Chimeric polypeptide

Abstract

To provide a vaccine for use in reducing the incidence and / or severity of periodontal pathogen Porphyromonas gingivalis (P. gingivalis) infection, or for treating P. gingivalis infection and disease in the target population. [Solution] A chimeric or fusion protein for inducing an immune response against P. gingivalis is provided, comprising a protein first polypeptide and a second polypeptide, wherein A) the first polypeptide contains 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, and B) the second polypeptide contains or consists of the amino acid sequence of the adhesion factor domain of Arg- or Lys-gingipain of P. gingivalis.

Description

Technical Field

[0001] The present invention relates to chimeric polypeptides useful for inducing an immune response against P. gingivalis, compositions containing them, and their use for the prevention and treatment of P. gingivalis-related conditions and diseases.

[0002] Related Applications This application claims priority from Australian Provisional Application No. 2022 / 900103, the entire content of which is incorporated herein by reference.

Background Art

[0003] If dental plaque remains accumulated around the teeth at the edge of the gum (gum margin), it causes inflammation of the gum (gingivitis). Chronic gingivitis can lead to the emergence of the periodontal pathogen Porphyromonas gingivalis (P. gingivalis) at the base of the periodontal pocket, potentially resulting in the development of chronic infections and severe diseases. This severe form of periodontal disease is called periodontitis and can lead to tooth loss through an approach by the immune system to eliminate the infection.

[0004] Chronic periodontitis is an inflammatory disease of the tooth supporting tissues, leading to the resorption of the alveolar bone and ultimately tooth loss. This disease is a major public health problem in all societies, with an estimated maximum of 30% of the adult population affected and 12 - 15% of the adult population affected by the severe form.

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

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

[0007] The magnitude of this public health problem suggests that a vaccine and means of providing it that offer a strong protective response against P. gingivalis infection are needed.

[0008] One problem was that it was unclear how to obtain a robust protective response against P. gingivalis infection, which has an excessive number of pathogenic factors to choose from.

[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 the target population.

[0010] Therefore, alternative and / or improved approaches to the design and manufacture of P. gingivalis vaccines, and alternative and / or improved vaccines produced from P. gingivalis, are needed.

[0011] Any reference to prior art in this specification does not constitute an admission or suggestion that such prior art forms part of the common general knowledge in any jurisdiction, or that such prior art can be reasonably expected to be understood, considered relevant, and / or combined with other prior art by those skilled in the art. [Overview of the project]

[0012] The present invention provides a chimeric or fusion protein for inducing an immune response against P. gingivalis, wherein the protein comprises a first polypeptide and a second polypeptide. A) The first polypeptide contains, 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 contains or consists of the amino acid sequence of the adhesion factor domain of Arg- or Lys-gingipain of P. gingivalis. The second polypeptide comprises a sequence of one or more adhesion factor-binding motifs (ABMs), preferably the ABMs correspond to part or all of the ABM between the DUF2436 domain and the cleaved adhesion factor domain (CAD) of P. gingivalis gingipain. Preferably, one or more ABMs include the sequence described in SEQ ID NO: 15 or 20 (ABM2) and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence that is at least 80% identical to them. More preferably, one or more ABMs include the sequence described 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 that is at least 80% identical to them. Most preferably, one or more ABMs contain the sequence described in SEQ ID NO: 16, or SEQ ID NO: 18 or 22, or SEQ ID NO: 27, 63, or 64, or a sequence that is at least 80% identical to them. The second polypeptide comprises a portion or all of a cleaved adhesion factor domain (CAD), preferably the amino acid sequence described in SEQ ID NO: 12 or 13, or a portion or all of a sequence of CAD having at least 80% identical to those sequences. The second polypeptide is a) an amino acid sequence substantially corresponding to the full length of the DUF2436 domain of Arg- or Lys-gingipain, preferably the amino acid sequence described in SEQ ID NO: 23, or a sequence that is at least 80% identical thereto, and / or b) Compared to a naturally occurring Arg- or Lys-gingipain sequence in the corresponding region, it contains one or more cysteine ​​amino acid substitutions in the adhesion factor domain, and / or c) Includes one or more amino acid motif substitutions selected from the following: i) Substitution of proline and / or asparagine residues in sequence PxxN at positions corresponding to or equivalent to residues 6-9 of sequence SEQ ID NO: 14 or 19 (ABM1), ii) Substitution of the motif NxFA with SxYQ in the sequence corresponding to or equivalent to residues 2-5 of sequence number 14 or 19 (ABM1), iii) Substitution of a second tyrosine residue at the position corresponding to or equivalent to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2), and a tryptophan residue at the position corresponding to or equivalent to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue.

[0013] Preferably, the chimeric or fusion protein comprises one or more further polypeptides containing, or comprising, the amino acid sequence of the Arg- or Lys-gingipaine active site of P. gingivalis, or a sequence that is at least 80% identical thereto. The one or more further polypeptides containing, or comprising the Arg- or Lys-gingipaine active site 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 further polypeptides containing, or comprising, the amino acid sequence of the Arg- or Lys-gingipaine active site of P. gingivalis, or a sequence that is at least 80% identical thereto. In such embodiments, the two further 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] One or more further polypeptides may be linked to the first or second polypeptide of the chimeric or fusion protein, preferably via a linker of 50 amino acids or less, or directly linked to the first or second polypeptide.

[0015] In any embodiment, the first polypeptide comprises or consists of amino acid sequences selected from the following group: SEQ ID NOs: 1-11, 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 them.

[0016] One or more further polypeptides preferably contain or consist of amino acid sequences selected from the following group: SEQ ID NOs: 1-11, 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 them.

[0017] In any embodiment, a first polypeptide and further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis contain, 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 an identical amino acid sequence. The first polypeptide and further polypeptides may be derived from the active site of a different gingipain (for example, from gingipain of a different strain of P. gingivalis). The first polypeptide and further polypeptides may have amino acid sequences derived from different gingipains (for example, one polypeptide may have an amino acid sequence of the active site derived from Kgp and another polypeptide may have an amino acid sequence of the active site derived from Rgp, or alternatively, one polypeptide may have an amino acid sequence of the active site derived from RgpA and another polypeptide may have an amino acid sequence of the active site 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, wherein the protein comprises a first polypeptide linked to a second polypeptide. A) The first polypeptide contains, 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 contains or consists of the amino acid sequence of the adhesion factor domain of Arg- or Lys-gingipain of P. gingivalis. The second polypeptide comprises a sequence of one or more adhesion factor-binding motifs (ABMs), preferably the ABMs correspond to part or all of the ABM between the DUF2436 domain and the cleaved adhesion factor domain (CAD) of P. gingivalis gingipain. Preferably, one or more ABMs comprise the sequence set forth in SEQ ID NO: 15 or 20 (ABM2), and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence that is at least 80% identical thereto. More preferably, one or more ABMs comprise the sequence 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 that is at least 80% identical thereto. Most preferably, one or more ABMs comprise the sequence set forth in any of SEQ ID NO: 16 or SEQ ID NO: 18 or 22, or a sequence that is at least 80% identical thereto. The second polypeptide a) comprises an amino acid sequence that substantially corresponds to the full length of the DUF2436 domain of Arg- or Lys-Zinjanthropus, preferably the amino acid sequence set forth in SEQ ID NO: 23, or a sequence that is at least 80% identical thereto.

[0019] As used herein, an amino acid sequence that substantially corresponds to the full length of the DUF2436 domain, or a sequence that is 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 Arg- or Lys-Zinjanthropus.

[0020] In a preferred embodiment, the amino acid sequence of the DUF2436 domain of Arg- or Lys-Zinjanthropus is the sequence set forth in SEQ ID NO: 23 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0021] Most preferably, the second polypeptide comprises, or consists of, the sequence set forth in SEQ ID NO: 33, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In an alternative embodiment, the second polypeptide comprises the sequence set forth in SEQ ID NO: 59, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto. In an alternative embodiment, the second polypeptide comprises the sequence set forth in SEQ ID NO: 61, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0022] Preferably, the chimeric or fusion protein comprises one or more additional polypeptides that comprise, or consist 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. One or more additional polypeptides that comprise, or consist 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 additional polypeptides may be linked to the first or second polypeptide of the chimeric or fusion protein, preferably via a linker of 50 amino acids or less, or may be linked directly to the first polypeptide.

[0023] In any embodiment, the first polypeptide comprises, or consists of, an amino acid sequence selected from the following group: SEQ ID NOs: 1-11, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0024] One or more further polypeptides preferably contain or consist of amino acid sequences selected from the following group: SEQ ID NOs: 1-11, 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 them.

[0025] In any embodiment, a first polypeptide and further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis contain, 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 an identical amino acid sequence. The first polypeptide and further polypeptides may be derived from the active site of a different gingipain (for example, from gingipain of a different strain of P. gingivalis). The first polypeptide and further polypeptides may have amino acid sequences derived from different gingipains (for example, one polypeptide may have an amino acid sequence of the active site derived from Kgp and another polypeptide may have an amino acid sequence of the active site derived from Rgp, or alternatively, one polypeptide may have an amino acid sequence of the active site derived from RgpA and another polypeptide may have an amino acid sequence of the active site derived from RgpB).

[0026] In a particularly preferred embodiment of a second aspect of the present invention, the chimeric or fusion protein contains, or comprises, an amino acid sequence described in any one of SEQ ID NOs. 55, 56, 58, or 60, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to them.

[0027] In a second aspect of the present invention, the present invention provides a chimeric or fusion protein for inducing an immune response against P. gingivalis, wherein the protein comprises a first polypeptide and a second polypeptide. A) The first polypeptide contains, 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 contains or consists of the amino acid sequence of the adhesion factor domain of Arg- or Lys-gingipain of P. gingivalis. The second polypeptide comprises a sequence of one or more adhesion factor-binding motifs (ABMs), preferably the ABMs correspond to part or all of the ABM between the DUF2436 domain and the cleaved adhesion factor domain (CAD) of P. gingivalis gingipain. Preferably, one or more ABMs include the sequence described in SEQ ID NO: 15 or 20 (ABM2) and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence that is at least 80% identical to them. More preferably, one or more ABMs include the sequence described 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 that is at least 80% identical to them. Most preferably, one or more ABMs contain the sequence described in SEQ ID NO: 16, or SEQ ID NO: 18 or 22, or SEQ ID NO: 27, 63, or 64, or a sequence that is at least 80% identical to them. The second polypeptide comprises a portion or all of a cleaved adhesion factor domain (CAD), preferably the amino acid sequence described in SEQ ID NO: 12 or 13, or a portion or all of a sequence of CAD having at least 80% identical to those sequences, and the second polypeptide comprises a) Compared to a naturally occurring Arg- or Lys-gingipain sequence in the corresponding region, it contains one or more cysteine ​​amino acid substitutions in the adhesion factor domain, and / or b) comprising one or more amino acid motif substitutions selected from the following: i) Substitution of proline and / or asparagine residues in sequence PxxN at positions corresponding to or equivalent to residues 6-9 of sequence number 14 or 19 (ABM1), ii) Substitution of the motif NxFA with SxYQ in sequences corresponding to or equivalent to residues 2-5 of sequence number 14 or 19 (ABM1), iii) Substitution of a second tyrosine residue at the position corresponding to or equivalent to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2), and a tryptophan residue at the position corresponding to or equivalent to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue.

[0028] One or more cysteine ​​amino acid substitutions may be substitutions to serine or valine residues. Preferably, one or more cysteine ​​substitutions may include one or more substitutions to serine residues.

[0029] In certain embodiments, only one cysteine ​​residue may be substituted. In other embodiments, two or three cysteine ​​residues may be substituted. In a particularly preferred embodiment, the cysteine ​​residues are substituted with a combination of valine and serine residues. In other embodiments, all substituted cysteine ​​residues are substituted with serine, or all substituted cysteine ​​residues are substituted with valine.

[0030] In a particularly preferred embodiment, the adhesion factor domain comprises the DUF2436 domain or a portion thereof, wherein the cysteine ​​residue in the DUF2436 domain is substituted with serine or valine, preferably serine. In other embodiments, the adhesion factor domain comprises the DUF2436 domain, wherein the cysteine ​​residue in the DUF2436 domain is unsubstituted, and preferably one or more cysteine ​​residues in the remaining portion of the adhesion factor domain are substituted.

[0031] Optionally, the adhesion factor domain contains or comprises the sequence described in Sequence ID No. 23 or a sequence that is at least 80% identical thereto, and the cysteine ​​residue is substituted with a serine or valine residue.

[0032] Optionally, the adhesion factor domain contains or comprises the sequence described in Sequence ID No. 25 or a sequence that is at least 80% identical thereto, and the cysteine ​​residue is substituted with a serine or valine residue.

[0033] Preferably, the adhesion factor domain contains or comprises the sequence described in SEQ ID NO: 33, SEQ ID NO: 59, or SEQ ID NO: 61, or a sequence that is at least 80% identical thereto, and one or more cysteine ​​residues are substituted with serine or valine residues.

[0034] In a particularly preferred embodiment of a second aspect of the present invention, a chimeric or fusion protein for inducing an immune response against P. gingivalis is provided, wherein the protein comprises a first polypeptide and a second polypeptide. A) The first polypeptide contains, 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 contains or consists of the amino acid sequence of the adhesion factor domain of Arg- or Lys-gingipain of P. gingivalis. The second polypeptide comprises a sequence of one or more adhesion factor-binding motifs (ABMs), preferably the ABMs correspond to part or all of the ABM between the DUF2436 domain and the cleaved adhesion factor domain (CAD) of P. gingivalis gingipain. Preferably, one or more ABMs include the sequence described 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 that is at least 80% identical to them. More preferably, one or more ABMs include the sequence described in SEQ ID NO: 16, or SEQ ID NO: 18 or 22, or SEQ ID NO: 27, 63, or 64, or a sequence that is at least 80% identical to those sequences. The second polypeptide is a) Compared to naturally occurring adhesion factor domain sequences, the sequence includes one or more cysteine ​​substitutions to serine amino acids, b) Proline substitutions and / or asparagine substitutions in sequence PxxN at positions corresponding to or equivalent to residues 6-9 of sequence (ABM1) of sequence SEQ ID NO: 14 or 19 (corresponding to or equivalent to residues 63-66 of sequence SEQ ID NO: 18 or 22).

[0035] Preferably, the chimeric or fusion protein comprises one or more further polypeptides containing, or comprising, the amino acid sequence of the Arg- or Lys-gingipain active site of P. gingivalis, or a sequence that is at least 80% identical thereto. The one or more further polypeptides containing, or comprising the Arg- or Lys-gingipain active site of P. gingivalis, may be located at the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, the C-terminus of the second polypeptide, or the N-terminus of the second polypeptide.

[0036] In a preferred embodiment, the second polypeptide comprises the amino acid sequence of the adhesion factor domain of Arg- or Lys-gingipain of P. gingivalis, wherein the adhesion factor domain comprises the sequence described in SEQ ID NO: 18 or 22 or a sequence that is at least 80% identical thereto, wherein one or both of the cysteine ​​residues in SEQ ID NO: 18 or 22 are substituted with serine residues, and the proline and / or asparagine residues in the sequence PxxN at positions 63-66 or equivalent in SEQ ID NO: 18 or 22 are substituted. Optionally, the proline residues are substituted with alanine residues, and / or the asparagine residues are substituted with either proline or alanine residues, preferably such that the sequence at positions 63-66 of SEQ ID NO: 18 or 22 is AxxP (e.g., AVQP), where proline is substituted with alanine and asparagine is substituted with proline.

[0037] In a particularly preferred embodiment, the second polypeptide comprises the amino acid sequence described in SEQ ID NO: 33, or a sequence that is at least 80% identical thereto, wherein one, two, or three cysteine ​​residues are substituted with serine residues. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 33, or at an equivalent position, is not substituted with either a serine or valine residue. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 33, or at an equivalent position, is not substituted with either a serine or valine residue, and the cysteine ​​residues at positions 208 and 222 of SEQ ID NO: 33, or at equivalent positions, are substituted with serine residues.

[0038] In a particularly preferred embodiment, the second polypeptide comprises the amino acid sequence described in SEQ ID NO: 33, or a sequence that is at least 80% identical thereto, wherein one, two, or three cysteine ​​residues are substituted with serine residues, and proline and asparagine residues at positions 235-238 in sequence PxxN, or equivalent positions, are substituted. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 33, or equivalent position, is not substituted with either a serine or valine residue. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 33, or equivalent position, is not substituted with either a serine or valine residue, the cysteine ​​residues at positions 208 and 222 of SEQ ID NO: 33, or equivalent positions, are substituted with serine residues, the proline residue at position 235, or equivalent position, is substituted with an alanine residue, and the asparagine residue at position 238, or equivalent position, is substituted with proline.

[0039] In a particularly preferred embodiment, the second polypeptide contains or comprises 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 any of them.

[0040] In any embodiment, the first polypeptide comprises or consists of amino acid sequences selected from the following group: SEQ ID NOs: 1-11, or sequences that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to them.

[0041] One or more further polypeptides preferably contain or consist of amino acid sequences selected from the following group: SEQ ID NOs: 1-11, 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 them.

[0042] In any embodiment, a first polypeptide and further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis contain, 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 an identical amino acid sequence. The first polypeptide and further polypeptides may be derived from the active site of a different gingipain (for example, from gingipain of a different strain of P. gingivalis). The first polypeptide and further polypeptides may have amino acid sequences derived from different gingipains (for example, one polypeptide may have an amino acid sequence of the active site derived from Kgp and another polypeptide may have an amino acid sequence of the active site derived from Rgp, or alternatively, one polypeptide may have an amino acid sequence of the active site derived from RgpA and another polypeptide may have an amino acid sequence of the active site derived from RgpB).

[0043] In a preferred embodiment of a second aspect of the present invention, the chimeric or fusion protein contains, or comprises, an amino acid sequence described in any of SEQ ID NOs: 49, 50, or 51, 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 to them.

