Methods of treatment

Abstract

The present invention relates to a method of treating or preventing neuropathology induced or associated with Porphyromonas gingivalis using a chimeric or fusion protein comprising a first polypeptide linked to a second polypeptide, wherein: A) the first polypeptide comprises or consists of the following: an amino acid sequence of an active site of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence at least 80% identical thereto; and B) the second polypeptide comprises or consists of the amino acid sequence of the adhesin domain of the Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis; wherein the second polypeptide comprises one or more sequences of an adhesin binding motif (ABM), and wherein the second polypeptide: a) does not comprise: a sequence of a cleaved adhesin domain (CAD) or a portion thereof; and / or b) comprising an amino acid sequence substantially corresponding to the full length of the DUF2436 domain of the Arg gingival protease or Lys gingival protease; and / or c) comprises one or more cysteine amino acid substitutions in the adhesin domain as compared to the naturally occurring Arg gingival protease or Lys gingival protease sequence in the corresponding region, and / or d) comprises one or more additional amino acid motif substitutions as defined herein.

Description

Technical Field

[0001] This invention relates to the prevention and / or treatment of Porphyromonas gingivalis (Porphyromonas gingivalis). P. gingivalis Methods that induce or are associated with neuropathology (such as dementia and related neurodegenerative symptoms).

[0002] Related applications

[0003] This application claims priority to Australian Provisional Application AU 2023902382, the entire contents of which are hereby incorporated by reference. Background Technology

[0004] If plaque accumulates around the teeth at the gingival margin, it causes inflammation of the gums (gingivitis). Chronic gingivitis can allow the periodontal pathogen Porphyromonas gingivalis to thrive at the base of the periodontal pockets, leading to chronic infection and the development of severe disease. This severe form of periodontitis is called periodontitis and can cause tooth loss during the immune system's attempts to eliminate the infection.

[0005] One-third of adults have moderate to severe periodontitis. Epidemiological studies have shown that periodontitis is associated with an increased risk of inflammatory diseases, including cardiovascular disease, certain cancers, premature birth, rheumatoid arthritis, and dementia.

[0006] Recent studies have linked chronic infections caused by *Porphyromonas gingivalis* to dementia and rheumatoid arthritis. For example, in one study, *Porphyromonas gingivalis* was found in 96% of brain samples from Alzheimer's disease (AD). Another study showed that chronic oral infections in mice with *Porphyromonas gingivalis* caused AD-associated brain plaques in humans, and that *Porphyromonas gingivalis* proteases can cleave amyloid precursors and tau protein to form AD-associated plaques and tangles.

[0007] It has been reported that many virulence factors contribute to the pathogenicity of Porphyromonas gingivalis, including: LPS, fimbriae, hemagglutinin, hemolysin, and extracellular hydrolases (especially Arg-X and Lys-X specific proteases), also known as "Porphyromonas gingivalis gingival proteases".

[0008] The importance of this public health issue necessitates a vaccine that provides a strong protective response against Porphyromonas gingivalis infection, as well as a method for delivering said vaccine.

[0009] There are currently no commercially approved vaccines for the prevention or reduction of the incidence and / or severity of Porphyromonas gingivalis infection, or for the treatment of Porphyromonas gingivalis infection and disease in subjects.

[0010] Therefore, there is a need for alternative and / or improved methods for designing and preparing Porphyromonas gingivalis vaccines, as well as alternative and / or improved vaccines produced by Porphyromonas gingivalis.

[0011] Reagents and methods are needed for the prevention and / or treatment of neuropathologies induced or associated with Porphyromonas gingivalis, such as neuropathologies that produce or lead to dementia.

[0012] References to any prior art in this specification do not imply an acknowledgment or implication that such prior art constitutes part of common common knowledge in any jurisdiction, nor do they imply that such prior art can be reasonably understood, regarded as related to, and / or combined with other prior art by a person skilled in the art. Summary of the Invention

[0013] In a first aspect, the present invention provides a method for preventing or treating neuropathology in a subject, wherein the neuropathology is related to *Porphyromonas gingivalis* (…). P. gingivalis The method comprises administering to the subject a therapeutically effective amount of a chimeric or fusion protein, the chimeric or fusion protein comprising a first polypeptide and a second polypeptide, wherein: A) The first polypeptide comprises or consists of the amino acid sequence of the active site of Arg gingivase or Lys gingivase from Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto; and B) The second polypeptide contains or is composed of the amino acid sequence of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis. The second polypeptide comprises a sequence of one or more adhesin-binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain and the cleaved adhesin domain (CAD) of Porphyromonas gingivalis gingivalis protease. And the second polypeptide described therein: a) Does not contain: sequences of cleaved adhesin domains (CAD) or portions thereof, preferably sequences of CAD having the sequence shown in SEQ ID NO: 12 or 13, or sequences that are at least 80% identical thereto; and / or b) Containing an amino acid sequence substantially corresponding in full length to the DUF2436 domain of Arg gingivase or Lys gingivase, preferably the amino acid sequence shown in SEQ ID NO: 23, or at least 80% identical thereto; and / or c) Contains one or more amino acid substitutions selected from the following: Compared to the naturally occurring Arg gingival protease or Lys gingival protease sequence in the corresponding region, one or more cysteine ​​amino acids in the adhesin domain are substituted; and / or One or more amino acid motifs selected from the following: i) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1) or located at positions equivalent to said residues; ii) Substitution of motif NxFA to SxYQ in sequences corresponding to residues 2 to 5 of the sequence corresponding to or located at positions equivalent to said residues; iii) The second tyrosine residue corresponding to or located at a position equivalent to the residue in SEQ ID NO: 15 or 20 (ABM2) and the tryptophan residue corresponding to or located at a position equivalent to the residue in SEQ ID NO: 14 or 19 (ABM1) are replaced by an alanine residue.

[0014] Optionally, the chimeric or fusion protein comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0015] This can prevent or treat Porphyromonas gingivalis-induced or related neuropathology in the subject.

[0016] The neuropathology may include pathologies associated with, caused by, or aggravated by the presence of *Porphyromonas gingivalis* gingival protease protein in neuronal (e.g., brain) tissue. *Porphyromonas gingivalis* gingival protease may include Lys gingival protease or Arg gingival protease, such as Kgp or Rgp, as further described herein.

[0017] Therefore, in a second aspect, the present invention also provides a method for preventing or reducing the deposition of *Porphyromonas gingivalis* gingival protease in the neuronal tissue of a subject, the method comprising administering to the subject a therapeutically effective amount of a chimeric or fusion protein, the chimeric or fusion protein comprising a first polypeptide and a second polypeptide, wherein: A) The first polypeptide comprises or consists of the amino acid sequence of the active site of Arg gingivase or Lys gingivase from Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto; and B) The second polypeptide contains or is composed of the amino acid sequence of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis. The second polypeptide comprises a sequence of one or more adhesin-binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain and the cleaved adhesin domain (CAD) of Porphyromonas gingivalis gingivalis protease. And the second polypeptide described therein: a) Does not contain: sequences of cleaved adhesin domains (CAD) or portions thereof, preferably sequences of CAD having the sequence shown in SEQ ID NO: 12 or 13, or sequences that are at least 80% identical thereto; and / or b) Containing an amino acid sequence substantially corresponding in full length to the DUF2436 domain of Arg gingivase or Lys gingivase, preferably the amino acid sequence shown in SEQ ID NO: 23, or at least 80% identical thereto; and / or c) Contains one or more amino acid substitutions selected from the following: Compared to the naturally occurring Arg gingival protease or Lys gingival protease sequence in the corresponding region, one or more cysteine ​​amino acids in the adhesin domain are substituted; and / or One or more amino acid motifs selected from the following: i) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1) or located at positions equivalent to said residues; ii) Substitution of motif NxFA to SxYQ in sequences corresponding to residues 2 to 5 of the sequence corresponding to or located at positions equivalent to said residues; iii) The second tyrosine residue corresponding to or located at a position equivalent to the residue in SEQ ID NO: 15 or 20 (ABM2) and the tryptophan residue corresponding to or located at a position equivalent to the residue in SEQ ID NO: 14 or 19 (ABM1) are replaced by an alanine residue.

[0018] Optionally, the chimeric or fusion protein comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0019] This prevents or reduces the deposition of Porphyromonas gingivalis gingivalis protease in the neuronal tissue of the subjects.

[0020] Subjects whose levels of Porphyromonas gingivalis gingivalis protease need to be prevented or need to be reduced may be subjects who are considered to be at risk of developing dementia or other neurodegenerative conditions, or subjects who have dementia or other neurodegenerative conditions.

[0021] Thirdly, the present invention also provides a method for delaying the onset of Porphyromonas gingivalis-induced or related neuropathology in a subject, the method comprising administering to the subject a therapeutically effective amount of a chimeric or fusion protein, the chimeric or fusion protein comprising a first polypeptide and a second polypeptide, wherein: A) The first polypeptide comprises or consists of the amino acid sequence of the active site of Arg gingivase or Lys gingivase from Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto; and B) The second polypeptide contains or is composed of the amino acid sequence of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis. The second polypeptide comprises a sequence of one or more adhesin-binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain and the cleaved adhesin domain (CAD) of Porphyromonas gingivalis gingivalis protease. And the second polypeptide described therein: a) Does not contain: sequences of cleaved adhesin domains (CAD) or portions thereof, preferably sequences of CAD having the sequence shown in SEQ ID NO: 12 or 13, or sequences that are at least 80% identical thereto; and / or b) Containing an amino acid sequence substantially corresponding in full length to the DUF2436 domain of Arg gingivase or Lys gingivase, preferably the amino acid sequence shown in SEQ ID NO: 23, or at least 80% identical thereto; and / or c) Contains one or more amino acid substitutions selected from the following: Compared to the naturally occurring Arg gingival protease or Lys gingival protease sequence in the corresponding region, one or more cysteine ​​amino acids in the adhesin domain are substituted; and / or One or more amino acid motifs selected from the following: i) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1) or located at positions equivalent to said residues; ii) Substitution of motif NxFA to SxYQ in sequences corresponding to residues 2 to 5 of the sequence corresponding to or located at positions equivalent to said residues; iii) The second tyrosine residue corresponding to or located at a position equivalent to the residue in SEQ ID NO: 15 or 20 (ABM2) and the tryptophan residue corresponding to or located at a position equivalent to the residue in SEQ ID NO: 14 or 19 (ABM1) are replaced by an alanine residue.

[0022] Optionally, the chimeric or fusion protein comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0023] This delays the onset of Porphyromonas gingivalis-induced or associated neuropathology in the subjects.

[0024] Neuropathology induced or associated with *Porphyromonas gingivalis* can include cognitive decline, cognitive impairment, or a decrease or alteration in motor function, or be related to these conditions. Neuropathology induced or associated with *Porphyromonas gingivalis* can include neurodegenerative diseases or symptoms, or pathologies that cause physical or chemical changes in neuronal tissues.

[0025] In any aspect of the invention, the neurodegenerative condition may be characterized by the presence of abnormal protein deposits in the brain, including amyloidosis, synucleinosis, or tau disease.

[0026] In any embodiment, neurodegenerative symptoms or conditions associated with, caused by, or induced by *Porphyromonas gingivalis* infection may include Alzheimer's disease (AD), Lewy-bodies disease (DLB), Huntington's disease, Creutzfeldt-Jakob disease (CJD), Gaucher disease type 3, or Parkinson's disease. Neurodegenerative disorders may be dementia such as mild cognitive and / or memory impairment, vascular dementia, frontotemporal dementia, or other forms of dementia not typically associated with the deposition of abnormal protein deposits.

[0027] Preferably, the neurodegenerative symptom or disease is Alzheimer's disease.

[0028] Fourthly, the present invention also provides a method for preventing or slowing the deposition of abnormal proteins in the neuronal tissue of a subject, preferably wherein the abnormal protein deposition is associated with or caused by *Porphyromonas gingivalis* infection, the method comprising administering to the subject a therapeutically effective amount of a chimeric or fusion protein, the chimeric or fusion protein comprising a first polypeptide and a second polypeptide, wherein: A) The first polypeptide comprises or consists of the amino acid sequence of the active site of Arg gingivase or Lys gingivase from Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto; and B) The second polypeptide contains or is composed of the amino acid sequence of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis. The second polypeptide comprises a sequence of one or more adhesin-binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain and the cleaved adhesin domain (CAD) of Porphyromonas gingivalis gingivalis protease. And the second polypeptide described therein: a) Does not contain: sequences of cleaved adhesin domains (CAD) or portions thereof, preferably sequences of CAD having the sequence shown in SEQ ID NO: 12 or 13, or sequences that are at least 80% identical thereto; and / or b) Containing an amino acid sequence substantially corresponding in full length to the DUF2436 domain of Arg gingivase or Lys gingivase, preferably the amino acid sequence shown in SEQ ID NO: 23, or at least 80% identical thereto; and / or c) Contains one or more amino acid substitutions selected from the following: Compared to the naturally occurring Arg gingival protease or Lys gingival protease sequence in the corresponding region, one or more cysteine ​​amino acids in the adhesin domain are substituted; and / or One or more amino acid motifs selected from the following: i) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1) or located at positions equivalent to said residues; ii) Substitution of motif NxFA to SxYQ in sequences corresponding to residues 2 to 5 of the sequence corresponding to or located at positions equivalent to said residues; iii) The second tyrosine residue corresponding to or located at a position equivalent to the residue in SEQ ID NO: 15 or 20 (ABM2) and the tryptophan residue corresponding to or located at a position equivalent to the residue in SEQ ID NO: 14 or 19 (ABM1) are replaced by an alanine residue.

[0029] Optionally, the chimeric or fusion protein comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0030] This prevents or slows down the deposition of abnormal proteins in the neuronal tissue of the subject.

[0031] The abnormal protein deposition can be an abnormal deposition of β-amyloid protein, phosphorylated tau protein, or α-synuclein.

[0032] Therefore, in a preferred embodiment, the method of the present invention also relates to a method for preventing or reducing the progression of amyloidosis, synucleinosis, or tau disease in a subject.

[0033] In addition, the method of the present invention also relates to a method for preventing or reducing the progression of neurodegenerative conditions selected from: Alzheimer's disease (AD), Lewy body disease (Lewy body dementia (DLB)), Huntington's disease, Creutzfeldt-Jakob disease (CJD), type 3 Gaucher disease, or Parkinson's disease, dementia such as mild cognitive and / or memory impairment, vascular dementia, frontotemporal dementia, or other forms of dementia that are generally not associated with the deposition of abnormal protein deposits.

[0034] Fifthly, the present invention also provides a method for reducing neuroinflammation, preferably neuroinflammation associated with or caused by *Porphyromonas gingivalis* infection, the method comprising administering to a subject a therapeutically effective amount of a chimeric or fusion protein, the chimeric or fusion protein comprising a first polypeptide and a second polypeptide, wherein: A) The first polypeptide comprises or consists of the amino acid sequence of the active site of Arg gingivase or Lys gingivase from Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto; and B) The second polypeptide contains or is composed of the amino acid sequence of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis. The second polypeptide comprises a sequence of one or more adhesin-binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain and the cleaved adhesin domain (CAD) of Porphyromonas gingivalis gingivalis protease. And the second polypeptide described therein: a) Does not contain: sequences of cleaved adhesin domains (CAD) or portions thereof, preferably sequences of CAD having the sequence shown in SEQ ID NO: 12 or 13, or sequences that are at least 80% identical thereto; and / or b) Containing an amino acid sequence substantially corresponding in full length to the DUF2436 domain of Arg gingivase or Lys gingivase, preferably the amino acid sequence shown in SEQ ID NO: 23, or at least 80% identical thereto; and / or c) Contains one or more amino acid substitutions selected from the following: Compared to the naturally occurring Arg gingival protease or Lys gingival protease sequence in the corresponding region, one or more cysteine ​​amino acids in the adhesin domain are substituted; and / or One or more amino acid motifs selected from the following: i) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1) or located at positions equivalent to said residues; ii) Substitution of motif NxFA to SxYQ in sequences corresponding to residues 2 to 5 of the sequence corresponding to or located at positions equivalent to said residues; iii) The second tyrosine residue corresponding to or located at a position equivalent to the residue in SEQ ID NO: 15 or 20 (ABM2) and the tryptophan residue corresponding to or located at a position equivalent to the residue in SEQ ID NO: 14 or 19 (ABM1) are replaced by an alanine residue.

[0035] Optionally, the chimeric or fusion protein comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease from Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto. This reduces neuroinflammation in the subjects.

[0036] In any embodiment of the fifth aspect of the invention, reducing neuroinflammation may comprise reducing the level or amount of any inflammatory markers in the subject's brain. Optionally, the reduction may comprise reducing the level of one or more of the following: IL-6, IL-1β, C-reactive protein (CRP), TNF-α and its receptors TNFR-I and TNFR-II, VCAM-I, d-dimer and sirtuin signaling, YKL-40, IL-7, IL-8, IL-15, IL-12, IP-10, ICAM-1, Flt-1, monocyte chemoattractant protein 1, nitric oxide (NO), COX-2, GM-CSF, etc.

[0037] In any embodiment of any aspect of the invention, the chimeric or fusion protein induces an immune response against *Porphyromonas gingivalis* or against *Porphyromonas gingivalis* gingival protease in the subject. Preferably, the induced immune response comprises a Th1-to-Th2 immune response.

[0038] It should be understood that, in any embodiment, the immune response induced by the application of the chimeric or fusion protein described herein is preferably antigen-specific. In any embodiment, the compositions, chimeric proteins, and methods of the present invention can be used to enhance the immune response (e.g., protective immune response) of a subject against *Porphyromonas gingivalis*.

[0039] In any aspect of the invention, the method of the invention may further comprise the administration of one or more of the following: an antimicrobial compound, an anti-inflammatory agent, or an additional immunogen to induce an immune response against Porphyromonas gingivalis or Porphyromonas gingivalis gingival protease.

[0040] The present invention also provides the use of a chimeric or fusion protein as defined herein for the preparation of a medicament for: ● Prevention or treatment of neuropathology in subjects, preferably wherein the neuropathology is related to or caused by Porphyromonas gingivalis infection; ● Prevents the accumulation of Porphyromonas gingivalis gingivalase in the neuronal tissue of subjects; ● Reduced the level of Porphyromonas gingivalis gingivalase in the neuronal tissue of the subjects; ● Delays the onset of neuropathology associated with Porphyromonas gingivalis; ● Prevents or slows down the deposition of abnormal proteins in the neuronal tissue of the subjects; ● To prevent or reduce the progression of amyloidosis, synucleinosis, or tau disease in subjects; ● To prevent the progression of or slow the rate of a neurodegenerative disease, optionally selected from Alzheimer's disease (AD), Lewy body disease (Lewy body dementia (DLB)), Huntington's disease, Creutzfeldt-Jakob disease (CJD), Gaucher disease type 3, or Parkinson's disease, dementia such as mild cognitive and / or memory impairment, vascular dementia, frontotemporal dementia, or other forms of dementia not typically associated with the deposition of abnormal protein deposits; or ● Reduce neuroinflammation in subjects, preferably neuroinflammation associated with or caused by Porphyromonas gingivalis infection; Optionally, neuropathologies induced or associated with Porphyromonas gingivalis include cognitive decline, cognitive impairment, or pathologies that cause physical or chemical changes in neuronal tissues; or neurodegenerative disorders as further defined herein.

[0041] This invention also provides chimeric or fusion proteins as defined herein, which are used for: ● Prevention or treatment of neuropathology in subjects, preferably wherein the neuropathology is related to or caused by Porphyromonas gingivalis infection; ● Prevents the accumulation of Porphyromonas gingivalis gingivalase in the neuronal tissue of subjects; ● Reduced the level of Porphyromonas gingivalis gingivalase in the neuronal tissue of the subjects; ● Delays the onset of neuropathology associated with Porphyromonas gingivalis; ● Prevents or slows down the deposition of abnormal proteins in the neuronal tissue of the subjects; ● To prevent or reduce the progression of amyloidosis, synucleinosis, or tau disease in subjects; ● To prevent the progression of or slow the rate of a neurodegenerative disease, optionally selected from Alzheimer's disease (AD), Lewy body disease (Lewy body dementia (DLB)), Huntington's disease, Creutzfeldt-Jakob disease (CJD), Gaucher disease type 3, or Parkinson's disease, dementia such as mild cognitive and / or memory impairment, vascular dementia, frontotemporal dementia, or other forms of dementia not typically associated with the deposition of abnormal protein deposits; or ● Reduce neuroinflammation in subjects, preferably neuroinflammation associated with or caused by Porphyromonas gingivalis infection; Optionally, neuropathologies induced or associated with Porphyromonas gingivalis include cognitive decline, cognitive impairment, or pathologies that cause physical or chemical changes in neuronal tissues; or neurodegenerative disorders as further defined herein.

[0042] In any method, use, or protein used according to the present invention, the subject may be any subject with or at risk of having *Porphyromonas gingivalis* infection. The subject may be a human. The subject may be a veterinary subject, such as a companion animal with or at risk of having *Porphyromonas gingivalis* infection.

[0043] In any embodiment of any aspect of the invention, the chimeric or fusion protein comprises a first polypeptide and a second polypeptide, wherein: A) The first polypeptide comprises or consists of the amino acid sequence of the active site of Arg gingivase or Lys gingivase from Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto; and B) The second polypeptide contains or is composed of the amino acid sequence of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis. The second polypeptide comprises a sequence of one or more adhesin-binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain and the cleaved adhesin domain (CAD) of Porphyromonas gingivalis gingivalis protease. Preferably, the one or more ABMs comprise sequences as shown in SEQ ID NO: 15 or 20 (ABM2) and / or SEQ ID NO: 17 or 21 (ABM3), or sequences that are at least 80% identical thereto. More preferably, the one or more ABMs comprise sequences as shown 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 sequences that are at least 80% identical thereto. Most preferably, the one or more ABMs comprise the sequence shown in any one of SEQ ID NO: 16 or SEQ ID NO: 18 or 22 or 27, or a sequence that is at least 80% identical thereto; And the second polypeptide described therein: a) A sequence that does not contain a cleaved adhesin domain (CAD), wherein the CAD preferably has the sequence shown in SEQ ID NO:12 or 13 or is at least 80% identical thereto; Optionally, the chimeric or fusion protein comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0044] In any further embodiment of any aspect of the invention, the chimeric or fusion protein comprises a first polypeptide linked to a second polypeptide, wherein: A) The first polypeptide comprises or consists of the amino acid sequence of the active site of Arg gingivase or Lys gingivase from Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto; and B) The second polypeptide contains or is composed of the amino acid sequence of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis. The second polypeptide comprises a sequence of one or more adhesin-binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain and the cleaved adhesin domain (CAD) of Porphyromonas gingivalis gingivalis protease. Preferably, the one or more ABMs comprise sequences as shown in SEQ ID NO: 15 or 20 (ABM2) and / or SEQ ID NO: 17 or 21 (ABM3), or sequences that are at least 80% identical thereto. More preferably, the one or more ABMs comprise sequences as shown in SEQ ID NO: 15 or 20 (ABM2), SEQ ID NO: 14 or 19 (ABM1), and / or SEQ ID NO: 17 or 21 (ABM2), or sequences that are at least 80% identical thereto. Most preferably, the one or more ABMs comprise the sequence shown in any one of SEQ ID NO: 16 or SEQ ID NO: 18 or 22, or a sequence that is at least 80% identical thereto; And the second polypeptide described therein: a) Does not contain: sequences of cleaved adhesin domains (CAD) or portions thereof, wherein the CAD preferably has the sequence shown in SEQ ID NO: 12 or 13 or a sequence that is at least 80% identical thereto; and b) Contains an amino acid sequence that substantially corresponds to the full length of the DUF2436 domain of Arg gingivase or Lys gingivase, preferably the amino acid sequence shown in SEQ ID NO: 23, or at least 80% identical to it. Optionally, the chimeric or fusion protein comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0045] As used herein, an amino acid sequence substantially corresponding to or at least 80% identical to the full length of the DUF2436 domain refers to a sequence comprising 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 gingivase or Lys gingivase.

[0046] The amino acid sequence of the DUF2436 domain of Arg gingivase or Lys gingivase is preferably the sequence shown 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 to it.

[0047] Most preferably, the second polypeptide comprises or consists of the sequence shown in SEQ ID NO: 34 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 alternative embodiments, the second polypeptide comprises or consists of the sequence shown in SEQ ID NO: 76 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.

[0048] In a particularly preferred embodiment of this invention, the chimeric or fusion protein comprises or is composed of the following: an amino acid sequence as shown in any of SEQ ID NO: 28, 50, 51, 52, 53 or 54, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to it.

[0049] In any further embodiment of any aspect of the invention, the chimeric or fusion protein comprises a first polypeptide and a second polypeptide, wherein: A) The first polypeptide comprises or consists of the amino acid sequence of the active site of Arg gingivase or Lys gingivase from Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto; and B) The second polypeptide contains or is composed of the amino acid sequence of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis. The second polypeptide comprises sequences of one or more adhesin-binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain and the cleaved adhesin domain (CAD) of *Porphyromonas gingivalis* gingival protease. Preferably, the one or more ABMs comprise sequences as shown in SEQ ID NO: 15 or 20 (ABM2) and / or SEQ ID NO: 17 or 21 (ABM3), or sequences that are at least 80% identical thereto. More preferably, the one or more ABMs comprise sequences as shown 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 sequences that are at least 80% identical thereto. Most preferably, the one or more ABMs comprise the sequence shown in any one of SEQ ID NO: 16, SEQ ID NO: 18, 22, or SEQ ID NO: 27, or a sequence that is at least 80% identical thereto; And said second polypeptide contains one or more amino acid substitutions selected from the following: a) One or more cysteine ​​amino acids in the adhesin domain are substituted compared to the naturally occurring Arg gingivase or Lys gingivase sequence in the corresponding region; and / or b) One or more amino acid motifs selected from the following: i) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1) or located at positions equivalent to said residues; ii) Substitution of motif NxFA to SxYQ in sequences corresponding to residues 2 to 5 of the sequence corresponding to or located at positions equivalent to said residues; iii) The substitution of a second tyrosine residue corresponding to or at an equivalent position to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue corresponding to or at an equivalent position to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue. Optionally, the chimeric or fusion protein comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

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

[0051] In a preferred embodiment of this invention, 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.

[0052] In a particularly preferred embodiment of this invention, the adhesin domain comprises the DUF2436 domain or a portion thereof, and the cysteine ​​residues in the DUF2436 domain are substituted with serine or valine, preferably serine. In other embodiments, the adhesin domain comprises the DUF2436 domain, and the cysteine ​​residues in the DUF2436 domain are not substituted, and preferably, one or more cysteine ​​residues in the remaining portion of the adhesin domain are substituted.

