domain (A), and the K1 domain (K1). Processing of the Kgp polyprotein on the surface of P. gingivalis to release the proteinase catalytic domain and the adhesin involves N-terminal truncation of the DUF domain by 38 amino acid residues. These extra N-terminal 38 residues were added to the construct to produce an intact DUF domain in place of the truncated domain (Dc), producing highly soluble recombinant proteins (e.g., DUF2436, KDA, and KKDAKK).

[0453] KDcAK1n also contains a C-terminally truncated K1 domain (K1n) again based on cell surface processing of the Kgp polyprotein to produce the A1 adhesin fragment. Adding the missing C-terminal sequence to K1n did not improve solubility as the protein was still expressed mainly as inclusion bodies by E. coli. Most constructs with partial or complete K1 domains showed some solubility issues, so we concluded that the complete DUF domain is a critical domain for expression of constructs as soluble proteins by E. coli and that inclusion of the K1 domain in chimeric variants should be avoided if possible. Furthermore, from the periodontal bone loss and epitope analysis data from animal studies, it seemed likely that the K1 domain would not add significantly to protection, especially when none of the protective antigens generated antisera that bound to the K1 epitope ABM5.

[0454] KDcAK1n contains four cysteine ​​residues, one in DUF, two in ABM, and one in K1. These cysteine ​​residues are thought to contribute to the problem of inclusion body formation. P. gingivalis is an obligate anaerobe and requires a highly reduced environment to be pathogenic. Known or modeled structures of the Kgp domain show that the cysteine ​​residues are reduced on the cell surface and are not involved in disulfide bridges. Expression in E. coli under more oxidizing conditions results in disulfide bridge formation between the denatured domain and inclusion body formation. Disulfide bridges between the denatured domains of the chimera are visible on non-reducing native-PAGE that change upon reduction.

[0455] The results presented herein showed that the folded ABM domain of Kgp multimerizes by beta-strand exchange to form a homogenous ladder independent of reducing agents (Figure 3, lanes 9 and 10). This novel beta-strand exchange multimerization is a key mechanism used by P. gingivalis to form a surface layer of gingipains not only on cells but also on outer membrane vesicles released into the host during disease progression. The present study characterizing the prior art chimeric KDcAK1n and domains clearly shows that there are two forms of multimers present in the original chimera: disulfide-bridged denatured domain-based multimers and beta-strand exchanged native ABM domain-based multimers. Attempts to separate and purify these different forms into specific dimers and larger molecular weight multimers were unsuccessful. Furthermore, the use of these different purified single species did not provide protection from periodontal bone loss in animal models because the single species are disulfide-locked denatured structures (Figure 6). This study suggests that soluble, defined, homogeneous recombinants with folded D and A domains free of disulfide bridges are superior to the mixture of denatured and native forms produced by solubilization of the chimeras from inclusion bodies in 8 M urea, which has low yields (Figures 6, 8, and 9).

[0456] These results suggest that the presence of cysteine ​​residues in the antigen may be problematic for expression and purification of soluble defined candidates for commercial development. Therefore, the possibility of mutating those cysteines to serines was explored. Mutation of the four cysteine ​​residues to four serine residues did not stop ABM(A) native domain beta-strand exchange multimerization, showing that the ABM domain still folded properly in the "4C" to "4S" mutant (Figure 3).

[0457] Furthermore, these results show that mutation of a key sequence in the ABM domain predicted to be the Pro hinge elbow to allow beta-strand inversion (PVQN>AVQP) eliminated beta-strand exchange multimerization (Figure 3). Thus, the combination of mutating the four Cys residues to Ser and the hinge region PVQN to AVQP eliminates multimerization under both oxidative and reductive conditions (Figure 3). In this regard, the mutant KDAK1nK-4S-AVQP protected against periodontal bone loss and generated good antibody titers against predicted protective epitopes (KAS, DUF, ABM) in an animal periodontitis model (Figure 7).

[0458] Examination of antibody responses against the various segments of the chimera (K, D, A, and K1) and the suspected important epitopes (KAS, ABM2, ABM3, EP1) using protected and non-protected sera did not provide any clear pattern, but it was apparent that those chimeric variants that were protected tended to show strong (IgG / IgG1) responses against whole P. gingivalis cells and known protective epitopes (KAS, DUF, ABM) such that protection may not be related to any one epitope, but more to a combination of epitopes required to ensure complete protection of periodontal bone loss. However, for those chimeric variants that provided protection, one epitope with the active site sequence KAS tended to stand out.