[0044] In a third aspect of the present invention, the present invention provides a chimeric or fusion protein for inducing an immune response against P. gingivalis, wherein the protein comprises a first polypeptide and a second polypeptide. A) The first polypeptide contains, 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 contains or consists of the amino acid sequence of the adhesion factor domain of Arg- or Lys-gingipain of P. gingivalis. The second polypeptide comprises a sequence of one or more adhesion factor-binding motifs (ABMs), preferably the ABMs correspond to part or all of the ABM between the DUF2436 domain and the cleaved adhesion factor domain (CAD) of P. gingivalis gingipain. Preferably, one or more ABMs include the sequence described in SEQ ID NO: 15 or 20 (AMB2) and / or SEQ ID NO: 17 or 21 (ABM3), or a sequence that is at least 80% identical to them. More preferably, one or more ABMs include the sequence described 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 that is at least 80% identical to them. Most preferably, one or more ABMs contain the sequence described in SEQ ID NO: 16 or SEQ ID NO: 18 or 22, or a sequence that is at least 80% identical to them. The second polypeptide comprises a portion or all of a cleaved adhesion factor domain (CAD), preferably the amino acid sequence described in SEQ ID NO: 12 or 13, or a portion or all of a sequence of CAD having at least 80% identical to those sequences. The second polypeptide is a) an amino acid sequence substantially corresponding to the full length of the DUF2436 domain of Arg- or Lys-gingipain, preferably the amino acid sequence described in SEQ ID NO: 23, or a sequence that is at least 80% identical thereto, Includes one or more amino acid substitutions selected from the following: b) One or more cysteine ​​amino acid substitutions in the adhesion factor domain compared to the naturally occurring Arg- or Lys-gingipain sequence in the corresponding region, and c) One or more amino acid motif substitutions selected from the following: i) Substitution of proline and / or asparagine residues in sequence PxxN at positions corresponding to or equivalent to residues 6-9 of sequence SEQ ID NO: 14 or 19 (ABM1), ii) Substitution of the motif NxFA with SxYQ in the sequence corresponding to or equivalent to residues 2-5 of sequence number 14 or 19 (ABM1), iii) Substitution of a second tyrosine residue at the position corresponding to or equivalent to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2), and a tryptophan residue at the position corresponding to or equivalent to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue.

[0045] One or more cysteine ​​amino acid substitutions may be substitutions to serine or valine residues. Preferably, one or more cysteine ​​substitutions may include one or more substitutions to serine residues.

[0046] In certain embodiments, only one cysteine ​​residue is substituted. In other embodiments, two or three cysteine ​​residues are substituted. In a particularly preferred embodiment, the cysteine ​​residues are substituted with a combination of valine and serine residues. In other embodiments, all substituted cysteine ​​residues are substituted with serine, or all substituted cysteine ​​residues are substituted with valine.

[0047] In a particularly preferred embodiment, the adhesion factor domain comprises a DUF2436 domain, where the cysteine ​​residue in the DUF2436 domain is substituted with serine or valine, preferably serine. In other embodiments, the adhesion factor domain comprises a DUF2436 domain, where the cysteine ​​residue in the DUF2436 domain is unsubstituted, and preferably one or more cysteine ​​residues in the rest of the adhesion factor domain are substituted.

[0048] Preferably, the adhesion factor domain contains or comprises the sequence described in SEQ ID NO: 33, SEQ ID NO: 59, or SEQ ID NO: 61, or a sequence that is at least 80% identical thereto, and one or more cysteine ​​residues are substituted with serine or valine residues.

[0049] According to this aspect of the present invention, the sequence PxxN at a position corresponding to or equivalent to residues 235-238 of SEQ ID NO: 33 includes substitutions of proline and asparagine residues. Preferably, the substitution is from PxxN to AxxP.

[0050] In a particularly preferred embodiment of this aspect of the present invention, the second polypeptide comprises the amino acid sequence described in SEQ ID NO: 33, or a sequence that is at least 80% identical thereto, wherein one, two, or three cysteine ​​residues are substituted with serine residues. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 33, or at an equivalent position, is not substituted with either a serine or valine residue. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 33, or at an equivalent position, is not substituted with either a serine or valine residue, and the cysteine ​​residues at positions 208 and 222 of SEQ ID NO: 33, or at equivalent positions, are substituted with serine residues.

[0051] In a particularly preferred embodiment, the second polypeptide comprises the amino acid sequence described in SEQ ID NO: 33, or a sequence that is at least 80% identical thereto, wherein one, two, or three cysteine ​​residues are substituted with serine residues, and proline and asparagine residues at positions 235-238 in sequence PxxN, or equivalent positions, are substituted. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 33, or equivalent position, is not substituted with either a serine or valine residue. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 33, or equivalent position, is not substituted with either a serine or valine residue, the cysteine ​​residues at positions 208 and 222 of SEQ ID NO: 33, or equivalent positions, are substituted with serine residues, the proline residue at position 235, or equivalent position, is substituted with an alanine residue, and the asparagine residue at position 238, or equivalent position, is substituted with proline.

[0052] In a particularly preferred embodiment, the second polypeptide contains or comprises 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 any of them.

[0053] In a particularly preferred embodiment, the chimeric or fusion protein contains, or comprises, a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence described in either SEQ ID NO: 52 or 53.

[0054] Preferably, the fusion protein chimera comprises one or more further polypeptides containing, or comprising, the amino acid sequence of the Arg- or Lys-gingipain active site of P. gingivalis, or a sequence that is at least 80% identical thereto. The one or more further polypeptides containing, or comprising, the Arg- or Lys-gingipain active site of P. gingivalis may be located at the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, the C-terminus of the second polypeptide, or the N-terminus of the second polypeptide.

[0055] In any embodiment, the first polypeptide comprises or consists of amino acid sequences selected from the following group: SEQ ID NOs: 1-11, 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 them.

[0056] One or more further polypeptides preferably contain or consist of amino acid sequences selected from the following group: SEQ ID NOs: 1-11, 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 them.

[0057] In any embodiment, a first polypeptide and further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis contain, 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 an identical amino acid sequence. The first polypeptide and further polypeptides may be derived from the active site of a different gingipain (for example, from gingipain of a different strain of P. gingivalis). The first polypeptide and further polypeptides may have amino acid sequences derived from different gingipains (for example, one polypeptide may have an amino acid sequence of the active site derived from Kgp and another polypeptide may have an amino acid sequence of the active site derived from Rgp, or alternatively, one polypeptide may have an amino acid sequence of the active site derived from RgpA and another polypeptide may have an amino acid sequence of the active site derived from RgpB).

[0058] In any embodiment of any aspect of the present invention, the chimeric or fusion protein consists of or is essentially composed of sequences of the first and second polypeptides as defined herein. Therefore, it will be understood that the chimeric or fusion protein includes a different arrangement or configuration of domains than that of those domains in naturally occurring gingipain polyprotein sequences. In other words, the first and second polypeptides and their domains have a different spatial configuration than naturally occurring gingipain polyproteins.

[0059] In any embodiment of any aspect of the present invention, the first and second polypeptides are linked. The first and second polypeptides may be linked directly, via a linker, or via polypeptide sequences of 100 amino acids or less, preferably 50 amino acids or less. Preferably, the first and second polypeptides are linked directly or 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 linked directly.

[0060] In any embodiment of any aspect of the present invention, the C-terminal residue of the first polypeptide may be linked to the N-terminal residue of the second polypeptide directly, via a linker, or via a polypeptide sequence of 50 amino acids or less. Alternatively, the N-terminal residue of the first polypeptide may be linked to the C-terminal residue of the second polypeptide directly, via a linker, or via a polypeptide sequence of 50 amino acids or less.

[0061] According to first to third aspects of the present invention, the DUF2436 domain and ABM domain derived from Arg- or Lys-gingipain can be directly linked or linked via a linker or polypeptide sequence. Preferably, the DUF2436 and ABM domains are linked via a short linker sequence containing 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 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.

[0062] In a preferred embodiment, the DUF2436 and ABM domains (each having representative amino acid sequences of SEQ ID NOs. 23 and 18, respectively (i.e., amino acid sequences without amino acid substitutions as described herein) can be linked via a peptide sequence having the amino acid sequence EVEDDP.

[0063] In any embodiment, further polypeptides may be attached directly or via a linker to a chimeric or fusion protein comprising the first and second polypeptides. In embodiments where further polypeptides are attached to the C-terminal region of the fusion protein via the second polypeptide, the C-terminus of the second polypeptide is preferably directly attached to the N-terminus of the further polypeptide. (For example, the C-terminus of the adhesion factor domain is preferably directly attached to the N-terminus of the active site amino acid sequence).

[0064] If two or more further polypeptides are included, copies of the further polypeptides can be obtained by directly linking them to each other or by linking them via a linker sequence.

[0065] In any embodiment of any aspect of the present 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 effect 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, e.g., 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 natural ginigipain protein sequence.

[0066] The present invention also provides nucleic acids that encode chimeric or fusion proteins as defined herein.

[0067] Preferably, the nucleic acid has a nucleotide sequence that encodes one or more of the amino acid sequences defined in Table 1 of this specification.

[0068] In any embodiment, such nucleic acids are contained in an expression construct in which the nucleic acids are operably ligated to a promoter. Such expression constructs may be vectors, such as plasmids, or viral vectors.

[0069] The present invention also provides cells comprising nucleic acids or nucleic acid vectors as described herein. Examples of cells of the present invention include bacterial cells, yeast cells, insect cells, or mammalian cells. Preferably, the cells are isolated, substantially purified, or recombinant.

[0070] The present invention also provides compositions comprising the chimeric or fusion proteins described herein, in combination with optionally pharmaceutically acceptable carriers.

[0071] The composition may also include adjuvants for enhancing the immune response to chimeric or fusion proteins.

[0072] Therefore, the present invention relates to a vaccine or immunostimulatory composition for inducing an immune response to P. gingivalis in a subject, wherein the composition is i) an immunogen in the form of a chimeric or fusion protein as described herein, ii) Further providing a vaccine or immunostimulatory composition comprising an adjuvant for enhancing the immune response to an immunogen in a subject.

[0073] Preferably, the sole immunogen provided in the composition, vaccine, or immunostimulatory composition of the present invention is a chimeric or fusion protein as described herein.

[0074] The present invention also provides a method for inducing an immune response to 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.

[0075] The present invention also provides a method for inducing a humoral immune response to P. gingivalis in a subject, the method comprising administering to the subject a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition as defined herein.

[0076] Preferably, the induced immune response includes a switch from a Th1 immune response to a Th2 immune response.

[0077] In any embodiment, it will be understood that the immune response induced by the administration of the chimeric or fusion proteins described herein, or vaccines or other compositions containing them, is preferably antigen-specific. Therefore, in preferred embodiments, the methods and compositions and chimeric proteins described herein are for inducing an immune response, preferably a protective immune response, to the P. gingivalis gingipain antigen.

[0078] In any embodiment, the compositions, chimeric proteins, and methods of the present invention may be used to enhance a target immune response (e.g., a protective immune response) against P. gingivalis.

[0079] The present invention also provides a method for immunizing a subject against P. gingivalis infection, comprising administering the subject a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition as defined herein.

[0080] In any embodiment, subjects who have received or been administered the chimeric or fusion protein of the present invention, or a composition or vaccine containing them, have a higher level of protection against infection by P. gingivalis, or an increased severity of one or more symptoms of P. gingivalis infection, compared to subjects who have not received the protein, composition, or vaccine.

[0081] Furthermore, the present invention provides a method for treating P. gingivalis infection in a subject, the method comprising administering a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition as defined herein to a subject in need thereof, thereby treating the P. gingivalis infection in the subject.

[0082] The present invention provides a method for reducing or minimizing the severity of symptoms associated with infection by P. gingivalis, comprising administering to an individual in need a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition as defined herein, wherein the symptoms are selected from the group consisting of swollen or puffy gums, bleeding gums, receding gums, periodontal pockets around the teeth, loss of supporting tissues of the teeth (periodontal ligaments, cementum, and / or alveolar bone), pus between the gums and teeth, and gingivitis.

[0083] The present invention also provides a method for treating P. gingivalis-related diseases in a subject, the method comprising administering a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition as defined herein to an individual in need thereof. Preferably, P. gingivalis-related diseases include periodontal disease.

[0084] The present invention also provides that a therapeutic method comprising the administration of the chimeric or fusion protein, composition, vaccine, or immunostimulatory composition of the present invention may further comprise the administration of one or more antimicrobial compounds and anti-inflammatory agents.

[0085] The present invention also, ● To induce an immune response (preferably a protective immune response) against P. gingivalis in the subject, ● To immunize the target against P. gingivalis infection, ● Treat P. gingivalis infection in the subjects, ● To minimize or reduce the severity of one or more symptoms of P. gingivalis infection, or ● To provide the use of chimeric or fusion proteins as defined herein in the manufacture of pharmaceuticals for use in treating P. gingivalis-related diseases in subjects.

[0086] The present invention also, ● To induce an immune response (preferably a protective immune response) against P. gingivalis in the subject, ● To immunize the target against P. gingivalis infection, ● Treat P. gingivalis infection in the subjects, ● To minimize or reduce the severity of one or more symptoms of P. gingivalis infection, or ● To provide a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as defined herein for use in treating P. gingivalis-related diseases in subjects.

[0087] Any method, use, or protein, composition, or vaccine for use according to the present invention may involve any subject that is infected with or at risk of being infected with P. gingivalis. The subject may be a human. The subject may be a veterinary subject such as a pet that is infected with or at risk of being infected with P. gingivalis.

[0088] The present invention also provides a method for obtaining antibodies directed towards P. gingivalis, the method comprising administering a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition of the present invention to a non-human animal, thereby generating antibodies directed towards P. gingivalis in the animal. Preferably, the method further comprises isolating the antibodies from the animal (for example, by extraction from the animal's blood) or from its eggs (if the animal is a bird species, preferably a chicken).

[0089] The present invention also provides an antibody preparation comprising an antibody directed toward P. gingivalis, wherein the antibody preparation is obtained by administering the chimeric or fusion protein, composition, vaccine, or immunostimulatory composition of the present invention to a non-human animal, thereby generating an antibody directed toward P. gingivalis in the animal, and by isolating the antibody from the animal or its eggs.

[0090] Antibodies targeted to P. gingivalis may be used therapeutically to eliminate or reduce P. gingivalis infection, or prophylactically to prevent or reduce the severity of P. gingivalis infection.

[0091] The present invention also provides a kit comprising a composition comprising a chimeric or fusion protein as defined herein, wherein the kit optionally comprises one or more cytokines and / or adjuvants in a sealed container.

[0092] Preferably, the kit includes a label or accompanying document indicating that the composition is used to immunize an individual, and optionally the label or accompanying document includes instructions for use.

[0093] Throughout this specification, unless otherwise required by context, the terms “comprise,” “comprises,” and “comprising” will be understood to mean that they encompass the steps or elements or groups of steps or elements described, but do not exclude any other steps or elements or groups of steps or elements. Thus, the use of terms such as “comprises” indicates that the enumerated elements are necessary or essential, while the other elements are optional and may or may not be present. “Consists of” means that it includes and is limited to everything that follows the phrase “consists of.” Thus, the phrase “consists of” indicates that the enumerated elements are necessary or essential, while the other elements may or may not be present. “Essentially consists of” means that it includes any elements enumerated after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or action identified in this disclosure for the enumerated elements. Therefore, the phrase "essentially consists of" indicates that the enumerated elements are necessary or essential, but other elements (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more 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 enumerated elements.

[0094] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will be given as examples and will become apparent from the following description with reference to the accompanying drawings. [Brief explanation of the drawing]

[0095] [Figure 1-1] A. Mouse periodontitis model - therapeutic vaccination. B. Schematic diagram showing the domain structure of Kgp gingipain polyprotein and its derived components for generating the chimeric or fusion protein of the present invention. [Figure 1-2] (As stated above.) [Figure 2]SDS-PAGE of E coli fractions expressing recombinant variants containing either full-length or cleaved DUF domains. Low temperature and low IPTG conditions were used. Full-length DUF is important for solubility. [Figure 3-1] Elimination of Multimerization A. Illustration of Kgp polyproteins showing the locations of ABM1 and ABM2 domains. B-D. Native PAGE analysis of purified recombinant protein variants subjected to electrophoresis in or without DTT. B: Study of "PVQN" mutant variants. C: Study of "PVQN" mutant variants with additional Cys>Ser mutations. D: Study of mutations in the loop preceding the "PVQN" motif (lanes 2-4), and study of 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. [Figure 3-2] (As stated above.) [Figure 3-3] (As stated above.) [Figure 3-4] (As stated above.) [Figure 4] Bone loss study. Statistical analysis - one-way ANOVA and post-hoc Dunnett T3. # indicates significant difference from the control load group. [Figure 5] Statistical analysis of bone loss studies - one-way ANOVA and post-hoc Dunnett T3. #p<0.01, ##p<0.05 (T-test) indicates significant difference from the control load group. [Figure 6] Bone loss study. S = soluble fraction, urea = urea used for purification, AC = affinity column purification, IB = inclusion body, batch = batch purification method. Statistical analysis: one-way ANOVA and post-hoc Dunnett T3. #p<0.01 indicates significant difference from the control loading group. [Figure 7]Research on bone loss after immunization in animals using various fusion protein constructs. Statistical analysis: One-way ANOVA and post-hoc Dunnett showed significant differences from the T3 control loading group (#p<0.05, #p<0.01, #p<0.001, #p<0.0001).

[0096] Sequence information [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Modes for carrying out the invention]

[0097] It will be understood that the present invention, as disclosed and defined herein, encompasses all of the two or more selective combinations of individual features referred to or evident from the text or drawings. All of these various combinations constitute various alternative embodiments of the present invention.

[0098] This document describes in detail certain embodiments of the present invention. While the present invention is described in conjunction with these embodiments, it will be understood that the invention is not intended to be limited to those embodiments. On the contrary, the present invention is intended to cover all alternatives, modifications, and equivalents that may fall within the scope of the invention as defined by the claims.

[0099] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in carrying out the present invention. The present invention is by no means limited to the methods and materials described herein. It will be understood that the present invention disclosed and defined herein encompasses all of two or more selective combinations of individual features referred to or evident from the text or drawings. All of these various combinations constitute various alternative embodiments of the present invention.

[0100] All patents and publications referenced herein are incorporated in their entirety by reference.

[0101] For the purposes of interpreting this specification, any term used in the singular form also includes its plural form, and vice versa.

[0102] In the process leading to the present invention, the inventors investigated various chimeric or fusion proteins for use in inducing an immune response against P. gingivalis, and methods for the large-scale production of such chimeras for use as vaccine candidates.

[0103] The inventors have identified various problems associated with the large-scale production and manufacturing of chimeric or fusion proteins derived from components of P. gingivalis gingipain. Firstly, the chimeric or fusion proteins described in the prior art are difficult to produce in sufficiently large quantities as soluble proteins. More specifically, since the chimeric proteins of the prior art are typically produced within E. coli inclusion bodies, it is difficult to produce large quantities of soluble protein for downstream clinical product development.