[0053] Optionally, the adhesin domain comprises or consists of the sequence shown in SEQ ID NO: 23 or at least 80% identical thereto, wherein the cysteine ​​residue at position 115 is replaced by a serine or valine residue.

[0054] Optionally, the adhesin domain comprises or consists of the sequence shown in SEQ ID NO: 25 or at least 80% identical thereto, wherein the cysteine ​​residue at position 77 is replaced by a serine or valine residue.

[0055] Preferably, the adhesin domain comprises or consists of the sequence shown in SEQ ID NO: 34 or SEQ ID NO: 76 or at least 80% identical thereto, wherein one or more cysteine ​​residues are substituted with serine or valine residues.

[0056] In a particularly preferred embodiment of this invention, the chimeric or fusion protein, the second polypeptide, is: a) Includes substitutions of one or more cysteine ​​residues with serine amino acids compared to the naturally occurring adhesin domain sequence; and b) Contains proline substitution and / or asparagine substitution in sequence PxxN corresponding to residues at positions 6 to 9 of the sequence of SEQ ID NO: 14 or 19 or located at positions equivalent to said residues.

[0057] In another preferred embodiment, the second polypeptide comprises the amino acid sequence of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, wherein the adhesin domain comprises the sequence shown in SEQ ID NO: 18 or 22 or at least 80% identical thereto; and wherein one or two cysteine ​​residues in SEQ ID NO: 18 or 22 are substituted with serine residues, and wherein proline and / or asparagine residues in sequence PxxN at positions 63 to 66 (corresponding to positions 6 to 9 in SEQ ID NO: 14 or 19) or equivalent positions in SEQ ID NO: 18 or SEQ ID NO: 22 are substituted. Optionally, the proline residue is substituted with an alanine residue, and / or the asparagine residue is substituted with a proline or alanine residue, preferably wherein the proline is substituted with alanine and the asparagine is substituted with proline, such that the sequence at positions 63 to 66 of SEQ ID NO: 18 or SEQ ID NO: 22 is AxxP (e.g., AVQP, SEQ ID NO: 85).

[0058] In a preferred embodiment, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 34 or 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: 34 or its equivalent is not substituted with a serine or valine residue. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 34 or its equivalent is not substituted with a serine or valine residue, while the cysteine ​​residues at positions 208 and 222 of SEQ ID NO: 34 or their equivalents are substituted with serine residues.

[0059] In another preferred embodiment of this invention, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 34 or at least 80% identical thereto, wherein one, two, or three cysteine ​​residues are substituted with serine residues, and proline and asparagine residues in sequence PxxN at positions 235 to 238 or equivalent thereto are substituted. Preferably, the cysteine ​​residue at position 115 or equivalent thereto in SEQ ID NO: 34 is not substituted with serine or valine residues. Preferably, the cysteine ​​residue at position 115 or equivalent thereto in SEQ ID NO: 34 is not substituted with serine or valine residues, while the cysteine ​​residues at positions 208 and 222 or equivalent thereto in SEQ ID NO: 34 are substituted with serine residues, and the proline residue at position 235 or equivalent thereto is substituted with alanine residue, and the asparagine residue at position 238 or equivalent thereto is substituted with proline.

[0060] In a particularly preferred embodiment of this invention, the second polypeptide comprises or is composed of the sequence shown in any of SEQ ID NO: 35 to 49 or the sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it.

[0061] The chimeric or fusion protein preferably comprises or consists of the following: an amino acid sequence as shown in any of SEQ ID NO: 55, 56 or 57, or a sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to it.

[0062] In any further embodiment of any aspect, the chimeric or fusion protein comprises a first polypeptide and a second polypeptide, wherein: A) The first polypeptide comprises or consists of the amino acid sequence of the active site of Arg gingivase or Lys gingivase from Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto; and B) The second polypeptide contains or is composed of the amino acid sequence of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis. The second polypeptide comprises a sequence of one or more adhesin-binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain and the cleaved adhesin domain (CAD) of Porphyromonas gingivalis gingivalis protease. Preferably, the one or more ABMs comprise sequences as shown 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 sequences that are at least 80% identical thereto. More preferably, the one or more ABMs comprise the sequence shown in any one of SEQ ID NO: 16 or SEQ ID NO: 18 or 22, or a sequence that is at least 80% identical thereto; And the second polypeptide described therein: a) Contains an amino acid sequence substantially corresponding to the full length of the DUF2436 domain of Arg gingivase or Lys gingivase, preferably the amino acid sequence shown in SEQ ID NO: 23, or at least 80% identical thereto; and One or more amino acids selected from the following are substituted: b) Compared to the naturally occurring Arg gingivase or Lys gingivase sequence in the corresponding region, one or more cysteine ​​amino acids in the adhesin domain are substituted; and / or d) Substitution of one or more amino acid motifs selected from the following: i) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1) or located at positions equivalent to said residues; ii) Substitution of motif NxFA to SxYQ in sequences corresponding to residues 2 to 5 of the sequence corresponding to or located at positions equivalent to said residues; iii) The substitution of a second tyrosine residue corresponding to or at an equivalent position to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue corresponding to or at an equivalent position to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue. Optionally, the chimeric or fusion protein comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0063] The one or more cysteine ​​amino acid substitutions may be substitutions that change to serine or valine residues. Preferably, the one or more cysteine ​​substitutions may include one or more substitutions that change to serine residues.

[0064] In some 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.

[0065] In a particularly preferred embodiment, the adhesin domain comprises the DUF2436 domain, and the cysteine ​​residues in the DUF2436 domain are substituted with serine or valine, preferably serine. In other embodiments, the adhesin domain comprises the DUF2436 domain, and the cysteine ​​residues in the DUF2436 domain are not substituted, and preferably, one or more cysteine ​​residues in the remaining portion of the adhesin domain are substituted.

[0066] Preferably, the adhesin domain comprises or consists of the sequence shown in SEQ ID NO: 34 or SEQ ID NO: 76 or at least 80% identical thereto, wherein one or more cysteine ​​residues are substituted with serine or valine residues.

[0067] According to this embodiment, the sequence PxxN corresponding to residues 235 to 238 of SEQ ID NO: 34, or located at positions equivalent to those residues, comprises substitutions for proline and asparagine residues. Preferably, the substitution is PxxN changing to AxxP.

[0068] Preferably, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 34 or 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: 34 or its equivalent is not substituted with a serine or valine residue. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 34 or its equivalent is not substituted with a serine or valine residue, while the cysteine ​​residues at positions 208 and 222 of SEQ ID NO: 34 or their equivalents are substituted with serine residues.

[0069] In a particularly preferred embodiment of this invention, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 34 or at least 80% identical thereto, wherein one, two, or three cysteine ​​residues are substituted with serine residues, and proline and asparagine residues in sequence PxxN at positions 235 to 238 or equivalent thereto are substituted. Preferably, the cysteine ​​residue at position 115 or equivalent thereto in SEQ ID NO: 34 is not substituted with serine or valine residues. Preferably, the cysteine ​​residue at position 115 or equivalent thereto in SEQ ID NO: 34 is not substituted with serine or valine residues, while the cysteine ​​residues at positions 208 and 222 or equivalent thereto in SEQ ID NO: 34 are substituted with serine residues, and the proline residue at position 235 or equivalent thereto is substituted with alanine residue, and the asparagine residue at position 238 or equivalent thereto is substituted with proline.

[0070] The second polypeptide preferably comprises or is composed of the following: a sequence as shown in any of SEQ ID NO: 35 to 49 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to it.

[0071] The chimeric or fusion protein more preferably comprises or consists of the sequence shown in any of SEQ ID NO: 62 to 63 or the sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it.

[0072] In any further embodiment of any aspect of the invention, the chimeric or fusion protein comprises a first polypeptide linked to a second polypeptide, wherein: A) The first polypeptide comprises or consists of the amino acid sequence of the active site of Arg gingivase or Lys gingivase from Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto; and B) The second polypeptide contains or is composed of the amino acid sequence of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis. The second polypeptide comprises sequences of one or more adhesin-binding motifs (ABMs), preferably wherein the ABM corresponds to part or all of the ABM between the DUF2436 domain and the cleaved adhesin domain (CAD) of *Porphyromonas gingivalis* gingival protease. Preferably, the one or more ABMs comprise sequences as shown in SEQ ID NO: 15 or 20 (ABM2) and / or SEQ ID NO: 17 or 21 (ABM3), or sequences that are at least 80% identical thereto. More preferably, the one or more ABMs comprise sequences as shown 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 sequences that are at least 80% identical thereto. Most preferably, the one or more ABMs comprise the sequence shown in any one of SEQ ID NO: 16 or SEQ ID NO: 18 or 22, or a sequence that is at least 80% identical thereto; And the second polypeptide described therein: a) Does not contain: a sequence of a cleaved adhesin domain (CAD) or a portion thereof, preferably a sequence of a CAD having the amino acid sequence shown in SEQ ID NO: 12 or 13, or a sequence at least 80% identical thereto; and b) Contains an amino acid sequence substantially corresponding to the full length of the DUF2436 domain of Arg gingivase or Lys gingivase, preferably the amino acid sequence shown in SEQ ID NO: 23, or at least 80% identical thereto; and It contains one or more amino acid substitutions: c) One or more cysteine ​​amino acids in the DUF2436 domain and the amino acid sequence of ABM are substituted compared to the naturally occurring Arg gingivase or Lys gingivase sequence in the corresponding region; and / or (preferably) as well. d) Substitution of one or more amino acid motifs selected from the following: i) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1) or located at positions equivalent to said residues; ii) Substitution of motif NxFA to SxYQ in sequences corresponding to residues 2 to 5 of the sequence corresponding to or located at positions equivalent to said residues; iii) The substitution of a second tyrosine residue corresponding to or at an equivalent position to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue corresponding to or at an equivalent position to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue. Optionally, the chimeric or fusion protein comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0073] The one or more cysteine ​​amino acid substitutions may be substitutions that change to serine or valine residues. Preferably, the one or more cysteine ​​substitutions may include one or more substitutions that change to serine residues.

[0074] Optionally, 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.

[0075] In a particularly preferred embodiment of this invention, the adhesin domain comprises the DUF2436 domain, and the cysteine ​​residues in the DUF2436 domain are substituted with serine or valine, preferably serine. In other embodiments, the adhesin domain comprises the DUF2436 domain, and the cysteine ​​residues in the DUF2436 domain are not substituted, and preferably, one or more cysteine ​​residues in the remainder of the adhesin domain are substituted.

[0076] Preferably, the adhesin domain comprises or consists of the sequence shown in SEQ ID NO: 34 or at least 80% identical thereto, wherein one or more cysteine ​​residues are substituted with serine or valine residues.

[0077] According to this embodiment, the sequence PxxN corresponding to residues 235 to 238 of SEQ ID NO: 34, or located at positions equivalent to those residues, comprises substitutions for proline and asparagine residues. Preferably, the substitution is PxxN changing to AxxP.

[0078] In a particularly preferred embodiment, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 34 or 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: 34 or its equivalent is not substituted with a serine or valine residue. Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 34 or its equivalent is not substituted with a serine or valine residue, while the cysteine ​​residues at positions 208 and 222 of SEQ ID NO: 34 or their equivalents are substituted with serine residues.

[0079] In a particularly preferred embodiment, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 34 or at least 80% identical thereto, wherein one, two, or three cysteine ​​residues are substituted with serine residues, and proline and asparagine residues in sequence PxxN at positions 235 to 238 or equivalent thereto are substituted. Preferably, the cysteine ​​residue at position 115 or equivalent thereto in SEQ ID NO: 34 is not substituted with serine or valine residues. Preferably, the cysteine ​​residue at position 115 or equivalent thereto in SEQ ID NO: 34 is not substituted with serine or valine residues, while the cysteine ​​residues at positions 208 and 222 or equivalent thereto in SEQ ID NO: 34 are substituted with serine residues, and the proline residue at position 235 or equivalent thereto is substituted with alanine residue, and the asparagine residue at position 238 or equivalent thereto is substituted with proline.

[0080] In a particularly preferred embodiment, the second polypeptide comprises or is composed of the sequence shown in any of SEQ ID NO: 35 to 49 or the sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it.

[0081] In a particularly preferred embodiment, the chimeric or fusion protein comprises or is composed of the following sequences as shown in any of SEQ ID NO: 58 to 61, 69, 70 to 75, 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 the sequences shown in SEQ ID NO: 58 to 61, 69, 70 to 75.

[0082] In a particularly preferred embodiment, the chimeric or fusion protein comprises or is composed of the following: a sequence as shown in any of SEQ ID NO: 69 or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it.

[0083] In any further embodiment of any aspect of the invention, the chimeric or fusion protein comprises a first polypeptide linked to a second polypeptide, wherein: A) The first polypeptide comprises or consists of the amino acid sequence of the active site of Arg gingivase or Lys gingivase from Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto; and B) The second polypeptide contains or is composed of the amino acid sequence of the adhesin domain of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis. The second polypeptide comprises sequences of one or more adhesin-binding motifs (ABMs), preferably wherein the ABM corresponds to part or all of the ABM between the DUF2436 domain and the cleaved adhesin domain (CAD) of *Porphyromonas gingivalis* gingival protease. Preferably, the one or more ABMs comprise sequences as shown in SEQ ID NO: 15 or 20 (ABM2) and / or SEQ ID NO: 17 or 21 (ABM3), or sequences that are at least 80% identical thereto. More preferably, the one or more ABMs comprise sequences as shown 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 sequences that are at least 80% identical thereto. Most preferably, the one or more ABMs comprise the sequence shown in any one of SEQ ID NO: 16 or SEQ ID NO: 18 or 22, or a sequence that is at least 80% identical thereto; And the second polypeptide described therein: a) Does not contain: a sequence of a cleaved adhesin domain (CAD) or a portion thereof, preferably a sequence of a CAD having the amino acid sequence shown in SEQ ID NO: 12 or 13, or a sequence at least 80% identical thereto; and And it contains one or more amino acid substitutions selected from the following: b) Compared to the naturally occurring Arg gingivase or Lys gingivase sequence in the corresponding region, one or more cysteine ​​amino acids in the amino acid sequence of the ABM are substituted; and / or c) One or more amino acid motifs selected from the following: i) Substitution of proline and / or asparagine residues in sequence PxxN (e.g., PVQN, SEQ ID NO: 86) corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1) or located at positions equivalent to said residues; ii) Substitution of motif NxFA in sequences corresponding to residues 2 to 5 of the sequence corresponding to or located at positions equivalent to said residues, such as NEFA, SEQ ID NO: 87, to SEYQ, SEQ ID NO: 88; iii) The substitution of a second tyrosine residue corresponding to or at an equivalent position to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue corresponding to or at an equivalent position to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) with an alanine residue. Optionally, the chimeric or fusion protein comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0084] It should be understood that, in this embodiment of the invention, the chimeric or fusion protein preferably does not contain an amino acid sequence corresponding to or derived therefrom the DUF2436 domain of Arg gingivase or Lys gingivase, as shown in SEQ ID NO: 23 or at least 80% identical thereto.

[0085] In a particularly preferred embodiment of this invention, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO:18 or at least 80% identical thereto, wherein one or two cysteine ​​residues are replaced with serine residues.

[0086] Preferably, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 18 or at least 80% identical thereto, wherein one or two cysteine ​​residues are replaced with serine residues, and wherein proline and asparagine residues in sequence PxxN at positions 63 to 66 or equivalent positions are replaced.

[0087] The first and second polypeptides can bind directly or via a linker. Preferably, the linker comprises a sequence of amino acids, preferably of about 1 amino acid residue to about 20 amino acid residues in length, preferably no more than about 5 amino acids, or no more than about 10 amino acids, or no more than about 15 amino acids in length.

[0088] The first polypeptide and the second polypeptide (having representative amino acid sequences of SEQ ID NO: 1 and 18, respectively) (i.e., amino acid sequences without the amino acid substitutions described herein) can be linked by a peptide sequence having the amino acid sequence [DM]EVEDDSP (SEQ ID NO: 89).

[0089] In a particularly preferred embodiment, the chimeric or fusion protein comprises or is composed of the following: a sequence as shown in any of SEQ ID NO: 80 or 81, or a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it.

[0090] In any embodiment or aspect described herein, the chimeric or fusion protein preferably comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease from *Porphyromonas gingivalis*, or a sequence at least 80% identical thereto. The one or more additional polypeptides comprising or consisting of the active site of Arg gingival protease or Lys gingival protease from *Porphyromonas gingivalis*, may be located at the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, or the N-terminus or C-terminus of the second polypeptide.

[0091] In any embodiment, the first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 11, or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same sequence.

[0092] The one or more additional polypeptides preferably comprise or consist of an amino acid sequence selected from or composed of the group consisting of SEQ ID NO: 1 to 11, 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 same sequence.

[0093] In any embodiment, the first polypeptide comprising or composed of the amino acid sequence of the active site of Arg gingivase or Lys gingivase from *Porphyromonas gingivalis*, and the other polypeptide comprising or composed of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the same amino acid sequence, or wherein the amino acid sequence is identical. The first polypeptide and the other polypeptide may be derived from the active site of a heterologous gingivase (e.g., gingivase from a different strain of *Porphyromonas gingivalis*). The first polypeptide and the other polypeptides may have amino acid sequences derived from different gingival proteases (e.g., one of the polypeptides has an amino acid sequence from the active site of Kgp and the other polypeptide has an amino acid sequence from the active site of Rgp; or alternatively, one of the polypeptides has an amino acid sequence from the active site of RgpA and the other polypeptide has an amino acid sequence from the active site of RgpB).

[0094] In any embodiment of any aspect of the invention, the chimeric or fusion protein comprises, or is substantially comprised of, the sequences of the first and second polypeptides as defined herein. Therefore, it should be understood that the chimeric or fusion protein includes an arrangement or configuration of domains that differ from the configuration of those domains in the naturally occurring gingival protease polyprotein sequence. In other words, the first and second polypeptides and their domains have a spatial configuration different from that of the naturally occurring gingival protease polyprotein.

[0095] In any embodiment of any aspect of the invention, the first polypeptide and the second polypeptide are linked. The first polypeptide and the second polypeptide may be linked directly, via a linker, or via a polypeptide sequence of no more than 100, preferably no more than 50 amino acids. Preferably, the first polypeptide and the second polypeptide are directly linked, or linked via a sequence of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. Most preferably, the first polypeptide and the second polypeptide are directly linked.

[0096] In any embodiment of any aspect of the invention, the C-terminal residue of the first polypeptide may be directly linked to the N-terminal residue of the second polypeptide, either via a linker or via a polypeptide sequence of no more than 50 amino acids. Alternatively, the N-terminal residue of the first polypeptide may be directly linked to the C-terminal residue of the second polypeptide, either via a linker or via a polypeptide sequence of no more than 50 amino acids.

[0097] In any aspect of the invention, the DUF2436 domain and ABM domain derived from Arg gingivase or Lys gingivase can bind directly, or via a linker, or via a polypeptide sequence. Preferably, the DUF2436 domain and ABM domain are linked by a short linker sequence comprising about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In a particularly preferred embodiment, the DUF domain and ABM domain are linked by a short linker sequence of no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0098] In a preferred embodiment, the DUF2436 domain and the ABM domain (having representative amino acid sequences of SEQ ID NO: 23 and 18, respectively) (i.e., amino acid sequences without the amino acid substitutions described herein) can be linked by a peptide sequence having the amino acid sequence EVEDDSP (SEQ ID NO: 90) (corresponding to the natural Porphyromonas gingivalis sequence).

[0099] In any embodiment, the additional polypeptide may bind directly or via a linker to the chimeric or fusion protein comprising the first polypeptide and the second polypeptide. In embodiments where the additional polypeptide binds to the C-terminal region of the fusion protein via the second polypeptide, preferably the C-terminus of the second polypeptide binds directly to the N-terminus of the additional polypeptide. (For example, the C-terminus of the adhesin domain preferably binds directly to the N-terminus of the active site amino acid sequence).

[0100] In cases involving more than one additional polypeptide (e.g., as illustrated in SEQ ID NO: 54), copies of the additional polypeptides may bind directly to each other or bind via a linker sequence.

[0101] In any embodiment of any aspect of the invention, the linker region is an amino acid sequence of no more than 15 amino acids, and preferably more than 2 amino acids. Suitable linkers used in protein constructs (including those with minimal impact on solubility) are known in the art. Useful linkers include glycine-serine (GlySer) linkers, which are well known in the art and comprise glycine and serine units in various combinations in various sequences. Examples include, but are not limited to, (GS), (GSGGS)n (SEQ ID NO: 91), (GGGS)n (SEQ ID NO: 92), and (GGGGS)n (SEQ ID NO: 93), where n is an integer of at least one, typically from 1 to 10, for example, from 1 to 8, from 1 to 6, or from 1 to 5. Other useful linkers include DSSG (SEQ ID NO: 94), DSSGAS (SEQ ID NO: 95), KLDSSG (SEQ ID NO: 96), or other linkers described herein. In some embodiments, the connector region may be derived from a natural gingival protease multiprotein sequence (such as the amino acid sequence EVEDDSP (SEQ ID NO: 90)).

[0102] The present invention also provides a method for inducing an immune response against *Porphyromonas gingivalis*, preferably against *Porphyromonas gingivalis* gingival protease protein, in a non-human animal model of neuropathology induced or associated with *Porphyromonas gingivalis*. Preferably, the non-human animal model is a rodent model.

[0103] In a preferred embodiment, the method comprises applying a preventative or therapeutically effective amount of the chimeric protein as described herein to the gingival margin of the maxillary molars of an animal. In any embodiment, the application is performed at least once, at least twice, or more times. In a particularly preferred embodiment, the application is performed at least 5 times, at least 10 times, at least 15 times, or at least 20 times. Optionally, the application is performed daily, every second day, every third day, every fourth day, or every fifth day for the desired total number of applications.

[0104] The present invention also provides a method for the following: ● Prevention or treatment of neuropathology in subjects, preferably wherein the neuropathology is related to or caused by Porphyromonas gingivalis infection; ● Prevents the accumulation of Porphyromonas gingivalis gingivalase in the neuronal tissue of subjects; ● Reduced the level of Porphyromonas gingivalis gingivalase in the neuronal tissue of the subjects; ● Delays the onset of neuropathology associated with Porphyromonas gingivalis; ● Prevents or slows down the deposition of abnormal proteins in the neuronal tissue of the subjects; ● To prevent or reduce the progression of amyloidosis, synucleinosis, or tau disease in subjects; or ● Reduce neuroinflammation in subjects, preferably neuroinflammation associated with or caused by Porphyromonas gingivalis infection; The method comprises administering an antibody or antibody preparation (such as a polyclonal antibody) targeting *Porphyromonas gingivalis* to the subject, wherein the antibody or antibody preparation is generated in a non-human animal by administering a chimeric or fusion protein as described herein, and optionally isolating the antibody from the animal (e.g., from the animal's blood) or from its eggs (in the case of an avian species, preferably a chicken).

[0105] Throughout this specification, unless the context otherwise requires, the terms “comprise,” “comprises,” and “comprising” will be understood to imply inclusion of the stated steps or elements or groups of steps or elements, but not to exclude any other steps or elements or groups of steps or elements. Thus, the use of the terms “comprise,” etc., indicates that the listed elements are necessary or mandatory, but other elements are optional and may or may not be present. “consisting of” means including and limited to anything following the phrase “consisting of.” Thus, the phrase “consisting of” indicates that the listed elements are necessary or mandatory, and other elements may be absent. “Substantially consisting of” means including any element listed following the phrase and is limited to other elements that do not interfere with or facilitate the activities or actions specified in this disclosure for the listed elements. Therefore, the phrase “consistent with…” indicates that the listed elements are required or mandatory, but other elements (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more additional amino acid residues at the N-terminus or C-terminus of the polypeptide sequence) are optional and may be present or absent, depending on whether they affect the activity or function of the listed elements.

[0106] Further aspects of the invention and further embodiments of the aspects described in the foregoing paragraphs will become apparent from the following description, which is given by way of example and with reference to the accompanying drawings. Attached Figure Description

[0107] Figure 1 A. Mouse periodontitis model - therapeutic vaccination. B. Schematic diagram showing the domain structure of the Kgp gingival protease polyprotein used to generate the chimeric or fusion protein of the present invention and its derived components.

[0108] Figure 2 A. *E. coli* expressing recombinant variants with or without a K1 domain and containing a full-length or truncated DUF domain ( E coli SDS-PAGE of fractions. Low temperature and low IPTG conditions were used. Full-length DUF is crucial for solubility. Full-length K1 is detrimental to solubility. B. Native PAGE of chimeric structures KDA and KDAK1 in the presence or absence of DTT. Adding a K1 domain to KDA reduces solubility and causes protein polymerization.

[0109] Figure 3 A. Diagrammatic representation of the Kgp polyprotein, showing the positions of the ABM1 and ABM2 domains. B. Native PAGE analysis of purified recombinant protein variants subjected to electrophoresis with or without DTT. B: Study of the “PVQN” mutant variant. C: Study of the “PVQN” mutant variant with an additional Cys>Ser mutation. D: Study of mutations in the “PVQN” motif proloop (lanes 2-4) and highly conserved hydrophobic Tyr and Trp residues (lanes 5-7) in ABM2 and ABM1, respectively. 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 strains expressing *E. coli*. F. Native PAGE analysis of small-scale purified recombinant variants. G. Various vaccine candidates: (i) 4-12% SDS gels and (ii) 3-12% natural gels. Lanes 1 and 2: KDAK-3S-AVQP (1: unreduced; 2: reduced); Lanes 3 and 4: KDAK-1V-2S-AVQP (3: unreduced; 4: reduced); Lanes 5 and 6: KDAK-1V-AVQP (5: unreduced; 6: reduced); Lanes 7 and 8: KDAK-AVQP (7: unreduced; 8: reduced); Lanes 9 and 10: KDcAK1n (9: unreduced; 10: reduced). The gels were stained with Coomassie blue.

[0110] Figure 4 Bone loss study. Statistical analysis - one-way ANOVA and post-hoc Dunnett T3. # Significant difference compared to the control stimulation group.

[0111] Figure 5Antigen isotype response to heat-inactivated whole cells of *Porphyromonas gingivalis* (individual serum). Primary = Unchallenged control. *Porphyromonas gingivalis* = Challenged control containing *Porphyromonas gingivalis*. Chimera = KDcAK1n antigen. Amox = Amoxicillin.

[0112] Figure 6 Bone loss study. Statistical analysis - one-way ANOVA and post-hoc Dunnett T3. # Significant difference compared to the control stimulation group.