[0459] Although there was no clear indication that any one epitope was more important such that the response to that epitope could be used as a surrogate or biomarker of protection induced by the vaccine, it was clear that the protective chimeric variants tended to generate good antibody responses against the protease active site sequence (KAS or K). Antibodies generated against the active site sequence have previously been shown to neutralize the proteolytic activity of gingipains (Kgp and RgpA / B), the major virulence factor of P. gingivalis. Therefore, in an approach to boost antibody titers against the active site and thus boost protection, we added an extra copy of the active site sequence (K) to KDA and KDAK1n, and both constructs showed improved protection in an animal periodontitis model (Figure 8). The soluble non-multimerizing variant KDAK1nK-4S-AVQP, carrying two copies of the KAS (K) motif, conferred good protection from periodontal bone loss induced by P. gingivalis. (Figure 9).

[0460] Other soluble, non-multimerizing mutants, including KDAK-3S-AVQP, KDAK-1V-2S-AVQP, and KDAK-2S-AVQP, also conferred good protection from periodontal bone loss induced by P. gingivalis (Figures 9-13).

Claims

1. A chimeric or fusion protein for inducing an immune response against P. gingivalis, the protein comprising a first polypeptide and a second polypeptide; A) the first polypeptide comprises or consists of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence at least 80% identical thereto; B) the second polypeptide comprises or consists of the amino acid sequence of the adhesin domain of Arg- or Lys-gingipain of P. gingivalis; the second polypeptide comprises the sequence of one or more adhesin binding motifs (ABMs); the second polypeptide is a) does not contain a truncated adhesin domain (CAD) sequence, such as the amino acid sequence set forth in SEQ ID NO: 12 or 13, or a sequence having at least 80% identity thereto; and b) comprising an amino acid sequence substantially corresponding to the entire length of the DUF2436 domain of Arg- or Lys-gingipain, or a sequence at least 80% identical thereto; and c) one or more cysteine ​​amino acid substitutions in the ABM compared to naturally occurring Arg- or Lys-gingipain sequences in the corresponding regions; and / or d) i) substitution of proline and / or asparagine residues in the sequence PVQN at positions corresponding to or equivalent to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1); ii) a substitution of the motif NEFA with SEYQ in the sequence at positions corresponding to or equivalent to residues 2 to 5 of the sequence of SEQ ID NO: 14 or 19 (ABM1); iii) a second tyrosine residue at a position corresponding to or equivalent to residue 5 of SEQ ID NO: 15 or 20 (ABM2), and a substitution of an alanine residue for the tryptophan residue at a position corresponding to or equivalent to residue 23 of SEQ ID NO: 14 or 19 (ABM1).

2. 2. The chimeric or fusion protein of claim 1, wherein the one or more ABMs comprise a sequence set forth in SEQ ID NO: 15 or 20, SEQ ID NO: 14 or 19, and / or SEQ ID NO: 17 or 21, or a sequence at least 80% identical thereto, preferably wherein the one or more ABMs comprise a sequence set forth in any of SEQ ID NO: 16 or SEQ ID NO: 18 or 22, or a sequence at least 80% identical thereto.

3. 2. The chimeric or fusion protein of claim 1, wherein the amino acid sequence substantially corresponding to the entire length of the DUF2436 domain of Arg- or Lys-gingipain is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the DUF2436 domain of Arg- or Lys-gingipain, and preferably the amino acid sequence of the DUF2436 domain of Arg- or Lys-gingipain is the sequence set forth in SEQ ID NO: 23 or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

4. 2. The chimeric or fusion protein of claim 1, wherein the second polypeptide comprises the sequence set forth in SEQ ID NO: 34, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

5. 2. The chimeric or fusion protein of claim 1, wherein the second polypeptide comprises a substitution of the proline and / or asparagine residue in the sequence PVQN of ABM1 of the P. gingivalis adhesin domain at a position corresponding to or equivalent to residues 6-9 of the sequence of SEQ ID NO: 14 or 19, preferably wherein the second polypeptide comprises the sequence AVQP in ABM1 at a position corresponding to or equivalent to residues 6-9 of the sequence of SEQ ID NO: 14 or 19.

6. 6. The chimeric or fusion protein of claim 5, wherein the second polypeptide comprises a substitution of the cysteine ​​residue in the DUF2436 domain and / or the second polypeptide comprises a substitution of one or more of the cysteine ​​residues in the ABM domain, preferably wherein the substitution of one or more cysteine ​​residues is a substitution with a serine or valine residue, more preferably wherein the substitution of one or more cysteine ​​residues is a substitution with a serine residue.