[0104] The reduced solubility when produced with E. coli also substantially contributes to the time and scale of production, as well as the decreased stability of the protein once solubilized and refolded from the inclusion body.

[0105] Furthermore, the gingipain-derived chimeric proteins described in the conventional technology also suffer from polymerization. The formation of beta sheets between domains in chimeric proteins not only makes the evaluation of the final vaccine product difficult from a regulatory standpoint, but also contributes to a reduction in the potential immune response when administered.

[0106] Accordingly, the present invention relates to improved designs of chimeric or fusion proteins for use in inducing an immune response against P. gingivalis, as well as methods and uses comprising the same.

[0107] In any embodiment, the chimeric or fusion protein of the present invention has improved solubility and / or stability compared to the chimeric or fusion proteins described in the prior art. This improved solubility and / or stability offers significant advantages with respect to the large-scale production of fusion proteins for use in clinical settings.

[0108] In alternative embodiments, the chimeric or fusion proteins of the present invention have a reduced tendency to aggregate and polymerize compared to conventional chimeric or fusion proteins. Aggregation and polymerization can hinder the large-scale production and manufacturing of therapeutic / preventive proteins. As a result, the reduced tendency to aggregate and / or polymerize of the chimeric proteins of the present invention represents a significant improvement over conventional chimeric proteins for use in inducing immune responses against P. gingivalis.

[0109] Furthermore, in any embodiment, the chimeric or fusion protein of the present invention has increased immunogenicity compared to the chimeric or fusion protein of the prior art.

[0110] Therefore, the inventors have identified various novel approaches to obtaining 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.

[0111] The inventors believe that one or more novel approaches to generating chimeric or fusion proteins for inducing an immune response against P. gingivalis, as described herein, may offer advantages including increased ease of production of suitable immunogenic compositions for inducing an immune response against P. gingivalis, improved shelf life, and / or improved immunogenicity.

[0112] Ginger pine The pathogenicity of P. gingivalis is primarily due to numerous surface-related pathogenic factors, including cysteine ​​proteinases (gingipain), cilia, heme-binding proteins, and outer membrane transport proteins. In particular, the extracellular Arg and Lys-specific proteinases "gingipain" (RgpA / B and Kgp) of P. gingivalis are involved as major pathogenic factors crucial for colonization, penetration into host tissues, dysregulation of the immune response, biological abnormalities, and disease.

[0113] Gingipain, particularly the Lys-specific proteinase Kgp, is essential for P. gingivalis to induce alveolar bone resorption in a mouse model of periodontitis. Gingipain is also found in gingival tissue at sites of severe periodontitis, at high concentrations proximal to subgingival plaque and at lower concentrations in deeper distal areas of gingival tissue. Lys-specific and Arg-specific proteinases have been shown to degrade various host proteins in vitro, such as fibrinogen, fibronectin, and laminin. Plasma host defense and regulatory proteinase inhibitors α-trypsin, α2-macroglobulin, anti-chymotrypsin, antithrombin III, and antiplasmin are also degraded by Lys- and Arg-proteinases derived from P. gingivalis. This has led to the development of a compelling mechanism to explain the important role that P. gingivalis plays in the development of chronic periodontitis.

[0114] The RgpA, RgpB, and Kgp genes all encode an N-terminal signal peptide of approximately 22 amino acids, an unusually long propeptide of approximately 200 amino acids, and a catalytic domain of approximately 480 amino acids. The C-terminus of the catalytic domain is a large hemagglutinin adherent factor (HA) domain consisting of an adherent factor-binding domain (ABM, or five different sequences described), an "unknown function domain" (referred to as DUF2436, IPR018832, defined as a conserved Pfam domain of unknown function), and a C-terminal adherent factor domain or a cleaved adherent factor domain (or CAD). The specific arrangements of ABM, DUF, and CAD vary between Kgp and RgpA / B.

[0115] Figure 1B shows the domain structure in the Kgp polyprotein. For example, Kgp includes (from N-terminus to C-terminus) a catalytic domain, a domain containing the first ABM (ABM1), DUF2436, ABM2, ABM1, and ABM3, two CAD domains (referred to as K1 and K2), a further domain containing ABM2 and ABM1, another CAD domain (referred to as K3), ABM2, and a C-terminal domain.

[0116] As used herein, references to ABM1, 2, and 3 will generally be understood to refer to the ABMs found in the order ABM2, ABM1, and ABM3 in the C-terminal sequence immediately preceding DUF2436 of Kgp, as shown in Figure 1B.

[0117] The catalytic domains of RgpB and RgpA share a high degree of sequence homology. However, RgpB lacks an HA domain and is located on the outer membrane in monomeric form. Some of the HA domains have been alternatively described as C-terminal adhesion domains or cleaved adhesion domains (CADs), and some are DUF ("domain of unknown function") 2436 domains (conserved Pfam domain of unknown function, IPR018832).

[0118] RgpA and Kgp precursor proteins are cleaved into multiple non-covalently associated domains, forming large outer membrane protein complexes. Therefore, in vivo, Arg and Lys-specific proteinases are found in cell-associated complexes of non-covalently associated proteinases and adherent factors. One such complex is designated as the RgpA-Kgp proteinase-adhesion factor complex (formerly referred to as the PrtR-PrtK proteinase-adhesion factor complex). The complex consists of a 45 kDa Arg-specific calcium-stabilized cysteine ​​proteinase and seven sequence-associated adherent factor domains.

[0119] As used herein, the Lys-gingipain catalytic domain may also be referred to as the KAS domain or PAS domain. As used herein, the Arg-gingipain catalytic domain may also be referred to as the RAS domain or PAS domain. Typically, the Lys-gingipain or Arg-gingipain catalytic domain is located in the N-terminal region of the protein, approximately 480 amino acids long. 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 listed in Table 1 as Sequence IDs 1-11.

[0120] As used herein, the Arg- or Lys-gingipaine adhesion factor domain of P. gingivalis will typically be understood to refer to the Arg- or Lys-gingipaine region that is the C-terminus of the catalytic domain or active site domain. The adhesion factor domain (also referred to as the HA domain) typically includes domains of unknown function (DUF) domains (in particular DUF2436, a conserved Pfam domain of unknown function, IPR018832), as well as several adhesion factor-binding motif (ABM) domains and cleaved adhesion factor domains (CAD).

[0121] First polypeptide The chimeric or fusion protein of the present invention comprises the amino acid sequence of the active site of P. gingivalis Arg-X or Lys-X proteinase (referred to herein as Arg- or Lys-gingipain, respectively), or a sequence that is at least 80% identical thereto, or a first polypeptide comprising such a sequence.

[0122] In any embodiment, the first polypeptide comprises or consists of amino acid sequences selected from the following group: SEQ ID NOs: 1-11, or sequences that are 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to them.

[0123] Improvement of immunogenicity Preferably, the chimeric or fusion protein comprises one or more further polypeptides containing, or comprising, the amino acid sequence of the Arg- or Lys-gingipain active site of P. gingivalis, or a sequence that is at least 80% identical thereto. The one or more further polypeptides containing, or comprising the Arg- or Lys-gingipain active site 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, preferably via a linker of 50 amino acids or less, or may be linked directly to the first polypeptide.

[0124] One or more further polypeptides preferably contain or consist of amino acid sequences selected from the following group: SEQ ID NOs: 1-11, 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 them.

[0125] In any embodiment, a first polypeptide and further polypeptides comprising or consisting of the amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis comprises or consists of the same amino acid sequence, 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.

[0126] In preferred embodiments, the chimeric or fusion protein of the present invention comprises two or fewer polypeptides comprising 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, or consisting of two or fewer, three or fewer, or four or fewer polypeptides. Preferably, the chimeric or fusion protein of the present invention comprises five, more preferably four, and most preferably three polypeptides comprising 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, or consisting of five or fewer polypeptides.

[0127] Second polypeptide The chimeric or fusion protein of the present invention comprises the amino acid sequence of the adhesion factor domain of the ArgX or Lys-X proteinase of P. gingivalis, or a sequence that is at least 80% identical thereto, or a second polypeptide comprising such a sequence.

[0128] The second polypeptide will typically contain at least one sequence corresponding to one or more adherent-binding motifs (ABMs), which are domains / motifs recognized in the adherent-binding domains (such as Kgp and RgpA) of the ArgX or Lys-X proteinases of P. gingivalis.

[0129] There are five defined ABMs (ABM1-5) for Kgp and RgpA. Exemplary sequences of these ABMs are shown in Table 1.

[0130] Typically, the second polypeptide comprises two or more ABMs, preferably the second polypeptide comprising at least ABM1 and ABM2, or at least 80% identical sequences to each of ABM1 and ABM2. The second polypeptide may also comprise the sequence of ABM3 or at least 80% identical sequences to it.

[0131] Furthermore, it will be understood that the placement of the ABM peptide in the second polypeptide does not need to correspond to the placement of the ABM peptide found in naturally occurring adhesion factor domains. For example, the ABM peptide can be placed in the second polypeptide in sequences (N-terminus to C-terminus) such as ABM1, ABM2, ABM3. Alternatively, the ABM peptide can be placed to reflect the placement in naturally occurring adhesion factor domains such as ABM2, ABM1, and ABM3, as is the case with adhesion factor domain 1 of Kgp.

[0132] Furthermore, the ABMs in the second polypeptide may be adjacent to each other or separated from each other by an amino acid sequence of 50 amino acids or less. Those skilled in the art will understand that the spacing between ABMs is not important in the design of the chimeric or fusion protein of the present invention.

[0133] In any embodiment, the second polypeptide comprises the amino acid sequence described in SEQ ID NO: 62, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0134] In any embodiment, the second polypeptide comprises the amino acid sequence described in SEQ ID NO: 16, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0135] In any embodiment, the second polypeptide comprises the amino acid sequence described in SEQ ID NO: 18, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0136] In any embodiment, the second polypeptide comprises the amino acid sequence described in SEQ ID NO: 22, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0137] In any embodiment, the second polypeptide comprises the amino acid sequence described in SEQ ID NO: 27, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0138] However, the inventors have found that when the chimeric or fusion protein of the present invention contains only a portion of the DUF2436 domain (i.e., the N-terminally cleaved DUF2436 domain as shown in SEQ ID NO: 25 or 27), the solubility of the chimeric or fusion protein of the present invention decreases.

[0139] Referring to the examples herein, the inventors recognized that prior art approaches to P. gingivalis vaccine design include the DUF2436 domain in an N-terminally cleaved form. While the cleaved 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 the production of soluble recombinant protein.

[0140] Therefore, in preferred embodiments, the second polypeptide in the chimeric or fusion protein of the present invention comprises residues 1 to 37 of the DUF2436 domain, and preferably the chimeric protein comprises an amino acid sequence substantially corresponding to the full length of the DUF2436 domain of Arg- or Lys-gingipain, or a sequence at least 80% identical thereto.

[0141] As used herein, an amino acid sequence substantially corresponding to the full 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 Arg- or Lys-gingipain.

[0142] Preferably, the DUF2436 domain contains the sequence described in Sequence ID No. 23, or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0143] Therefore, in a preferred embodiment, the second polypeptide comprises the amino acid sequence described in any one of SEQ ID NOs: 33, 59, or 61, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

[0144] The inventors have also demonstrated that various domains in dinidipain tend to polymerize, and that polymerization or aggregation contributes to reduced solubility and immunogenicity of chimeric or fusion proteins. Therefore, polymer formation affects the ease of large-scale production and use as a vaccine of chimeric proteins derived from dinidipain sequences, but it may also affect the generation of protective responses.

[0145] In particular, the inventors identified two mechanisms for multimerization: the formation of disulfide crosslinks between cysteine ​​residues and beta-chain exchange between ABM domains. Cross-referencing the schematic diagram showing the various domains of Kgp in Figure 1B, the inventors hypothesized that ABM1 may interact with the next available adjacent ABM2 during the folding of the Kgp polyprotein in vivo. Separately, the inventors show that co-expression of recombinant ABM1 and ABM2 as distinct proteins was able to form a stable beta-sheet complex structure.

[0146] Therefore, in order to reduce the polymerization of the chimeric protein proposed for use according to the present invention, the inventors 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.

[0147] The inventors have identified that one or more of the following modifications contribute to reducing the multimerization of chimeric fusion proteins derived from Kgp and Rgp polyproteins: c) Compared to the naturally occurring Arg- or Lys-gingipain sequence in the corresponding region, the amino acid sequence of the DUF2436 domain and / or the ABM domain contains one or more cysteine ​​amino acid substitutions, and / or preferably, and d) Includes one or more amino acid motif substitutions selected from the following: i) Substitution of proline and / or asparagine residues in sequence PxxN at positions corresponding to or equivalent to residues 6-9 of sequence number 14 or 19 (ABM1), ii) Substitution of the motif NxFA with SxYQ in sequences corresponding to or equivalent to residues 2-5 of sequence number 14 or 19 (ABM1), iii) Substitution of a second tyrosine residue at the position corresponding to or equivalent to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2), and a tryptophan residue at the position corresponding to or equivalent to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue.

[0148] Therefore, in a particularly preferred embodiment of the present invention, the second polypeptide comprises one or more cysteine ​​amino acid substitutions compared to a naturally occurring adhesion factor domain sequence.

[0149] Cysteine ​​residues are found (at least) in the DUF2436 domain, ABM2, and the K1 CAD sequence in the P. gingivalis Kgp sequence. Therefore, it will be understood that the present invention intends to create chimeric or fusion proteins in which one or more, two or more, or three or more cysteine ​​residues are substituted to reduce, minimize, or eliminate the formation of disulfide crosslinks.

[0150] Cysteine ​​amino acid substitutions may be substitutions to serine or valine residues. Preferably, one or more cysteine ​​substitutions include one or more substitutions to serine residues.

[0151] 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 adhesion factor 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 described in SEQ ID NO: 18 or 22. 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 a serine residue or a valine residue, preferably a serine residue.

[0152] An exemplary sequence of the DUF2436 domain + ABM2 + 1 + 3 region is described in Sequence ID No. 33. Exemplary sequences of the DUF2436 domain + ABM2 + 1 region, and of the DUF2436 domain + ABM2 and 3, are described in Sequence ID Nos. 59 and 61, respectively. In a preferred embodiment of the present invention, one or both of the cysteine ​​residues at positions 208 and 222 or equivalent positions in Sequence ID No. 33 are optionally substituted with a serine residue or a valine residue, preferably a serine residue.

[0153] In alternative embodiments, cysteine ​​residues in the DUF2436 domain are substituted with serine or valine residues, preferably serine residues, and one or both cysteine ​​residues in the adhesion factor domain region between the C-terminus of the DUF2436 domain and the N-terminus of the K1CAD domain may be substituted. An exemplary sequence of the region between the DUF domain and the CAD domain is described in SEQ ID NO: 18. In preferred embodiments 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 described in SEQ ID NO: 33. In preferred embodiments of the present invention, the cysteine ​​residue at position 115, and 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.

[0154] Therefore, in a particularly preferred embodiment, the chimeric or fusion protein of the present invention comprises an amino acid sequence corresponding to any one of the sequences described in SEQ ID NOs: 34 to 48, or a sequence that is at least 80% identical thereto (excluding cysteine ​​substitutions).

[0155] The inventors further demonstrated that polymerization can also be reduced by mutations in conserved motifs present in ABM domains 2, 1, and 3.

[0156] In one example, the inventors investigated modifications to the N-terminal domain of the adhesion factor domain ABM1. More specifically, they found that substitution of the motif PXXN (e.g., PVQN in ABM1 of P. gingivalis Kgp as described in residues 6-9 of SEQ ID NO: 14 or 19) substantially contributes to the reduction of multimerization between ABM domains and the reduction of beta-chain exchange.

[0157] Therefore, in preferred embodiments, the chimeric or fusion protein of the present invention comprises modification of the ABM1 PXXN motif in the region of the chimeric or fusion protein corresponding to the adhesion factor domain of P. gingivalis gingipain. Accordingly, the second polypeptide preferably comprises proline substitutions and asparagine substitutions in the PxxN sequence at positions 6-9 of the sequence of SEQ ID NO: 14 or 19 that defines ABM1, or at equivalent positions.

[0158] The proline amino acid substitution is preferably a substitution to an alanine residue.

[0159] The asparagine amino acid substitution may be a substitution for a proline residue or an alanine residue. Preferably, the asparagine residue is substituted for a proline residue. In other embodiments, the asparagine residue is not substituted.

[0160] In further examples, the inventors considered the motif NEFA in the sequence of ABM1, which is defined as residues 2-5 of Sequence IDs 14 and 19 as herein. As further shown in the examples, modification of SEQY with the motif NEFA by substituting asparagine, phenylalanine, and alanine residues with serine, glutamine, and tyrosine, respectively, significantly reduces polymerization.

[0161] In another embodiment, the inventors determined that substitution of a tyrosine residue in ABM2 at a position corresponding to or equivalent to the residue at position 5 of SEQ ID NO: 15, and a tryptophan residue in ABM1 at a position corresponding to or equivalent to the residue at position 23 of SEQ ID NO: 14 or 19, also significantly reduced polymerization.

[0162] Finally, the inventors found that polymerization was substantially eliminated through a combination of cysteine ​​modification and one or more of the following substitutions: i) Substitution of proline and / or asparagine residues in sequence PxxN at positions corresponding to or equivalent to residues 6-9 of sequence number 14 or 19 (ABM1), ii) Substitution of the motif NxFA with SxYQ in sequences corresponding to or equivalent to residues 2-5 of sequence number 14 or 19 (ABM1), iii) Substitution of a second tyrosine residue at the position corresponding to or equivalent to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2), and a tryptophan residue at the position corresponding to or equivalent to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue.

[0163] 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 modification of the PxxN motif of ABM1 to AxxP eliminated the polymerization of the resulting recombinant chimeric protein.

[0164] Therefore, combining the cysteine ​​modification of the ABM1 domain and the modification of the PxxN motif, the present invention provides a chimeric or fusion protein as described herein, wherein the second polypeptide of the chimeric protein corresponds to the region of the adhesion factor domain of P. gingivalis Arg or Lys gingipain and contains, or consists of, the amino acid sequence described in SEQ ID NO: 33, or a sequence that is at least 80% identical thereto, wherein one, two, or three cysteine ​​residues are substituted with serine residues, and / or proline and asparagine residues in the PxxN sequence at positions 235-238 or equivalent are substituted.

[0165] Preferably, the cysteine ​​residue at residue 115 of SEQ ID NO: 33, or at an equivalent position, is not substituted with either a serine or valine residue.