[0113] Figure 7 Statistical analysis of bone loss study - univariate ANOVA and post-hoc Dunnett T3. #p < 0.01; ##p < 0.05 (T-test) showed a significant difference from the control stimulation group.

[0114] Figure 8 Bone loss study. S = soluble fraction; urea = urea used for purification; AC = affinity column purification; IB = inclusion bodies; batch = batch purification method. Statistical analysis: one-way ANOVA and post-hoc Dunnett T3. #p < 0.01 significantly different from the control stimulation group.

[0115] Figure 9 Bone loss in animals following immunization was studied using various fusion protein constructs. Statistical analysis: univariate ANOVA and post-hoc Dunnett T3. Significant differences were observed compared to the control stimulation group. # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001.

[0116] Figure 10 Bone loss was studied in animals after immunization using various fusion protein constructs. Statistical analysis: univariate ANOVA and post-hoc Dunnett T3. ### (p < 0.05); #### (p < 0.01); Significant difference was observed compared to the control stimulation group.

[0117] Figure 11 Bone loss was studied in animals after immunization using various fusion protein constructs. Statistical analysis: univariate ANOVA and post-hoc Dunnett T3. The results showed a significant difference (p < 0.05) compared to the control stimulation group.

[0118] Figure 12Bone loss following immunization was studied in animals using various fusion protein constructs. Except for KDcAK1n, all tested antigens contained "AVQP" substitutions; i.e., KDAK-3S = KDAK-3S-AVQP; KDAK-1V2S = KDAK-1V2S-AVQP; KDAK-3S-Tag = KDAK-3S-AVQP-Tag; Statistical analysis included univariate ANOVA and post-hoc Dunnett T3. (p < 0.05) There was a significant difference from the original group (i.e., the control group without stimulation).

[0119] Figure 13 Purity of the final product of *Porphyromonas gingivalis* antigen tested in Example 4. PAGE analysis of His-tagged antigens (A and B) and untagged KDAK-3S-AVQP (C). (A) SDS-PAGE; (B) Natural PAGE or natural gel. (C) SDS- and natural PAGE or natural gel with heated (95°C) or unheated sample loaded. R: Sample reduction on gel; NR: No sample reduction on gel; M: Protein standard. His-tagged KDAK-3S-AVQP was purified in parallel with other His-tagged proteins.

[0120] Figure 14 Bone loss was studied in animals after immunization using various fusion protein constructs. Statistical analysis - one-way ANOVA and post-hoc Dunnett T3. # (p < 0.05 compared with the original control (i.e., control not challenged); ## (p < 0.05 compared with the infected control (i.e., control challenged)).

[0121] Figure 15 Antibody titers against heat-inactivated whole cells of *Porphyromonas gingivalis* in mouse serum (individual). (A) Total IgG titer; (B) IgG1 subtype titer; (C) IgG2a subtype titer.

[0122] Figure 16 The exemplary fusion protein of the present invention exhibits a dose-response trend with periodontal bone loss protection using Kgpcat cross-reactive Ab titers.

[0123] Figure 17 A schematic diagram of the timeline of *Porphyromonas gingivalis*-induced neuropathological models in each mouse group, on a weekly basis. Orange boxes represent intraperitoneal (IP) and subcutaneous (SC) vaccinations with 200 μg of recombinant KDAK-3S-AVQP (SEQ ID NO: 69), green boxes represent *Porphyromonas gingivalis* challenge feeding protocols, beige boxes represent sham feeding protocols, and red boxes represent the number of mice at the end of the experiment and those sacrificed. The numbers above the axes represent the weeks.

[0124] Figure 18 Establishment of a neuropathological model induced by *Porphyromonas gingivalis*: A. Levels of RgpA protein in the brains of *Porphyromonas gingivalis*-fed and sham-fed mice 3 weeks after feeding. B. Levels of β-amyloid protein in the brains of *Porphyromonas gingivalis*-fed and sham-fed mice 3 weeks after feeding. C. Bone loss measured 6 weeks after *Porphyromonas gingivalis* feeding. T-test. = P < 0.05 D. Levels of RgpA protein in the brains of mice fed and sham-fed with *Porphyromonas gingivalis* after 6 weeks. E. Levels of β-amyloid protein in the brains of mice fed and sham-fed with *Porphyromonas gingivalis* after 6 weeks. F. Comparison of *Porphyromonas gingivalis*-induced neuropathology at 6 weeks compared to 3 weeks of *Porphyromonas gingivalis* feeding. Left inset: Mean number of positive cells (RgpA or Aβ) per brain. Right inset: Per mm 2 The mean number of positive cells (RgpA or Aβ). t-test, = P < 0.05, = P < 0.01

[0125] Figure 19 Immunogenicity of the chimeric protein named KDAK-3S-AVQP (SEQ ID NO: 69) in mouse models of neuropathology induced or associated with *Porphyromonas gingivalis*. A. Total IgG titer. B. IgG1 titer. C. Heat-inactivated *Porphyromonas gingivalis* IgG1 titer. D. Heat-inactivated *Porphyromonas gingivalis* total IgG1. E. Kgpcat total IgG titer. F. Kgpcat IgG1 titer. = P ≤ 0.05, = P ≤ 0.01, = P ≤ 0.001, =P ≤ 0.0001

[0126] Figure 20 Confirmation of bone loss protection using KDAK-3S-AVQP in a mouse model of Porphyromonas gingivalis-induced neuropathology. Data shown represent bone loss at 6 weeks. = P ≤ 0.05, = P ≤ 0.01, =P ≤ 0.001, = P ≤ 0.0001

[0127] Figure 21Brain infiltration of *Porphyromonas gingivalis* in mice, as measured by detection of gingival protease (RgpA) protein. (A) Sham-fed and unvaccinated; (B) Sham-fed and vaccinated with KDAK-3S-AVQP; (C) Unvaccinated and fed *Porphyromonas gingivalis*; and (D) Vaccinated with KDAK-3S-AVQP and fed *Porphyromonas gingivalis*.

[0128] Figure 22 Detection of β-amyloid protein in the brains of mice: (A) sham-fed and unvaccinated; (B) sham-fed and vaccinated with KDAK-3S-AVQP; (C) unvaccinated and fed with Porphyromonas gingivalis; and (D) vaccinated with KDAK-3S-AVQP and fed with Porphyromonas gingivalis.

[0129] Figure 23 Detection of phosphorylated tau in the brain of mice: (A) sham-fed and unvaccinated; (B) sham-fed and vaccinated with KDAK-3S-AVQP; (C) unvaccinated and fed with Porphyromonas gingivalis; and (D) vaccinated with KDAK-3S-AVQP and fed with Porphyromonas gingivalis.

[0130] Figure 24 Neuroinflammation in the brain of mice (IL-6 detection) (A) sham-fed and unvaccinated; (B) sham-fed and vaccinated with KDAK-3S-AVQP; (C) unvaccinated and fed with Porphyromonas gingivalis; and (D) vaccinated with KDAK-3S-AVQP and fed with Porphyromonas gingivalis.

[0131] Figure 25 Neuroinflammation in the brain of mice (IL-1β detection) (A) sham-fed and unvaccinated; (B) sham-fed and vaccinated with KDAK-3S-AVQP; (C) unvaccinated and fed with Porphyromonas gingivalis; and (D) vaccinated with KDAK-3S-AVQP and fed with Porphyromonas gingivalis.

[0132] Figure 26 An overview of the efficacy of KDAK-3S-AVQP vaccination in a mouse model of Porphyromonas gingivalis-induced neuropathology. A. Percentage of RgpA-positive cells. B. Percentage of β-amyloid cells. C. Percentage of phosphorylated tau positive pixels (regions). D. Percentage of IL-6-positive cells. E. Percentage of IL-1β-positive cells. = P≤ 0.05, = P ≤ 0.01, = P ≤ 0.001, = P ≤ 0.0001

[0133] Figure 27 A. An overview of the immunohistochemical results of β-amyloid (Aβ) staining in the brains of mice (primary, fed with *Porphyromonas gingivalis*, and fed with *Porphyromonas gingivalis* followed by vaccination with KDAK-3S-AVQP adjuvanted with alum). The results show the percentage of amyloid β-positive cells. B. Confirmation of bone loss protection using KDAK-3S-AVQP in the corresponding treatment model of A. = P ≤ 0.05, = P ≤ 0.01, =P ≤ 0.001, = P ≤ 0.0001.

[0134] Figure 28 Co-localization of Kgp gingival protease and β-amyloid protein in brain tissue of mice orally infected with Porphyromonas gingivalis. A. Mice fed with Porphyromonas gingivalis. B. Negative control (sham feeding).

[0135] Sequence information

[0136] Table 1: Sequence Information Detailed Implementation

[0137] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or apparent in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0138] Reference will now be made to certain embodiments of the invention. Although the invention will be described in conjunction with embodiments, it should be understood that it is not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents that may be included within the scope of the invention as defined in the claims.

[0139] Those skilled in the art will recognize that many methods and materials are similar to or equivalent to those described herein and can be used to practice this invention. This invention is by no means limited to the methods and materials described. It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or apparent in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0140] All patents and publications mentioned in this article are incorporated herein by reference in their entirety.

[0141] For the purposes of interpreting this specification, terms used in the singular will also include the plural form, and vice versa.

[0142] In the work that led to this invention, the inventors investigated various chimeric or fusion proteins for inducing an immune response against Porphyromonas gingivalis, and methods for the large-scale production of such chimeras for use as vaccine candidates.

[0143] The inventors have identified various problems associated with the large-scale preparation and production of chimeric or fusion proteins containing components of *Porphyromonas gingivalis* gingival protease. First, chimeric or fusion proteins described in the prior art are difficult to produce as soluble proteins in sufficiently high quantities. More specifically, prior art chimeric proteins are typically produced in inclusion bodies in *E. coli*, which makes the development of large quantities of soluble proteins for downstream clinical products challenging.

[0144] The reduced solubility when produced in E. coli also significantly increases the preparation time and scale, and the protein has poor stability once it is dissolved and refolded from the inclusion bodies.

[0145] Furthermore, the gingival protease-derived chimeric proteins described in the prior art are also subject to multimerization. From a regulatory perspective, the formation of β-sheets between the domains in chimeric proteins makes the evaluation of the final vaccine product challenging and may lead to a reduced immune response once administered.

[0146] An improved design for a chimeric or fusion protein to induce an immune response against *Porphyromonas gingivalis* has been identified. The inventors then sought to determine whether the chimeric or fusion protein could be used to treat, prevent, or reduce the progression of *Porphyromonas gingivalis*-induced or associated neuropathologies, such as the formation of amyloid plaques in the brain and / or phosphorylation of tau protein, which are known to be associated with the onset of neurodegenerative diseases, including dementia, Alzheimer's disease, and related pathologies.

[0147] A wealth of preclinical, epidemiological, clinical, and other data support the role of the oral pathogen *Porphyromonas gingivalis* in the progression of Alzheimer's disease (AD) in a subgroup of patients with active periodontitis. Multiple *Porphyromonas gingivalis* biomolecules, including nucleic acids, lipopolysaccharide (LPS), and surface proteases (gingival proteases), have been identified in multiple regions of the human AD brain, including the hippocampus, cortical gray matter, basal forebrain, and hypothalamus. These biomolecules co-localize with neurons, tau tangles, and intracellular Aβ pathology in the brains of AD patients, and the brain burden of gingival proteases is significantly associated with AD diagnosis, tau proteinopathy, and ubiquitin pathology. High serum anti-*Porphyromonas gingivalis* immunoglobulin G (IgG) titers have been reported as a risk factor for AD development, increasing AD incidence and impairing delayed memory and calculation abilities. A recent study of 20 AD patients and 20 patients with other types of dementia detected elevated levels of anti-*Porphyromonas gingivalis* IgG antibodies in the cerebrospinal fluid of both groups.

[0148] Gingival protease

[0149] The pathogenicity of *Porphyromonas gingivalis* is attributed to numerous surface-associated virulence factors, including cysteine ​​proteases (gingival proteases), pili, heme-binding proteins, and outer membrane transporters. Specifically, the extracellular Arg and Lys-specific proteases of *Porphyromonas gingivalis*, 'gingival proteases' (RgpA / B and Kgp), are considered key virulence factors, crucial for colonization, invasion of host tissues, dysregulation of the immune response, ecological imbalance, and disease.

[0150] Gingival proteases, particularly the Lys-specific protease Kgp, are crucial for *Porphyromonas gingivalis* to induce alveolar bone resorption in a mouse model of periodontitis. High concentrations of gingival proteases have also been found in gingival tissue near subgingival plaques and in distal sites deep within the gingival tissue at sites of severe periodontitis. Lys-specific and Arg-specific proteases have been shown to degrade a variety of host proteins in vitro, such as fibrinogen, fibronectin, and laminin. Plasma host defense and regulatory protease inhibitors α-trypsin, α2-macroglobulin, antichymotrypsin, antithrombin III, and antifibrinolysin have also been degraded by Lys and Arg proteases from *Porphyromonas gingivalis*. This leads to the development of a compelling mechanism to explain the key role *Porphyromonas gingivalis* plays in the development of chronic periodontitis.

[0151] 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-adhesin (HA) domain, which includes an adhesin-binding domain (ABM, of which five distinct sequences have been described), a "domain of unknown function" (designated DUF2436, defined as a conserved Pfam domain of unknown function; IPR018832), and a C-terminal adhesin domain or a cleaved adhesin domain (or CAD). The specific arrangement of ABM, DUF, and CAD differs between Kgp and RgpA / B.

[0152] The architecture of domains in Kgp multiproteins Figure 1 As shown in B. For example, Kgp contains (from N-terminus to C-terminus): a catalytic domain, a first ABM (ABM1), DUF2436, a domain containing ABM2, ABM1, and ABM3, two CAD domains (called K1 and K2), another domain containing ABM1 and ABM2, another CAD domain (called K3), ABM2, and a C-terminal domain.

[0153] As used herein, references to ABM 1, 2, and 3 will be understood to generally refer to the ABMs found in the sequence ABM2, ABM1, and ABM3 in the C-terminus of DUF2436 immediately following Kgp, such as... Figure 1 As described by B.

[0154] The catalytic domains of RgpB and RgpA share a high degree of sequence homology. However, RgpB lacks the HA domain and is located on the outer membrane in monomeric form. Some of the HA domains have been alternatively described as C-terminal adhesin domains or cleaved adhesin domains (CAD), and some are DUF (“domain of unknown function”) 2436 domains (conserved Pfam domain of unknown function; IPR018832).

[0155] RgpA and Kgp precursor proteins are cleaved into multiple domains that remain non-covalently associated, forming a large outer membrane protein complex. Thus, in vivo, Arg-specific and Lys-specific proteases have been found in cell-associated complexes of non-covalently associated proteases and adhesins. One such complex has been named the RgpA-Kgp protease-adhesin complex (previously known as the PrtR-PrtK protease-adhesin complex). This complex consists of a 45 kDa Arg-specific, calcium-stabilized cysteine ​​protease and seven sequence-associated adhesin domains.

[0156] As used herein, the Lys gingivase catalytic domain may also be referred to as the KAS domain or PAS domain. Similarly, the Arg gingivase catalytic domain may also be referred to as the RAS domain or PAS domain. Typically, the catalytic domains of Lys or Arg gingivase are located in the region approximately 480 amino acids from the N-terminus of the protein. The active sites within the catalytic domain are typically located at amino acid residues 426-446 (for RgpA) and 432-453 (for Kgp). Exemplary active site peptides found within the catalytic domains, such as SEQ ID NO: 1-11, are listed in Table 1.

[0157] As used herein, the adhesin domain of Arg gingivase or Lys gingivase from *Porphyromonas gingivalis* will be understood to generally refer to the region of Arg gingivase or Lys gingivase located at the C-terminus of the catalytic or active site domain. The adhesin domain (also known as the HA domain) typically contains a domain of unknown function (DUF) (particularly the conserved unfunctional Pfam domain of DUF 2436; IPR018832), several adhesin-binding motif (ABM) domains, and a cleaved adhesin domain (CAD).

[0158] First polypeptide

[0159] The chimeric or fusion protein of the present invention comprises a first polypeptide comprising or consisting of the amino acid sequence of the active site of Arg-X protease or Lys-X protease of Porphyromonas gingivalis (also referred to herein as Arg gingival protease or Lys gingival protease, respectively), or at least 80% identical thereto.

[0160] In any embodiment, the first polypeptide comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to 11, or the same sequence as 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0161] Enhanced immunogenicity

[0162] Preferably, the chimeric or fusion protein comprises one or more additional polypeptides, which comprise or consist of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease from *Porphyromonas gingivalis*, or a sequence at least 80% identical thereto. The one or more additional polypeptides comprising or consisting of the active site of Arg gingival protease or Lys gingival protease from *Porphyromonas gingivalis*, or thereof, may be located at the N-terminus of the first polypeptide, the C-terminus of the first polypeptide, the N-terminus of the second polypeptide, or the C-terminus of the second polypeptide. The one or more additional polypeptides may be linked to the first polypeptide or the second polypeptide of the chimeric or fusion protein, preferably through a linker of no more than 50 amino acids, or directly linked to the first polypeptide.

[0163] The one or more additional polypeptides preferably comprise or consist of an amino acid sequence selected from or composed of the group consisting of SEQ ID NO: 1 to 11, 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 same sequence.

[0164] In any embodiment, the first polypeptide comprising or composed of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease of Porphyromonas gingivalis and the other polypeptides comprising or composed of the following: identical amino acid sequences or sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to each other.

[0165] In a preferred embodiment, the chimeric or fusion protein of the present invention comprises no more than two, three, or four polypeptides, said polypeptides comprising or consisting of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease from *Porphyromonas gingivalis*, or a sequence that is at least 80% identical thereto. Preferably, the chimeric or fusion protein of the present invention has fewer than five polypeptides, more preferably fewer than four, and most preferably fewer than three, said polypeptides comprising or consisting of the amino acid sequence of the active site of Arg gingival protease or Lys gingival protease from *Porphyromonas gingivalis*, or a sequence that is at least 80% identical thereto.

[0166] Second polypeptide

[0167] The chimeric or fusion protein of the present invention comprises a second polypeptide, the second polypeptide comprising or consisting of the following: an amino acid sequence of the adhesin domain of ArgX protease or Lys-X protease of Porphyromonas gingivalis, or a sequence that is at least 80% identical thereto.

[0168] It should be understood that the second polypeptide will typically contain at least one sequence corresponding to one or more of an adhesin-binding motifs (ABMs), which are identified domains / motifs within the adhesin domains of ArgX or Lys-X proteases (such as Kgp and RgpA) of Porphyromonas gingivalis.

[0169] Five ABMs (ABM1-5) have been defined for Kgp and RgpA. Exemplary sequences of these ABMs are shown in Table 1.

[0170] Typically, the second polypeptide contains more than one ABM, preferably wherein the second polypeptide contains at least ABM1 and ABM2, or at least 80% identical sequences to each of ABM1 or ABM2. The second polypeptide may also contain the sequence of ABM3, or at least 80% identical sequences thereto.

[0171] It should also be understood that the arrangement of the ABM peptides in the second polypeptide does not need to correspond to the arrangement of ABM peptides found in naturally occurring adhesin domains. For example, in the second polypeptide, the ABM peptides can be arranged sequentially (N-terminus to C-terminus) as ABM1, ABM2, ABM3, etc. Alternatively, the ABM peptides can be arranged to reflect the arrangement in naturally occurring adhesin domains (such as ABM2, ABM1, and ABM3), as present in adhesin domain 1 of Kgp.

[0172] Furthermore, the ABMs in the second polypeptide can be arranged continuously or separated from each other by an amino acid sequence of no more than 50 amino acids. Those skilled in the art will understand that the spacing between ABMs is not important in the design of the chimeric or fusion proteins of the present invention.

[0173] In any embodiment, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 16 or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it.

[0174] In any embodiment, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 18 or SEQ ID NO: 27, 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 same sequence.

[0175] In any embodiment, the second polypeptide comprises an amino acid sequence as shown in SEQ ID NO: 22 or SEQ ID NO: 27, 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 same sequence.

[0176] Increased solubility

[0177] The inventors have determined that prior art methods, including simply selecting various domains found in naturally occurring gingival proteases for chimeric or fusion proteins, typically produce proteins that are poorly expressed in recombinant systems and tend to be expressed as insoluble proteins (e.g., in inclusion bodies of E. coli).

[0178] Referring to examples in this specification, the inventors recognized that the sequence shown in SEQ ID NO: 13 (i.e., a truncated form of the CAD domain) represents how Kgp polyproteins undergo proteolytic processing and assembly on cell surfaces. Therefore, the inclusion of such sequences is considered crucial for inducing an immune response against *Porphyromonas gingivalis*. However, the inventors surprisingly found that, when present, expression of this sequence in *Escherichia coli* leads to poor solubility and the formation of inclusion bodies of the recombinant protein.

[0179] The inventors attempted to correct this problem by including the complete sequence of CAD in the fusion protein. Surprisingly, this did not improve solubility; in fact, it resulted in even lower solubility.

[0180] Therefore, the inventors have found that sequences including those corresponding to one or more cleaved adhesin domains (CAD) or portions thereof help reduce the solubility of the proposed chimeric protein and vaccine compositions derived from gingivase sequences.

[0181] Therefore, in a particularly preferred embodiment of the invention, the second polypeptide does not contain the sequence shown in SEQ ID NO: 13 or a sequence that is at least 80% identical to it.

[0182] Alternatively, the second polypeptide does not contain the sequence shown in SEQ ID NO: 12 or at least 80% identical to it or a portion thereof.

[0183] The inventors have also discovered that the solubility of the chimeric or fusion protein of the present invention decreases when it contains only a portion of the DUF2436 domain (i.e., the N-terminal truncated DUF2436 domain).

[0184] Referring to the examples in this specification, the inventors recognize that prior art methods for designing *Porphyromonas gingivalis* vaccines include a truncated N-terminus of the DUF2436 domain. While the truncated DUF2436 domain represents how Kgp polyproteins undergo proteolytic processing and assembly on the cell surface, the inventors have found that providing the full-length sequence of the DUF2436 domain (i.e., including the N-terminal portion of the DUF2436 domain) significantly improves the production of soluble recombinant proteins.

[0185] Therefore, in a preferred embodiment, the second polypeptide in the chimeric or fusion protein of the present invention comprises residues 1 to 37 of the DUF2436 domain, preferably wherein the chimeric protein comprises an amino acid sequence substantially corresponding to or at least 80% identical to the full length of the DUF2436 domain of Arg gingivase or Lys gingivase.

[0186] As used herein, an amino acid sequence substantially corresponding to the full length of the DUF2436 domain, or a sequence that is at least 80% identical to it, refers to a sequence comprising 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 gingivase or Lys gingivase.

[0187] Preferably, the DUF2436 domain contains the sequence shown in SEQ ID NO: 23 or a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it.

[0188] Reduced polymerization

[0189] The inventors have also determined that various domains in gingivase have a tendency to polymerize, which helps reduce the solubility of chimeric or fusion proteins and reduces immunogenicity upon polymerization or aggregation. Therefore, polymer formation has an impact on the ease of large-scale production of chimeric proteins derived from gingivase sequences and their use as vaccines, but may also affect the generation of protective responses.

[0190] Specifically, the inventors have identified two mechanisms for polymer formation: the formation of disulfide bridges between cysteine ​​residues and β-chain exchange between ABM domains. (Through cross-referencing) Figure 1 Figure B illustrates various domains of Kgp. The inventors hypothesize that ABM1 may interact with its next available neighbor, ABM2, during the in vivo folding of the Kgp multiprotein. Specifically, the inventors demonstrate that co-expression of recombinant ABM1 and ABM2 as separate proteins can form a stable β-sheet complex.

[0191] Therefore, in order to reduce the multimerization of the chimeric protein proposed for use according to the present invention, in addition to targeting specific motifs within ABM 2 and 1, the inventors also target cysteine ​​residues in the DUF and ABM domains (which may be responsible for β-sheet formation).

[0192] The inventors have identified one or more of the following modifications that help reduce multimerization of chimeric fusion proteins derived from Kgp and Rgp multiproteins: c) Substitution of one or more cysteine ​​amino acids in the amino acid sequence containing the DUF2436 domain and ABM compared to the naturally occurring Arg gingivase or Lys gingivase sequence in the corresponding region; and / or d) Contains one or more amino acid motif substitutions selected from the following: i) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1) or located at positions equivalent to said residues; ii) Substitution of motif NxFA to SxYQ in sequences corresponding to residues 2 to 5 of the sequence corresponding to or located at positions equivalent to said residues; iii) The second tyrosine residue corresponding to or located at a position equivalent to the residue in SEQ ID NO: 15 or 20 (ABM2) and the tryptophan residue corresponding to or located at a position equivalent to the residue in SEQ ID NO: 14 or 19 (ABM1) are replaced by an alanine residue.

[0193] Therefore, in a particularly preferred embodiment of the invention, the second polypeptide contains one or more cysteine ​​amino acid substitutions compared to the naturally occurring adhesin domain sequence.

[0194] Cysteine ​​residues were found (at least) in the DUF2436 domain of the *Porphyromonas gingivalis* Kgp sequence, in ABM 2, and in the K1 CAD sequence. Therefore, it should be understood that the present invention contemplates 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 bridging.

[0195] Cysteine ​​amino acid substitutions can be substitutions that change to serine or valine residues. Preferably, one or more cysteine ​​substitutions comprise one or more substitutions that change to serine residues.

[0196] In a particularly preferred embodiment, the cysteine ​​residues in the DUF2436 domain are unsubstituted, while one or more cysteine ​​residues in the region of the adhesin domain between the C-terminus of the DUF2436 domain and the N-terminus of the K1 CAD domain may be substituted. An exemplary sequence of the region between the DUF domain and the CAD domain is shown in SEQ ID NO: 18. In a preferred embodiment of the invention, one or both cysteine ​​residues at positions 36 and 50 or equivalent are substituted, optionally substituted with serine or valine residues, preferably substituted with serine residues. An exemplary sequence of the DUF2436 domain plus ABM2+1+3 region is shown in SEQ ID NO: 34. In a preferred embodiment of the invention, one or both cysteine ​​residues at positions 208 and 222 or equivalent are substituted, optionally substituted with serine or valine residues, preferably substituted with serine residues.