7. 7. The chimeric or fusion protein of claim 6, wherein the sequence of the one or more ABM domains is set forth in SEQ ID NO: 18 and the one or more cysteine ​​residues correspond to residues at positions 36 and 50 of SEQ ID NO: 18 or 22 or equivalent positions thereof.

8. 2. The chimeric or fusion protein of claim 1, wherein the second polypeptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 35 to 49, preferably wherein the second polypeptide comprises or consists of an amino acid sequence set forth in SEQ ID NOs: 42 or 44 to 49.

9. the first polypeptide is linked to the second polypeptide, located at the N-terminus of the second polypeptide, directly connected to said second polypeptide; or linked to the second polypeptide via a linker, e.g., a peptide linker; Optionally, the peptide linker comprises at least 2 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, preferably the linker comprises 50 amino acids or less; or the peptide linker is 25 amino acids or less, 20 amino acids or less, 15 amino acids or less, or 10 amino acids or less; The chimeric or fusion protein of claim 1.

10. the first polypeptide comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 1-11, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; Optionally, the first polypeptide comprises or consists of an amino acid sequence set forth in SEQ ID NO: 1 or 2, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; The chimeric or fusion protein of claim 1.

11. the chimeric or fusion protein comprises one or more additional polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis, or a sequence at least 80% identical thereto; Optionally, the one or more further polypeptides are located at the N-terminus of the first polypeptide, at the C-terminus of the first polypeptide, at the N-terminus of the second polypeptide, at the C-terminus of the second polypeptide; Optionally, the one or more further polypeptides are directly linked to the first or second polypeptide of the chimeric or fusion protein. Or, the one or more further polypeptides are linked to the first or second polypeptide by a peptide linker; Preferably, the one or more further polypeptides comprise or consist of an amino acid sequence selected from the group of SEQ ID NOs: 1 to 11, or a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto; More preferably, the first polypeptide and the one or more further polypeptides are identical or 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, the first polypeptide and the one or more additional polypeptides comprise the amino acid sequence of the active site of a heterologous gingipain of P. gingivalis; Preferably, the first polypeptide and the one or more further polypeptides comprise the amino acid sequence of the active site of Lys-gingipain. Or, the first polypeptide comprises the amino acid sequence of the active site of Lys-gingipain, and the one or more further polypeptides comprise the amino acid sequence of the active site of Arg-gingipain; The chimeric or fusion protein of claim 1.

12. the chimeric or fusion protein comprises or consists of an amino acid sequence set forth in any one of SEQ ID NOs: 58, 59, 60, 61, 69, 70, 71, 72, 73, 74, or 75, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% thereof; Preferably, the chimeric or fusion protein comprises or consists of the amino acid sequence set forth in SEQ ID NO: 58 or 59, SEQ ID NO: 60 or 61, SEQ ID NO: 69, or SEQ ID NO: 72 or 74. The chimeric or fusion protein of claim 1.

13. the protein comprises additional amino acid residues to facilitate expression in a recombinant expression system and / or to facilitate purification of the protein; Optionally, the additional amino acids comprise 1, 2, 3, 4, or 5 amino acids in the N-terminal region, optionally including an N-terminal methionine and / or alanine residue; Optionally, the additional amino acids include 1, 2, 3, 4, or 5 amino acids in the C-terminal region, preferably to facilitate purification. The chimeric or fusion protein of claim 1.

14. A nucleic acid encoding the chimeric or fusion protein of claim 1, or a vector or construct comprising said nucleic acid.

15. 10. A composition comprising the chimeric or fusion protein of claim 1, preferably said composition being a vaccine composition and comprising one or more adjuvants for enhancing the immune response to said chimeric or fusion protein.

16. Induce an immune response against P. gingivalis in a subject; immunize a subject against P. gingivalis infection; - treating a P. gingivalis infection in a subject; or - Use of a chimeric or fusion protein according to claim 1 in the manufacture of a medicament for minimising or reducing the severity of one or more symptoms of P. gingivalis infection in a subject.

17. Induce an immune response against P. gingivalis in a subject; immunize a subject against P. gingivalis infection; - treating a P. gingivalis infection in a subject; or - The chimeric or fusion protein of claim 1 or the composition of claim 15 for use in minimizing or reducing the severity of one or more symptoms of P. gingivalis infection in a subject.

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