[0166] Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 33, or at an equivalent position, is not substituted with either a serine or valine residue; the cysteine ​​residues at positions 208 and 222 of SEQ ID NO: 33, or at equivalent positions, are substituted with serine residues; the proline residue at position 235, or at an equivalent position, is substituted with an alanine residue; and the asparagine residue at position 238, or at an equivalent position, is substituted with proline.

[0167] Linking of the first and second polypeptides In the chimeric or fusion protein of the present invention, the C-terminal residue of the first polypeptide may be covalently bonded to the N-terminal residue of the second polypeptide (corresponding to the adhesion factor domain polypeptide), or the N-terminal residue of the first peptide may be covalently bonded to the C-terminal residue of the second polypeptide (corresponding to the adhesion factor domain polypeptide). In this arrangement, the first peptide and the adhesion factor domain polypeptide are said to be "directly linked" or "adjacent."

[0168] In other embodiments, the chimeric or fusion protein includes a linker for linking the first peptide to the adhesion factor domain polypeptide. The linker may be any linker capable of linking the peptide to the polypeptide, including both amino acid and non-amino acid linkers.

[0169] Preferably, the linker is non-immunogenic. Typically, the linker is composed of amino acids and can therefore be called a peptide linker.

[0170] The linker is typically a peptide having a length of up to 20 amino acids, but may be longer. The terms “linked to” or “fused to” refer to a covalent bond, such as a peptide bond, formed between two parts. 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 amino acids or more. For example, the chimeric or fusion proteins provided herein may include an amino acid sequence of the P. gingivalis gingipain active domain, such as between the N-terminus of a second polypeptide and the C-terminus of a first polypeptide, or may include a linker between a first polypeptide consisting of the same and a second polypeptide corresponding to the adhesion factor domain of P. gingivalis gingipain. Such a linker has the advantage of increasing the likelihood that the different polypeptides of the fusion protein will fold independently and act as expected. A suitable linker may be up to 50 amino acids long, but is preferably less than 20, less than 15, or less than 5 amino acids. The linker may function to position the first peptide and the adhesion factor domain polypeptide closer together than is typically observed in P. gingivalis trypsin-like enzymes. Alternatively, it may create a gap between the first polypeptide and the second polypeptide (corresponding to the adhesion factor domain polypeptide).

[0171] Suitable linkers for use in protein constructs, including those that have minimal effect on solubility, are known in the art. The linker may be any linker known to those skilled in the art and may be a flexible linker (such as one containing repeats of glycine and serine residues), a rigid linker (such as one containing repeats of glutamic acid and lysine residues and adjacent alanine residues), and / or a cleavable linker (such as a sequence sensitive 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.

[0172] 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 at least an integer of 1, typically between 1 and about 10, for example, between 1 and about 8, 1 and about 6, or 1 and about 5.

[0173] In some embodiments, the peptide linker may comprise amino acids glycine and serine of varying lengths and combinations. In some embodiments, the peptide linker may comprise the sequences Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS), or Gly-Gly-Gly-Ser (GGGGS) and their variations or repeats. In some embodiments, the peptide linker may comprise the amino acid sequence GGGGS (a 6-amino acid 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 the 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 having linkers of such lengths are included within the scope of the present invention. Similarly, the linker can be a series of repeating glycine residues separated by serine residues. For example, (GGGGS)3 (i.e., the linker may include the amino acid sequence GGGGSGGGGSGGGGS(G4S)3) and its variations.

[0174] In one embodiment, the peptide linker may comprise the amino acid sequence GGGGS (a linker with a length of 6 amino acids) 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 the 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.

[0175] Other useful linkers include DSSG, DSSGAS, KLDSSG, and their variations. Examples of other suitable linkers are described in Chen et al., (2013) Advanced Drug Delivery Reviews, 65:1357-1369.

[0176] Chimeric or fusion proteins and recombinant proteins The chimeric or fusion proteins of the present invention can be prepared by any of many conventional techniques, but typically the polypeptides are produced using recombinant techniques.

[0177] For recombinant polypeptides, the DNA fragment encoding the desired peptide can be subcloned into a suitable vector using well-known molecular genetic techniques (see, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1982) and 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 (e.g., those manufactured by Clontech, Palo Alto, Calif., Amersham Pharmacia Biotech Inc., Piscataway, NJ, InVitrogen, Carlsbad, Calif., etc.). Polymerase chain reaction can optionally be used in nucleic acid manipulation.

[0178] A “fragment” is a portion of the polypeptide of the present invention that has substantially the same functional activity as the polypeptide or substantially the same biological function or activity as the polypeptide, as can be determined using the assay described herein.

[0179] The “amino acid sequence identity percentage (%)” or “identity percentage (%)” with respect to a polypeptide sequence, i.e., the polypeptide of the present invention as defined herein, is defined as the percentage of amino acid residues in a candidate sequence that is identical to an amino acid residue in a particular polypeptide of the present invention, after aligning the sequences and introducing gaps as necessary to achieve the maximum sequence identity percentage, and without considering any conservative substitutions as part of the sequence identity. In this specification, a reference to a variant having “at least x% sequence identity” to an enumerated sequence means that the variant is at least x% identical to the enumerated sequence.

[0180] In various aspects and embodiments of the present invention, a defined polypeptide is described by referring to a variant having at least 80% homology or more to a reference sequence. The homology percentage (%) generally refers to the polypeptide of the present invention as defined herein, which is 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 as necessary to achieve the maximum sequence identity percentage, and without considering any conservative substitutions as part of the sequence identity.

[0181] The amino acids glycine, alanine, valine, leucine, and isoleucine are often substituted for each other (amino acids with aliphatic side chains). Of these possible substitutions, glycine and alanine are preferably used for substitution (because they have relatively short side chains), and valine, leucine, and isoleucine are preferably used for substitution (because they have larger, hydrophobic aliphatic side chains). Other amino acids that are often substituted for each other include phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0182] Substitutions of this nature are often referred to as "conservative" or "semi-conservative" amino acid substitutions.

[0183] Deletions or insertions of amino acids can also be made to the native sequence of P. gingivalis proteins. Therefore, for example, amino acids may 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, because they can reduce the total length and molecular weight of the polypeptide while still retaining its activity. This can reduce the amount of polypeptide required for a particular purpose, for example, by lowering the dosage level.

[0184] Amino acid insertions into the sequence of natural polypeptides may also be performed. This may be done to alter the properties of the polypeptide for use in the present invention (for example, to improve antigenicity).

[0185] Amino acid alterations can be performed using any suitable technique, for example, site-directed mutagenesis or solid-phase synthesis.

[0186] It should be understood that amino acid substitutions or insertions within the scope of this invention may be performed using naturally occurring or non-natural amino acids. Regardless of whether natural or synthetic amino acids are used, it is preferable that only L-amino acids are present.

[0187] A person skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm (non-limiting examples described below) necessary to achieve the maximum alignment over the entire length of the sequences being compared. When amino acid sequences are aligned, the amino acid sequence identity percentage of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, a given amino acid sequence A may be expressed as having or containing a particular amino acid sequence identity percentage to, with, or against a given amino acid sequence B) can be calculated as follows: amino acid sequence identity percentage = X / Y100, where X is the number of amino acid residues scored as identical by the alignment of A and B by the 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 amino acid sequence identity percentage of A to B is not equal to the amino acid sequence identity percentage of B to A.

[0188] When calculating identity percentages, exact matches are typically counted. Determining identity percentages between two sequences can be achieved using mathematical algorithms. A non-restrictive example of a mathematical algorithm used for comparing two sequences is the algorithm of Karlin and Altschul (1990) Proc.Natl.Acad.Sci.USA 87:2264, modified as described in Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-5877. Such algorithms are incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J.MoI.Biol.215:403. To obtain gap 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, iterative searches can be performed using PSI-Blast to detect intermolecular distance relationships. See Altschul et al. (1997) above. When using BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of each program (e.g., BLASTX and BLASTN) may be used. Alignment may be performed manually by the survey. Another non-restrictive example of a mathematical algorithm used for sequence comparison 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, and thus provide 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 amino acid sequence alignment using ClustalW, the amino acid identity percentage can be evaluated.Non-exclusive examples of software programs useful for ClustalW alignment analysis include GENEDOC® or JalView (http: / / www.jalview.org / ). GENEDOC® allows for the evaluation of amino acid (or DNA) similarity and identity between multiple proteins. Another non-exclusive example of a mathematical algorithm used for sequence comparison is the algorithm in Myers and Miller (1988) CABIOS 4:11-17. Such algorithms are 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 using the ALIGN program to compare amino acid sequences, the PAM120 weight residue table, 12 gap length penalties, and 4 gap penalties may be used.

[0189] Polypeptides may optionally include amino and carboxyl termini. Polypeptides may comprise D-amino acids, L-amino acids, or mixtures of D-amino acids and L-amino acids. However, D-type amino acids are particularly preferred because polypeptides composed of D-amino acids are expected to retain their biological activity more effectively in vivo.

[0190] As used herein, the term “conservative substitution” refers to replacing an amino acid present in the natural sequence of a peptide with a naturally occurring or non-natural amino acid or peptide mimetic having similar stereochemical properties. If the side chain of the naturally occurring amino acid being substituted is either polar or hydrophobic, the conservative substitution must be a naturally occurring, non-natural amino acid, or peptide mimetic that is also polar or hydrophobic (in addition to having the same stereochemical properties as the side chain of the substituted amino acid).

[0191] Tables of conserved amino acid substitutions that provide functionally similar amino acids are well known to those skilled in the art. The following six groups are examples of amino acids that can be considered to be conserved substitutions of 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).

[0192] Conservative substitutions using naturally occurring amino acids can be determined by noting that naturally occurring amino acids are typically grouped according to their properties, and therefore substitutions of charged amino acids with sterically similar uncharged amino acids are considered conservative substitutions. It is also possible to use amino acid analogs (synthetic amino acids) known in the art to create conservative substitutions using unnatural amino acids. Peptide mimes of naturally occurring amino acids are well described in literature known to those skilled in the art, and non-natural or unnatural amino acids are further described below. If the substitution affects the conservation of the substitution, the substituted amino acid must have the same or similar functional groups in its side chain as the original amino acid.

[0193] As used herein, the terms “non-conservative substitution” or “non-conservative residue” refer 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 stereochemical properties. Therefore, the side chain of the substituted amino acid may be significantly larger (or smaller) than the side chain of the substituted natural amino acid and / or may have a functional group with significantly different electronic properties than the substituted amino acid. 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 conserved.

[0194] Non-conservative amino acid substitutions can result from changes in: (a) the structure of the amino acid backbone in the substitution region, (b) the charge or hydrophobicity of the amino acid, or (c) the volume of the amino acid side chain. Generally, substitutions that are expected to cause the greatest changes in protein properties are those in: (a) a hydrophilic residue being replaced by (or thereby) a hydrophobic residue, (b) proline being replaced by (or thereby) any other residue, (c) a residue with a bulky side chain, such as phenylalanine, being replaced by (or thereby) a residue without a side chain, such as glycine, or (d) a residue with a positively charged side chain, such as lysyl, arginyl, or histadyl, being replaced by (or thereby) a negatively charged residue, such as glutamyl or aspartyl.

[0195] Modification of the natural amino acid sequence to produce mutant polypeptides, such as by insertion, deletion, and / or substitution, can be carried out by various means known to those skilled in the art. For example, site-directed mutations can be introduced by ligating a synthetic oligonucleotide containing the modification site into an expression vector. Alternatively, oligonucleotide-induced site-directed 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. Patents 4,518,584 and 4,737,462. A preferred means for introducing mutations is the QuikChange Site-Directed Mutagenesis Kit (Stratagene, LaJolla, Calif.).

[0196] Any suitable expression vector (e.g., those described in Pouwels et al., Cloning Vectors: A Laboratory Manual (Elsevier, NY: 1985)) and a corresponding suitable host can be used for the production of recombinant polypeptides. Expression hosts include, but are not limited to, bacterial species within the genera Escherichia, Bacillus, Pseudomonas, and Salmonella, mammalian or insect cell lines including baculoviruses (e.g., those described in Luckow et al., Bio / Technology 6:47 (1988)), and established cell lines such as COS-7, C127, 3T3, CHO, HeLa, and BHK cell lines. Those skilled in the art will recognize that the selection of the expression host has a derivational effect on the type of polypeptide produced. For example, the glycosylation of polypeptides produced in yeast cells or mammalian cells (e.g., COS-7 cells) differs from the glycosylation of polypeptides produced in bacterial cells such as Escherichia coli.

[0197] Alternatively, the polypeptides of the present invention may be synthesized using standard peptide synthesis techniques well known to those skilled in the art (e.g., summarized in Bodanszky, Principles of Peptide Synthesis (Springer-Verlag, Heidelberg: 1984)). Specifically, the polypeptides may be synthesized using solid-phase synthesis procedures (see, for example, Merrifield, J. Am. Chem. Soc. 85:2149-54 (1963), Barany et al., Int. J. Peptide Protein Res. 30:705-739 (1987), and U.S. Patent No. 5,424,398). If necessary, this may be done using an automated peptide synthesizer. Removal of the t-butyloxycarbonyl (t-BOC) or 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking group and separation of the polypeptide from the resin may be achieved, for example, by acid treatment at low temperatures. Next, the polypeptide-containing mixture can be extracted, for example, with dimethyl ether to remove non-peptidic organic compounds, and the synthesized polypeptide can be extracted from the resin powder (for example, with about 25% w / v acetic acid). After polypeptide synthesis, further purification (for example, using high-performance liquid chromatography (HPLC)) can be optionally performed to remove any incomplete polypeptide or free amino acids. Amino acid and / or HPLC analysis may be performed on the synthesized polypeptide to verify its identity. For other applications of the present invention, it may be preferable to produce polypeptides as part of a larger fusion protein by the method described herein or other genetic means, or as part of a larger conjugate via physical or chemical conjugations, etc., as known to those skilled in the art and described herein.

[0198] In any embodiment of the present invention, the chimeric or fusion protein of the present invention may contain additional amino acid residues to promote expression in recombinant expression systems and / or to promote protein purification. Thus, the proteins defined herein may contain additional amino acids in the N-terminal region, such as 1, 2, 3, 4, or 5 amino acids. Typically, the additional amino acids include an N-terminal methionine to promote expression in recombinant expression systems, although it will typically be understood that such an N-terminal residue is cleaved after translation of the protein. In certain embodiments, the N-terminal amino acids include at least methionine and alanine residues.

[0199] Furthermore, the chimeric or fusion protein according to the present invention may preferably contain additional amino acids, such as 1, 2, 3, 4, or 5 amino acids, in the N-terminal or C-terminal region to facilitate purification. It will be understood that such amino acid residues may facilitate the inclusion of purification tags (such as histidine tags) in the protein. Such residues may be omitted if an untagged version of the protein is produced.

[0200] The polypeptides of the present invention may also be modified by conjugating or fusing them to another portion to facilitate purification, 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 present invention may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, linkage to cell ligands or other proteins, etc.

[0201] nucleic acid Nucleic acid molecules encoding any of the chimeric or fusion proteins or polypeptides of the present invention are also within the scope of the present invention. Nucleic acids are useful, for example, in the production of the polypeptides of the present invention and as therapeutic agents. They may be administered to cells in culture or in vivo and may contain secretory signals that direct or promote the secretion of the polypeptides of the present invention from the cells. Expression vectors and host cells containing or comprising the nucleic acids of the present invention are also within the scope of the present invention (as further described below). The nucleic acids of the present invention may be referred to as “isolated,” but by definition, the polypeptides of the present 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 present invention may be “purified,” “substantially purified,” “isolated,” “recombinant,” or “synthesized,” but do not necessarily have to be so in order to be distinguished from naturally occurring substances.

[0202] An "isolated" nucleic acid molecule is a nucleic acid molecule that has been identified and separated from a nucleic acid molecule of at least one contaminant associated with the natural source of the nucleic acid. An isolated nucleic acid molecule is in a form or situation other than that which is found in nature. Therefore, isolated nucleic acid molecules are distinguished from nucleic acid molecules present in natural cells. However, isolated nucleic acid molecules typically include nucleic acid molecules contained in cells expressing KGP, for example, the nucleic acid molecules are located in different chromosomal positions than those in natural cells.

[0203] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to nucleotides in any polymerized form of deoxyribonucleotides or ribonucleotides, or analogs thereof, of any length. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotides of the present invention may be provided in isolated or purified form. A nucleic acid sequence that “encodes” a selected polypeptide is a nucleic acid molecule that, when placed under the control of an appropriate regulatory sequence, is transcribed in vivo (in the case of DNA) and translated into a polypeptide (in the case of mRNA). 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 the purposes of the present invention, such nucleic acid sequences include, but are not limited to, cDNA derived from viral, prokaryotic, or eukaryotic mRNA, genomic sequences derived from viral or prokaryotic DNA or RNA, and synthetic DNA sequences. The transcription termination sequence can be located 3' to the coding sequence.

[0204] The polynucleotides of the present invention can be synthesized according to methods well known in the art, as described by example in Sambrook et al (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).

[0205] The polynucleotide molecules of the present invention may be provided in the form of an expression cassette containing a control sequence operably linked to an inserted sequence, thereby enabling in vivo expression of the polypeptide of the present invention in a target subject. These expression cassettes are then typically provided in a vector (e.g., a plasmid or recombinant viral vector) suitable for use as a reagent in nucleic acid immunization. Such expression cassettes may be administered directly to a host subject. Alternatively, a vector containing the polynucleotide of the present invention may be administered to a host subject. Preferably, the polynucleotide is prepared and / or administered using a gene vector. A suitable vector may be any vector capable of carrying a sufficient amount of genetic information and enabling the expression of the polypeptide of the present invention.

[0206] Accordingly, the present invention includes an expression vector comprising such a polynucleotide sequence. Accordingly, the present invention provides a vector for use in the prevention or treatment of inflammatory diseases or conditions, comprising a polynucleotide sequence encoding the polypeptide of the present invention and optionally one or more further polynucleotide sequences encoding different polypeptides as defined herein.

[0207] Furthermore, it will be understood that the compositions and products of the present invention may comprise mixtures of polypeptides and polynucleotides. Accordingly, the present invention provides compositions or products as defined herein, wherein, instead of any one of the polypeptides, the present invention provides a polynucleotide capable of expressing said polypeptide.

[0208] Expression vectors are routinely constructed in the field of molecular biology and, for example, to enable the expression of the peptide of the present invention, may involve the use of plasmid DNA, as well as appropriate initiators, promoters, enhancers, and other elements such as polyadenylation signals, which may be necessary and positioned in the correct orientation. Other suitable vectors will be apparent to those skilled in the art. For further examples in this regard, see Sambrook et al.