[0197] 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 region of the adhesin domain between the C-terminus of the DUF2436 domain and the N-terminus of the K1 CAD domain may be substituted. An exemplary sequence of the region between the DUF domain and the CAD domain is shown in SEQ ID NO: 18. In a preferred embodiment of the invention, one or both cysteine ​​residues at positions 36 and 50 or equivalent are substituted, optionally substituted with serine or valine residues, preferably substituted with serine residues. An exemplary sequence of the DUF2436 domain plus the ABM2+1+3 region is shown in SEQ ID NO: 34. In a preferred embodiment of the invention, one or both cysteine ​​residues at position 115 and at positions 208 and 222 or equivalent are substituted, optionally substituted with serine or valine residues, preferably substituted with serine residues.

[0198] Therefore, in a particularly preferred embodiment, the chimeric or fusion protein of the present invention comprises an amino acid sequence corresponding to or at least 80% identical to the sequence shown in any of SEQ ID NO: 35 to 49 (except for cysteine ​​substitutions therein). In a particularly preferred embodiment, the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 49 as a second polypeptide.

[0199] The inventors further determined that mutations in the conserved motifs present in ABM domains 2, 1, and 3 also reduce multimerization.

[0200] In one instance, the inventors considered modification of the domain at the N-terminus of the adhesin domain in ABM1. More specifically, substitution of the motif PxxN (e.g., PVQN in ABM1 of Porphyromonas gingivalis Kgp, as shown in SEQ ID NO: 14) was found to significantly contribute to reducing the reduction in polymerization between ABM domains and the reduction in β-chain exchange.

[0201] Therefore, in a preferred embodiment, the chimeric or fusion protein of the present invention includes a modification of the ABM1 PxxN motif in the region of the chimeric or fusion protein corresponding to the adhesin domain of Porphyromonas gingivalis gingival protease. Therefore, the second polypeptide preferably comprises proline and asparagine substitutions in sequence PxxN corresponding to positions 6 to 9 of the sequence defining ABM1 in SEQ ID NO: 14 or 19, or at positions equivalent to those positions.

[0202] The proline amino acid substitution is preferably a substitution of alanine residues.

[0203] The asparagine amino acid substitution can be a substitution of a proline residue or an alanine residue. Preferably, the asparagine residue is substituted with a proline residue. In other embodiments, the asparagine residue is unsubstituted.

[0204] In another example, the inventors considered the motif NEFA in the sequence of ABM1 in the Kgp / Rgp multiprotein. This sequence is defined at residues 2 to 5 of SEQ ID NO: 14 and 19 herein. As further demonstrated in the examples, modifying the motif NEFA to SEQY by substituting the asparagine, phenylalanine, and alanine residues with serine, glutamine, and tyrosine, respectively, significantly reduced multimerization.

[0205] In another instance, the inventors determined that the substitution of tyrosine residues in ABM2 corresponding to or located at position 5 of SEQ ID NO: 15 or 20, and tryptophan residues in ABM1 corresponding to or located at position 23 of SEQ ID NO: 14 or 19, with alanine residues also significantly reduced polymerization.

[0206] Finally, the inventors discovered that through cysteine ​​modification... and Combinations of one or more of the following substitutions almost eliminate polymerization: i) Substitution of proline and / or asparagine residues in sequence PxxN corresponding to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1) or located at positions equivalent to said residues; ii) Substitution of motif NxFA to SxYQ in sequences corresponding to residues 2 to 5 of the sequence corresponding to or located at positions equivalent to said residues; iii) The second tyrosine residue corresponding to or located at a position equivalent to the residue in SEQ ID NO: 15 or 20 (ABM2) and the tryptophan residue corresponding to or located at a position equivalent to the residue in SEQ ID NO: 14 or 19 (ABM1) are replaced by an alanine residue.

[0207] 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 modifications to replace ABM1 with the PXXN motif of AXXP, eliminated the multimerization of the resulting recombinant chimeric protein.

[0208] In summary, through cysteine ​​modification and modification of the PXXN motif of the ABM1 domain, the present invention thus provides a chimeric or fusion protein as described herein, wherein the second polypeptide of the chimeric protein corresponding to the region of the adhesin domain of *Porphyromonas gingivalis* Arg gingival protease or Lys gingival protease comprises or consists of the following: an amino acid sequence as shown in SEQ ID NO: 34 or a sequence 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 sequence PXXN at positions 235 to 238 or equivalent thereto are substituted.

[0209] Preferably, the cysteine ​​residue at position 115 of SEQ ID NO: 34 or its equivalent position is not substituted with a serine or valine residue.

[0210] Preferably, the cysteine ​​residue at position 115 or equivalent of SEQ ID NO: 34 is not substituted with a serine or valine residue, while the cysteine ​​residues at positions 208 and 222 or equivalent of SEQ ID NO: 34 are substituted with serine residues, and the proline residue at position 235 or equivalent of SEQ ID NO: 34 is substituted with an alanine residue, and the asparagine residue at position 238 or equivalent of SEQ ID NO: 34 is substituted with proline.

[0211] The linking of the first polypeptide and the second polypeptide

[0212] In the chimeric or fusion proteins of the present invention, the C-terminal residues of the first peptide may be covalently linked to the N-terminal residues of the second peptide (corresponding to the adhesin domain peptide), or the N-terminal residues of the first peptide may be covalently linked to the C-terminal residues of the second peptide (corresponding to the adhesin domain peptide). In this arrangement, the first peptide and the adhesin domain peptide are referred to as "directly linked" or "adjacent".

[0213] In other embodiments, the chimeric or fusion protein includes a connector for linking the first peptide to the adhesin domain polypeptide. The connector can be any connector capable of binding the peptide to the polypeptide, including both amino acid connectors and non-amino acid connectors.

[0214] Preferably, the linker is non-immunogenic. Typically, the linker contains amino acids and can therefore be called a peptide linker.

[0215] Linkers are typically peptides of up to 20 amino acids in length, although they may be longer. The terms “linked with” or “fused with” refer to a covalent bond (e.g., a peptide bond) formed between two parts. Therefore, in the context of this invention, the length of a linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 or more amino acids. For example, the chimeric or fusion proteins provided herein may comprise a linker between a first polypeptide and a second polypeptide (e.g., between the N-terminus of the second polypeptide and the C-terminus of the first polypeptide), the first polypeptide comprising or consisting of the amino acid sequence of or composed of the active domain of *Porphyromonas gingivalis* gingivalis, and the second polypeptide corresponding to the adhesin domain of *Porphyromonas gingivalis* gingivalis. An advantage of such linkers is that they make it more likely that the different polypeptides of the fusion protein will fold independently and exhibit the expected behavior. A suitable linker can be up to 50 amino acids long, although less than 20, 15, or 5 amino acids are preferred. The linker can serve to bring the first peptide and the adhesin domain peptide closer together in spatial arrangement than is typically observed in *Porphyromonas gingivalis* trypsin-like enzymes. Alternatively, it can separate the first peptide from the second peptide (corresponding to the adhesin domain peptide).

[0216] Suitable linkers used in protein constructs (including linkers with minimal impact on solubility) are known in the art. Linkers can be any linker known to those skilled in the art and can be flexible linkers (e.g., linkers containing repeating sequences of glycine and serine residues), rigid linkers (e.g., linkers containing glutamic and lysine residues flanking alanine repeating sequences), and / or cleavable linkers (e.g., sequences susceptible to protease cleavage). Examples of such linkers are known to those skilled in the art and are described, for example, in Chen et al., (2013) *Advanced Drug Delivery Reviews*, 65: 1357-1369.

[0217] Useful linkers include glycine-serine (GlySer) linkers, which are well known in the art, and comprise glycine and serine units in various combinations in a sequence. Examples include, but are not limited to, (GS), (GSGGS)n (SEQ ID NO: 91), (GGGS)n (SEQ ID NO: 92), and (GGGGS)n (SEQ ID NO: 93), where n is an integer of at least one, typically from 1 to 10, such as from 1 to 8, from 1 to 6, or from 1 to 5.

[0218] In some embodiments, the peptide linker may comprise amino acids glycine and serine of various lengths and combinations. In some aspects, the peptide linker may comprise the sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS, SEQ ID NO: 92), or Gly-Gly-Gly-Gly-Ser (GGGGS, SEQ ID NO: 93), as well as variants or repeating sequences thereof. In some aspects, the peptide linker may comprise the amino acid sequence GGGGS (a linker of 6 amino acids in length, SEQ ID NO: 93) or even longer. The linker may be a series of repeating glycine and serine residues (GS) of varying lengths, i.e., (GS)n, where n is any number from 1 to 15 or greater. For example, the linker may be (GS)3 (i.e., GSGSGS, SEQ ID NO: 97) or longer (GS)11 or longer. It should be understood that n may be any number including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or greater. Fusion proteins having linkers of such lengths are included within the scope of this invention. Similarly, the linker can be a series of repeating glycine residues separated by serine residues. For example, (GGGGS)3 (i.e., the linker can contain the amino acid sequence GGGGSGGGGSGGGGS, (G4S)3, SEQ ID NO: 98) and its variants.

[0219] In one embodiment, the peptide linker may comprise the amino acid sequence GGGGS (a linker of 6 amino acids in length, SEQ ID NO: 93) or even longer. The linker may be a series of repeating glycine and serine residues (GS) of varying lengths, i.e., (GS)n, where n is any number from 1 to 15 or greater. For example, the linker may be (GS)3 (i.e., GSGSGS, SEQ ID NO: 97) or longer (GS)11 or longer. It should be understood that n may be any number including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or greater.

[0220] Other useful adapters include DSSG (SEQ ID NO: 94), DSSGAS (SEQ ID NO: 95), KLDSSG (SEQ ID NO: 96), and their variants. Examples of other suitable adapters are described in Chen et al., (2013) Advanced Drug Delivery Review, 65: 1357-1369.

[0221] Chimeric or fusion proteins and recombinant proteins

[0222] The chimeric or fusion proteins of the present invention can be prepared by any of a variety of conventional techniques, although recombinant techniques are typically used to prepare peptides.

[0223] For recombinant peptides, well-known molecular genetic techniques can be used to subclone the DNA fragment encoding the desired peptide into a suitable vector (see, for example, Maniatis et al., *Molecular Cloning: A Laboratory Manual*, 2nd edition (Cold Spring Harbor, 1982); Sambrook et al., *Molecular Cloning: A Laboratory Manual*, 2nd edition (Cold Spring Harbor, 1989). The fragment can be transcribed, and the peptide is subsequently translated in vitro. Commercially available kits can also be used (e.g., those prepared by Clontech, Palo Alto, California; Amersham Pharmacia Biotech Inc., Piscataway, NJ; InVitrogen, Carlsbad, California, etc.). Polymerase chain reaction can optionally be used to manipulate nucleic acids.

[0224] A “fragment” is a part of the polypeptide of the present invention that retains substantially similar functional activity or substantially the same biological function or activity as the polypeptide, which can be determined using the assays described herein.

[0225] Any amino acid sequence or functional equivalent of a polypeptide mentioned herein should be understood as comprising a polypeptide having substantially the same properties as the defined reference sequence. For example, a functional equivalent of a protein comprising the sequence of SEQ ID NO: 69 should be understood as a protein having less than 100% sequence identity with the sequence of SEQ ID NO: 69 (e.g., being at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to it), but comprising substantially the same ability as the protein of SEQ ID NO: 69, namely, to prevent or treat neuropathology, prevent the deposition of Porphyromonas gingivalis gingivalis protease or reduce its level in the neuronal tissue of a subject, delay the onset of Porphyromonas gingivalis-induced or associated neuropathology, or prevent or slow the deposition of abnormal proteins in the neuronal tissue of a subject.

[0226] As used herein, "to prevent or treat a specific condition with substantially the same capacity as the reference protein" should be understood to mean being able to treat or prevent a specific condition at a level of at least about 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the reference protein.

[0227] The "percentage of amino acid sequence identity (%)" or "percentage of identical amino acids (%)" for a polypeptide sequence (i.e., the polypeptide of the present invention as defined herein) is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific polypeptide of the present invention, after sequence alignment and the introduction of vacancies (if necessary) to achieve the maximum percentage of sequence identity, and without considering any conservative substitutions as part of sequence identity. A variant mentioned herein that has "at least x% sequence identity" with the stated sequence means that the variant is at least x% identical to the stated sequence.

[0228] In various aspects and embodiments of the invention, the defined polypeptide is described by reference to a variant having at least 80% homology or higher with a reference sequence. Homology percentage (%) generally refers to the polypeptide of the invention as defined herein, defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in a specific polypeptide of the invention, after sequence alignment and the introduction of vacancies (if necessary) to achieve the maximum sequence identity percentage, and without considering any conserved substitutions as part of sequence identity.

[0229] The amino acids glycine, alanine, valine, leucine, and isoleucine (amino acids with aliphatic side chains) can generally be substituted for each other. Among these possible substitutions, glycine and alanine are preferred to be substituted for each other (because of their relatively short side chains), and valine, leucine, and isoleucine are preferred to be substituted for each other (due to their larger hydrophobic aliphatic side chains). Other amino acids that can frequently be substituted for each other include: phenylalanine, tyrosine, and tryptophan (amino acids with aromatic side chains); lysine, arginine, and histidine (amino acids with basic side chains); aspartic acid and glutamic acid (amino acids with acidic side chains); asparagine and glutamine (amino acids with amide side chains); and cysteine ​​and methionine (amino acids with sulfur-containing side chains).

[0230] This type of substitution is often referred to as "conservative" or "semi-conservative" amino acid substitution.

[0231] Amino acid deletions or insertions can also be made relative to the native sequence of *Porphyromonas gingivalis* proteins. Thus, for example, amino acids that do not substantially affect the activity of the peptide, or at least do not eliminate such activity, can be deleted. Such deletions can be advantageous, especially for longer peptides, because the overall length and molecular weight of the peptide can be reduced while still maintaining its activity. This allows for a reduction in the amount of peptide required for a specific purpose, for example, by lowering the dosage level.

[0232] Amino acid insertions relative to the natural polypeptide sequence can also be performed. This can alter the properties of the polypeptide used in this invention (e.g., to enhance antigenicity).

[0233] Amino acid changes can be performed using any suitable technique, such as by using site-directed mutagenesis or solid-state synthesis.

[0234] It should be understood that amino acid substitutions or insertions within the scope of this invention can be made using naturally occurring or non-natural amino acids. Regardless of whether natural or synthetic amino acids are used, it is preferred that only L-amino acids are present.

[0235] Those skilled in the art can determine appropriate parameters for measuring alignments, including any algorithm required to achieve maximum alignment across the full length of the sequences being compared (non-limiting examples described below). When aligning amino acid sequences, the percentage of amino acid sequence identity between a given amino acid sequence A and (and, or against) a given amino acid sequence B (which may alternatively be expressed as a given amino acid sequence A having or containing a certain percentage of amino acid sequence identity with (and, or against) a given amino acid sequence B) can be calculated as: Amino acid sequence identity percentage = X / Y × 100, where X is the number of amino acid residues that are rated as identical matches by the alignment procedure or algorithm for A and B, and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the percentage of amino acid sequence identity between A and B will not be equal to the percentage of amino acid sequence identity between B and A.

[0236] When calculating the percentage of identity, exact matches are typically counted. The determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms. A preferred, non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm described in Karlin and Altschul (1990) Proceedings of the National Academy of Sciences (PNAS) 87:2264, as modified in Karlin and Altschul (1993) PNAS 90:5873-5877. Such algorithms are incorporated into the BLASTN and BLASTX procedures described in Altschul et al. (1990) Journal of Molecular Biology 215:403. To obtain vacancy-free alignments for comparative purposes, vacancy-free BLAST (in BLAST 2.0) can be used, as described below: Altschul et al. (1997) Nucleic Acids Research. 25:3389. Alternatively, PSI-Blast can be used for iterative retrieval to detect distance relationships between two molecules. See Altschul et al. (1997) Ibid. When using BLAST, vacancy-free BLAST, and PSI-Blast procedures, the default parameters of the respective procedures (e.g., BLASTX and BLASTN) can be used. Alignments can also be performed manually by inspection. Another non-limiting example of a mathematical algorithm for sequence comparison is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Research 22:4673-4680). ClustalW compares multiple sequences and aligns the entire amino acid or DNA sequence, and thus can provide data on sequence conservation for the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA / amino acid analysis software packages, such as the ALIGNX module of the Vector NTI program suite (Ingenie, Carlsbad, California). After aligning amino acid sequences using ClustalW, the percentage of amino acid identity can be assessed. Non-limiting examples of software programs useful for analysis using ClustalW alignments are GENEDOC™ or JalView (http: / / www.jalview.org / ). GENEDOC™ allows for the assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non-limiting example of a mathematical algorithm used for sequence comparison is the algorithm of Myers and Miller (1988), *Computer Applications in the Biosciences (CABIOS)*, 4:11-17.This type of algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When comparing amino acid sequences using the ALIGN program, the PAM 120 weighted residue table, vacancy length penalty 12, and vacancy penalty 4 can be used.

[0237] Peptides ideally contain both an amino terminus and a carboxyl terminus. Peptides may contain D-amino acids, L-amino acids, or a mixture of D-amino acids and L-amino acids. However, the D-form of the amino acid is particularly preferred because peptides containing D-amino acids are expected to retain a greater degree of their biological activity in vivo.

[0238] As used herein, the term "conservative substitution" refers to replacing an amino acid present in the natural sequence of a peptide with a naturally occurring or non-naturally occurring amino acid or a peptide mimic with similar stereochemical properties. If the side chain of the natural amino acid to be replaced is polar or hydrophobic, the conservative substitution should be the use of a naturally occurring amino acid, a non-naturally occurring amino acid, or a polar or hydrophobic peptide mimic moiety (other than having the same stereochemical properties as the side chain of the replaced amino acid).

[0239] Conservative amino acid substitutions that provide functionally similar amino acids are well known to those skilled in the art. The following six groups are examples of amino acids that can be considered as conserved substitutions for each other: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0240] Since naturally occurring amino acids are typically grouped according to their properties, conservative substitutions of naturally occurring amino acids can be determined, taking into account the fact that replacing a charged amino acid with a spatially similar, uncharged amino acid is considered a conservative substitution. To generate conservative substitutions from non-natural amino acids, amino acid analogs (synthetic amino acids) well-known in the art can also be used. Peptide analogs of naturally occurring amino acids are well documented in the literature known to those skilled in the art, and non-natural or unnatural amino acids are further described below. When influencing conservative substitutions, the substituted amino acid should have the same or similar functional groups in its side chain as the original amino acid.

[0241] As used herein, the phrase “non-conservative substitution” or “non-conservative residue” refers to the replacement of an amino acid present in a parent sequence with another naturally occurring or non-naturally occurring amino acid that has different electrochemical and / or steric properties. Therefore, the side chain of the substituted amino acid can be significantly larger (or smaller) than the side chain of the substituted natural amino acid and / or can have functional groups with significantly different electronic properties than the substituted amino acid. Examples of such non-conservative substitutions include the substitution of alanine with phenylalanine or cyclohexylmethylglycine, the substitution of glycine with isoleucine, or the substitution of aspartic acid with -NH-CH[(-CH2)5-COOH]-CO-. Non-conservative substitutions include any mutation that is not considered conserved.

[0242] Non-conservative amino acid substitutions can result from changes in: (a) the structure of the amino acid backbone in the substituted region; (b) the charge or hydrophobicity of the amino acid; or (c) a large portion of the amino acid side chain. Substitutions that are generally expected to produce the greatest changes in protein properties are: (a) a hydrophilic residue replacing (or being replaced by) a hydrophobic residue; (b) proline replacing (or being replaced by) any other residue; (c) a residue with a large side chain (e.g., phenylalanine) replacing (or being replaced by) a residue without a side chain (e.g., glycine); or (d) a residue with a positively charged side chain (e.g., lysyl, arginyl, or histidine) replacing (or being replaced by) an electronegative residue (e.g., glutamyl or aspartic).

[0243] Alterations to the natural amino acid sequence to produce mutant peptides (e.g., through insertion, deletion, and / or substitution) can be performed in a variety of ways known to those skilled in the art. For example, site-specific mutations can be introduced by ligating a synthetic oligonucleotide containing the modified site into an expression vector. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be used, such as those disclosed in Walder et al., *Gene* 42:133 (1986); Bauer et al., *Gene* 37:73 (1985); Craik, *Biotechniques*, 12-19 (January 1995); and U.S. Patent Nos. 4,518,584 and 4,737,462. A preferred method for introducing mutations is the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, California).

[0244] Any suitable expression vector (e.g., as described in Pouwels et al., *Cloning Vectors: A Laboratory Manual*, Elsevier, NY: 1985)) and a corresponding suitable host can be used to produce recombinant peptides. Expression hosts include, but are not limited to, *Escherichia coli* and *Bacillus*. Bacillus ), Pseudomonas spp. Pseudomonas Salmonella ( Salmonella Bacterial species, mammalian or insect host cell systems, including baculovirus systems (e.g., as described in Luckow et al., Bio / Technology 6:47 (1988)), and established cell lines such as COS-7, C127, 3T3, CHO, HeLa, and BHK cell lines. Those skilled in the art will understand that the choice of expression host influences the type of polypeptide produced. For example, the glycosylation of polypeptides produced in yeast or mammalian cells (e.g., COS-7 cells) will differ from that produced in bacterial cells (e.g., E. coli).

[0245] Alternatively, the polypeptides of the present invention can be synthesized using standard peptide synthesis techniques well known to those skilled in the art (e.g., summarized in Bodanszky, Principles of Peptide Synthesis (Springer-Verlag, Heidelberg: 1984)). Specifically, the polypeptides can be synthesized using solid-phase synthesis (see, for example, Merrifield, *Journal of the American Chemical Society* 85: 2149-54 (1963); Barany et al., *International Journal of Peptide and Protein Research* 30: 705-739 (1987); and U.S. Patent No. 5,424,398). Automated peptide synthesizers can be used if desired. For example, the removal of the tert-butyloxycarbonyl (t-BOC) or 9-fluorenylmethoxycarbonyl (Fmoc) amino acid blocking groups and the separation of the polypeptide from the resin can be accomplished by acid treatment at low temperature. The polypeptide-containing mixture can then be extracted, for example, with dimethyl ether to remove non-peptide organic compounds, and the synthesized polypeptide can be extracted from the resin powder (e.g., with about 25% w / v acetic acid). Following polypeptide synthesis, further purification (e.g., using high-performance liquid chromatography (HPLC)) can optionally be performed to eliminate any incomplete polypeptides or free amino acids. The synthesized polypeptide can be subjected to amino acid and / or HPLC analysis to verify identity. For other applications according to the invention, it may be preferred to produce the polypeptide as part of a larger fusion protein (such as by the methods described herein or other genetic means) or as part of a larger conjugate (such as by physical or chemical conjugation, as known to those skilled in the art and described herein).

[0246] In any embodiment of the invention, the chimeric or fusion protein of the invention may contain additional amino acid residues to facilitate expression in a recombinant expression system and / or to facilitate protein purification. Therefore, the protein as defined herein may include additional amino acids, such as one, two, three, four, or five amino acids in the N-terminal region. Typically, the additional amino acid will include an N-terminal methionine to facilitate expression in a recombinant expression system, although it should be understood that such N-terminal residues are typically cleaved post-translationally. In some embodiments, the N-terminal amino acid includes at least methionine and alanine residues.

[0247] Additionally, the chimeric or fusion proteins according to the invention may include additional amino acids, such as one, two, three, four, or five amino acids, in the N-terminal or C-terminal region, preferably to facilitate purification. It should be understood that such amino acid residues can facilitate the inclusion of purification tags (such as histidine tags) in the protein. Such residues may not be included when producing an untagged version of the protein.

[0248] The peptides of the present invention can also be modified, conjugated, or fused with another portion to facilitate purification, increase the in vivo half-life of the peptide, or be used in immunoassays using methods known in the art. For example, the peptides of the present invention can be modified by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, or linkage with cellular ligands or other proteins.

[0249] Nucleic acid

[0250] Nucleic acid molecules encoding any of the chimeric or fusion proteins or polypeptides of the present invention are also within the scope of the present invention. For example, nucleic acids can be used to prepare the polypeptides of the present invention and as therapeutic agents. The nucleic acids can be applied to cells in cultures or in vivo and can include secretion signals that direct or promote the secretion of the polypeptides of the present invention from cells. Expression vectors and host cells containing or including the nucleic acids of the present invention are also within the scope of the present invention (further described below). Although the nucleic acids of the present invention may be referred to as “isolated,” by definition, the polypeptides of the present invention are not wild-type polypeptides and therefore are not encoded by naturally occurring nucleic acids. Therefore, while the polypeptides and nucleic acids of the present invention may be “purified,” “substantially purified,” “isolated,” “recombinant,” or “synthetic,” this is not necessary to distinguish them from naturally occurring materials.

[0251] "Isolated" nucleic acid molecules are those identified and separated from at least one contaminant nucleic acid molecule typically associated with the natural source of nucleic acids. Isolated nucleic acid molecules differ from those found in nature or in their environment. Therefore, isolated nucleic acid molecules are distinct from those present in natural cells. However, isolated nucleic acid molecules include those typically found in cells expressing Kgp, wherein, for example, the nucleic acid molecule is located at a chromosomal location different from its position in natural cells.

[0252] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, namely deoxyribonucleotides or ribonucleotides, or similar substances. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotides of the present invention may be provided in isolated or purified form. The nucleic acid sequence “encoding” a selected polypeptide is a nucleic acid molecule that, when placed under the control of appropriate regulatory sequences, is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo. The boundaries of the coding sequence are defined by a start codon at the 5' (amino) end and a translation stop codon at the 3' (carboxyl) end. For the purposes of this invention, such nucleic acid sequences may include, but are not limited to, cDNA from viral, prokaryotic, or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and even synthetic DNA sequences. The transcription termination sequence may be located at the 3' end of the coding sequence.

[0253] The polynucleotides of the present invention can be synthesized according to methods well known in the art, as described by way of example in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Press).

[0254] The polynucleotide molecules of the present invention can be provided in the form of expression cassettes, which include control sequences operatively linked to an insert sequence, thereby allowing the polypeptides of the present invention to be expressed in vivo in targeted subjects. In turn, these expression cassettes are typically provided within a vector (e.g., a plasmid or recombinant viral vector) suitable for use as a reagent for nucleic acid immunization. Such expression cassettes can be administered directly to a host subject. Alternatively, a vector containing the polynucleotides of the present invention can be administered to a host subject. Preferably, the polynucleotides are prepared and / or administered using a genetic vector. A suitable vector can be any vector capable of carrying a sufficient amount of genetic information and allowing expression of the polypeptides of the present invention.

[0255] Therefore, the present invention includes expression vectors comprising such polynucleotide sequences. Thus, the present invention provides a vector for the prevention or treatment of inflammatory diseases or symptoms, the vector comprising a polynucleotide sequence encoding a polypeptide of the present invention and optionally one or more additional polynucleotide sequences encoding different polypeptides as defined herein.

[0256] Furthermore, it should be understood that the compositions and products of the present invention may comprise a mixture of polypeptides and polynucleotides. Therefore, the present invention provides a composition or product as defined herein, wherein any one of the polypeptides is replaced with a polynucleotide capable of expressing the polypeptide.

[0257] Expression vectors are routinely constructed in the field of molecular biology and may involve, for example, the use of plasmid DNA and appropriate initiators, promoters, enhancers, and other elements, such as polyadenylation signals, which may be necessary and correctly oriented to allow expression of the peptides of the present invention. Other suitable vectors will be apparent to those skilled in the art. As another example in this regard, see Sambrook et al.

[0258] Therefore, the polypeptides of the present invention can be provided by delivering such vectors into cells and allowing transcription from the vectors. Preferably, the polynucleotides of the present invention, or those used in the vectors in the present invention, are operatively linked to a control sequence capable of providing expression of the coding sequence carried out by the host cell, i.e., the vector is an expression vector.

[0259] "Operationally linked" refers to an arrangement of elements in which the components described herein are configured to perform their usual functions. Therefore, in the presence of a suitable enzyme, a given regulatory sequence (such as a promoter) operably linked to a nucleic acid sequence can influence the expression of that sequence. The promoter need not be adjacent to the sequence, as long as it serves to guide its expression. Thus, for example, an intermediate untranslated but still transcribed sequence can exist between the promoter sequence and the nucleic acid sequence, and the promoter sequence and coding sequence can still be considered "operationally linked."

[0260] Various expression systems have been described in the art, each typically consisting of a vector containing a gene or nucleotide sequence of interest operatively linked to expression control sequences. These control sequences include transcription promoter sequences and transcription initiation and termination sequences. The vectors of the present invention can be, for example, plasmids, viral or phage vectors, provided with an origin of replication, a promoter optionally for the expression of the polynucleotide, and optionally a regulatory factor for the promoter. A “plasmid” is a vector in the form of an extrachromosomal genetic element. The vector may contain one or more optional marker genes, such as an ampicillin resistance gene in a bacterial plasmid or a resistance gene in a fungal vector. The vector can be used in vitro, for example, for the production of DNA or RNA, or for transfection or transformation of host cells, such as mammalian host cells. The vector can also be adapted for in vivo use, for example, to allow in vivo expression of polypeptides.

[0261] A promoter is a nucleotide sequence that initiates and regulates the transcription of a polynucleotide encoding a polypeptide. Promoters can include inducible promoters (where the expression of a polynucleotide sequence operatively linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressive promoters (where the expression of a polynucleotide sequence operatively linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. The terms "promoter" or "control element" are intended to include both the full-length promoter region and the functional (e.g., controlling transcription or translation) segments of these regions.

[0262] The polynucleotides, expression cassettes, or vectors according to the invention may additionally include a signal peptide sequence. The signal peptide sequence is typically inserted in a manner operatively linked to a promoter such that the signal peptide is expressed and promotes the secretion of a polypeptide encoded by a coding sequence, which is also operatively linked to the promoter.

[0263] Typically, signal peptide sequences encode 10 to 30 amino acids, such as peptides of 15 to 20 amino acids. These amino acids are usually predominantly hydrophobic. Under normal circumstances, the signal peptide targets the growth polypeptide chain carrying the signal peptide to the endoplasmic reticulum (ER) of the expressing cell. The signal peptide is cleaved in the ER, allowing the polypeptide to be secreted via the Golgi apparatus.

[0264] Immunogenicity and vaccine composition

[0265] The present invention further provides compositions comprising chimeric or fusion proteins as defined herein, and the use of such compositions and chimeric or fusion proteins in immunogenic or vaccine compositions for the treatment or prevention of neuropathology induced or associated with Porphyromonas gingivalis infection.

[0266] As used herein, the term "vaccine composition" is defined as a composition intended to induce an immune response against an antigen (immunogen) within the composition to protect or treat an organism from disease. Therefore, compositions used according to the present invention may also be referred to as "immunostimulatory compositions."

[0267] As used herein, the terms “immunostimulatory composition,” “vaccine composition,” and “immunogenic composition” are generally used interchangeably.

[0268] In addition to one or more peptides of the present invention as therapeutic or prophylactic active ingredients, the compositions or vaccines of the present invention may suitably include pharmaceutically acceptable carriers, excipients, diluents, adjuvants, mediators, buffers, or stabilizers. Such carriers include, but are not limited to, saline, buffered saline, dextran, liposomes, water, glycerol, polyethylene glycol, ethanol, and combinations thereof.

[0269] Immunostimulatory compositions or vaccine compositions may be adapted for administration via any suitable route, such as parenteral (including subcutaneous, intramuscular, intravenous, or intradermal, or by injection into cerebrospinal fluid), oral (including buccal or sublingual), nasal, local (including buccal, sublingual, or transdermal), vaginal, or rectal routes. Such compositions may be prepared by any method known in the pharmaceutical field, such as by mixing peptides with carriers or excipients under sterile conditions. Typically, vaccine compositions are adapted for administration via subcutaneous, intramuscular, intravenous, or intradermal routes, and are usually administered by injection. Alternatively, vaccine compositions may be adapted for oral or nasal administration.

[0270] Immunostimulatory compositions or vaccine compositions suitable for parenteral administration can be aqueous and non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickeners. Excipients that can be used in injectable solutions include, for example, water, alcohols, polyols, glycerol, and vegetable oils. The compositions can be present in single-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored under lyophilized (freeze-dried) conditions where a sterile liquid carrier, such as water for injection, is only required to be added immediately before use. Temporary injectable solutions and suspensions can be prepared from sterile powders, granules, and tablets.

[0271] Immunostimulatory or vaccine compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, or lozenges; as powders or granules; as solutions, syrups, or suspensions (in aqueous or non-aqueous liquids; or as edible foams or pastes; or as emulsions).

[0272] Suitable excipients for use in tablets or hard gelatin capsules include lactose, corn starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients for use with soft gelatin capsules include, for example, vegetable oils, waxes, fats, semi-solid or liquid polyols.

[0273] For the preparation of solutions and syrups, excipients that can be used include, for example, water, polyols, and sugars. For the preparation of suspensions, oils (e.g., vegetable oils) can be used to provide oil-in-water or water-in-oil suspensions.

[0274] Where the carrier is a solid, suitable immunostimulatory or vaccine composition for nasal administration comprises coarse powder, for example, in the particle size range of 20 to 500 micrometers, which is administered by sniffing, i.e., rapidly inhaled through the nasal passages from a powder container near the nose. Where the carrier is a liquid, suitable compositions for administration as nasal sprays or drops may contain an aqueous or oil solution of the active ingredient.

[0275] Compositions suitable for inhalation administration include fine particulate dust or mist, which can be generated by various types of metered-dose pressurized aerosols, nebulizers, or blowers.

[0276] Immunostimulatory or vaccine compositions suitable for transdermal administration can be presented as discrete patches designed to maintain close contact with the recipient's epidermis for extended periods. For example, the active ingredient can be delivered from the patch via iontophoresis, as generally described in Pharmaceutical Research 3(6):318 (1986).

[0277] Compositions suitable for topical application can be formulated as ointments, creams, suspensions, lotions, powders, solutions (e.g., mouthwashes), pastes, gels, sprays, aerosols, or oils. For infections of the eyes or other external tissues (e.g., the mouth and skin), the composition can be applied as a topical ointment or cream. When formulated into an ointment, the active ingredient can be used with a paraffin-based ointment base or a water-miscible ointment base. Alternatively, the active ingredient can be formulated into a cream with an oil-in-water or water-in-oil base. Pharmaceutical compositions suitable for topical application to the eyes can comprise eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier, particularly an aqueous solvent. Pharmaceutical compositions suitable for topical application in the oral cavity can comprise lozenges, soft lozenges, or mouthwashes.

[0278] Immunostimulatory or vaccine compositions may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, salts (the substances of the present invention may be provided in the form of pharmaceutically acceptable salts), buffers, coating agents, or antioxidants.

[0279] In addition to chimeric or fusion proteins as defined herein, the vaccine compositions of the present invention may also contain one or more other prophylactic or therapeutic active agents.

[0280] The chimeric or fusion proteins used in the vaccine compositions of the present invention may or may not be lyophilized.

[0281] In addition to peptides as defined herein, the vaccine compositions of the present invention may also include pharmaceutically acceptable adjuvants. Adjuvants are added to enhance the immunogenicity of the vaccine composition.

[0282] Suitable adjuvants for inclusion in vaccine compositions are known in the art and include incomplete Freund's adjuvant, complete Freund's adjuvant, Freund's adjuvant with MDP (muramyldipeptide), alum (aluminum hydroxide), alum plus Bordatella pertussis and immunostimulatory complex (ISCOM, typically a matrix containing Quil A viral protein), QS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, alethine, MPC-026, Adjuvax, CpG ODN, betafectin, and MF59.

[0283] The vaccine composition of the present invention may also include one or more co-stimulatory molecules or be administered therewith.

[0284] The dosage of the vaccine composition of the present invention can vary within a range, depending on the age and condition of the individual to be treated, and the physician will ultimately determine the appropriate dosage to be used.

[0285] This dose can be repeated at an appropriate frequency. For example, an initial dose of vaccine can be administered, followed by a booster dose at a later date.

[0286] For administration to mammals (particularly humans), the expected daily dose of the active agent will be from 1 μg / kg to 10 mg / kg body weight, typically from about 10 μg / kg to 1 mg / kg body weight. In any case, the physician will determine the most suitable actual dose for the individual based on factors including age, weight, sex, and response. The above doses are examples of general cases. Of course, there may be situations where higher or lower doses are justified, and such dose ranges are within the scope of this invention.

[0287] The vaccine compositions of the present invention can be administered by any convenient route as described herein, such as intramuscular, intravenous, inhalation, intraperitoneal, or oral routes, or by injection into the cerebrospinal fluid.

[0288] The vaccine compositions of the present invention may be provided in unit dosage forms, typically in sealed containers, and may be provided as part of a kit. Such kits typically (though not necessarily) include instructions for use. They may include various of the aforementioned unit dosage forms.

[0289] Therefore, in another aspect, the present invention provides a kit comprising the vaccine composition of the present invention and one or more cytokines and / or adjuvants in a sealed container.

[0290] Neuropathology induced or associated with Porphyromonas gingivalis

[0291] The present invention provides methods and compositions for treating or preventing neuropathology associated with, caused by, or induced by Porphyromonas gingivalis infection, or for delaying its onset.

[0292] Furthermore, the present invention provides a method for reducing the level of Porphyromonas gingivalis gingival proteases (such as RgpA and / or KgpA) in the neuronal tissue (e.g., brain tissue) of a subject.

[0293] The present invention also relates to a method for reducing neuroinflammation in a subject, wherein the neuroinflammation may be caused by or related to Porphyromonas gingivalis infection.

[0294] Neuropathology induced or associated with Porphyromonas gingivalis can include cognitive decline, neurocognitive impairment, or pathology causing physical or chemical changes in neuronal tissue; or neurodegenerative disorders as further defined herein.

[0295] In any embodiment, the chemical change may include inflammation in neurogenic tissue.

[0296] In any respect, the neurodegenerative symptoms may be characterized by the presence of abnormal protein deposits in the brain, including amyloidosis, synucleinosis, or tau disease. Neurodegenerative symptoms or conditions may include Alzheimer's disease (AD), Lewy body disease (Lewy body dementia (DLB)), Huntington's disease, Creutzfeldt-Jakob disease (CJD), Gaucher disease type 3, or Parkinson's disease. Neurodegenerative diseases may be vascular dementia, frontotemporal dementia, or other forms of dementia that are not typically associated with the deposition of abnormal protein deposits.

[0297] As used herein, cognitive impairment or neurocognitive impairment (NCD) refers to mental health and / or neurological disorders that primarily affect cognitive abilities, including learning, memory, perception, and / or problem-solving. Neurocognitive impairment includes delirium as well as mild and severe neurocognitive impairment (formerly known as dementia). It is defined as an acquired deficit of cognitive ability (rather than a developmental deficit), typically representing a decline in cognitive abilities and possibly with underlying brain lesions. The DSM-5 defines six key areas of cognitive function: executive function, learning and memory, sensorimotor function, language, complex attention, and social cognition.

[0298] Although Alzheimer's disease accounts for the majority of neurocognitive impairment cases, there are various medical conditions that affect mental functions such as memory, thinking, and reasoning abilities, including frontotemporal degeneration, vascular dementia, Huntington's disease, Lewy body disease, traumatic brain injury (TBI), Parkinson's disease, prions, and dementia / neurocognitive problems caused by viral infections.

[0299] Neurocognitive impairment is classified as mild or severe based on the severity of symptoms. Skilled personnel will be familiar with standard methods for identifying subjects at risk of dementia or related neurocognitive impairment, or for diagnosing subjects with neurodegenerative diseases, including any of the methods described herein.

[0300] Similarly, skilled personnel will be familiar with standard methods used to identify whether a patient has an intellectual disability, such as those related to the neurocognitive disorders described in this article. Such methods may include a variety of behavioral and cognitive tests.

[0301] For example, in the case of neurodegenerative diseases, although there are currently no definitive diagnostic methods for many such conditions (including AD), those skilled in the art should be familiar with collecting information to predict possible diagnostic outcomes, thereby identifying patients suitable for treatment according to the present invention. For example, the diagnosis of AD typically involves collecting the following information: ● Complete medical history and family medical history; ● Basic medical examinations (blood and urine tests; assessment of biomarkers in blood or cerebrospinal fluid); ● Neuropsychological and intellectual function tests (e.g., memory tests, problem-solving ability tests, language ability tests, Mini-Mental State Examination (MMSE)); ● Interview with family members to gain a comprehensive understanding of the patient's behavior; ● Perform medical imaging of the brain (e.g., MRI, CT, SPECT, or PET scans) to identify areas containing plaques, rule out other brain lesions and dementia subtypes, and predict the transition from the prodromal stage (mild cognitive impairment) to AD; ● Genetic factors (family history of AD); ● Environmental factors (exposure to aluminum, zinc and other metals).

[0302] In the context of this invention, further testing to determine the likelihood or risk of *Porphyromonas gingivalis*-induced or associated neuropathology may include a diagnosis of *Porphyromonas gingivalis* infection. This can be achieved by detecting the presence of detectable levels of *Porphyromonas gingivalis* in the subject's saliva. Such tests are well known to those skilled in the art and may include various methods using polymerase chain reaction (PCR), such as qPCR, RT-PCR, and ddPCR, to detect *Porphyromonas gingivalis* DNA and / or RNA in saliva, on the tongue, in periodontal pockets (including subgingival pockets), or in other parts of the oral cavity. Other detection methods may include lateral flow detection using antibodies and antigen detection, as described in O'Brien-Simpson et al., 2017, which is incorporated herein by reference. Examples of PCR / qPCR-based methods are described in WO 2020 / 069397, which is also incorporated herein by reference. In other known examples of saliva tests, the BANA test is commercially available in the dental field for testing for proteases of *Porphyromonas gingivalis* and other oral bacteria. The BANA test is a small plastic card with two separate reagent substrates, shown as strips on the card. The lower white reagent substrate is impregnated with N-benzoyl-DL-arginine-β-naphthylamide (BANA). A subgingival plaque sample is applied to the lower substrate, and then distilled water is applied to the upper substrate. The lower substrate is then folded back to contact the upper substrate. The upper buffer reagent substrate contains a chromogenic diazo reagent, which reacts with one of the hydrolysis products of the enzyme reaction to produce a blue color. The reaction occurs when the plastic strip is inserted into an incubator set to 35 degrees Celsius for 5 minutes. The BANA substrate detects at least three different oral bacteria, but not specifically *Porphyromonas gingivalis*. The BANA test can be used to identify individuals at risk of *Porphyromonas gingivalis*-induced or associated neuropathology, or those eligible for treatment according to the invention. Alternatively, BANA substrates can be replaced with RgpA, RgpB, and / or Kgp-specific substrates in similar forms or liquid assays. Reagents that bind to active gingival proteases are known in the art, including, as examples in WO 2017 / 083433, as non-limiting.

[0303] In another embodiment, determining the likelihood of Porphyromonas gingivalis-induced or associated neurodegenerative or neurocognitive symptoms may include a history of Porphyromonas gingivalis infection.

[0304] The National Institute of Neurological Disorders and Stroke (NINCDS) and the Alzheimer's Disease and Related Disorders Association (ADRDA, now known as the Alzheimer's Association) established the most commonly used NINCDS-ADRDA diagnostic criteria for Alzheimer's disease in 1984, and it was extensively updated in 2007. These criteria require the confirmation of cognitive impairment and suspected dementia syndrome through neuropsychological testing in order to make a clinical diagnosis of possible or probable AD. Definitive diagnosis requires histopathological confirmation, including microscopic examination of brain tissue. Good statistical reliability and validity have been demonstrated between the diagnostic criteria and final histopathological confirmation. The eight intellectual domains most commonly impaired in AD are: memory, language, perception, attention, motor skills, orientation, problem-solving, and executive function. These domains are equivalent to the NINCDS-ADRDA Alzheimer's disease criteria listed in the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR) published by the American Psychiatric Association.

[0305] Alzheimer's disease (AD) progresses through several stages, ranging from mild amnesia to severe dementia. The course and rate of progression of the disease vary from person to person. The duration from symptom onset to death can range from 5 to 20 years.

[0306] Early symptoms of dementia are often subtle and may not appear immediately. Some common symptoms may include: ● Gradual and frequent memory loss ● Chaos ● Personality changes ● Indifference and withdrawal ● Loss of the ability to complete daily tasks.

[0307] The initial symptoms of Alzheimer's disease (AD) (pre-dementia) are often mistaken for aging or stress. Detailed neuropsychological testing can reveal mild cognitive difficulties, which can be detected as early as eight years before a person meets the clinical diagnostic criteria for AD. These early symptoms can affect the most complex activities of daily life. The most obvious deficit is short-term memory loss, manifested as difficulty remembering recently learned facts and an inability to acquire new information.

[0308] Subtle problems with executive functions such as attention, planning, flexibility, and abstract thinking, or impaired semantic memory (memory of meaning and conceptual relationships), may also be symptoms of the early stages of Alzheimer's disease (AD). Apathy can be observed at this stage and is the most persistent neuropsychiatric symptom throughout the disease. Depressive symptoms, irritability, and a decline in the ability to perceive mild memory difficulties are also common symptoms. The preclinical stage of the disease is also known as mild cognitive impairment (MCI). This is often considered a transitional phase between normal aging and dementia. MCI can present with a variety of symptoms, and when memory loss is the primary symptom, it is called "amnestic MCI" and is often considered a prodromal phase of Alzheimer's disease.

[0309] For people with Alzheimer's disease (AD), a gradual decline in learning and memory abilities eventually leads to a diagnosis. In rare cases, difficulties with language, executive function, perception (agnosia), or motor execution (apraxia) are more prominent than memory problems. AD affects all memory capacities differently. Earlier life memories (epistemological memories), learned facts (semantic memories), and implicit memories (memories of how the body does things, such as eating with a fork or drinking from a cup) are less affected than new facts or memories.

[0310] The main characteristics of language problems are a reduced vocabulary and decreased fluency, leading to a general decline in spoken and written language abilities. In the early stages, patients with Alzheimer's disease are usually able to express basic concepts adequately. They may experience some coordination and planning difficulties (apraxia) when performing fine motor tasks such as writing, drawing, or dressing, but these difficulties are often unnoticed. As the disease progresses, patients with AD can usually continue to complete many tasks independently, but may require assistance or supervision in activities that demand the highest cognitive abilities.

[0311] As the condition gradually worsens, it eventually hinders the patient's ability to live independently, rendering them unable to perform most common daily activities. The inability to recall vocabulary leads to significant speech difficulties, resulting in frequent mispronunciation (mapping). Reading and writing abilities also gradually diminish. With the passage of time and the progression of AD, coordination of complex motor sequences declines, increasing the risk of falls. At this stage, memory problems worsen, and the patient may be unable to recognize close relatives. Previously intact long-term memories are impaired.

[0312] As neurodegenerative / neurocognitive decline progresses, behavioral and neuropsychiatric changes become more prevalent. Common manifestations include wandering, irritability, and mood swings, leading to crying, unplanned outbursts of provocative behavior, or resistance to care. Evening syndrome may also occur. Approximately 30% of patients with Alzheimer's disease experience illusions and other delusional symptoms. Subjects may also lose awareness of their own disease progression and limitations (agnosia). Urinary incontinence may occur. These symptoms can be stressful for family members and caregivers, and transferring patients from home care to other long-term care facilities can alleviate this stress.

[0313] This invention can also be used to improve or slow the decline in cognitive function in subjects with or suspected of having Lewy body dementia. Lewy body dementia (DLB) is a type of dementia characterized by visual hallucinations and symptoms resembling "Parkinson's disease." Parkinson's disease is characterized by symptoms including tremors, muscle rigidity, and a blank facial expression. Visual hallucinations in DLB are typically vivid images of people or animals, and these hallucinations often occur when the patient is about to fall asleep or has just woken up. Other prominent symptoms include problems with attention, organization, problem-solving, and planning (executive function), as well as difficulties with visuospatial function.

[0314] While imaging studies cannot always diagnose DLB, some signs are particularly common. Individuals with DLB typically show occipital lobe hypoperfusion on SPECT scans or decreased occipital lobe metabolism on PET scans. Generally, diagnosing DLB is relatively straightforward, and brain scans are not always necessary unless the condition is complex.

[0315] In a preferred embodiment of the invention, the cognitive impairment is caused by, or attributed to, Alzheimer's disease. In another embodiment, the cognitive impairment is caused by, or attributed to, mild cognitive impairment (MCI). In yet another embodiment, the cognitive impairment is caused by, or attributed to, dementia.

[0316] As used herein, the terms “treat symptoms associated with cognitive decline or loss” or “counteract cognitive decline” or “treat cognitive impairment” or “delay the progression of cognitive impairment” or “improve cognitive function” or “counteract cognitive decline” as used throughout this specification mean promoting cognitive function (making the subject’s impaired cognitive function closer to that of a similarly age-matched normal, unimpaired subject, including a state of cognitive decline compared to normal subjects) and maintaining cognitive function (ensuring that normal or impaired cognitive function does not decline, or is not lower than the cognitive function observed at the subject’s first visit or diagnosis, for example, reaching the expected decline without treatment).

[0317] In one embodiment of the invention, the subject receiving treatment has normal cognitive function, which is improved after administration of the chimeric or fusion protein according to the methods currently described.

[0318] In another embodiment, subjects exhibited age-related cognitive impairment before treatment but showed improvement in cognitive function after treatment.

[0319] This invention also provides methods and compositions for inducing a humoral immune response against *Porphyromonas gingivalis* in a subject. The humoral response may be for the purpose of directly obtaining protective / therapeutic antibodies against *Porphyromonas gingivalis* in an individual requiring protection / therapy. For example, for subjects considered at risk of *Porphyromonas gingivalis*-induced or associated neuropathology or dementia, the compositions and proteins described herein may be provided to obtain a protective effect against *Porphyromonas gingivalis*-induced or associated neuropathology or dementia.

[0320] As used herein, the term "treatment" or "treating" a subject includes the application or administration of the compositions of the present invention (or the application or administration of proteins as described herein to cells or tissues derived from the subject) to a subject for the purpose of delaying, slowing, stabilizing, curing, healing, alleviating, relieving, altering, remedying, reducing the deterioration of a disease or condition, its symptoms, or its risk (or susceptibility). The term "treatment" refers to any indicator of successful treatment or improvement of an injury, pathology, or condition, including any objective or subjective parameter such as reduction; relief; reduction of the rate of deterioration; reduction of the severity of the disease; stabilization, reduction of symptoms, or making the injury, pathology, or condition more tolerable for the subject; slowing the rate of deterioration or decline; making the late stages of deterioration less debilitating; or improving the physical or mental health of the subject.

[0321] As used herein, “preventing” or “prevention” means at least reducing the likelihood of acquiring a disease or condition (i.e., preventing a subject who may be exposed to or susceptible to a disease but has not yet experienced or displayed symptoms of the disease from developing at least one of the clinical symptoms of the disease). This document provides biological and physiological parameters for identifying such subjects, and these parameters are well known to physicians. For example, preventive or preventative measures may be taken for patients considered at risk, such as those diagnosed with dementia or other cognitive or neurodegenerative conditions or other conditions described herein.

[0322] The vaccine compositions of the present invention can be administered to subjects who need them most, such as children or the elderly or individuals at risk of exposure to *Porphyromonas gingivalis* in the context of human patients. The vaccine compositions of the present invention can also be administered to subjects suspected of having or diagnosed with *Porphyromonas gingivalis* infection (e.g., determined according to standard methods for determining *Porphyromonas gingivalis* infection, such as using saliva-based tests or signs or symptoms of one or more periodontal diseases).

[0323] In any embodiment, the method of the present invention may comprise administering a chimeric or fusion protein as described herein to a subject who is believed to have or is suspected of having an age-related neurogenic disease (such as dementia) or a neurodegenerative condition (such as Alzheimer's disease or any other neurodegenerative disease as disclosed herein). In such embodiments, the method may help prevent the further development of neurogenic or neurodegenerative conditions (e.g., by reducing or preventing the accumulation of gingival proteases or other abnormal protein deposits in neurogenic tissues).

[0324] The compositions and methods of the present invention are equally extended to use in both human medicine and / or veterinary medicine, in the production of diagnostic agents or other therapeutic agents.

[0325] As used herein, the term "subject" should be understood to mean any animal, including humans, such as mammals. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, a subject can be a human. In other instances, a subject can be a veterinary subject, such as a companion animal (cat, dog, guinea pig, etc.).

[0326] As used herein, the terms “subject,” “individual,” and “patient” are used interchangeably.

[0327] Those skilled in the art will be familiar with methods for determining successful vaccination / immunization with chimeric or fusion proteins or compositions as described herein. For example, those skilled in the art will be familiar with methods for quantifying antibodies produced after immunization and / or for quantifying the extent of humoral (Th2) responses induced after immunization.

[0328] Just as those skilled in the art will be familiar with the methods for identifying subjects requiring treatment according to the present invention, they will also be able to determine whether the treatment was successful. Standard techniques known to those skilled in the art can be used to determine whether treatment with the chimeric or fusion protein of the present invention was successful. For example, in determining whether treatment with the protein described herein was successful in treating neuropathology (such as symptoms of cognitive impairment or neurodegenerative diseases), those skilled in the art can utilize standard cognitive tests to determine whether cognitive abilities have improved, or whether there are other signs of neuropathology induced by one or more *Porphyromonas gingivalis* species.