[0209] Therefore, the polypeptides of the present invention may be provided by delivering such vectors to cells and enabling transcription from the vectors to occur. Preferably, the polynucleotides for use in the present invention, or in vectors, are operably ligated to a control sequence that can provide expression of the coding sequence by a host cell, i.e., the vector is an expression vector.

[0210] "Operatively linked" refers to an arrangement of elements configured so that the components described in this way perform their normal functions. Therefore, a given regulatory sequence, such as a promoter, operationally linked to a nucleic acid sequence can achieve expression of that sequence if the appropriate enzyme is present. A promoter does not need to be adjacent to a sequence, as long as it functions to direct its expression. For example, an intervening untranslated but transcribed sequence can exist between the promoter sequence and the nucleic acid sequence, and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0211] Many expression systems have been described in the art, each typically comprising a vector containing a gene or nucleotide sequence of interest operably ligated to an expression regulatory sequence. These regulatory sequences include transcription promoter sequences and transcription start and stop sequences. The vectors of the present invention may be plasmids, viruses, or phage vectors having, for example, 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 gene element. The vector may contain one or more selectable marker genes, for example, an ampicillin resistance gene in the case of a bacterial plasmid, or a resistance gene in the case of a fungal vector. The vector may be used, for example, in vitro for the production of DNA or RNA, or for the transfection or transformation of host cells, for example, mammalian host cells. The vector may also be adapted for in vivo use, for example, to enable in vivo expression of polypeptides.

[0212] A “promoter” is a nucleotide sequence that initiates and regulates the transcription of a polynucleotide encoding a polypeptide. Promoters may include inductive promoters (where the expression of a polynucleotide sequence operably ligated to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressive promoters (where the expression of a polynucleotide sequence operably ligated to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. The terms “promoter” or “regulatory element” are intended to include full-length promoter regions and functional segments of these regions (e.g., those that regulate transcription or translation).

[0213] The polynucleotide, expression cassette, or vector according to the present invention may further comprise a signal peptide sequence. The signal peptide sequence is generally inserted via an operable linkage to a promoter such that the signal peptide is expressed and facilitates the secretion of the polypeptide encoded by the sequence also being encoded by an operable linkage to the promoter.

[0214] Typically, signal peptide sequences encode peptides of 10–30 amino acids, e.g., 15–20 amino acids. Often, the amino acids are primarily hydrophobic. In a typical scenario, the signal peptide targets the endoplasmic reticulum (ER) of the expressing cell, where it cleaves a growing polypeptide chain. Within the ER, the signal peptide is cleaved, allowing for the secretion of the polypeptide via the Golgi apparatus.

[0215] Immunogenicity and vaccine composition The present invention further provides compositions comprising chimeric or fusion proteins as defined herein, and the use of such chimeric or fusion proteins in immunogenic or vaccine compositions for the treatment or prevention of P. gingivalis infection.

[0216] As used herein, the term "vaccine composition" is defined as a composition used to induce an immune response to an antigen (immunogen) within the composition in order to protect or treat an organism against a disease.

[0217] As used herein, the terms “immunostimulating composition,” “vaccine composition,” and “immunogenic composition” may generally be used interchangeably.

[0218] The immunostimulatory composition or vaccine of the present invention may suitably contain, in addition to one or more peptides of the present invention as therapeutic or prophylactic active ingredients, pharmaceutically acceptable carriers, excipients, diluents, adjuvants, vehicles, buffers, or stabilizers. Such carriers include, but are not limited to, physiological saline, buffered physiological saline, dextrose, liposomes, water, glycerol, polyethylene glycol, ethanol, and combinations thereof.

[0219] Immunostimulatory compositions or vaccine compositions may be adapted for administration via any suitable route, e.g., parenteral (including subcutaneous, intramuscular, intravenous, or intradermal injection, or injection into cerebrospinal fluid), oral (including buccal or sublingual), nasal, topical (including buccal, sublingual, or transdermal), vaginal, or rectal routes. Such compositions may be prepared by any method known in the field of pharmacy, for example, by mixing peptides with carriers or excipients under sterile conditions. Typically, vaccine compositions are adapted for administration by subcutaneous, intramuscular, intravenous, or intradermal routes, usually by injection. Alternatively, vaccine compositions may be adapted for oral or nasal administration.

[0220] Immunostimulatory compositions or vaccine compositions suitable for parenteral administration may be aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation substantially isotonic with the blood of the target recipient, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners. Excipients that may be used in injectable solutions include, for example, water, alcohol, polyols, glycerin, and vegetable oils. Compositions may be supplied in unit-dose or multi-dose containers, such as 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, such as water for injection, immediately before use. Instantaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0221] Immunostimulant or vaccine compositions suitable for oral administration may be provided as separate units such as capsules, tablets, or lozenges, as powders or granules, as solutions, syrups, or suspensions (in aqueous or non-aqueous liquids, or as edible foam or whipped cream, or as emulsions).

[0222] Suitable excipients for tablets or hard gelatin capsules include lactose, corn starch or its derivatives, stearic acid or its salts. Suitable excipients for use with soft gelatin capsules include, for example, vegetable oils, waxes, fats, semi-solid or liquid polyols.

[0223] Excipients that can be used for the preparation of solutions and syrups 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.

[0224] Immunostimulant or vaccine compositions adapted for nasal administration, where the carrier is solid, are administered by nasal inhalation, i.e., by rapid inhalation through the nasal cavity from a container of powder held near the nose, and include, for example, a coarse powder having a particle size in the range of 20 to 500 microns. Preferred compositions, where the carrier is liquid for administration as a nasal spray or nasal drops, may include an aqueous or oily solution of the active ingredient.

[0225] Compositions suitable for inhalation administration include particulate dust or mist that can be generated by various types of metered-pressure aerosols, nebulizers, or inhalers.

[0226] Immunostimulant or vaccine compositions adapted for transdermal administration may be provided as separate patches intended to remain in close contact with the recipient's epidermis over an extended period. For example, the active ingredient may be delivered from the patch by ion electrophoresis, as generally described in Pharmaceutical Research. 3(6):318 (1986).

[0227] Compositions suitable for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions (e.g., mouthwashes), pastes, gels, sprays, aerosols, or oils. In the case of infections of the eyes or other external tissues, such as the mouth and skin, the composition may be applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be used with either a paraffinic or water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream having an oil-in-water or water-in-oil base. Pharmaceutical compositions suitable for topical administration to the eyes may include eye drops, in which the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent. Pharmaceutical compositions suitable for topical administration to the mouth may include lozenges, pastes, or mouthwashes.

[0228] The immunostimulant or vaccine composition may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts (the substance of the present invention itself may be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents, or antioxidants.

[0229] The vaccine composition of the present invention may also contain one or more other prophylactic or therapeutically active agents in addition to the chimeric or fusion protein as defined herein.

[0230] The chimeric or fusion protein for use in the vaccine composition of the present invention may or may not be freeze-dried.

[0231] The vaccine composition of the present invention may also contain a pharmaceutically acceptable adjuvant in addition to the peptide as defined herein. The adjuvant is added to enhance the immunogenicity of the vaccine composition.

[0232] Suitable adjuvants for inclusion in vaccine compositions are known in the art and include incomplete Freund's adjuvant, complete Freund's adjuvant, Freund's adjuvant having MDP (muramyl dipeptide), alum (aluminum hydroxide), alum + Bordatella pertussis and immunostimulatory complex (ISCOM, typically a Quil A matrix containing viral proteins), QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, β-aretin, MPC-026, Adjuvax, CpG ODN, Betafectin, and MF59.

[0233] The vaccine composition of the present invention may also contain or be administered together with one or more costimulatory molecules.

[0234] The dosage of the vaccine composition of the present invention can vary over a wide range depending on the age and condition of the individual being treated, and the physician will ultimately determine the appropriate dosage to be used.

[0235] This dosage can be repeated frequently as needed. For example, the initial dose of the vaccine may be administered, followed by a subsequent dose of Boosoo.

[0236] For administration to mammals, particularly humans, the expected daily dose of the activator is 1 μg / kg to 10 mg / kg body weight, typically around 10 μg / kg to 1 mg / kg body weight. In any case, the physician will determine the most appropriate actual dose for the individual, depending on factors including the individual's age, weight, sex, and response. The above doses are examples of average cases. Of course, there may be cases where higher or lower doses are required, and such cases are within the scope of this invention.

[0237] The vaccine composition of the present invention may be administered by any convenient route described herein, such as intramuscularly, intravenously, by inhalation, intraperitoneally, or orally, or by injection into the cerebrospinal fluid.

[0238] The vaccine composition of the present invention may be provided in unit dosage forms, generally in sealed containers, and may be provided as part of a kit. Such kits usually (but not necessarily) include instructions for use. Multiple unit dosage forms may be included.

[0239] Therefore, in yet another embodiment, the present invention provides a kit of parts comprising the vaccine composition of the present invention and one or more cytokines and / or adjuvants in a sealed container.

[0240] Methods for immunizing a subject using a chimeric or fusion protein of the subject. The present invention provides methods and compositions for treating or preventing infection, or minimizing the possibility of infection by P. gingivalis, in individuals where such treatment or prevention is necessary, the methods comprising administering the fusion or chimeric protein of the present invention.

[0241] The present invention further provides methods and compositions for inducing a humoral immune response to P. gingivalis in a subject. The humoral response may be for the purpose of directly obtaining protective / therapeutic anti-P. gingivalis antibodies in an individual requiring protection / treatment. Alternatively, the humoral response may be for the purpose of generating antibodies that can therefore be directly administered to an individual requiring treatment / protection with the antibodies, and thus isolated from the subject (or its eggs).

[0242] Therefore, the present invention includes methods and compositions for preventing infection with P. gingivalis in individuals, minimizing the likelihood of infection, and / or reducing the severity and duration of P. gingivalis infection.

[0243] The present invention also provides a method for obtaining antibodies directed towards P. gingivalis, the method comprising administering a chimeric or fusion protein, composition, vaccine, or immunostimulatory composition of the present invention to a non-human animal, thereby generating antibodies directed towards P. gingivalis in the animal. Preferably, the method further comprises isolating the antibodies from the animal (for example, from the animal's blood) or from the animal's eggs (for example, when generating IgY antibodies from chickens).

[0244] The present invention also provides an antibody preparation comprising an antibody directed toward P. gingivalis, wherein the antibody preparation is obtained by administering the chimeric or fusion protein, composition, vaccine, or immunostimulatory composition of the present invention to a non-human animal, thereby generating an antibody directed toward P. gingivalis in the animal, and by isolating the antibody from the animal or its eggs.

[0245] Antibodies targeted to P. gingivalis may be used therapeutically to eliminate or reduce P. gingivalis infection, or prophylactically to prevent or reduce the severity of P. gingivalis infection.

[0246] As used herein, the terms "treatment" or "treating" of a subject includes the application or administration of a composition of the invention to the subject (or the application or administration of a compound of the invention to cells or tissues derived from the subject) for the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, reducing, modifying, improving (remedying), reducing exacerbation, ameliorating, improving, or affecting a disease or condition, a symptom of a disease or condition, or a risk (or susceptibility) of a disease or condition. The term "treating" includes remission; remission; reduction in the rate of exacerbation; reduction in the severity of a disease; stabilization, reduction, or making an injury, pathology, or condition more tolerable to the subject; slowing the rate of exacerbation or debilitation; preventing the ultimate point of exacerbation from causing debilitation; or any indicator of success in the treatment or improvement of an injury, pathology, or condition, including objective or subjective parameters such as improving the physical or mental health of the subject.

[0247] As used herein, "preventing" or "prevention" is intended to refer to at least reducing the likelihood of the risk (or susceptibility) of acquiring a disease or disorder (i.e., in a subject who may be exposed to the disease or has a predisposition thereto but has not yet experienced or manifested any of the clinical symptoms of the disease, preventing the onset of at least one of the clinical symptoms of the disease). Biological and physiological parameters for identifying such subjects are provided herein and are also well known to physicians.

[0248] The vaccine composition of the present invention can be administered to a subject who is believed to be most in need thereof, for example, a child or an elderly person or an individual at risk of exposure to P. gingivalis. The vaccine composition of the present invention can also be administered to a subject suspected of being infected with P. gingivalis or diagnosed as being infected with P. gingivalis.

[0249] The compositions and methods of the present invention are equally extended to use in both human and / or veterinary medicine, the generation of diagnostic agents, or the generation of other therapeutic reagents.

[0250] As used herein, the term "subject" shall mean any animal including a human, e.g., a mammal. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject can be a human. In a further example, the subject can be a veterinary subject, e.g., a pet (such as a cat, dog, guinea pig, etc.). As used herein, the terms "subject", "individual", and "patient" can be used interchangeably.

[0251] One skilled in the art will be proficient in methods for determining successful vaccination / immunization with the chimeric or fusion proteins or compositions described herein. For example, one skilled in the art will be proficient in methods for quantifying antibodies generated after immunization and / or methods for quantifying the degree of the humoral (Th2) response induced after immunization.

[0252] Kit In another embodiment, a kit or product is provided that includes one or more proteins, polypeptides or polynucleotides of the present invention, and / or the above-described immunogenic composition.

[0253] In yet another aspect, the present invention provides a kit of parts that includes the vaccine composition of the present invention and one or more adjuvants for separate, sequential, or simultaneous administration to a subject.

[0254] In other embodiments, kits are provided for use in the above-described therapeutic or prophylactic applications, the kits comprising - a container holding the protein, polypeptide, polynucleotide, or immunogenic composition of the present invention, - a label or package insert having instructions for use.

[0255] In any embodiment, the kit may contain one or more additional active principles or active ingredients for inducing an immune response to P. gingivalis in a subject.

[0256] A kit or “product” may include a container and a label or accompanying information on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and blister packs. Containers may be formed from a variety of materials, such as glass or plastic. Containers may hold a therapeutic composition effective for treating a condition and may have a sterile access port (for example, the container may be an intravenous solution bag or vial with a stopper that can be pierced by a subcutaneous needle). The label or accompanying information indicates that the therapeutic composition is used to treat a selected condition. In one embodiment, the label or accompanying information includes instructions for use indicating that the therapeutic or preventive composition may be used to treat an inflammatory disease or condition described herein.

[0257] The kit may comprise (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 present invention may further comprise a document describing the composition, and the other active ingredient may be used to treat disorders resulting from inflammatory diseases or conditions described herein, or to prevent complications. Alternatively or additionally, the kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. This may further comprise other desirable materials, from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0258] In any embodiment, the therapeutic composition may be provided in the form of a disposable or reusable device comprising a container for holding the therapeutic, prophylactic, or immunogenic composition. In one embodiment, the device is a syringe, an auto-injector, or a nanopatch. The device may hold 0.1 to 2 mL of the therapeutic or immunogenic composition. The therapeutic or prophylactic composition may be provided in the device ready for use, or requiring mixing, dissolution, resuspension, or addition of further components.

[0259] It will be understood that the present invention, as disclosed and defined herein, encompasses all of two or more selective combinations of individual features referred to or evident from the text or drawings. All of these various combinations constitute various selective embodiments of the present invention.

[0260] The following embodiments are presented to more fully illustrate some embodiments of the present invention. However, they should not be construed as limiting the broad scope of the present invention. Those skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the present invention. [Examples]

[0261] The following examples illustrate a series of in vitro and in vivo studies related to the chimeric or fusion protein of the present 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 study, and Example 3 describes the results of the in vivo study.

[0262] Example 1: Method 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 essentially expressed from the pET28 vector (or pDUET-1 for rABM1+rABM2 co-expression from a single vector) by induction with isopropyl β-D-thiogalactosidase (IPTG) as follows:

[0263] Nucleic acids encoding various chimeric and component proteins (including active site and adhesion factor domains) were generated by DNA splicing via standard PCR or duplication extension ("SOEing") PCR using P. gingivalis W50 genomic DNA templates and specifically designed oligonucleotide primers. The PCR fragments or SOEn PCR fragments were purified, ligated to the cloning vector pGEMTeasy or pBHA, and transformed into chemically competent E. coli α-Gold cells (Bioline, New South Wales, Australia).

[0264] DNA sequences encoding a single additional KAS (i.e., active site) residue, either containing a "DSSG" amino acid linker region or without a linker, were sequentially added one at a time to the recombinant chimeric and Kgp adherent factor domains, essentially as follows: Restriction enzyme sites were introduced to the ends of the DNA insertion fragments in the chimeric and adherent factor variant mother clones by PCR using oligonucleotide primers containing restriction enzyme-specific nucleotides. Synthetic DNA fragments encoding individual KAS sequences with corresponding restriction sequences were ligated to the insertion ends of the DNA constructs from the mother clone, and the recombinant clones were purified. Subsequently, if a second or third additional KAS was added, further restriction sequences were introduced to the ends of these cloned insertions, 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 sub-fragments were exchanged via ligation to produce a large number of variants (including those exemplified in Table 1).

[0265] The residues within ABM1 and ABM2, as well as the cysteine residues, were mutated using the QuickChange II Site Directed Mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions.

[0266] 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). The mutations were further verified by DNA sequencing of the entire insert. The verified constructs were then subjected to restriction digestion with the selected enzymes and the inserts were cloned into the relevant pET expression vectors in E. coli α-select chemically competent cells, followed by cloning into the E. coli expression host, BL21-CodonPlus(DE3)RIPL (Stratagene, Australia).

[0267] Investigation of chimeric features affecting the solubility of recombinant proteins, small scale Single colony transformants were used to inoculate 2 mL of Luria Bertani (LB) broth containing 30 μg / ml kanamycin in an orbital shaker at 37°C overnight. This inoculum was then used to inoculate 2 ml of LB containing 30 μg / ml kanamycin (1:100). The cell culture with OD600 = 0.6 - 1.0 was induced with 1 mM IPTG at 37°C for 2 hours. The cell culture was centrifuged, the pellet was resuspended in 250 μl of PBS, sonicated briefly using a CPX750 sonicator (Cole Parmer Instrument Company, USA), and centrifuged.

[0268] The solubility of recombinant proteins was evaluated by analyzing the total lysate, soluble (supernatant), and insoluble (pellet) fractions by SDS-PAGE. 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 to 30°C) and lower IPTG levels (0.01 to 0.5 mM) to determine the optimal conditions for enhancing solubility. All recombinant protein inductions were scaled up (20 to 200 ml) under optimal conditions to test solubility in medium to large-scale conditions. Cultures (200 mL) were subjected to IPTG induction at various temperatures (e.g., 5 hours at 30°C or 16 hours at 16°C).