[0329] In some embodiments, “improvement” may refer to a decrease in the scale or subscale score of the Neuropsychiatric Scale for Nursing Homes (NPI-NH) or the Alzheimer’s Disease Collaborative Study-Clinical Global Impression Change (ADSC-CGIC). For example, improvement may refer to a decrease in a patient’s total NPI-NH score from 50 to 40. In some embodiments, the improvement may optionally refer to one or more patients.

[0330] As used herein, the terms “improvement,” “improved,” and “improves” in relation to the clinical setting refer to a clinically relevant effect that, after a specified time period, represents a change in baseline of greater than approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 200%, 300%, 400%, or 500%. In some embodiments, improvement refers to a significant improvement in efficacy in a single patient after administration of the protein, compared to baseline (i.e., prior to administration of the chimeric or fusion protein described herein). In other embodiments, improvement refers to a higher percentage of patients exhibiting efficacy after a specified time period compared to placebo or no treatment, thus demonstrating therapeutic efficacy. In various embodiments, the percentage of patients demonstrating an effective effect increased by more than about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 200%, 300%, 400%, or 500% compared to placebo or no treatment. In some embodiments, the specific time period is about two weeks, four weeks, or six weeks. In one embodiment, the specific time period is six weeks.

[0331] As used herein, the terms “reduction,” “reduced,” and “reduces” in relation to the clinical setting refer to a clinically relevant effect of less than approximately 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 200%, 300%, 400%, or 500% compared to baseline or to placebo or no treatment after a specified time period. In some embodiments, the specified time period is approximately two weeks, four weeks, or six weeks. In one embodiment, the specified time period is six weeks.

[0332] In any embodiment, the method of the present invention further includes treating the subject as described herein with one or more adjunctive therapies. In some instances, the adjunctive therapy includes treatment to reduce the Porphyromonas gingivalis load in the subject's oral cavity. For example, in some embodiments, the adjunctive therapy may include subgingival scaling and root planing (SRP) to remove the Porphyromonas gingivalis oral biofilm from the subject, antibiotic treatment to reduce bacterial load / infection, or anti-inflammatory drugs to reduce swelling and inflammation. The adjunctive therapy may also include therapies or treatments targeting neuropathology, such as treatment for dementia, neurodegenerative diseases, cognitive decline, or abnormal protein deposits in the brain, including amyloidosis, synucleinosis, or tau proteinosis. In any embodiment, the adjunctive therapy may include therapies or treatments targeting Alzheimer's disease (AD), Lewy body disease (Lewy body dementia (DLB)), Huntington's disease, Creutzfeldt-Jakob disease (CJD), Gaucher disease type 3, or Parkinson's disease.

[0333] Reagent test kit

[0334] In another embodiment, a kit or article is provided that includes one or more proteins, peptides, or polynucleotides available for use according to the invention in any of the methods described above.

[0335] In another aspect, the present invention provides a kit comprising a vaccine composition for use according to the method of the present invention and one or more adjuvants for administration to subjects alone, subsequently or simultaneously.

[0336] In other embodiments, a kit is provided for use in the above-described therapeutic or preventative applications, the kit comprising: - A container for containing the protein, polypeptide, polynucleotide, or immunogenic composition of the present invention; - Includes a label or packaging insert with an instruction manual.

[0337] In any embodiment, the kit may contain one or more additional active principles or active ingredients for inducing an immune response against Porphyromonas gingivalis in a subject.

[0338] The kit or "article" may include a container and a label or packaging insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. The container may be formed from a variety of materials, such as glass or plastic. The container contains a therapeutic composition for the effective treatment of a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial with a stopper that can be punctured by a hypodermic needle). The label or packaging insert indicates that the therapeutic composition is intended for the treatment of the selected condition. In one embodiment, the label or packaging insert includes instructions for use and indicates that the therapeutic or prophylactic composition may be used to treat the inflammatory disease or condition described herein.

[0339] The kit may comprise (a) a therapeutic or prophylactic composition; and (b) a second container containing a second active principle or active ingredient. The kit in this embodiment of the invention may further include a packaging insert indicating that the composition and other active ingredients can be used to treat a condition or prevent complications arising from the inflammatory disease or condition described herein. Alternatively or additionally, the kit may further comprise a second (or third) container containing a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextran solution. It may further include other materials desired from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0340] In any embodiment, the therapeutic composition may be provided in the form of a disposable or reusable device, including a container for containing a therapeutic, prophylactic, or immunogenic composition. In one embodiment, the device is a syringe, an autoinjector, or a nanopatch. The device may contain 0.1 to 2 mL of the therapeutic or immunogenic composition. The therapeutic or prophylactic composition may be provided in the device in a state ready for use or in a state requiring mixing, dissolution, resuspension, or the addition of additional components.

[0341] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more features mentioned or apparent in the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0342] The following examples are described to more fully illustrate some embodiments of the invention. However, these examples should not in any way be construed as limiting the broad scope of the invention. Those skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0343] Example

[0344] The following examples describe a series of in vitro and in vivo studies related to the chimeric or fusion proteins used in accordance with the present invention.

[0345] Example 1 describes the materials and methods used in the studies described in Examples 2 and 3, which respectively describe the results of in vitro and in vivo studies related to the development and initial characterization of chimeric or fusion proteins.

[0346] Examples 4 and 5 involve follow-up experiments related to establishing a mouse model of Porphyromonas gingivalis-induced neuropathology and testing chimeric proteins in prevention studies.

[0347] Example 6 involves the testing of chimeric proteins in a preliminary therapeutic model of neuropathology induced by Porphyromonas gingivalis.

[0348] Example 1: Materials and methods for the initial development and characterization of chimeric or fusion proteins for generating immune responses against Porphyromonas gingivalis.

[0349] 1.1 Materials and methods for in vitro studies

[0350] Expression of recombinant proteins

[0351] Recombinant proteins (i.e., the various chimeric or fusion proteins as described herein) are expressed from the pET28 vector (or pDUET-1 co-expressed from a vector of rABM1 + rABM2) by induction with isopropyl β-D-thiogalactosidase (IPTG), as follows.

[0352] Using *Porphyromonas gingivalis* W50 genomic DNA templates and specially designed oligonucleotide primers, nucleic acids encoding different chimeras and chimeric components, as well as fusion proteins (including active sites and adhesin domains), were generated via standard PCR or DNA overlap extension splicing (“SOEing”) PCR. The PCR fragments or SOEing PCR fragments were purified and ligated into cloning vectors pGEMTeasy or pBHA, and transformed into chemically competent *Escherichia coli* α-Gold cells (Bioline, New South Wales, Australia).

[0353] DNA sequences encoding a single additional KAS (i.e., active site) residue (containing the “DSSG” amino acid linker region or without a linker) are sequentially added to the recombinant chimera and Kgp adherein domain, one at a time, essentially as follows: Restriction enzyme sites are introduced to the ends of DNA inserts in the chimeric and adherein variant parent clones via PCR using oligonucleotide primers containing nucleotides specific to the restriction enzyme. Synthetic DNA fragments encoding a single KAS sequence with the corresponding restriction sequence are ligated to the inserts of DNA constructs from the parent clone, and the recombinant clones are purified. Subsequently, if a second or third additional KAS is added, additional restriction sequences are introduced to the ends of these clonal inserts, and additional DNA fragments encoding KAS sequences with different restriction sites are ligated one at a time. Parent clone constructs with single, double, or tetralinear KAS coding sequences are subjected to specific restriction digestion and can then be ligated by interchanging subfractions to generate various variants (including those illustrated in Table 1).

[0354] According to the manufacturer’s instructions, residues in ABM1 and ABM2, as well as cysteine ​​residues, were mutated using the QuickChange II site-directed mutagenesis kit (Stratagene, La Jolla, California).

[0355] The integrity of each insert in the cloning vectors pGEMTeasy and pBH1 was confirmed by DNA sequencing (Applied Genetics and Diagnostics Facility, The University of Melbourne). Mutations were further verified by DNA sequencing of the entire insert. The verified constructs were then subjected to restriction digestion with a selectase, and the inserts were cloned into the relevant pET expression vector in *E. coli* α-selective chemocompetent cells, and subsequently into the *E. coli* expression host BL21-CodonPlus(DE3)RIPL (Stratagene, Australia).

[0356] A study on the characteristics of chimeras that affect the solubility of recombinant proteins; small-scale...

[0357] Using a single colony transformant, 2 mL of Luria Bertani (LB) broth containing 30 μg / mL kanamycin was inoculated overnight at 37°C on an orbital shaker. This inoculum was then used to inoculate 2 mL of LB broth containing 30 μg / mL kanamycin (1:100). Cell cultures with OD600 = 0.6–1.0 were induced for 2 hours at 37°C with 1 mM IPTG. The cell cultures were centrifuged, and the pellet was resuspended in 250 μL PBS and briefly sonicated and centrifuged using a CPX750 sonicator (Cole Parmer Instrument Company, USA).

[0358] The solubility of recombinant proteins was assessed by SDS-PAGE analysis of total lysates, soluble fractions (supernatant), and insoluble fractions (precipitate). Recombinant proteins that remained insoluble or exhibited low solubility under these initial pilot expression conditions were expressed at various lower temperatures (16°C to 30°C) and lower IPTG levels (0.01–0.5 mM) to determine the optimal conditions for improving solubility. Under optimal conditions, all recombinant protein inductions were scaled up (20–200 mL) to test medium- to large-scale solubility. Cultures (200 mL) were subjected to IPTG induction at different temperatures (e.g., 5 h at 30°C or 16 h at 16°C).

[0359] Cells were collected and resuspended in lysis buffer (20 mM sodium phosphate at pH 8, 500 mM NaCl, 0.5% v / v Triton X-100, 5 mg / ml DNAseI, 1X protease inhibitor mixture, 1 mg / ml lysozyme, 10 mM imidazole). The cell resuspended cells were incubated at 4°C for 1 hour, and then the clarified lysis buffer was centrifuged (8,000 mL / min). (30 minutes, 4°C) to collect supernatant (soluble) fractions and precipitate (insoluble) fractions, and analyze by SDS-PAGE and natural-PAGE.

[0360] In-gel HIS staining of recombinant proteins

[0361] According to the manufacturer's instructions, the recombinant protein subjected to SDSPAGE was stained using the Invision In-Gel His Staining Kit (Inviscon). This staining is highly specific for His-tagged proteins. This procedure was used to identify N-terminal degradation of recombinant proteins because all candidates contained a C-terminal His tag.

[0362] Large-scale purification of chimeras for animal model studies

[0363] The purification conditions for each recombinant protein are detailed in the following sections.

[0364] Protein expression and cell lysis

[0365] As previously described, all recombinant proteins of vaccine candidates identified for use as animal models were expressed in *E. coli* BL21(DE3) as recombinant C-terminal His-tagged fusion proteins or “untagged” proteins. Cells were grown at 37°C in LB, TB, or modified M9 minimal medium supplemented with 50 μg / mL kanamycin. Cultures with OD600 = 0.8–1.0 were induced with 0.4 mM IPTG at 37°C or 34°C for 2–4 h, at 25°C for 12 h, or at 16°C for 16–20 h. Unless otherwise specified, after collection by centrifugation at 8000 g at 4°C, cells were lysed on ice for 1.5 h in lysis buffer [0.35 mg / mL lysozyme, 40 μg / mL TBS150 (50 mM Tris·Cl, 150 mM NaCl at pH 7.5) with DNase I], 1% Triton X-100, and an EDTA-free protease inhibitor (Complete ULTRA tablets, Sigma-Aldrich). The cell lysate was clarified by centrifugation at 23,500 g for 40 min at 4°C.

[0366] The initiator Met residues (aa 1) (starting from the translation sequence MA) on all recombinant proteins shown in Table 1 were removed in vivo by an E. coli N-terminal methionine processing enzyme (which cleaves methionine when the penultimate residue is a small residue). Mass spectrometry analysis of KDAK and KDAK variant recombinant proteins confirmed this.

[0367] Purification of His-tagged proteins from soluble fractions under non-denaturing conditions

[0368] 1. Ni affinity chromatography

[0369] 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 a TBS300 loading buffer (50 mM Tris·Cl, 300 mM NaCl, pH 7.5) with 10 mM imidazole and 1% Triton X-100. The column was thoroughly washed with loading buffer and then thoroughly washed with 20 mM imidazole in TBS300. Bound proteins were eluted using a 20–350 mM imidazole gradient in TBS300, and absorbance was monitored at 280 nm. Peak fractions were analyzed by SDS-PAGE 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 protein size. The resulting protein solution was stored at -70 °C for further purification.

[0370] 2. Anion exchange chromatography

[0371] The online analysis tool ProtParam, ExPASy (https: / / web.expasy.org / protparam / ), predicted the isoelectric points (pI) of all candidate proteins to be within the acidic range, thus allowing for further purification using anion-exchange chromatography if necessary. In short, the concentrated protein from Ni affinity purification was 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. The protein was eluted with a 20–400 mM NaCl gradient in Tris buffer, and absorbance was monitored at 280 nm. The target protein in the peak fraction was validated by SDS- and / or native PAGE, and concentrated using an Amicon filter unit with an appropriate molecular weight cutoff. The protein solution was stored at -70°C for further purification using size exclusion chromatography or dialysis, or buffer exchange.

[0372] 3. Size exclusion chromatography

[0373] Size exclusion chromatography was performed at 4°C on a HiLoad Superdex 200 or HiLoad Superdex 75 column (GE Healthcare). Proteins from additional steps of Ni affinity purification or anion exchange chromatography were loaded onto appropriate columns and eluted in TBS150 or TBS100 buffer (100 mM NaCl in 50 mM Tris.Cl at pH 7.5), with absorbance monitored at 280 nm. Target proteins in the peak fractions were validated by SDS-PAGE and / or native PAGE. A second or additional round of size exclusion purification was performed to separate individual oligomeric species, such as dimers. Fractions containing optimal protein purity were pooled and concentrated. To prepare mixtures of protein homopolymers, the target protein fractions were simply pooled and concentrated. If necessary, protein identity was confirmed by determining the molar mass of the intact protein using liquid chromatography-electrospray ionization spectrometry (LC-MS, Agilent Technologies) or by sequence analysis using liquid chromatography-Orbitrap tandem mass spectrometry (LC-MS / MS, Agilent Technologies). Protein concentration was quantified by determining the absorbance at 280 nm using the calculated extinction coefficient before submission for animal model experiments.

[0374] Purification of His-tagged proteins from inclusion bodies

[0375] 1. Under oxidizing conditions

[0376] Following cell lysis (as described for purification from soluble fractions), insoluble precipitates were washed twice with TBS150. Proteins expressed as inclusion bodies were dissolved for 1 hour at room temperature on a rolling platform in TBS300 or PBS500 (pH 7.4) with 8 M urea. The protein extract was centrifuged at 20,000 g and filtered through a 0.22 μm filter unit, and then loaded onto a HisTrap affinity column in urea-buffered TBSU (8 M urea plus 20 mM imidazole in TBS300) or PBSU (8 M urea, 20 mM NaPi, 500 mM NaCl plus 20 mM imidazole at pH 7.8). The column was thoroughly washed with the same 8 M urea buffer. An additional wash with PBSU at pH 6.5 was applied to the column containing the protein loaded in PBSU. To remove urea, the target protein was eluted using a gradient of 20–500 mM imidazole in PBSU. When using loose Ni-NTA resin (Thermofisher), the urea extract was mixed with the resin for 2 hours with gentle agitation in PBSU. After thorough washing with PBSU (pH 7.8, and then pH 6.5), the bound protein was eluted from the resin with 500 mM imidazole in the same buffer. The eluted protein was stepwise dialyzed into 6 M, 4 M, and then 2 M urea phosphate buffer (Fisher Biotec, Australia) in dialysis tubes with a molecular weight cutoff of 3.5 kDa. Before submission for animal model experiments, the protein in the 2 M urea buffer was further dialyzed to saline-only and maintained at 4°C, or submitted to the 2 M urea buffer without further dialysis.

[0377] 2. Under reducing conditions

[0378] Proteins were extracted from inclusion bodies with 6 M urea buffer in the presence of 5 mM TCEP and loaded onto a HisTrap column in the same buffer. With 2 M TCEP present, the column was washed with 4 M urea buffer containing 2 M TCEP, and the target protein refolded on the column as the urea concentration gradient decreased from 4 M to 0 M. The protein was then eluted with imidazole in an increasing concentration gradient of 20–350 mM in a TBS300 containing 2 mM TCEP. The eluted protein was then concentrated in a reduction buffer (2 mM TCEP in a TBS150) for size exclusion chromatography, following the same procedure described in the section on purifying protein dimers or polymers from soluble fractions under non-denaturing conditions. After analysis by SDS- and native PAGE, mass spectrometry, and quantification, the samples were stored in the reduction buffer at -70°C or submitted for animal model experiments.

[0379] Purification of His-tagged proteins from soluble fractions under oxidative and denaturing conditions

[0380] The cell lysate was clarified by centrifugation at 20,000 g to remove debris and insoluble material, and then immediately denatured to a final concentration of 8 M by adding urea solution to PBS500 and stirring at room temperature for 1 hour. The resulting lysate was then centrifuged at 20,000 g for 40 minutes at room temperature. The target protein was purified using a HisTrap Ni affinity column or loose Ni-NTA resin, following the same method described above in the section on purification from inclusion bodies under oxidative conditions. The purified antigen in saline was stored at 4°C before submission for animal model experiments.

[0381] Purification of unlabeled candidates from soluble fractions

[0382] Anion exchange chromatographyThis is the first chromatographic step in the purification of the untagged protein. Similar to its His-tagged counterpart, the pI of the untagged candidate was predicted to be in 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 lysate was filtered through a 0.22 μm filter and loaded onto an anion exchange HiTrap Q column (GE Healthcare) in buffer AA (20 mM NaPi at pH 6.5, supplemented with a 0.05x protease inhibitor mixture). After washing with 10 column volumes of buffer AA, the bound protein was eluted with a NaCl gradient of 0–100% buffer BA (1 M NaCl in 20 mM NaPi at pH 6.5), and absorbance was monitored at 280 nm. The target protein in the peak fraction was validated by SDS- and / or native PAGE. In the second round, the optimal fraction from the first round of anion exchange was pooled, diluted 6-fold with buffer AA, and then reloaded onto an anion exchange HiTrap Q column (GE Healthcare) in the same buffer. Bound proteins were eluted with a NaCl gradient of 0–50% buffer BA, and absorbance was monitored at 280 nm. Target proteins in the peak fraction were validated by SDS-PAGE and / or native PAGE, and the optimal fraction was concentrated using an Amicon filter unit with a 10 kDa molecular weight cutoff. The protein solutions were stored at -20°C for further purification using size exclusion chromatography.

[0383] First round of size exclusion chromatography Size exclusion chromatography (SLC) was performed at room temperature on a HiLoad Superdex 200 column (GE Healthcare). Proteins purified by anion exchange chromatography were loaded onto the SLC column and eluted in AS buffer (1 M (NH₄)₂SO₄, 50 mM NaPi, supplemented with a 0.05x protease inhibitor mixture at pH 7.0), with absorbance monitored at 280 nm. Target proteins in the peak fractions were validated by SDS-PAGE and / or native PAGE. Fractions with optimal protein purity were pooled for further purification using hydrophobic interaction chromatography (HACE).

[0384] Hydrophobic interaction chromatographyThe pooled fractions of target proteins from buffer AS of the first round of size exclusion chromatography were filtered through a 0.22 μm membrane and loaded at 4 °C into a HiTrap Phenyl HP column pre-equilibrated in buffer AH (1 M (NH4)2SO4, 50 mM NaPi, pH 7). Bound proteins were eluted in buffer BH (50 mM NaPi, pH 7) with a gradient of ionic strength decreasing from 800 mM to 200 mM (NH4)2SO4, and absorbance was monitored at 280 nm. Target proteins in the peak fractions were validated by SDS-PAGE and / or native PAGE, and the optimal fractions were pooled and concentrated using an Amicon filter unit for further purification or a buffer exchange step in the second round of size exclusion chromatography.

[0385] Second round size exclusion chromatography The concentrated sample from the purification step involving hydrophobic interactions was loaded onto a size exclusion column as used in the first round of size exclusion chromatography and eluted in BTS buffer (20 mM Bis-Tris, pH 6.5, 150 mM NaCl). Peak fractions were analyzed by SDS- and / or native PAGE. Fractions of optimal purity were pooled and concentrated. After identification and quantification by liquid chromatography-electrospray ionization mass spectrometry (LC-MS, Agilent Technologies), the antigen was aliquoted and stored at -80°C for future use.

[0386] Protein identification and quantification of unlabeled candidates

[0387] In addition to SDS- or native PAGE validation, protein identity was confirmed, if necessary, by determining the molar mass of the intact protein using LC-MS or by sequence analysis using Orbitrap tandem mass spectrometry (LC-MS / MS, Agilent Technologies). Protein concentration was quantified by determining the absorbance at 280 nm using the calculated extinction coefficient (Table 3). In cases where buffer exchange was required before submission for animal model experiments, Superdex 200 size size exclusion columns (GE Healthcare) or Zeba™ rotary desalting columns (Thermo Fisher Scientific) were used.

[0388] 1.2 Materials and Methods for In Vivo Studies

[0389] Bacterial culture in a mouse model of periodontitis

[0390] *Porphyromonas gingivalis* strain W50 (serotype C) was obtained from the Oral Health Cooperative Research Centre, The Melbourne Dental School, University of Melbourne, Australia. *Porphyromonas gingivalis* W50 was grown on horse blood agar (HBA) supplemented with 10% v / v lysed horse blood (37°C) in an anaerobic N2 atmosphere containing 5% CO2 at the MK3 anaerobic workstation (Don Whitley Scientific Ltd., Adelaide, Australia) (20 g / L HBA; Oxoid Ltd., Hampshire, UK). Colonies were inoculated into a starting culture consisting of 20 mL of sterile brain heart extract (37 g / L BHI; Oxoid, Hampshire, UK) supplemented with 5 mg / L heme and 0.5 mg / L cysteine ​​[McKee et al. (1986), Infection and Immunity (…]. Infect Immun [52: 349-355] and anaerobic incubation (24 h, 37 °C). The absorbance of batch cultures at OD650 nm was monitored using a spectrophotometer (model 295E, Perkin-Elmer, Germany). Bacterial cells were collected during the late exponential growth phase by centrifugation (7,000 g, 20 min, 4 °C). Bacterial purity was usually confirmed by Gram staining [Slots (1982). In: Host-Parasite Interaction in Periodontal Disease, Genco, RJ and Merganhagan, SE (eds.). Washington DC: American Society for Microbiology. pp. 27-45].

[0391] Preparation of heat-inactivated bacteria

[0392] Cultures of *Porphyromonas gingivalis* W50 were collected (6,500 g, 4°C) and washed once with phosphate-buffered saline (PBS) (pH 7.4: 0.01 M Na₂HPO₄, 1.5 mM KH₂PO₄, and 0.15 M NaCl), followed by centrifugation (7,000 g, 20 min, 4°C) to precipitate the cells. The bacterial cells were resuspended in PBS and heated to 65°C for 15 min. The suspension was centrifuged (7,000 g, 20 min, 4°C) and resuspended in sterile PBS, and this was repeated once. After the second wash, the supernatant was discarded, and the cell pellet was resuspended in sterile PBS to obtain 2 × 10⁶ cells / mL. 10 Cell density was determined by cells / mL, and protein concentration was determined using Biorad protein assay dye reagent concentrate (Life Science, NSW, Australia).

[0393] Mouse periodontitis model

[0394] The mouse periodontitis experiment was based on the model of Baker et al. (1994). (Archives of Oral Biology) Arch Oral Biol Modified as described in 39:1035-1040, and performed as previously described by O'rien-Simpson et al. (J. Immunol, 2005, 175:3980-3989). On day 0, mice (female BALB / c; 6–8 weeks old, 10 mice / group) were intraorally inoculated with *Porphyromonas gingivalis*, which was prepared by four doses of *Porphyromonas gingivalis* W50 [suspended in 20 μL of PG buffer (pH 7.4, 50 mM Tris-HCl, 150 mM NaCl, 10 mM MgSO4, and 14.3 mM β-mercaptoethanol) containing 2% w / v carboxymethyl cellulose (CMC, Sigma, New South Wales, Australia)]. 10 The inoculum consisted of [1 live *Porphyromonas gingivalis* W50 cells] administered at two-day intervals. The inoculum was prepared anaerobically and then immediately applied to the gingival margin of the maxillary molars. The number of live bacteria in each inoculum was verified by flow cytometry and CFU counting on blood agar. The animal groups consisted of: an orally administered *Porphyromonas gingivalis* W50 (infected control) group, a non-bacterial inoculation control group, and an immunized group. For therapeutic vaccination of periodontitis models (… Figure 1Mice were immunized on day 19 following an initial oral administration of 50 μg of the vaccine candidate in saline / alum (Alhydrogel; 2% aluminum hydroxide wet gel suspension; Invivogen, Inc.). Mice received a second immunization (50 μg in saline / alum) subcutaneously on day 40. On day 62, mice were euthanized by exsanguination via cardiac puncture. The maxilla was removed and bisected along the midline, with 10 halves used to determine alveolar bone loss. Serum was used to determine antibody profiles using ELISA.

[0395] Measurement results of alveolar bone loss in the maxilla of mice

[0396] The maxillae with bone loss to be examined were boiled in deionized water (1 minute), mechanically demineralized, and immersed in 2% w / v potassium hydroxide (16 hours, 25°C). The maxillae were washed twice with deionized water (25°C), dried (1 hour, 37°C), and stained with 0.5% w / v methylene blue aqueous solution. Digital images of the buccal side of the maxillae were taken using Olympus DP12 digital cameras mounted on a dissecting microscope to assess horizontal bone loss, using OLYSIA BioReport software version 3.2 (Olympus Australia Pty Ltd, New South Wales, Australia). The maxillae were oriented so that the buccal and lingual molar cusps overlapped. Images were taken in micrometers per frame to allow measurements to be standardized for each image. Horizontal bone loss was defined as loss occurring in a horizontal plane perpendicular to the alveolar ridge, resulting in a decrease in ridge height. The visible area from the cementum-enamel junction (CEJ) to the alveolar ridge (ABC) of each molar was measured using OLYSIA BioReport software version 3.2 imaging software, and the results were obtained in mm. 2 Total visible CEJ-ABC area in mm². *Porphyromonas gingivalis*-induced alveolar bone loss in mm² was calculated by subtracting the total visible CEJ-ABC area of ​​the uninoculated (NC) group from the total visible CEJ-ABC area of ​​each experimental group. Alveolar bone loss measurements were determined twice in a randomized and blinded protocol. Data are presented as mean + / - standard deviation (in mm²). 2 The results were expressed in units of 1, and the analysis was performed using one-way ANOVA and Dunnett's T3 post-hoc test.

[0397] Pre-screening of vaccine candidates using enzyme-linked immunosorbent assay (ELISA).

[0398] Vaccine candidates were pre-screened using several monoclonal antibodies against *Porphyromonas gingivalis* to determine whether key domains and epitopes present in the vaccine construct were accessible to the antibodies, thus allowing for an in vivo antibody response. The antigens to be screened were coated onto flat-bottomed polyethylene microtiter plates (Dynatech Laboratories, McLean, VA, US) in 0.1 M PBS (pH 7.4) at 4°C for 16 hours. The following antibody dilutions were used in these experiments: a 1 / 4000 dilution of goat anti-mouse antibody; and IgG (M8642) antibody (Sigma, NSW, Australia). A 1 / 4000 dilution of horseradish peroxidase-conjugated porcine anti-goat IgG antibody (M5420; Sigma, NSW, Australia) was used to develop the ELISA assay.

[0399] Use ELISA to determine subclass antibodies in serum.

[0400] ELISA was performed to evaluate subclass antibodies in serum, as described by Pathirana et al. (2007). *Infection and Immunity* 75: 1436-1442, using HK W50 cells, domain subunits, or epithelial cells in 0.1 M PBS (pH 7.4) at 1 μg / mL to coat wells of flat-bottomed polyethylene microplates (Microtiter; Danetek Laboratories, McLean, VA, USA) for 16 hours at 4°C. In these experiments, the following antibody dilutions were used: a 1 / 4000 dilution of goat anti-mouse antibody; and IgG (M8642), IgG1 (M8770), and IgG2a (M4434) antibodies (Sigma, NSW, Australia). A 1 / 4000 dilution of horseradish peroxidase-conjugated porcine anti-goat IgG antibody (M5420; Sigma, NSW, Australia) was used to develop the ELISA assay. For epitope ELISA, biotinylated peptides were conjugated to pre-blocked streptavidin-coated flat plates (Pierce; Thermo Fisher Scientific) at 10 μg / mL. After incubation with serum, an ELISA was developed using porcine anti-goat IgG antibodies conjugated with 1 / 4000 goat anti-mouse IgG and 1 / 4000 horseradish peroxidase. All densitometric measurements were performed at 405 nm on a Wallac VICTOR3 1420 multi-tag counter (PerkinElmer).

[0401] Example 2: Results of in vitro studies

[0402] Study 1: Effect of chimeric components on the solubility of recombinant proteins

[0403] (In WO 2010 / 022463) A previously reported chimera consists of a KAS(K) peptide conjugated to an N-terminal truncated DUF2436 domain (Dc), an adhesin domain containing ABMs213(A), and a C-terminal truncated CAD domain (a truncated K1 domain, referred to as K1n). This chimera is referred to as "KDcAK1n" (SEQ ID NO: 64, and may also be referred to herein as "original chimera" or "chimera" in comparison to the chimeric and fusion proteins of the present invention). The KDcAK1n protein is produced as an inclusion body in E. coli and exhibits poor solubility and stability.

[0404] The truncated DUF and K1 domains of the A1 adhesin fragment represent how the Kgp polyprotein undergoes native proteolytic processing and assembly on the surface of *Porphyromonas gingivalis* cells during infection. Therefore, the truncated DUF and K1 domains are promising candidates for inclusion in vaccines to elicit an immune response to gingival proteases.

[0405] Extensive attempts were made to generate soluble KDcAK1n. Variations in growth medium, growth conditions, IPTG concentration, *E. coli* expression strain, induction temperature, cell growth stage at induction, addition of growth stabilizers, lysis buffer, and protein storage buffer were systematically investigated. Enhanced expression of the soluble recombinant protein was observed in small-scale expression under low temperature and IPTG induction. At small scales, KDcAK1n, as a soluble protein, accounted for approximately 30–50% of the total recombinant protein expression. However, soluble recombinant expression could not be scaled up to cultures larger than 10 ml, resulting in inclusion body formation in any attempt to scale up.

[0406] The effect of the K1 domain on solubility

[0407] All intact DUF, ABM213, ABM21, and DUF-ABM213 domains of the Kgp multiprotein are expressed as highly soluble and stable recombinant proteins. However, the intact K1 domain alone is insoluble and is expressed only as inclusion bodies (IBs).

[0408] Expression of the KAS motif conjugated to the ABM213 domain (i.e., "KA") or the KAS motif conjugated to both the DUF domain and the ABM213 domain (i.e., "KDA") produces a highly soluble and stable recombinant protein.

[0409] However, as Figure 2 As shown, adding a C-terminal truncated K1 domain (i.e., the K1n fragment) or a complete K1 domain to the recombinant KA or KDA (KAK1 or KDAK1) will result in a decrease in solubility.

[0410] Both KDAK1n (SEQ ID NO: 65) and KDAK1 (SEQ ID NO: 66) proteins (i.e., containing a K domain, an adhesin domain containing DUF2436 and ABM, and a truncated or full-length CAD domain) require more demanding growth conditions to achieve soluble expression, and their levels are significantly lower than those of KDA (i.e., the same protein except for the truncated or full-length CAD).

[0411] Based on the results of the K1 domain expression, it is clear that the K1 (CAD) domain is the cause of poor solubility of the recombinant chimera.

[0412] Effect of DUF domain on protein solubility

[0413] Truncating the DUF domain in the chimeric protein construct resulted in the elimination of soluble protein expression. This result suggests that a complete DUF domain containing an additional 38 N-terminal residues is desirable for optimizing the soluble expression and efficient stability of recombinant proteins.

[0414] Therefore, a construct expressing a chimeric variant with an extended Dc domain (a chimera with Dc extended by 38 N-terminal residues) was created. This recombinant was named KDAK1n, and results showed that the chimera with the intact DUF domain exhibited significantly improved solubility. Large-scale expression of KDAK1n with 0.2–0.5 mM IPTG at 30 °C yielded good yields of soluble recombinant protein.

[0415] The Influence of K1 and Full-Length DUF Domains on Solubility

[0416] Despite the existence of this soluble protein expression at this scale, including K1n / K1 in KDAK1n / KDAK1 does appear to be more problematic than the preparation of KDA variants lacking K1n / K1, so these variants involving K1 may not be very desirable for scaling up and commercial development.

[0417] In summary: ● The full-length DUF domain is desirable for the solubility of recombinant variants. Figure 2 A) ● The K1 domain (whether full-length or truncated) is largely responsible for the aggregation and insolubility of variants.

[0418] ● In the absence of a full-length DUF domain (i.e., DUFc), variants containing K1 and K1n are completely insoluble. Figure 2 A).

[0419] ● It is evident that the solubility of variants containing K1 and K1n is very low under very mild induction conditions, even in the presence of a full-length DUF domain. However, this solubility is so low that approximately 80–90% of all recombinant proteins are expressed as inclusion bodies.

[0420] ● Medium-scale purification of native PAGE gel analysis of soluble KDAK1 recombinants revealed disordered aggregation of the recombinants (i.e., the KDA variants were not a neat and uniform sequence ladder). Small-scale purification of these variants using mini-Nickle spinning columns (at low protein concentrations) did not induce similar aggregation; however, small-scale purification of truncated DUF variants also revealed that the characteristic sequence ladder of the protein on native PAGE was non-uniform, indicating disordered aggregation.

[0421] ● Variants containing K1 and K1n domains tend to aggregate more readily after freeze-thaw cycles at higher concentrations and in purified samples. The K1 domain is responsible for this instability. However, all variants lacking either K1 or K1n are very stable during freeze-thaw cycles and at higher concentrations (e.g., above 30 mg / ml).

[0422] Variants containing full-length or truncated K1 domains are highly insoluble, and the low levels of soluble recombinant proteins produced after purification are prone to instability and aggregation. It is not feasible to efficiently scale up soluble K1-containing variants.

[0423] Therefore, extending DUF to its full length and / or removing the K1 (or K1n) domain (preferably both) is important for the solubility and stability of recombinant proteins.

[0424] Study 2: Factors influencing polymerization

[0425] Native PAGE analysis of purified soluble DUF-ABM213 and ABM213, as well as recombinant ABM21 proteins, revealed extensive, even ladder-like, indications of multimerization. Multimerization was also evident in prior art KDcAK1n chimeras, via intermolecular disulfide bond formation between denatured domains. These disulfide-linked multimers were most pronounced in KDcAK1n chimeras purified from inclusion bodies.

[0426] During the study of recombinant chimeras and Kgp adhesins, it became clear that the multimerization of recombinant proteins occurred through the interaction between the ABM1 and ABM2 motifs. Recombinant proteins containing ABM2(1), ABM2(2), and ABM3 domains (see reference). Figure 3(The schematic diagram in A) can readily form multimers. Subsequent expression of the smaller recombinant protein encoding ABM2(1), ABM1(2), and ABM3 (named rABM213) and the recombinant protein encoding ABM2(1) and ABM1(2) (named rABM21) demonstrates that both recombinants can readily undergo multimerization. This multimerization is evident by the presence of a neat band sequence ladder on the native PAGE.

[0427] A method is proposed whereby the ABM1 domain interacts with the ABM2 domain during the folding of Kgp multi-domain multiproteins to form an FnIII-like stable β-sheet complex. The inventors hypothesize that during the folding of Kgp multiproteins, ABM1(1) will interact with its next available neighbor, ABM2(1), and ABM1(2) will interact with ABM2(2), etc. (e.g.) Figure 3 (A schematic depiction).

[0428] In separate studies, the inventors demonstrated that co-expression of rABM1 and rABM2 as separate proteins can interact to form stable β-sheet complexes. rABM2(1) is capable of forming stable structures with either rABM1(1) or rABM1(2). More specifically, NMR spectral analysis of the folded ABM domains indicated that the domains are polymerized through β-chain exchange involving ABM1 and ABM2 motifs. These results collectively provide evidence that chimeras and variants containing ABM domains will polymerize through interactions between the ABM1 and ABM2 motifs of properly folded ABM domains.

[0429] Subsequently, the inventors aim to mutate specific residues of ABM1 or ABM2 in the recombinant ABM21 (rABM21) protein, which covers residues 878-968 on the Kgp W50 multiprotein.

[0430] BLAST analysis of Kgp ABM1 and ABM2 sequences from all available bacterial genomes revealed that the submotifs within the ABM1 and ABM2 motifs are highly conserved across all phyla. Subsequent alignment of the ABM1 and ABM2 sequences with all ORFs within the W83 genome revealed that these residues are similarly highly conserved in *Porphyromonas gingivalis* genes. The “PVQN” motif, containing conserved proline residues, is highly conserved across phyla, including *Porphyromonas gingivalis*.

[0431] Molecular modeling of the proposed tertiary fold using an online program revealed that this “PVQN” sequence is located at the beginning of the β-sheet structure, and that the N-terminal sequence immediately following this motif (“NEFA”) is most likely an unstructured “loop”.

[0432] The inventors mutated residues within the PVQN motif of rABM21, as well as mutant residues within the loop immediately preceding the PVQN. The residues within the loop were targeted because the size and shape of the loop preceding the β-sheet structure are thought to influence the strength of adjacent β-sheet folding. The number and position of proline residues within such "loop" structures are reported to correlate with interactions with adjacent β-sheets.

[0433] 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 to become SxYQ into the sequence of the motif NxFA in ABM1 (see the SSEYQ variant mentioned in Table 2 below).

[0434] The mutated rABM21 variant was purified and its ability to polymerize was evaluated. The inventors sought to identify mutations that would produce stable, soluble recombinant molecules that could be purified into stable monomers.

[0435] Table 2: Overview of recombinant protein solubility and multimer-forming ability on native-PAGE with and without DTT. (N / T = Not tested)

[0436] All recombinant proteins (WT rABM213, rABM21, and rABM21 mutant variants) produced very high levels of expression of highly soluble recombinant proteins under harsh induction conditions (summarized in Table 2).

[0437] Purified recombinant protein subjected to natural Page analysis ( Figure 3 BE) indicates that rABM21 can form polymers through two mechanisms: (1) polymerization of the ABM1 motif “PVQN”, and (2) through disulfide bonds.

[0438] The highly conserved “PVQN” motif mutation in rABM21 to AVQN or AVQA does not eliminate multimerization.

[0439] The mutation of “PVQN” to AVQP (SEQ ID NO: 85) resulted in a significant reduction in multimerization in the presence of DTT, and a slight reduction in multimerization in the absence of DTT, indicating that the change from PVQN (SEQ ID NO: 86) to AVQP (SEQ ID NO: 85) alone has some effect on reducing multimerization.

[0440] The AVQA mutation, combined with the substitution of one or both cysteine ​​residues in the ABM domain for serine, slightly reduces polymerization. Mutating “PVQN” to AVQP (SEQ ID NO: 85) and substituting the cysteine ​​residues in the ABM domain... One or two The mutation of cysteine ​​residues to serine together causes the near or complete elimination of rABM21 polymerization.

[0441] Direct manipulation of residues in the ABM1 “PVQN” motif preceding the modeled “β-sheet region” and mutation of the modeled “loop” region also reduced multimerization, but did not eliminate it completely. More specifically, “SSEYQ” substitution in ABM1 (e.g., modifying the sequence SNEFA (SEQ ID NO: 99) immediately following the highly conserved PVQN motif at the N-terminus to SSEYQ (SEQ ID NO: 100)) resulted in a significant reduction in multimerization.

[0442] Mutations in Tyr-889 and Trp-964 residues (in ABM2 and 1, respectively) also together cause the elimination of rABM21 multimerization.

[0443] Manipulating the “PVQN” motif to AVQP (SEQ ID NO: 85), along with the substitution of cysteine ​​residues within the ABM domain, enables the expression and purification of highly soluble monomeric proteins using the E. coli pET expression system.

[0444] All chimeric candidates consisting of the original sequence with two Cys residues within the complete ABM21 domain exhibited extensive step-like multimerization. Figure 3 B and C). However, the substitution of these two Cys residues did not completely eliminate the step-multiplying ( Figure 3 Lanes C 15 and 16), although substitution of one Cys residue combined with substitution of AVQP (SEQ ID NO: 85) provides a significant reduction in multimerization. Complete elimination of ladder-like multimerization can only be achieved by substituting two ABM cysteine ​​residues to change motif PVQN to AVQP (SEQ ID NO: 85).

[0445] Interestingly, under reducing conditions, both KDAK-1V-AVQP (SEQ ID NO: 70) and KDAK-AVQP (SEQ ID NO: 71) showed significantly reduced polymerization, regardless of whether only the DUF domain cysteine ​​residues were changed or not, while retaining two ABM cysteine ​​residues. The two proteins shared essentially identical curves on native gels (reduced or unreduced). Figure 3(G). Clearly, the DUF cysteine ​​residues do not contribute to step-merging, but the presence of the two ABM cysteine ​​residues is crucial for the formation of step-mergeners in both variants. This suggests that the DUF domain cysteine ​​residues are not a significant inducing factor for mechanization.

[0446] Although changes in cysteine ​​residues and the PVQN motif disenchanted step-merging, an equilibrium between monomeric and oligomeric states persisted under certain conditions. Analysis using size exclusion chromatography revealed that this equilibrium was temperature, pH, and concentration-dependent. Increasing temperature, decreasing pH, and decreasing concentration favored the monomeric state. The presence of the His tag was not the cause of the protein state equilibrium. SEC-MALS analysis showed that monomers were overwhelmingly present in solutions at concentrations of 2 mg / mL or lower, where the lead candidate exhibited high stability.

[0447] Study 3: Active Site (KAS) Motif

[0448] Additional KAS motifs were engineered into the recombinant chimeric variants to determine whether this would affect immunogenicity. A single KAS or two consecutive KAS residues were added to the N-terminus or both ends of the variant. Additionally, a linear sequence of four KAS residues containing a DSSG linker sequence between each KAS motif was added to the N-terminus of the selected variant.

[0449] Purification curves and His gel staining analysis of the recombinant proteins revealed that additional KAS residues caused a degree of instability and degradation in the recombinant proteins. The 4x linear KAS variant showed the greatest degradation and instability, indicating that proteolytic processing occurred in the additional KAS sequences. Subsequent removal of the DSSG linker between adjacent KAS sequences did not improve stability (Table 3).

[0450] Therefore, it can be concluded that when multiple consecutive KAS sequences are expressed as linear sequences, these variants are readily processed by protein hydrolysis. Soluble variants with a single KAS at one or both ends are relatively stable and not easily degraded, and their purification yield is not easily reduced.

[0451] Study 4: Large-scale protein yield and stability

[0452] Most recombinant proteins expressed as soluble proteins and purified under non-denaturing conditions exhibited relatively high yields of their final products (>10 mg / L culture). Some even reached yields exceeding 20 mg / L culture. Specifically, when expressed in 2 YT-rich medium, the His-tagged KDAK-1V2S-AVQP protein (SEQ ID NO:59, excluding the His tag) yielded 45 mg / L culture. The purified final products of untagged KDAK-1V2S-AVQP (SEQ ID NO: 59) and KDAK-3S-AVQP (SEQ ID NO: 69) showed the most significant yields, producing over 80 and 60 mg / L culture, respectively, when expressed in premium broth (TB), several times higher than the yields of cells grown in LB or 2 YT media. This significant increase in target protein production is attributed to the high cell growth rate and high cell density when cells were grown in TB.

[0453] When these same recombinant proteins are purified from inclusion bodies or from soluble fractions under conditions that intentionally denature them (e.g., urea), the yields are much lower (2.1–3.6 mg / L culture). It should be noted that denatured proteins exhibit low binding affinity to Ni-affinity resins under denaturing conditions and low solubility when finally equilibrated in non-denaturing buffer. Furthermore, variants purified from their denatured form are unstable in unbuffered saline, regardless of whether the protein was initially expressed as a soluble or insoluble form, and regardless of whether a pre-packed column or loose Ni-NTA resin was used for purification. These results suggest that, where denaturants are not required, it may be desirable to reduce or avoid their use.

[0454] Low expression temperatures of 16°C have been shown to effectively improve the soluble expression of poorly soluble proteins, such as KDAK1 (SEQ ID NO: 66). However, even at low temperatures of 4°C, enrichment on the column in low ionic strength buffer during anion exchange purification significantly reduces yield due to column aggregation or precipitation. For these proteins, purification bypassing the anion exchange step yields higher yields; for example, the yield of KDAK1 doubled when produced in this manner. KDAK1n (SEQ ID NO: 65) was also found to be expressed as a soluble protein, but it appeared unstable in solution during subsequent purification, with a yield of only 2.3 mg / L culture.

[0455] Given that K1 is insoluble and the addition of K1 or K1n to KDA or DA reduces the solubility of recombinant variants in large-scale purification, as mentioned above, the K1 region may be the cause of instability in K1-containing chimeric variants. Therefore, excluding K1 is preferred if the domain is not necessary for protection.

[0456] Although His staining of the gel confirmed degradation in regions containing multiple KAS(K) residues, no negative impact of Ni affinity chromatography on yield was observed. For example, the three protective antigens KKDAK1nKK (SEQ ID NO: 78), KKDAKK (SEQ ID NO: 54), and KDAK1nK-4S-AVQP (SEQ ID NO: 63) contained more than 90% of the expected full-length protein species in their final products, with yields ranging from 12 to 21 mg / L of culture (Table 3).

[0457] Reducing conditions were found to favor the extraction of insoluble KDcAK1n from inclusion bodies using urea. Higher concentrations of urea were required under non-reducing conditions than under reducing conditions. Clearly, disulfide bond formation under denaturing conditions negatively impacted protein solubility. Interestingly, native PAGE gel analysis of the KDcAK1n chimera relative to a chimera with all four cysteine ​​residues mutated to serine (SEQ ID NO: 56) showed that the elimination of disulfide bonds in the mutated KDcAK1n chimera resulted in a clearer sequence ladder formation on native PAGE, consistent with β-chain exchange polymerization occurring only. Figure 3 E). Furthermore, under the same non-reductive denaturation conditions, the chimeric mutant KDcAK1n PVQN>AVQP / 4 Cys> Ser variant (SEQ ID NO: 57) exhibited higher solubility in urea than KDcAK1n.

[0458] Some soluble proteins, such as the His-tagged KDAK-1V2S-AVQP, can reach concentrations of at least 40 mg / mL. Two lead untagged candidates, SEQ ID NO: 59 and SEQ ID NO: 69 (KDAK-1V2S-AVQP and KDAK-3S-AVQP), were concentrated to concentrations exceeding 30 mg / mL and 16 mg / mL, respectively, in a buffer solution at pH 6.5, and these concentrations have not yet reached their maximum values.

[0459] In contrast, the original C-terminal His-tagged chimera (KDcAK1n) purified from inclusion bodies and its variant KDcAK1n-4S were the least stable in unbuffered saline. The highest achievable concentrations of KDcAK1n and KDcAK1n-4S in saline were 1.35 mg / mL and 2.3 mg / mL, respectively. Clearly, nonspecific disulfide bond formation is one of the factors contributing to the significant protein aggregation and low solubility of the chimera. Removal of Cys residues in KDcAK1n-4S disables intermolecular disulfide bond formation and increases protein solubility.

[0460] When proteins are expressed under the same conditions, the C-terminal His tag has no significant effect on protein production and solubility.

[0461] In the purification of the untagged candidates, although anion exchange chromatography enriched the target protein, this step was ineffective in separating the target protein from the target protein of the *E. coli* host due to the tight binding affinity to the resin, as most *E. coli* proteins are acidic in the pI range of 4–7, and many of these proteins have pIs close to those of the target protein. Interestingly, however, size exclusion chromatography with 1 M (NH₄)₂SO₄ buffer at room temperature appeared to be the key step and provided efficient separation of the target protein from the *E. coli* target protein. Following further purification via hydrophobic interactions and a second round of size exclusion chromatography, both lead untagged candidates were purified to 99% purity with high homogeneity, as evidenced by SDS- / native PAGE and SEC-MALS analyses.

[0462] Table 3: Overview of recombinant Kgp chimeras and fragments expressed in Escherichia coli

[0463] Study 5: Assessing the Importance of the DUF and KAS Domains

[0464] In this study, the inventors tested various constructs (antigens) to determine the importance of the DUF domain in the chimeric fusion protein and compared their solubility / stability with that of constructs containing an active site sequence derived from arginine-dependent gingivase. The resulting constructs were: KDAK-3S-AVQP; KDAK-2S-AVQP (His tag), KAK-2S-AVQP (His tag), and KDAR-3S-AVQP (His tag).

[0465] Protein production and yield

[0466] All antigens were expressed well. The remaining cysteine ​​residue in the DUF domain of the KDAK-2S-AVQP construct did not appear to affect protein solubility.

[0467] The candidate KDAR-3S-AVQP, which contains RAS, has lower solubility than other antigens, indicating that the RAS sequence interferes with the protein structure. However, it can still be used in subsequent mouse model experiments and can be purified from the soluble fraction under non-reducing conditions.

[0468] All antigens were purified from soluble fractions under non-reducing conditions. Three His-tagged proteins were purified by Ni affinity, anion exchange, and size exclusion chromatography. KDAK-3S-AVQP (untagged) was purified by anion exchange, hydrophobic interaction, and size exclusion chromatography. The purified antigens were produced in high yields. Among the three His-tagged proteins, KDAK-2S-AVQP-His6 was produced in the highest yield (>160 mg / L culture, 20 mg / g wet cells).

[0469] When proteins are expressed under the same conditions, no effect of the C-terminal His tag on protein production and solubility was found, indicating that the presence of the His tag does not interfere with the core structure of the protein.

[0470] In contrast, the KDAR-3S-AVQP-His6 antigen had a lower yield due to its lower solubility (55 mg / L culture, 7.2 mg / g wet cells). The antigen with the smallest molecular weight, KAK-2S-AVQP-His6, had a yield of 106 mg / L culture (14 mg / g wet cells). The identity of all antigens was confirmed by intact protein MS spectroscopy, with the first Met residue missing.

[0471] For His-tagged proteins, the peak intensity in the elution curve of His-tagged proteins at the Ni affinity purification step reflects the higher solubility of KAS antigens relative to RAS-containing antigens. However, when the RAS antigen is pure, it is also highly soluble and can be concentrated to at least 15 mg / mL.

[0472] Although these proteins have so far been concentrated to a range of 15-33 mg / mL, their high solubility may allow for even further concentrations.

[0473] Oligomerization

[0474] On SDS gels of anion-exchange fractions of KAK-2S-AVQP-His6 and KDAR-3S-AVQP-His6, small bands were observed below each major full-length target protein, indicating slight degradation of both proteins. Size exclusion chromatography largely removed the degradation products, yielding high-quality final products for each protein. Figure 13 However, the builder without the DUF domain (KAK-2S-AVQP-His6) degraded more severely, exhibiting greater persistence to its final product. Figure 13 This indicates that the DUF domain may provide a certain level of protection against degradation.

[0475] Two species in KAK-2S-AVQP-His6 and a ladder-like species in KDAR-3S-AVQP-His6 with molecular weights lower than monomeric form co-eluted in a single main peak of AIEX, as seen in previous Ni affinity chromatography.

[0476] The slight asymmetry of the main peak in the size exclusion tail curves of both KAK-2S-AVQP-His6 and KDAR-3S-AVQP-His6 can also indicate the presence of degradation products.

[0477] This type of degradation of KDAK-2S-AVQP-His6 is not clearly visible (e.g. Figure 13 The presence of RAS appears to destabilize the protein, while the DUF domain makes it more stable.

[0478] Despite the possibility of slight degradation, all final products, except for KAK-2S-AVQP-His6, exhibited high purity and homogeneity, supported by SEC-MALS analysis. For all these proteins, SEC-MALS showed a predominant presence of monomers in solutions of 2 mg / mL (data not shown). Based on SDS-PAGE analysis, KDAK-3S-AVQP and KDAK-2S-AVQP-His6 appeared to have higher quality among the four antigens, and KAK-2S-AVQP-His6 appeared to be the most heterogeneous, although minor species (e.g., KAK-2S-AVQP-His6) were not distinguished in SEC-MALS analysis. Figure 13 ).