[0269] Cells were collected 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 hour, and the clarified lysate was then centrifuged (8,000 g, 30 min, 4°C). The supernatant (soluble) and pellet (insoluble) fractions were collected and analyzed by SDS-PAGE and native-PAGE.

[0270] In-gel HIS staining of recombinant proteins Recombinant proteins subjected to SDS-PAGE were stained using the Invision In Gel His Stain Kit (Invitrogen) according to the manufacturer's instructions. This staining is highly specific to His-tagged proteins. This procedure was used to identify N-terminal degradation of recombinant proteins, as all candidates contain a C-terminal His tag.

[0271] Large-scale purification of chimeras for research in animal models The purification conditions for each recombinant protein are described in detail in the following section.

[0272] Protein expression and cell lysis All recombinant proteins identified for use as vaccine candidates in animal models were expressed in E. coli BL21(DE3) as recombinant C-terminal His-tagged fusion proteins or as "untagged" proteins, as described above. Cells were grown at 37°C in LB medium, TB medium, or modified M9 minimal medium supplemented with 50 μg / mL kanamycin. Cultures with an OD600 of 0.8–1.0 were induced with 0.4 mM IPTG at 37°C or 34°C for 2–4 hours, at 25°C for 12 hours, or at 16°C for 16–20 hours. Cells were collected by centrifugation at 8000g at 4°C. Unless otherwise instructed, the cells were lysed on ice for 1.5 hours in lysis buffer [0.35 mg / mL lysozyme, TBS150 (50 mM Tris·Cl, 150 mM NaCl, 40 μg / mL DNAseI in pH 7.5)] + 1% TritonX-100 and a protease inhibitor without EDTA (Complete ULTRA tablets, Sigma-Aldrich). The cell lysates were clarified by centrifugation at 23,500g at 4°C for 40 minutes.

[0273] The initiating Met residue (aa1) on all recombinant proteins shown in Table 1, starting with the translated sequence MA, is removed in vivo by an E. coli N-terminal methionine-processing enzyme that cleaves methionine if the second-to-last residue is a small residue. This was confirmed by mass spectrometry of KDAK and KDAK variant recombinant proteins.

[0274] Purification of His-tagged proteins from soluble fractions under non-denaturing conditions 1. Ni-affinity chromatography The cell lysates were clarified by centrifugation at 20,000 g at 4°C to remove cell debris. After filtration through a 0.22 μm filter, the lysates were 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 thoroughly washed with loading buffer, then 20 mM imidazole in TBS300. The 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 proteins were concentrated using an Amicon filter unit 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.

[0275] 2. Anion exchange chromatography The isoelectric points (pI) of all candidate proteins were predicted to be in the acidic range using the online analysis tool ProtParam-ExPASy (https: / / web.expasy.org / protparam / ), and anion exchange chromatography was applied for further purification as needed. Briefly, concentrated proteins from Ni-affinity purification were diluted in buffer A (50 mM Tris·Cl, 20 mM NaCl, pH 7.5) to reduce ionic strength, and then loaded onto an anion exchange HiTrap Q column (GE Healthcare) in buffer A. Proteins were eluted in Tris buffer under a 20–400 mM NaCl gradient while monitoring absorbance at 280 nm. Target proteins in the peak fraction were validated by SDS-PAGE and / or native PAGE and concentrated using an Amicon filter unit with appropriate molecular weight cutoff. Protein solutions were stored at -70°C for further purification or buffer exchange using size exclusion chromatography or dialysis.

[0276] 3. Size exclusion chromatography Size exclusion chromatography was performed at 4°C on a HiLoad Superdex 200 or HiLoad Superdex 75 column (GE Healthcare). Proteins from further steps of Ni affinity purification or anion exchange chromatography were loaded onto the appropriate column and eluted in TBS150 or TBS100 buffer (100 mM NaCl in 50 mM Tris·Cl, pH 7.5) while monitoring absorbance at 280 nm. Target proteins in the peak fraction were validated by SDS-PAGE and / or native PAGE. A second or further round of size exclusion purification was performed for the isolation of single oligomer species such as dimers. The fractions containing the best protein purity were pooled and concentrated. The target protein fraction was simply pooled and concentrated to prepare a mixture of protein homomultimers. Protein identity was confirmed, if necessary, by determining the 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). Protein concentration was quantified by determining the absorbance at 280 nm using a calculated decay coefficient before submitting to animal model experiments.

[0277] Purification of His-tagged proteins from inclusion bodies 1. Under oxidative conditions After cell lysis (as described for purification from the soluble fraction), the insoluble pellet was washed twice with TBS150. The proteins expressed as inclusion bodies were solubilized with 8M urea at room temperature for 1 hour on a rolling platform in TBS300 or 20mM NaPi, 500mM NaCl, pH 7.4 (PBS500). 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 (8M urea + 20mM imidazole in TBS300) or PBSU (8M urea, 20mM NaPi pH 7.8, 500mM NaCl + 20mM imidazole). The column was thoroughly washed with the same 8M urea buffer. Further washing with PBSU at pH 6.5 was applied to the column containing the loaded protein in PBSU. To remove urea, the target protein was eluted with a gradient of 20–500 mM imidazole in PBSU. When using loose Ni-NTA resin (Thermofisher), the urea extract was mixed with the resin in PBSU with gentle agitation for 2 hours. After thorough washing 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 in 6 M, 4 M, then 2 M urea phosphate buffer in dialysis tubing with a molecular weight cutoff of 3.5 kDa (Fisher Biotec, Australia). The protein in 2 M urea buffer was further dialyzed to physiological saline alone and maintained at 4°C before submission to animal model experiments, or otherwise submitted in 2 M urea buffer without further dialysis.

[0278] 2. Under reduction conditions The protein was extracted from the inclusion body 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 while decreasing the urea concentration gradient from 4 to 0 M. The protein was then eluted in TBS300 + 2 mM TCEP while increasing the heimidazole concentration gradient from 20 to 350 mM. The eluted protein was then concentrated for size exclusion chromatography in reducing buffer (2 mM TCEP in TBS150) using the same method as described in the section for purifying protein dimers or macromers from soluble fractions under non-denaturing conditions. After analysis using SDS-PAGE and native PAGE, mass spectrometry and quantification, the samples were stored in reducing buffer at -70°C or submitted for animal model experiments.

[0279] Purification of His-tagged proteins from soluble fractions under oxidative and denaturing conditions The cell lysates were clarified by centrifugation at 20,000 g to remove residues and insoluble materials. The lysates were then immediately denatured by adding urea solution to PBS500 to a final concentration of 8 M while stirring at room temperature for 1 hour. The resulting lysates were then centrifugated at 20,000 g for 40 minutes at room temperature. The target proteins were purified using HisTrap Ni-affinity columns or loose Ni-NTA resins using the same approach as described in the section on purification from inclusion bodies under oxidation conditions. The purified antigens in physiological saline were stored at 4°C and then submitted to animal model experiments.

[0280] Purification of untagged candidates from soluble fractions Anion exchange chromatography This is the first chromatographic step for purifying untagged proteins. Similar to the His-tagged counterparts, the pI of untagged candidate proteins was also predicted to be within the acidic range using the online analysis tool ProtParam-ExPASy (https: / / web.expasy.org / protparam / ). Therefore, anion exchange chromatography was applied and performed in two rounds. In the first round, the clarified cell lysates were 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 supplemented with a 0.05 × protease inhibitor cocktail, pH 6.5). After washing with 10 column volumes of buffer AA, bound proteins were eluted with a NaCl gradient of 0–100% buffer BA (1 M NaCl in 20 mM NaPi pH 6.5) and absorbance at 280 nm was monitored. Target proteins in the peak fraction were validated 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. The bound proteins were eluted with a NaCl gradient of 0–50% buffer BA, and absorbance at 280 nm was monitored. Target proteins in the peak fractions were validated by SDS-PAGE and / or native PAGE, and the best fractions were concentrated using an Amicon filter unit with a molecular weight cutoff of 10 kDa. The protein solutions were stored at -20°C and further purified by size exclusion chromatography.

[0281] Size exclusion chromatography in the first round was performed at room temperature on a HiLoad Superdex200 column (GE Healthcare). Proteins purified by anion exchange chromatography were loaded onto the size exclusion column and eluted in AS buffer (1M (NH4)2SO4 supplemented with a 0.05× protease inhibitor cocktail, 50mM NaPi pH 7.0) while monitoring absorbance at 280nm. Target proteins in the peak fraction were validated by SDS-PAGE and / or native PAGE. The fraction with the best protein purity was pooled for further purification using hydrophobic interaction chromatography.

[0282] Hydrophobic Interaction Chromatography The pooled fraction of the target protein in buffer AS from the first round of size exclusion chromatography was 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. The bound protein was eluted in buffer BH (50 mM NaPI, pH 7) with a gradient decreasing the ionic intensity of (NH4)2SO4 from 800 mM to 200 mM, while monitoring absorbance at 280 nm. The target protein in the peak fraction was validated by SDS-PAGE and / or native PAGE, and the best fraction was pooled for the next purification or buffer exchange step of the second round of size exclusion chromatography and concentrated using an Amicon filter unit.

[0283] Second round of 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 BTS buffer (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 antigens were divided and stored at -80°C for future use.

[0284] Identification of proteins and quantification of untagged candidates In addition to SDS-PAGE or native PAGE validation, protein identity was confirmed, as needed, by determining the intact protein molar mass using LC-MS or by sequence analysis using liquid chromatography-Orbitrap tandem mass spectrometry (LC-MS / MS, Agilent). Protein concentration was quantified by determining the absorbance at 280 nm using the calculated decay coefficient (Table 3). If buffer exchange was required before submission to animal model experiments, Superdex 200 size exclusion columns (GE Healthcare) or Zeba® spin desalting columns (ThermoFisher Scientific) were used.

[0285] 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 of the Oral Health Cooperative Research Centre, The Melbourne Dental School, University of Melbourne, Australia. P. gingivalis W50 was grown in an anaerobic N2 atmosphere containing 5% CO2 at an MK3 anaerobic workstation (Don Whitley Scientific Ltd., Adelaide, Australia) on equine blood agar (HBA) supplemented with 10% v / v dissolved equine blood (37°C) (20 g / L HBA, Oxoid Ltd., Hampshire, UK). Colonies were inoculated into a starter medium consisting of 20 mL of sterile cerebral and cardiac infusion medium (37 g / L BHI, Oxoid Ltd., Hamsphire, UK) 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 hours, 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 minutes, 4°C). Bacterial purity was routinely confirmed by Gram staining [Slots (1982). In: Host-Parasite Interaction in Periodontal Disease, Genco, R.Jand Merganhagan, SE (eds). Washington DC: American Society for Microbiology. pp. 27-45.].

[0286] Preparation of heat-killing bacteria P. gingivalis W50 cultures were collected (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 pelletized by centrifugation (7,000 g, 20 min, 4°C). The bacterial cells were resuspended in PBS and heated at 65°C for 15 minutes. The suspension was centrifuged (7,000 g, 20 min, 4°C), resuspended in sterile PBS, and this process was repeated once. After the second wash, the supernatant was discarded, and the cell pellet was resuspended in sterile PBS and divided into 2 × 10⁶ cells. 10 Cell density was obtained in cells / mL, and protein concentration was determined using Biorad Protein Assay Dye Reagent Concentrate (Life Science, NSW, Australia).

[0287] Mouse periodontitis model The mouse periodontitis experiment was modified from Baker et al.'s mode (1994). Arch Oral Biol 39:1035-1040) and carried out as previously described by O'Brien-Simpson et al. (2005 J Immunol 175:3980-3989). On day 0, mice (female BALB / c, 6-8 weeks old, 10 mice / group) were given 1 × 10¹⁶ doses of P. gingivalis W50 suspended in 20 μL of 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) at 2-day intervals. 10P. gingivalis consisting of [number of viable P. gingivalis W50 cells] was orally inoculated. The inoculation was prepared anaerobically and then immediately applied to the gingival margin of the maxillary molars. The number of viable cells in each inoculation was verified by flow cytometry and counted CFU on blood agar. The animal groups consisted of orally inoculated P. gingivalis W50 (infection control), non-bacterial inoculation control, and an immunized group. For therapeutic vaccination, periodontitis model mice (Figure 1) were first orally inoculated with 50 μg of vaccine candidate in saline / alum (alhydrogel, 2% aluminum hydroxide wet gel suspension, Invivogen), and then immunized via the intraperitoneal route on day 19. Mice received a second immunization (50 μg in saline / alum) via the subcutaneous route on day 40. On day 62, mice were killed by inducing bleeding by cardiac puncture. The maxilla was removed, cut in half along the midline, and divided into 10 sections to determine alveolar bone loss. Antibody profiles were determined using serum and ELISA.

[0288] Measurement of alveolar bone loss in the mouse maxilla To examine bone loss, the maxilla was boiled in deionized water (1 minute), mechanically disassembled, and immersed in 2% w / v potassium hydroxide (16 hours, 25°C). The maxilla was washed twice with deionized water (25°C), dried (1 hour, 37°C), and stained with a 0.5% w / v methylene blue aqueous solution. Buccal digital images of the maxilla were captured with an Olympus DP12 digital camera mounted on a dissecting microscope using OLYSIA BioReport software version 3.2 (Olympus Australia Pty Ltd, New South Wales, Australia) to assess horizontal bone loss. The maxilla was positioned so that the apex of the buccal and lingual molars overlapped. Images were captured with micrometers in the frame to allow for standardization of measurements for each image. Horizontal bone loss was defined as loss occurring in the 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, in mm². 2 The total visible CEJ-ABC area was obtained. P. gingivalis-induced alveolar bone loss (mm 2 Alveolar bone loss was calculated by subtracting the total visible CEJ-ABC area of ​​the unvaccinated (NC) group from the total visible CEJ-ABC area of ​​each experimental group. Alveolar bone loss was determined twice using randomized and blinded protocols. Data were expressed as mean + / - standard deviation (mm²). 2 The results were expressed as follows and analyzed using one-way ANOVA and Dunnetts T3 post-hoc test.

[0289] Pre-screening of vaccine candidates using enzyme-linked immunosorbent assay (ELISA) Vaccine candidates were pre-screened using several monoclonal antibodies against P. gingivalis to determine whether the key domains and epitopes present in the vaccine constructs were accessible to the antibodies, thus enabling the generation of an in vivo antibody response. The antigens to be screened were coated on flat-bottomed 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; a 1 / 4000 dilution of IgG (M8642) antibody (Sigma, New South Wales, Australia). An ELISA experiment was developed using a 1 / 4000 dilution of horseradish peroxidase conjugate porcine anti-goat IgG antibody (M5420, Sigma, New South Wales, Australia).

[0290] Determination of subclass antibodies in serum using ELISA As described in Pathirana et al. (2007). Infect Immun 75:1436-1442), ELISA was performed using a solution (1 μg / mL) of either a domain subunit or epitope of HK W50 cells in 0.1 M PBS (pH 7.4) to coat wells of flat-bottom polyvinyl microtiter plates (Microtiter, Dynatech Laboratories, McLean, VA, US) for 16 hours at 4°C to evaluate antibodies to subclasses of the region. The following antibody dilutions were used in these experiments: 1 / 4000 dilutions of goat anti-mouse, IgG (M8642), IgG1 (M8770), and IgG2a (M4434) antibodies (Sigma, New South Wales, Australia). An ELISA experiment was developed using a 1 / 4000 dilution of horseradish peroxidase-conjugated porcine anti-goat IgG antibody (M5420, Sigma, New South Wales, Australia). For the epitope ELISA, a biotinylated peptide was conjugated at 10 μg / mL to a pre-blocked streptavidin-coated flat-bottom plate (Pierce, Thermo-Fisher). After incubation with serum, the ELISA was developed with 1 / 4000 dilution of goat anti-mouse IgG and 1 / 4000 dilution of horseradish peroxidase-conjugated porcine anti-goat IgG antibody. All optical density measurements were performed at 405 nm using a Wallac VICTOR3 1420 Multilabel counter (Perkin Elmer).

[0291] Example 2: Results of in vitro studies Study 1: Chimeric components that affect the solubility of recombinant proteins One previously reported chimera (in WO2010 / 022463) consists of a KAS(K) peptide conjugated to an N-terminally cleaved DUF2436 domain (Dc), an adhesion factor domain containing ABM213(A), and a C-terminally cleaved CAD domain (referred to as a cleaved K1 domain, K1n). This chimera is referred to as "KDcAK1n" (SEQ ID NO: 54) and may also be referred to herein as the "original chimera" or "chimera" in comparison to the chimeras and fusion proteins of the present invention. The KDcAK1n protein is produced as an inclusion body in E. coli and has low solubility and low stability.

[0292] The cleaved DUF and K1 domains of the A1 adhesion factor fragment represent how the Kgp polyprotein is spontaneously proteolytically processed and assembled on the P. gingivalis cell surface during infection. Therefore, the cleaved DUF and K1 domains are obvious candidates for inclusion in vaccines to generate an immune response against gingipain.

[0293] Extensive attempts were made to produce soluble KDcAK1n. Variations in growth media, growth conditions, IPTG concentration, E. coli expression strains, induction temperature, cell growth stage during induction, addition of growth stabilizers, lysis buffer, and protein preservation buffer were systematically investigated. Enhanced soluble recombinant protein expression was observed in small-scale expression under low temperature and IPTG induction. In small-scale cultures, KDcAK1n produced approximately 30-50% of total recombinant protein expression as soluble protein. Nevertheless, the soluble recombinant could not be scaled up to culture volumes exceeding 10 ml, and attempts to increase the scale resulted in inclusion body formation.

[0294] Effect of DUF domain on protein solubility Cleavage of the DUF domain in the chimeric protein construct resulted in the exclusion of soluble protein expression. This result indicates that a complete DUF domain containing 38 extra N-terminal residues is desirable for optimizing soluble protein expression and efficient stabilization of recombinant proteins.

[0295] Therefore, we constructed a construct expressing a chimeric variant with an extended Dc domain (a chimera with a Dc extended by 38 N-terminal residues). This recombinant was designated KDAK1n (e.g., SEQ ID NO: 55), and the results show that the solubility of the chimeric molecule is significantly enhanced by the complete DUF domain. Large-scale expression of KDAK1n in 0.2–0.5 mM IPTG at 30°C resulted in a good yield of soluble recombinant protein expression.

[0296] To summarize: ●The full-length DUF domain is desirable for the solubility of recombinant variants (Figure 2A). Therefore, extending the DUF to its full length is important for the solubility and stability of recombinant proteins.

[0297] Study 2: Factors affecting polymerization Native PAGE analysis of purified soluble DUF-ABM213 and ABM213-ABM21 recombinant proteins reveals broad, homogeneous laddering indicative of multimerization. Multimerization is also evident in conventionally produced KDcAK1n chimeras, via the formation of intermolecular disulfide bonds between denatured domains. This disulfide bond multimerization was most pronounced in KDcAK1n chimeras purified from inclusion bodies.

[0298] In the course of research on recombinant chimeras and Kgp adhesion factors, it was revealed that recombinant protein multimerization occurs via interactions between ABM1 and ABM2 motifs. (See schematic diagram in Figure 3A) Recombinant proteins containing ABM2(1), ABM2(2), and ABM3 domains were able to readily form multimers. Subsequent expression of smaller recombinant proteins encoding ABM2(1), ABM1(2), and ABM3 (designated rABM213), as well as recombinant proteins encoding ABM2(1) and ABM1(2) (designated rABM21), showed that both recombinants were able to readily multimerize. This multimerization was evident from the presence of an orderly ladder of bands on native PAGE.

[0299] It has been proposed that during the folding of the Kgp multidomain polyprotein, the ABM1 domain interacts with the ABM2 domain to form a stable FnIII-like beta-sheet complex structure. The inventors hypothesized that ABM1(1) interacts with its next available adjacent ABM2(1), and ABM1(2) interacts with ABM2(2), etc., during the folding of the Kgp polyprotein (as schematically shown in Figure 3A).

[0300] In a separate study, the inventors demonstrated that co-expression of rABM1 and rABM2 as distinct proteins can 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 spectroscopy of the folded ABM domain showed that the domain multimerizes via beta-chain exchange involving ABM1 and ABM2 motifs. These results provide evidence that chimeric and variants containing the ABM domain multimerize via interactions between the ABM1 and ABM2 motifs of a properly folded ABM domain.

[0301] Next, the inventors aimed to mutate specific residues of ABM1 or ABM2 within recombinant ABM21 (rABM21) protein, encompassing residues 878-968 on the Kgp W50 polyprotein.

[0302] BLAST analysis of Kgp ABM1 and ABM2 sequences in all available bacterial genomes revealed that submotifs within the ABM1 and ABM2 motifs are highly conserved across all phyla. Subsequently, alignment of the ABM1 and ABM2 sequences against all ORFs in the W83 genome revealed that these residues are similarly highly conserved in the P. gingivalis gene. The "PVQN" motif with conserved proline residues was highly conserved across the entire Phyla, including P. gingivalis.

[0303] The proposed molecular modeling of the tertiary folding using an online program revealed that this "PVQN" sequence is located at the start of the beta-sheet structure, and that the sequence immediately following the N-terminus of this motif is most likely an unstructured "loop."

[0304] The inventors mutated residues within the PVQN motif of rABM21, as well as mutant residues within the loop immediately preceding the PVQN. They targeted residues within the loop because it was hypothesized that the size and shape of the loop preceding the beta-sheet structure could influence the strength of adjacent beta-sheet folding. The number and position of proline residues within such "loop" structures have been reported to be related to adjacent beta-sheet interactions.

[0305] In addition, the inventors mutated the highly conserved hydrophobic residues Y(878) and W(968) present in ABM2 and ABM1, respectively. The inventors also introduced a substitution of SxYQ into the sequence of the motif NxFA in the ABM1 sequence (see the SSEYQ variant listed in Table 2 below).

[0306] Mutant rABM21 variants were purified, and their ability to polymerize was evaluated. The inventors sought to identify mutations that would result in stable, soluble recombinant molecules that could be purified as stable monomers. [Table 2]

[0307] All recombinant proteins (WT rABM213, rABM21, and rABM21 mutant variants) resulted in very high levels of soluble recombinant protein expression under harsh induction conditions (summarized in Table 2).

[0308] Purified recombinant proteins subjected to native PAGE analysis (Figures 3B-E) demonstrated that rABM21 can form multimers via two mechanisms: (1) multimerization via the ABM1 motif "PVQN", and (2) multimerization via disulfide bonds.

[0309] Mutations of the highly conserved "PVQN" motif in rABM21 to AVQN or AVQA did not rule out multimerization.

[0310] 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 influence on the decrease in multimerization.

[0311] AVQA mutations combined with serine substitution of one or both cysteine ​​residues in the ABM domain resulted in a slight decrease in multimerization.

[0312] Mutations of one or both cysteine ​​residues in the ABM domain to serine, along with mutations of "PVQN" to AVQP, resulted in the near or complete elimination of rABM21 multimerization.

[0313] Manipulation and mutation of residues within the modeled "loop" region immediately preceding the ABM1 "PVQN" motif in the modeled "beta sheet region" could also reduce, but could not completely eliminate, multimerization. More specifically, "SSEYQ" substitutions in ABM1 (e.g., modification of the sequence SNEFA to SSEYQ immediately N-terminus of the highly conserved PVQN motif) resulted in a significant reduction in multimerization.

[0314] Mutations in the Tyr-889 and Trp-964 residues (in ABM2 and ABM1, respectively) also resulted in the exclusion of rABM21 multimerization.

[0315] Manipulation of the "PVQN" motif into AVQP, along with substitution of cysteine ​​residues within the ABM domain, enabled the expression and purification of highly soluble monomer proteins using the E. coli pET expression system.

[0316] All chimeric candidates composed of the original sequence with two intact cysteine ​​residues in the ABM21 domain showed extensive ladder multimerization (Figure 3B and C). However, while substitution of one cysteine ​​residue in combination with AVQP substitution resulted in a significant reduction in multimerization, substitution of these two cysteine ​​residues did not result in complete elimination of ladder multimerization (Figure 3C lanes 15 and 16). Complete elimination of ladder multimerization was achieved only by the removal of two ABM cysteine ​​residues + mutation of the motif PVQN to AVQP. This suggests that cysteine ​​residues in the DUF domain (which were not substituted in these experiments) are not significant contributors to multimerization.

[0317] Ladder multimerization was negated by mutations in cysteine ​​residues and the PVQN motif, but an equilibrium between monomeric and oligomeric states still existed under certain conditions. Analysis by size exclusion chromatography revealed that this type of equilibrium was temperature, pH, and concentration-dependent. Increased temperature, decreased pH, and reduced concentration were found to favor the monomeric state. The presence of the His tag did not affect the protein state equilibrium. SEC-MALS analysis showed the overwhelming presence of monomers in solution at concentrations of 2 mg / mL or lower, exhibiting high stability, for the main candidate.

[0318] Study 3: Active Site (KAS) Motifs We manipulated recombinant chimeric variants with additional KAS motifs to determine if this affected immunogenicity. A single KAS or two consecutive KASs were added to the variant at their N-terminus or both ends. Furthermore, 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.

[0319] 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 most pronounced in the 4× linear KAS variant, suggesting that the proteolytic process occurred within the additional KAS sequence. Subsequent removal of the DSSG linker between adjacent KAS sequences did not improve stability.

[0320] Therefore, we concluded that these variants tend to undergo proteolytic treatment when multiple consecutive KAS sequences are expressed in a linear sequence. Soluble variants with a single KAS at one or both ends were relatively stable, less prone to degradation, and did not significantly impair the purified yield.

[0321] Study 4: Large-scale protein yield and stability Most recombinant proteins expressed as soluble proteins and purified under non-denaturing conditions yielded relatively high yields of their final product (>10 mg / L culture). Some of these reached yields exceeding 20 mg / L culture.

[0322] The yield of these same recombinant proteins was significantly 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). It was noted that denatured proteins exhibited low binding affinity to 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 protein was initially expressed as a soluble or insoluble form, or whether purification was performed using a pre-packed column or a loose Ni-NTA resin. These results suggest that it may be desirable to reduce or avoid the use of denaturants when they are not required.

[0323] Low expression temperatures of 16°C were proven effective in improving the soluble expression of proteins such as KDAK1 (SEQ ID NO: 56). For these proteins, purification bypassing the anion exchange step resulted in higher yields; for example, the yield of KDAK1 was twice as high when produced by this method. It was also found that KDAK1n (SEQ ID NO: 55) was expressed as a soluble protein.

[0324] His staining of the gel demonstrated degradation within regions containing multiple KAS(K) residues, but no negative impact on yield was observed by Ni-affinity chromatography. For example, two protective antigens, KKDAK1nKK (SEQ ID NO: 60) and KDAK1nK-4S-AVQP (SEQ ID NO: 53), yielded over 90% of the expected full-length protein species in the final product in the range of 12–21 mg / L cultures.

[0325] Reducing conditions were found to be preferable for extracting insoluble KDcAK1n from inclusion bodies using urea. Under non-reducing conditions, a higher concentration of urea was required than under reducing conditions. Clearly, the formation of disulfide bonds under denaturing conditions negatively affected the protein's solubility. Interestingly, native PAGE gel analysis of a KDcAK1n chimera versus a chimera (SEQ ID NO: 50) in which all four cysteine ​​residues were mutated to serine showed that the elimination of disulfide bonds in the mutated KDcAK1n chimera resulted in a more pronounced ladder formation on native PAGE, consistent with the occurrence of only beta-chain exchange multimerization. Furthermore, the chimeric mutant KDcAK1n PVQN>AVQP / 4Cys>Ser variant (SEQ ID NO: 51) showed higher solubility than KDcAK1n under the same non-reducing denaturing conditions in urea.

[0326] Under the same conditions, the C-terminal His tag was found not to have a significant effect on protein production or solubility when the protein was expressed.

[0327] In the purification of untagged candidates, anion exchange chromatography enriched the target protein, but this step failed to efficiently separate the target protein from that of the E. coli host due to the close binding affinity to the resin, as most E. coli proteins are acidic in the pI range of 4–7, and many of them have a pI close to that of the target protein. Interestingly, however, size exclusion chromatography at room temperature using 1M (NH4)2SO4 buffer appeared to be a crucial step, providing very efficient separation of the target protein from that of the E. coli. Following further purification in a second round of hydrophobic interaction and size exclusion chromatography, both major untagged candidates were purified to 99% purity with high homogeneity, as evidenced by SDS-PAGE / native PAGE and SEC-MALS analysis. [Table 3]

[0328] Example 3: Animal Research Prevention of P. gingivalis bone loss in a mouse model of periodontitis 1. Experiment 1 Antigens tested: KDcAK1n 50 μg, KDcAK1n 0.5 μg, KDAK1 50 μg, KDAK1 0.5 μg. All antigens were absorbed onto alum in PBS (pH 7.4).

[0329] KDcAK1n: The inclusion bodies were batch purified with Ni-NTA resin, followed by dialyzing to 2M urea-PBS under non-reducing conditions.

[0330] KDAK1: Anion exchange chromatography with Ni-affinity (pre-packed column) and gradient elution was performed on the soluble fraction, followed by dialysis to PBS under non-reducing conditions.

[0331] P. gingivalis-induced alveolar bone loss in the mouse maxilla KDcAK1n protected against bone loss in animal models at both concentrations tested (not shown).

[0332] Antibody response Serum antibody subclass responses in immunized mice in a periodontitis model were investigated by ELISA. Antiserum was used to search for the absorbed antigen of thermally killed P. gingivalis strain W50. Antibody responses were expressed as the obtained ELISA titer minus 3 times the background level, with each titer representing the mean ± sd of 10 individual mice (data not shown). KDcAK1n induced the most potent total IgG and IgG1 responses against all P. gingivalis cells, followed by KDAK1. There were no significant differences in IgG and IgG1 antibody responses at 50 μg among the tested antigens.

[0333] 2. Experiment 2 Antigens tested: KDcAK1n 50 μg, KDcAK1n 0.5 μg, KDAK1 50 μg, KDAK1 0.5 μg. All antigens were absorbed onto alum in physiological saline (pH 7.4).

[0334] The antigen was purified using the same method as in Experiment 1.

[0335] P. gingivalis-induced alveolar bone loss in the mouse maxilla In this experiment, the same antigens as in Experiment 1 were investigated, but they were absorbed into alum using saline instead of PBS. The bone loss results were reflected in Experiment 1 (data not shown), showing that only KDcAK1n showed protection, and that using PBS or saline for alum preparation did not affect the experimental results.

[0336] Antibody response All antigens tested induced similar total IgG and IgG1 responses to whole-cell P. gingivalis. KDAK1 induced a stronger IgG2a response compared to KDcAK1n (did not show).

[0337] 3. Experiment 3 Antigens tested: All antigens except KDcAK1n were fractionated as described: KDcAK1n (dimer), KDcAK1n (multimer), KDcAK1n (conventional chimera), KDcAK1n-4S-AVQP.

[0338] KDcAK1n-4S-AVQP, KDcAK1n dimer and polymer: Urea extraction was performed from the inclusion bodies, followed by Ni-affinity column purification, and then gel filtration of two KDcAK1n samples under reducing conditions.

[0339] KDcAK1n: Purified in the same manner as in Experiment 1. Pre-screening of vaccine candidates containing mAbs against the P. gingivalis epitope In an attempt to develop a pre-screening assay to determine antigen suitability for animal models, antigens were screened by ELISA using mAbs against KAS2, ABM2, ABM3, and EP1. 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 KDcAKln-4S-AVQP bound to ABM2 mAb, but KDcAK1n dimers and KDcAK1n polymers did not—indicating that the epitopes recognized by this mAb are not accessible in these constructs.

[0340] P. gingivalis-induced alveolar bone loss in the mouse maxilla KDcAK1n-4S-AVQP provided moderate protection against P. gingivalis-induced bone loss, though not to the same extent as KDcAK1n (Figure 4).

[0341] Antibody response Serum antibody subclass responses in immunized mice in a periodontitis model were investigated by ELISA. Using antiserum, the thermally killed P. gingivalis strain W50 was searched for as an adsorbed antigen. All antigens tested produced varying degrees of IgG response. The KDcAK1n multimer produced a potent IgG1 isotype response.

[0342] Analysis of unprotected purified dimeric and multimeric antigens Analysis of single KDA dimer and polymer species purified by anion exchange and gel filtration, which did not provide protection in Experiment 3, using reduced and non-reduced native PAGE and SDS PAGE, revealed that in both cases the purified single species consisted of disulfide-crosslinked modified domains locked to stable species. This analysis explains why these molecular weight species are stable and can be purified by anion exchange and gel filtration chromatography.

[0343] 4. Experiment 4 Antigens tested: KKDA1nKK, KDAK1n, KDAK1nK-4S-AVQP.

[0344] With the exception of KDAK1nK-4S-AVQP, which does not use anion exchange chromatography, all antigens were purified from the soluble fraction under non-reducing conditions using Ni-affinity column chromatography, anion exchange chromatography, and gel filtration chromatography.

[0345] Alum absorption The ability of a 2% alhydrogel to absorb antigens was tested by incubation, gently mixing the antigens with alum at 4°C for 60 minutes. The alum was then pelletized, and the Bradford protein assay was performed before and after alum adsorption of the antigens. All antigens bound to Amphibacterium spp. at a rate of 91.7%–99.3%. SDS-PAGE gels were also run on samples before and after alum absorption, and the results were consistent with the protein determinations in the Bradford assay.

[0346] Pre-screening of vaccine candidates containing mAbs against the P. gingivalis epitope 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 and KDAK1n did not—indicating that the epitopes recognized by this mAb are not accessible in these constructs.

[0347] P. gingivalis-induced alveolar bone loss in the mouse maxilla KKDAK1nKK and KDAK1nK-4S-AVQP protected against P. gingivalis-induced bone loss (Figure 5).

[0348] Antibody response Serum antibody subclass responses in immunized mice in a periodontitis model were investigated by ELISA. Using antiserum, the heat-killed P. gingivalis strain W50 was searched for as an adsorbed antigen. All antigens tested produced varying degrees of IgG response. Protected KKDAK1nKK induced robust total IgG and IgG1 responses against heat-killed P. gingivalis. Interestingly, KDAK1nK-4S-AVQP, protected from bone loss, resulted in a lower antibody response to P. gingivalis in all cells than some of the lower antigens.

[0349] Pooled serum samples were used to explore for P. gingivalis domains adsorbed onto ELISA plates. Total IgG responses to ABM21 (multimer and dimer) and ABM213 (multimer and dimer) were generated to varying degrees for all antigens. The IgG response to DUF2436 was evident only for antigens containing the complete DUF domain (KKDAK1nKK, KDAK1n, and KDAK1nK-4S-AVQP). Pooled serum samples were also used to explore for P. gingivalis epitope peptides, and a clear trend of high KAS titers was observed in protected antiserum, but again, no clear pattern was observed between protected and unprotected.

[0350] 5. Experiment 5 Tested antigens All antigens were purified under non-reducing conditions using Ni-affinity chromatography followed by dialysis to physiological saline.

[0351] KDcAK1n inclusion bodies were purified using urea and affinity columns (IB, urea, AC), KDAK1 soluble fraction was purified using urea and affinity columns (S, urea, AC), KDAK1 inclusion bodies were purified using urea and affinity columns (IB, urea, AC), and KDAK1 inclusion bodies were purified using urea and batch purification methods (IB, urea, batch).

[0352] Pre-screening of vaccine candidates containing mAbs against the P. gingivalis epitope.

[0353] 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 the ABM2 mAb, indicating (not indicating) that the epitope recognized by this mAb is not accessible in these constructs.

[0354] 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), and KDAK1(IB, urea, batch) provided the best protection, demonstrating significant protection against bone loss (Figure 6).

[0355] Unfractionated affinity-purified antigen provided protection against bone loss, and particularly good protection was observed with affinity-purified soluble KDA. However, it is noteworthy that this protection was partially lost upon treatment with urea under oxidative conditions, which would promote disulfide crosslinking of the denatured D and A domains. Again, these results suggest that urea should be avoided, as disulfide crosslinking of the denatured domains may disrupt antigen-induced protection, and the preparation of soluble folded domains is preferable.

[0356] 6. Experiment 6 Tested antigens All antigens were purified under non-reducing conditions using Ni-affinity chromatography followed by dialysis to physiological saline. The antigens tested were: KDcAK1n-inclusion bodies purified using urea and affinity columns (IB, urea, AC), KDAK1nK, KDAK1n-4S-AVQP, and KKDAK1nKK.

[0357] P. gingivalis-induced alveolar bone loss in the mouse maxilla All antigens tested provided protection against P. gingivalis-induced bone loss (Figure 7).

[0358] 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 the components of a chimeric vaccine that are most effective in promoting ease of production (such as components that contribute to solubility, stability, and reduced tendency to polymerize), as well as in inducing an immune response to P. gingivalis and / or reducing P. gingivalis-induced alveolar bone loss.

[0359] While effective in preventing periodontal bone loss in animal periodontitis models, the conventional vaccine KDcAK1n is expressed as an inclusion body by E. coli and exhibits variable solubility and stability.

[0360] KDcAK1n was based on the fusion of the active site sequence (KAS or K) of Lys-specific gingipain Kgp with a treated adhesion factor fragment (A1) of the Kgp polyprotein. Structural analysis of the Kgp polyprotein domain revealed that the A1 adhesion factor fragment found on the cell surface contains three distinct structural domains: the DUF domain (D), the ABM domain (A), and the K1 domain (K1). Treatment of the Kgp polyprotein on the surface of P. gingivalis to release the proteinase catalytic domain and adhesion factor resulted in N-terminal cleavage of the DUF domain by 38 amino acid residues. Adding these extra N-terminal 38 residues to the construct produced a complete DUF domain instead of the cleaved domain (Dc), resulting in a highly soluble recombinant protein.

[0361] KDcAK1n contains four cysteine ​​residues: one in DUF, two in ABM, and one in K1. These cysteine ​​residues 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 indicate that the cysteine ​​residues are reduced on the cell surface and are not involved in disulfide crosslinking. Expression in E coli under more oxidative conditions results in disulfide crosslinking between the denatured domain and inclusion body formation. Disulfide crosslinking between the denatured domains of the chimeric strain can be observed on non-reducing native-PAGE, where it changes upon reduction.

[0362] The results presented herein demonstrate that the folded ABM domain of Kgp multimerizes via beta-chain exchange to form a homogeneous ladder independent of the reducing agent (Figure 3). This novel beta-chain exchange multimerization is a key mechanism used by P. gingivalis to form the surface layer of gingipain not only on cells but also on outer membrane vesicles released to the host during disease progression. This study characterizing the conventional chimeric KDcAK1n and domain clearly demonstrates the presence of two forms of multimers in the original chimera: a disulfide-bridged denatured domain-based multimer and a beta-chain exchange native ABM domain-based multimer. Attempts to isolate 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 result in protection from periodontal bone loss in animal models because the single species is a disulfide-locked denatured structure (Figure 4). This study suggests that soluble, defined, homogeneous recombinants with folded D and A domains that do not have disulfide crosslinks are superior to a mixture of modified and native forms produced by solubilization of chimeras from inclusion bodies in 8M urea, which yields low returns (Figure 7).

[0363] These results suggest that the presence of cysteine ​​residues in the antigen may pose a problem for the expression and purification of soluble, defined candidates for commercial development. Therefore, the possibility of mutating these cysteines to serine was explored. Mutation of four cysteine ​​residues to four serine residues did not halt the ABM(A) native domain beta-chain exchange multimerization, demonstrating that the ABM domain still folded properly in the "4C" to "4S" variant (Figure 3).

[0364] Furthermore, these results indicate that a key sequence mutation in the ABM domain (PVQN>AVQP), predicted to be the Pro-hinge elbow enabling β-chain inversion, eliminated β-chain exchange multimerization (Figure 3). Thus, the combination of mutating 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 produced good antibody titers against protective epitopes (KAS, DUF, ABM) predicted in animal periodontitis models (Figure 5).

[0365] Testing of antibody responses to various segments of the chimeric cells (K, D, A, and K1) and suspected key epitopes (KAS, ABM2, ABM3, EP1) using protected and unprotected serum did not provide any clear patterns. However, it was evident that these protected chimeric variants tended to show a strong (IgG / IgG1) response to whole P. gingivalis cells and known protective epitopes (KAS, DUF, ABM), suggesting that protection may not be related to any single epitope, but rather to a combination of epitopes required to ensure complete protection against periodontal bone loss. However, for the protective chimeric variants, there tended to be a prominent single epitope associated with the active site sequence KAS.

[0366] While there was no clear indication that any single epitope was more important so that the response to that epitope could be used as a substitute or biomarker for vaccine-induced protection, it was evident that protective chimeric variants tended to produce a good antibody response against protease active site sequences (KAS or K). Antibodies generated against active site sequences have previously been shown to neutralize the proteolytic activity (Kgp and RgpA / B) of gingipain, a major pathogenic factor of P. gingivalis. Therefore, in an approach to enhance antibody titers against the active site and thus enhance protection, the inventors added an extra copy of the active site sequence (K)KDAK1n, and this construct showed improved protection in an animal model of periodontitis (Figure 6). The soluble non-multimerized mutant KDAK1nK-4S-AVQP, having two copies of the KAS(K) motif, provided good protection from periodontal bone loss induced by P. gingivalis (Figure 6).

Claims

1. A chimeric or fusion protein for inducing an immune response against P. gingivalis, wherein the protein comprises a first polypeptide and a second polypeptide. A) The first polypeptide contains, or comprises, 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 contains or consists of the amino acid sequence of the adhesion factor domain of Arg- or Lys-gingipain of P. gingivalis. The second polypeptide comprises a sequence of one or more adhesion factor-binding motifs (ABMs), The second polypeptide comprises a portion or all of the sequence of the cleaved adhesion factor domain (CAD), and the second polypeptide comprises a) an amino acid sequence substantially corresponding to the full length of the DUF2436 domain of Arg- or Lys-gingipain, or a sequence that is at least 80% identical thereto, b) Compared to naturally occurring Arg- or Lys-gingipain sequences in the corresponding region, the sequence contains one or more cysteine ​​amino acid substitutions in the adhesion factor domain, c) i) Substitution of proline and / or asparagine residues in sequence PxxN at positions corresponding to or equivalent to residues 6-9 of sequence SEQ ID NO: 14 or 19 (ABM1), ii) Substitution of motif NxFA with SxYQ in sequences corresponding to or equivalent to residues 2-5 of sequence number 14 or 19 (ABM1), iii) The chimeric or fusion protein comprising one or more amino acid motif substitutions selected from: a second tyrosine residue at a position corresponding to or equivalent to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2), and a substitution of a tryptophan residue at a position corresponding to or equivalent to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue.

2. The chimeric or fusion protein according to claim 1, wherein one or more ABMs include the sequence described in SEQ ID NO: 15 or 20, SEQ ID NO: 14 or 19, and / or SEQ ID NO: 17 or 21, or a sequence that is at least 80% identical to those thereto.

3. The chimeric or fusion protein according to claim 1 or 2, wherein one or more ABMs include the sequence described in SEQ ID NO: 16 or SEQ ID NO: 18 or 22, or a sequence that is at least 80% identical to them.

4. The chimeric or fusion protein according to any one of claims 1 to 3, wherein the second polypeptide comprises a portion or all of the sequence of CAD having at least 80% the same amino acid sequence as described in SEQ ID NO: 12 or 13 or a sequence identical thereto thereto.

5. The chimeric or fusion protein according to any one of claims 1 to 4, wherein the amino acid sequence substantially corresponding to the full length of the DUF2436 domain of Arg- or Lys-gingipain is a sequence that 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.

6. The chimeric or fusion protein according to claim 5, wherein the amino acid sequence of the DUF2436 domain of Arg- or Lys-gingipain is the sequence described in SEQ ID NO: 23, or is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

7. The chimeric or fusion protein according to any one of claims 1 to 6, wherein the second polypeptide comprises the sequence described in SEQ ID NO: 33, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

8. The chimeric or fusion protein according to any one of the prior claims, wherein the second polypeptide does not involve substitution of a cysteine ​​residue in the DUF2436 domain.

9. The chimeric or fusion protein according to any one of claims 1 to 7, wherein the second polypeptide comprises the substitution of the cysteine ​​residue in the DUF2436 domain.

10. The chimeric or fusion protein according to claim 9, wherein the substitution of the cysteine ​​residue is a substitution of a serine or valine residue.

11. The chimeric or fusion protein according to claim 10, wherein the substitution of the cysteine ​​residue is a substitution of a serine residue.

12. The chimeric or fusion protein according to any one of claims 1 to 11, wherein the second polypeptide comprises substitution of one or more cysteine ​​residues in the ABM domain.

13. The chimeric or fusion protein according to claim 12, wherein the substitution of one or more cysteine ​​residues is a substitution of a serine residue or a valine residue.

14. The chimeric or fusion protein according to claim 13, wherein the substitution of one or more cysteine ​​residues is a substitution of serine residues.

15. The chimeric or fusion protein according to any one of claims 12 to 14, wherein the sequence of one or more ABM domains is described in SEQ ID NO: 18 or 22, and the one or more cysteine ​​residues correspond to the residues at positions 36 and 50 of SEQ ID NO: 18 or 22.

16. The chimeric or fusion protein according to any one of the prior claims, wherein the second polypeptide comprises substitutions of proline and / or sparagine residues in the sequence PxxN of the ABM1 of the P. gingivalis adhesion factor domain at positions corresponding to or equivalent to residues 6-9 of the sequence of SEQ ID NO: 14 or 19.

17. The chimeric or fusion protein according to claim 16, wherein the second polypeptide comprises the sequence AxxN, AxxA, or AxxP in ABM1 at a position corresponding to or equivalent to residues 6-9 of the sequence of SEQ ID NO: 14 or 19.

18. The chimeric or fusion protein according to any one of the prior claims, wherein the second polypeptide comprises or consists of the amino acid sequence described in any one of SEQ ID NOs: 33 to 48.

19. The chimeric or fusion protein according to claim 18, wherein the second polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 39 or 40.

20. The chimeric or fusion protein according to claim 17, wherein the second polypeptide comprises or consists of the amino acid sequence described in SEQ ID NO: 47 or 48.

21. The chimeric or fusion protein according to any one of claims 1 to 15, wherein the second polypeptide comprises the substitution of the motif NxFA in the ABM1 of the P. gingivalis adhesion factor domain at a position corresponding to or equivalent to residues 2 to 5 of SEQ ID NO: 14 or 19 with SxYQ.

22. The chimeric or fusion protein according to any one of claims 1 to 15, wherein the second polypeptide comprises substitution of the second tyrosine residue of ABM2 at a position corresponding to or equivalent to the residue at position 5 of SEQ ID NO: 15 or 20, and the tryptophan residue of ABM1 at a position corresponding to or equivalent to the residue at position 23 of SEQ ID NO: 14 or 19 with an alanine residue.

23. The chimeric or fusion protein according to any one of claims 1 to 22, wherein the first polypeptide is linked to the second polypeptide.

24. The chimeric or fusion protein according to any one of claims 1 to 23, wherein the first polypeptide is located at the N-terminus of the second polypeptide.

25. The chimeric or fusion protein according to any one of claims 1 to 24, wherein the first polypeptide is directly linked to the second polypeptide.

26. The chimeric or fusion protein according to any one of claims 1 to 24, wherein the first polypeptide is linked to the second polypeptide via a linker.

27. The chimeric or fusion protein according to claim 26, wherein the linker is a peptide linker.

28. The chimeric or fusion protein according to claim 27, wherein the peptide linker comprises at least 2 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, and at least 20 amino acids, preferably the linker comprises 50 amino acids or less.

29. The chimeric or fusion protein according to claim 27 or 28, wherein 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.

30. The chimeric or fusion protein according to any one of claims 1 to 29, wherein the first polypeptide contains, or comprises, an amino acid sequence described in any one of Sequence IDs 1 to 11, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

31. The chimeric or fusion protein according to claim 30, wherein the first polypeptide contains, or comprises, an amino acid sequence described in SEQ ID NO: 1 or 2, or a sequence that is identical to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of those sequences.

32. The chimeric or fusion protein according to any one of claims 1 to 31, wherein the chimeric or fusion protein comprises 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, or one or more further polypeptides comprising the same.

33. The chimeric or fusion protein according to claim 32, wherein one or more further polypeptides comprising or consisting of the active site of Arg- or Lys-gingipain of P. gingivalis are located at the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, the C-terminus of the second polypeptide, and the N-terminus of the second polypeptide.

34. The chimeric or fusion protein according to claim 33, wherein one or more further polypeptides are directly linked to the first or second polypeptide of the chimeric or fusion protein.

35. The chimeric or fusion protein according to claim 34, wherein one or more further polypeptides are linked to the first or second polypeptide by a peptide linker.

36. The chimeric or fusion protein according to claim 35, wherein the peptide linker comprises at least 2 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, and at least 20 amino acids, preferably the linker comprises 50 amino acids or less.

37. The chimeric or fusion protein according to claim 35 or 36, wherein 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.

38. The chimeric or fusion protein according to any one of claims 32 to 37, wherein the one or more further polypeptides preferably comprises, or consist of, amino acid sequences selected from the group of sequences identical to SEQ ID NOs. 1 to 11, 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 thereto.

39. The chimeric or fusion protein according to claim 37, wherein the first polypeptide and the one or more further polypeptides are identical to each other, or are identical by at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

40. The chimeric or fusion protein according to claim 38, wherein the first polypeptide and the one or more further polypeptides contain the amino acid sequence of the active site of a heterologous gingipain of P. gingivalis.

41. The chimeric or fusion protein according to claim 38, wherein the first polypeptide and the one or more further polypeptides include the amino acid sequence of the active site of Lys-gingipain.

42. The chimeric or fusion protein according to claim 38, wherein the first polypeptide comprises the amino acid sequence of the active site of Lys-gingipain, and one or more further polypeptides comprise the amino acid sequence of the active site of Arg-gingipain.

43. The chimeric or fusion protein according to any one of claims 1 to 42, wherein the chimeric or fusion protein comprises, or consists of, the amino acid sequence described in either one of SEQ ID NOs: 52 or 53, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the sequence thereto.

44. The chimeric or fusion protein according to any one of claims 1 to 43, wherein the protein comprises additional amino acid residues to promote expression in a recombinant expression system and / or to promote purification of the protein.

45. The chimeric or fusion protein according to claim 44, wherein the additional amino acids optionally include an N-terminal methionine and / or alanine, and the N-terminal region comprises 1, 2, 3, 4, or 5 amino acids.

46. The chimeric or fusion protein according to claim 44 or 45, wherein the additional amino acids include 1, 2, 3, 4, or 5 amino acids in the C-terminal region.

47. A nucleic acid encoding a chimeric or fusion protein according to any one of claims 1 to 46.

48. A vector or construct comprising the nucleic acid described in claim 47.

49. A host cell comprising the nucleic acid described in claim 47, or the vector or construct described in claim 48.

50. A composition comprising a chimeric or fusion protein according to any one of claims 1 to 46.

51. The composition according to claim 50, wherein the composition is a vaccine composition and comprises one or more adjuvants for enhancing the immune response to the chimeric or fusion protein.

52. A vaccine or immunostimulatory composition comprising a chimeric or fusion protein according to any one of claims 1 to 46, and optionally an adjuvant for enhancing the immune response to the chimeric or fusion protein.

53. A method for inducing an immune response to P. gingivalis in a subject, the method comprising administering to a subject requiring such response a chimeric or fusion protein according to any one of claims 1 to 46, a composition according to claim 50 or 51, or a vaccine or immunostimulatory composition according to claim 52.

54. A method for inducing a humoral immune response to P. gingivalis in a subject, wherein the method comprises administering to the subject a chimeric or fusion protein according to any one of claims 1 to 46, a composition according to claim 50 or 51, or a vaccine or immunostimulatory composition according to claim 52.

55. The method according to claim 54, wherein the induced immune response includes switching from a Th1 immune response to a Th2 immune response.

56. A method for immunizing a subject against P. gingivalis infection, the method comprising administering to the subject a chimeric or fusion protein according to any one of claims 1 to 46, a composition according to claim 50 or 51, or a vaccine or immunostimulatory composition according to claim 52.

57. A method for treating P. gingivalis infection in a subject, the method comprising administering to a subject in need thereof a chimeric or fusion protein according to any one of claims 1 to 46, a composition according to claim 50 or 51, or a vaccine or immunostimulatory composition according to claim 52.

58. A method for reducing or minimizing the severity of symptoms associated with infection by P. gingivalis, comprising administering to an individual in need of such treatment a chimeric or fusion protein according to any one of claims 1 to 46, a composition according to claim 50 or 51, or a vaccine or immunostimulatory composition according to claim 52. The method wherein the symptoms are selected from the group consisting of swollen or puffy gums, bleeding gums, receding gums, periodontal pockets around the teeth, loss of supporting tissues of the teeth (periodontal ligaments, cementum, and / or alveolar bone), pus between the gums and teeth, and gingivitis.

59. The method according to claim 57 or 58, further comprising administering one or more of an antimicrobial compound and an anti-inflammatory agent.

60. ● To induce an immune response to P. gingivalis in the subjects, ● To immunize the target against P. gingivalis infection, ● Treat P. gingivalis infection in the subjects, or ● Use of a chimeric or fusion protein according to any one of claims 1 to 46 in the manufacture of a pharmaceutical product for minimizing or reducing the severity of one or more symptoms of P. gingivalis infection.

61. ● To induce an immune response to P. gingivalis in the subjects, ● To immunize the target against P. gingivalis infection, ● Treat P. gingivalis infection in the subjects, or ● A chimeric or fusion protein according to any one of claims 1 to 46, a composition according to claim 50 or 51, or a vaccine or immunostimulatory composition according to claim 52, for use in minimizing or reducing the severity of one or more symptoms of P. gingivalis infection.

62. A method for obtaining antibodies targeted to P. gingivalis, the method comprising administering to a non-human animal a chimeric or fusion protein according to any one of claims 1 to 46, a composition according to claim 50 or 51, or a vaccine or immunostimulatory composition according to claim 52, thereby generating antibodies targeted to P. gingivalis in the animal.

63. The method according to claim 62, further comprising isolating the antibody from the animal or the egg of the animal.

64. An antibody preparation comprising an antibody directed towards P. gingivalis, wherein the antibody preparation is obtained by administering to a non-human animal a chimeric or fusion protein according to any one of claims 1 to 46, a composition according to claim 50 or 51, or a vaccine or immunostimulatory composition according to claim 52, thereby generating an antibody directed towards P. gingivalis in the animal, and isolating the antibody from the animal or its eggs.

65. A kit comprising a composition comprising a chimeric or fusion protein according to any one of claims 1 to 46, a composition according to claim 50 or 51, or a vaccine or immunostimulatory composition according to claim 3523, wherein the kit optionally comprises one or more cytokines and / or adjuvants in a sealed container, preferably the kit comprises a label or accompanying document indicating that the composition is used to immunize an individual, and optionally the label or accompanying document comprises instructions for use.

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