[0479] Example 3: Animal Research

[0480] Bone loss in *Porphyromonas gingivalis* in a mouse model of periodontitis prevention

[0481] 1. Experiment 1

[0482] The antigens tested were: KDcAK1n 50 μg; KDcAK1n 0.5 μg; KDA 50 μg; KDA 0.5 μg; KDAK1 50 μg; and KDAK1 0.5 μg. All antigens were adsorbed onto alum in PBS (pH 7.4).

[0483] KDcAK1n: derived from inclusion bodies, purified in batches using Ni-NTA resin, and then dialyzed into 2 M urea PBS under non-reducing conditions.

[0484] KDA and KDAK1: derived from soluble fractions, subjected to gradient elution by Ni affinity (pre-packed column) and anion exchange chromatography under non-reducing conditions, followed by dialyzing into PBS.

[0485] Alveolar bone loss in the maxilla of mice induced by Porphyromonas gingivalis

[0486] KDcAK1n and KDA both demonstrated protective effects against bone loss in animal models. KDcAK1n protected against bone loss at both tested concentrations, while KDA only provided protection at 50 μg. Figure 4 KDAK1 data is not shown.

[0487] Antibody response

[0488] Serum antibody subclass responses in immunized mice in a periodontitis model were detected by ELISA. Antiserum was used to detect heat-inactivated *Porphyromonas gingivalis* strain W50 as an adsorbed antigen. Antibody responses were expressed as the obtained ELISA titer minus three times the background level, where each titer represents the mean ± standard deviation of 10 individual mice. Figure 5 KDcAK1n (in) Figure 5 The total IgG and IgG1 responses against whole cells of *Porphyromonas gingivalis* induced by KDA (referred to as "chimera") and KDAK1 were the strongest, followed by KDAK1. At 50 μg, there were no significant differences in IgG and IgG1 antibody responses among the tested antigens.

[0489] 2. Experiment 2

[0490] The antigens tested were: KDcAK1n 50 μg; KDcAK1n 0.5 μg; KDA 50 μg; KDA 0.5 μg; KDAK1 50 μg; and KDAK1 0.5 μg. All antigens were adsorbed onto alum in saline solution (pH 7.4).

[0491] The antigen was purified in the same manner as in Experiment 1.

[0492] Alveolar bone loss in the maxilla of mice induced by Porphyromonas gingivalis

[0493] This experiment examined the same antigen as in Experiment 1; however, saline was used instead of PBS to adsorb it onto alum. This reflects the results of Experiment 1 and... Figure 4 The bone loss results (data not shown) show that only KDcAK1n and KDA showed protection, indicating that the use of PBS or saline for alum preparation had no effect on the experimental results.

[0494] Antibody response

[0495] All tested antigens induced similar total IgG and IgG1 responses against whole-cell Porphyromonas gingivalis. KDA and KDAK1 induced a stronger IgG2a response (not shown) compared to KDcAK1n.

[0496] 3. Experiment 3

[0497] Antigens tested: All antigens except KDcAK1n were fractionated as described: KDcAK1n (dimer); KDcAK1n (multimer); KDcAK1n (chimery, primitive); KDcAK1n-4S-AVQP; KDA (dimer); KDA (multimer).

[0498] KDcAK1n-4S-AVQP, KDcAK1n dimer and polymer: Urea was extracted from inclusion bodies under reducing conditions, purified by Ni affinity column, followed by gel filtration, using two KDcAK1n samples.

[0499] KDA dimers and polymers: derived from soluble fractions, purified by Ni affinity column under non-reducing conditions, followed by anion exchange and gel filtration.

[0500] KDcAK1n: Purified according to Experiment 1.

[0501] Vaccine candidates were pre-screened using mAbs targeting epitopes of Porphyromonas gingivalis.

[0502] To develop pre-screening assays to determine the suitability of antigens for animal models, antigens were screened by ELISA using mAbs targeting KAS2, ABM2, ABM3, and EP1. All vaccine candidates were able to bind to the KAS2, ABM3, and EP1 mAbs, as shown by large titration curves compared to the negative control muBM4 mAb. While KDcAK1n and KDcAK1n-4S-AVQP bound to the ABM2 mAb, the KDcAK1n dimer, KDcAK1n multimer, KDA dimer, and KDA multimer did not bind – indicating that the epitopes recognized by these mAbs are inaccessible in these constructs (not shown).

[0503] Alveolar bone loss in the maxilla of mice induced by Porphyromonas gingivalis

[0504] Apart from KDcAK1n with a 2x His tag, no graded antigen (including KDcAK1n with a single His tag) provides protection against Porphyromonas gingivalis-induced bone loss. Figure 6 Although there is a trend (not significant) that the purified dimeric species (KDcAK1n) of a single His tag chimera has a protective effect.

[0505] Antibody response

[0506] Serum antibody subclass responses in immunized mice in a periodontitis model were detected by ELISA. Antiserum was used to detect heat-inactivated *Porphyromonas gingivalis* strain W50 as an adsorbed antigen. All tested antigens elicited varying degrees of IgG responses. KDcAK1n and KDA multimers produced strong IgG1 isotype responses.

[0507] Collected serum samples were used to detect *Porphyromonas gingivalis* domains (not shown) adsorbed onto the ELISA plate. All antigens, except for KDcAK1n-4S-AVQP and KDA dimer, elicited a total IgG response against ABM21 (multimer and dimer) and ABM213 (multimer and dimer).

[0508] The IgG response to DUF2436 was evident only in the KDA (dimer and multimer), which was the only primary antigen with an intact DUF domain tested in the model. Pooled serum samples were also used to probe *Porphyromonas gingivalis* epitope peptides (not shown). No clear pattern was observed between protective and non-protective sera.

[0509] Analysis of non-protective purified dimer and multimer antigens

[0510] Analysis of the single KDA dimer and multimer species purified by anion exchange and gel filtration without protection in Experiment 3 using reduced and non-reduced natural and SDS-PAGE revealed that, in both cases, the purified single species consisted of denatured domains crosslinked with disulfide, which were locked within stable species by the disulfide. This analysis explains why these molecular weight species are stable and can be purified by anion exchange and gel filtration chromatography.

[0511] 4. Experiment 4

[0512] Antigens tested: KKDA1nKK; KDAK1n; KDAK1nK-4S-AVQP; KKDAKK.

[0513] Except for KDAK1nK-4S-AVQP, all antigens were purified from the soluble fractions under non-reducing conditions using Ni affinity column, anion exchange, and gel filtration chromatography, without the use of anion exchange chromatography.

[0514] alum adsorption

[0515] The ability of Alhydrogel 2% to adsorb antigens was tested by incubating the antigens with alum at 4°C for 60 minutes and gently mixing. The alum was precipitated, and the antigens before and after alum adsorption were then subjected to the Bradford protein assay. All antigens bound to alum at percentages ranging from 91.7% to 99.3%. SDS-PAGE gels were also run on samples before and after alum adsorption, and the results were consistent with the Bradford protein assay (not shown).

[0516] Vaccine candidates were pre-screened using mAbs targeting epitopes of Porphyromonas gingivalis.

[0517] All vaccine candidates were able to bind to KAS2, ABM3, and EP1 mAb, as shown by large titration curves compared to the negative control muBM4 mAb. While KDAK1nK-4S-AVQP bound to ABM2 mAb, KKDA1nKK, KDAK1n, and KKDAKK did not bind – indicating that the epitopes recognized by these mAbs are inaccessible in these constructs (not shown).

[0518] Porphyromonas gingivalis Induced alveolar bone loss in the maxilla of mice

[0519] KKDAK1nKK, KDAK1nK-4S-AVQP, and KKDAKK provide protection against Porphyromonas gingivalis-induced bone loss. Figure 7 ).

[0520] Antibody response

[0521] Serum antibody subclass responses in immunized mice in a periodontitis model were detected by ELISA. Antiserum was used to detect heat-inactivated *Porphyromonas gingivalis* strain W50 as an adsorbed antigen. All tested antigens elicited varying degrees of IgG responses. Protective KKDAK1nKK and KKDAKK induced robust total IgG and IgG1 responses against heat-inactivated *Porphyromonas gingivalis*. Interestingly, KDAK1nK-4S-AVQP, which protects against bone loss, showed a lower antibody response against whole-cell *Porphyromonas gingivalis* compared to some weaker protective antigens (KDAK1n).

[0522] Pooled serum samples were used to probe *Porphyromonas gingivalis* domains adsorbed onto ELISA plates. All antigens elicited total IgG responses to varying degrees against ABM21 (multimer and dimer) and ABM213 (multimer and dimer). IgG responses to DUF2436 were only evident in antigens containing intact DUF domains (KKDAK1nKK, KDAK1n, KDAK1nK-4S-AVQP, and KKDAKK). Pooled serum samples were also used to probe *Porphyromonas gingivalis* epitope peptides, and although there was a clear trend toward high KAS titers in protective antisera, no clear pattern between protected and unprotected antisera (not shown) was observed.

[0523] 5. Experiment 5

[0524] Antigens being tested

[0525] All antigens were purified under non-reducing conditions using Ni affinity chromatography and then dialyzed into saline.

[0526] KDcAK1n - from inclusion bodies, purified using urea and affinity column (IB, urea, AC); KDA - from soluble fraction and affinity column (S, AC); KDA - from soluble fraction, purified using urea and affinity column (S, urea, AC); KDA - from soluble fraction, purified using urea and batch purification method (S, urea, batch); KDAK1 - from soluble fraction, purified using urea and affinity column (S, urea, AC); KDAK1 - from inclusion bodies, purified using urea and affinity column (IB, urea, AC); KDAK1 - from inclusion bodies, purified using urea and batch purification method (IB, urea, batch).

[0527] Vaccine candidates were pre-screened using mAbs targeting epitopes of Porphyromonas gingivalis. .

[0528] All candidates were able to bind to KAS2, ABM3, and EP1 mAb, as shown by large titration curves compared to the negative control muBM4 mAb. No antigen bound to ABM2 mAb, indicating that the epitope recognized by this mAb is inaccessible in these constructs (not shown).

[0529] Porphyromonas gingivalis Induced alveolar bone loss in the maxilla of mice

[0530] KDcAK1n(IB, urea, AC), KDA(S, AC), KDAK1(S, urea, AC), KDAK1(IB, urea, AC), KDAK1(IB, urea, batch), and KDA(S, urea, batch) showed significant protection against bone loss, with KDcAK1n(IB, urea, AC), KDA(S, AC), and KDAK1(IB, urea, batch) providing the best protection. It should be noted that the significant protection observed with KDA(S, AC) was lost upon denaturation with urea (e.g., KDA(S, urea, AC)). Figure 8 ).

[0531] Notably, unfractionated affinity-purified antigens provided protection against bone loss, particularly with good protection observed using affinity-purified soluble KDA. However, this protection was partially lost upon treatment with urea under oxidative conditions (which promoted disulfide cross-linking of the denatured D and A domains). These results further demonstrate that disulfide cross-linking of the denatured domains can disrupt antigen-induced protection; therefore, urea should be avoided, and the preparation of soluble folded domains should be preferred.

[0532] 6. Experiment 6

[0533] Antigens being tested

[0534] All antigens were purified under non-reducing conditions using Ni affinity chromatography, followed by dialyzing into saline. The antigens tested were: KDcAK1n - derived from inclusion bodies, purified using urea and affinity columns (IB, urea, AC); KDAK-3S-AVQP, KDAK1nK, KDAK1n-4S-AVQP, KDAK, KKDAKK, KKDAK1nKK.

[0535] Porphyromonas gingivalis Induced alveolar bone loss in the maxilla of mice

[0536] All antigens tested were protective against bone loss induced by Porphyromonas gingivalis. Figure 9 ).

[0537] 7. Experiment 7

[0538] Antigens being tested

[0539] In this experiment, the inventors tested several new constructs based on 4S, 3S, 4S-AVQP and 3S-AVQP mutations: KDcAK1n (existing technology chimera), KDcAK1n-4S, KDAK, KDAK-3S, KDAK-3S-AVQP, and KDAK1nK-4S-AVQP.

[0540] All antigens were His-tagged and purified using affinity chromatography. Antigens were adsorbed onto alum by adding an equal volume of alum to the protein at a weight ratio. The alum:protein compound was adjusted to an appropriate concentration (0.5 mg / mL) for intraperitoneal or subcutaneous injection before incubation at 4°C and continuous mixing. Mice were then injected intraperitoneally (vaccination 1) or subcutaneously (vaccination 2). All antigens were purified under non-reducing conditions using Ni affinity chromatography, followed by dialyzing into saline.

[0541] Porphyromonas gingivalis Induced alveolar bone loss in the maxilla of mice

[0542] Compared to the original (unstimulated) control, animals injected with KDcAK1n, KDAK-3S-AVQP, and KDAK did not show significant bone loss. Figure 10 KDAK1nK-4S-AVQP provided partial protection. Infected control mice showed significant levels of alveolar bone loss. Figure 10 ).

[0543] The results showed statistically significant differences from the control group (###p < 0.05, ####p < 0.01, ###), univariate ANOVA and post-hoc Dunnett T3 ( Figure 10 ).

[0544] 8. Experiment 8

[0545] Antigens being tested

[0546] In this experiment, the inventors tested several new constructs based on different arrangements of 1V, 1V-2S, 3S and AVQP mutations: KDcAK1n (pre-existing chimera), KDAK-3S-AVQP, KDAK-1V-2S-AVQP, KDAK-1V-AVQP and KDAK-AVQP.

[0547] All antigens were His-tagged and purified using affinity chromatography. The antigens were adsorbed onto alum by adding an equal volume of alum to the protein at a weight ratio. The alum:protein compound was adjusted to an appropriate concentration (0.5 mg / mL) for intraperitoneal or subcutaneous injection before incubation at 4°C and continuous mixing. Mice were then administered intraperitoneally (vaccination 1) or subcutaneously (vaccination 2).

[0548] Porphyromonas gingivalis Induced alveolar bone loss in the maxilla of mice

[0549] Mice immunized with chimeric (KDcAK1n), KDAK-3S-AVQP, KDAK-1V-2S-AVQP, KDAK-1V-AVQP, or KDAK-AVQP showed significantly less bone loss than infected control mice. Figure 11 Compared with the original mice, the infected control mice showed a significant level of alveolar bone loss. (Statistical analysis was performed using one-way ANOVA and post-hoc Dunnett T3.) = p<0.05; compared with the control stimulation group).

[0550] 9. Experiment 9

[0551] Antigens being tested

[0552] In this experiment, the inventors tested several constructs with different arrangements of 1V-2S, 3S and AVQP mutations at different concentrations, and introduced antigens purified without using the His tag.

[0553] The study also included groups to determine the effect of alum on the antigenicity of proteins. All His-tagged antigens were purified using affinity chromatography, and antigens without His tags were purified using a combination of anion exchange chromatography, size exclusion chromatography, and hydrophobic interaction chromatography, as described in the Methods section. Where appropriate, antigens were adsorbed onto alum using a constant concentration (100 μg / dose), regardless of protein concentration (100 μg, 50 μg, and 25 μg). The alum:protein compound was adjusted to an appropriate antigen concentration (1.0 mg / mL, 0.5 mg / mL, or 0.25 mg / mL) for intraperitoneal and subcutaneous injections before incubation at 4°C and continuous mixing. Mice were then administered intraperitoneally (vaccination 1) and subcutaneously (vaccination 2).

[0554] The antigens tested were: KDcAK1n - His-tagged, derived from inclusion bodies, purified using urea and affinity columns (IB, urea, AC); KDAK-3S-AVQP untagged (100 μg, 50 μg, and 25 μg doses); KDAK-1V-2S-AVQP untagged (100 μg, 50 μg, and 25 μg doses); KDAK-3S-AVQP His-tagged (100 μg dose); and KDAK-3S-AVQP untagged and alum-free.

[0555] Porphyromonas gingivalis Induced alveolar bone loss in the maxilla of mice

[0556] like Figure 12As shown, mice treated with 100 μg of the prior art chimera (KDcAK1n) did not exhibit significant bone loss compared to the original control. Furthermore, mice treated with all concentrations of KDAK-3S-AVQP (100 μg, 50 μg, and 25 μg) did not show significant levels of alveolar bone loss compared to the original control mice.

[0557] Mice vaccinated with KDAK-1V-2S-AVQP were protected from alveolar bone loss at the highest dose of 100 μg, but significant bone loss did occur when vaccinated with 50 μg and 25 μg or protein.

[0558] Removing the His tag from KDAK-3S-AVQP did not significantly affect the protective efficacy. However, removing alum from the vaccine resulted in higher levels of bone loss in both vaccines.

[0559] Antibody response

[0560] Serum antibody subclass responses in immunized mice in a periodontitis model were detected by ELISA. Antiserum was used to detect heat-inactivated *Porphyromonas gingivalis* strain W50 as an adsorbed antigen. All tested antigens elicited a total IgG response to *Porphyromonas gingivalis*, with KDA(S, urea, AC), followed by KDAK1(IB, urea, batch) and KDcAK1n(IB, urea, AC) showing a strong IgG1 response to whole *Porphyromonas gingivalis* cells. Only KDAK1(IB, urea, AC) induced a strong IgG2a response to whole *Porphyromonas gingivalis* cells.

[0561] Pooled serum samples were used to probe the *Porphyromonas gingivalis* domains adsorbed onto ELISA plates. All antigens elicited total IgG responses to varying degrees against ABM21 (multimer and dimer) and ABM213 (multimer and dimer), with KDA(S, urea, AC), KDcAK1n-1His(IB, urea, AC), and KDAK1(IB, urea, batch) consistently inducing the strongest responses, while KDA(S, AC) induced the weakest responses against either ABM21 or ABM213 (multimer and dimer). Interestingly, KDA(S, urea, AC), KDA(S, AC), and KDAK1(IB, urea, AC) induced the strongest responses against the DUF2436 domain (not shown).

[0562] Collected serum samples were also used to detect *Porphyromonas gingivalis* epitope peptides. KDA(S, urea, AC), KDAK1(IB, urea, batch), and KDAK1(IB, urea, AC) induced strong IgG responses to the KAS and ABM3 epitopes. KDA(S, urea, AC) also induced IgG antibodies against the EP1 and ABM2a epitopes. Except for KDAK1(S, urea, AC), all antigen groups induced responses to the EP1 epitope. No protective antigen produced antiserum (not shown) binding to the K1 epitope ABM5.

[0563] 10. Experiment 10

[0564] Antigens being tested

[0565] In this experiment, the inventors further tested the in vivo activity of the constructs previously tested in Examples 6, 7, 8 and 9, and compared the activity with that of constructs that do not contain the DUF domain and constructs that contain an active site sequence derived from arginine-dependent gingivase.

[0566] The constructs used in the tests were: KDAK-3S-AVQP (previously tested in Experiments 6, 7, 8 and 9); KDAK-2S-AVQP (His tag), KAK-2S-AVQP (His tag), and KDAR-3S-AVQP (His tag).

[0567] Porphyromonas gingivalis Induced alveolar bone loss in the maxilla of mice

[0568] Two concentrations of the antigen KDAK-3S-AVQP were used in this experiment: 100 μg / mouse as previously tested and a two-fold increase to 200 μg / mouse. A dose-dependent increase in protection against alveolar bone loss was observed, with mice receiving the 200 μg dose experiencing less bone loss than the 100 μg group. Figure 14 ).

[0569] Restoring one cysteine ​​residue (the difference between KDAK-3S-AVQP and KDAK-2S-AVQP) did not cause a significant difference in alveolar bone loss between the two variants. Figure 14 ).

[0570] Similarly, removal of the D domain (the difference between KDAK-2S-AVQP and KAK-2S-AVQP) did not significantly affect the observed alveolar bone loss. Figure 14 However, the KAK-2S-AVQP variant has a higher mean and may reach significance, thus having a greater impact in studies (e.g., more animals).

[0571] When compared with other variants at equivalent vaccine doses, changing the C-terminal KAS to RAS group (the difference between KDAK-3S-AVQP and KDAR-3S-AVQP) significantly increased mean alveolar bone loss. Figure 14 This underscores the importance of the KAS sequence in providing protection against bone loss in this model. Nevertheless, the mean bone loss generated by the KDAR-3S-AVQP antigen was still lower than that of the infected control animals, and thus still provides evidence that the RAS domain can be used in the context of chimeric fusion protein antigens as described herein.

[0572] Antibody response

[0573] The serum antibody subclass responses of immunized mice in a periodontitis model to several different antigens were examined by ELISA. These antigens included whole *Porphyromonas gingivalis*, a purified RgpA / Kgp complex, a purified catalytic domain Kgpcat, and several different protective epitopes including KAS, as well as the ABM domain, and protective epitopes of ABM3 and ABM2. (The RgpA / Kgp complex and the purified catalytic domain Kgpcat were purified from *Porphyromonas gingivalis* and confirmed to be folded and enzymatically active at the time of assay.)

[0574] Initially, antiserum was used to detect heat-inactivated *Porphyromonas gingivalis* as an adsorbed antigen. Antibody response was expressed as the obtained ELISA titer minus twice the background level, where each titer represents the mean ± standard deviation of 10 individual mice. All tested antigens elicited an IgG response to *Porphyromonas gingivalis*, with the strongest responses observed following vaccination with KDAR-3S-AVQP and KDAK-3S-AVQP 200 μg. The induced immune response was predominantly an IgG1 immune response, which was associated with a protective immune response (…). Figure 15 ).

[0575] Antibody titers against purified RgpA / Kgp protease complex (data not shown) and recombinant Kgpcat from *Porphyromonas gingivalis* (data not shown) were also measured. In both cases, the KDAK-3S-AVQP vaccine elicited a strong, dose-dependent, and primarily IgG1 immune response. Compared to the other two tested vaccines, KAK-2S-AVQP and KDAR-3S-AVQP, the KDAK-2S-AVQP vaccine induced higher levels of IgG1 antibodies against both the RgpA / Kgp protease complex and recombinant Kgpcat.

[0576] To further determine responsiveness to protective epitopes, serum samples were pooled and used to detect *Porphyromonas gingivalis*-specific biotinylated peptides bound to streptavidin plates. For each antigen tested, very high titers against the KAS2 peptide were observed. However, KDAK-3S-AVQP induced the highest levels of anti-KAS2 antibodies in a dose-dependent, IgG1-dominant manner. Among the three previously untested vaccines, similar to the responses to Kgpcat and RgpA / Kgp complexes, vaccination with KDAK-2S-AVQP induced the highest levels of anti-KAS2 antibodies.

[0577] Compared to anti-KAS2 antibodies, vaccination with all proteins induced lower levels of anti-ABM2 antibodies; however, the immune response remained strong and was predominantly IgG1. Vaccination with KDAK-3S-AVQP induced a dose-dependent IgG1-dominant immune response, while KAK-2S-AVQP induced the strongest observed immune response against ABM2.

[0578] Ultimately, dose-response data for the protection of KDAK-3S-AVQP against *Porphyromonas gingivalis*-induced bone loss from experiments 9 and 10 were closely correlated with antibody titers that cross-reacted with purified native Kgpcat protease. Figure 16 ).

[0579] Overview and discussion of the results presented in Examples 2 and 3

[0580] The work described in this report compared the production and efficacy of various vaccine candidates to identify components of chimeric vaccines that facilitate production (e.g., components that help improve solubility, stability and reduce the tendency to polymerize) and are most effective in inducing an immune response against Porphyromonas gingivalis and / or reducing Porphyromonas gingivalis-induced alveolar bone loss.

[0581] Although it effectively prevents periodontal bone loss in animal models of periodontitis, the existing vaccine KDcAK1n is expressed as an inclusion body in Escherichia coli and exhibits variable solubility and stability.

[0582] KDcAK1n is based on the fusion of the active site sequence (KAS or K) of the Lys-specific gingival protease Kgp with a processed adhesin fragment (A1) of the Kgp multiprotein. Structural analysis of the Kgp multiprotein domains now clearly shows that the A1 adhesin fragment found on the cell surface contains three discrete structural domains: the DUF domain (D), the ABM domain (A), and the K1 domain (K1). Processing of the Kgp multiprotein releasing protease catalyzes the domains and adhesin on the surface of *Porphyromonas gingivalis* involves truncating the N-terminus of the DUF domain by 38 amino acid residues. Adding these additional 38 N-terminal residues to the construct to produce the intact DUF domain instead of the truncated domain (Dc) yields highly soluble recombinant proteins (e.g., DUF2436, KDA, and KKDAKK).

[0583] KDcAK1n also includes a C-terminally truncated K1 domain (K1n) that is again based on cell surface processing of the Kgp polyprotein to produce an A1 adhesin fragment. Adding the missing C-terminal sequence to the complete K1 domain in K1n did not improve solubility, as the protein was still primarily expressed as inclusion bodies in *E. coli*. Since most constructs with partial or complete K1 domains exhibited some solubility issues, it was concluded that the complete DUF domain is a key domain for *E. coli* to express constructs as soluble proteins, and that the inclusion of the K1 domain in chimeric variants should be avoided if possible. Furthermore, data from periodontal bone loss and epitope analysis in animal experiments suggest that the K1 domain may not significantly enhance protection, especially in the absence of a protective antigen generating antiserum that binds to the K1 epitope ABM5.

[0584] KDcAK1n contains four cysteine ​​residues: one in DUF, two in ABM, and one in K1. These cysteine ​​residues are thought to be the cause of the inclusion body formation problem. *Porphyromonas gingivalis* is an obligate anaerobe and requires a highly reducing environment to become pathogenic. Known or modeled structures of the Kgp domains indicate that the cysteine ​​residues are reduced on the cell surface and do not participate in disulfide bonds. Expression in *E. coli* under more oxidative conditions induces the formation of disulfide bridges between the denatured domains and the formation of inclusion bodies. The disulfide bridges between the denatured domains of the chimera can be seen on non-reduced native PAGE (which changes upon reduction).

[0585] The results presented in this paper demonstrate that the folded ABM domains of Kgp undergo β-chain exchange polymerization to form a uniform sequence ladder independent of the reducing agent. Figure 3Lanes 9 and 10). This novel β-chain exchange polymerization is an important mechanism used by *Porphyromonas gingivalis* to form the surface layer of gingival protease, not only on the cell surface but also on the outer membrane vesicles released into the host during disease progression. This work characterizing the prior art chimera KDcAK1n and its domains clearly demonstrates the presence of two forms of polymers in the original chimera: a polymer based on the disulfide-bridged denatured domain and a polymer based on the β-chain exchange native ABM domain. Attempts to isolate and purify these different forms into specific dimers and larger molecular weight polymers have been unsuccessful. Furthermore, because these different purified single species are disulfide-locked denatured structures, the use of said single species in animal models does not produce protection against periodontal bone loss ( Figure 6 This work demonstrates that soluble, well-defined, and homogeneous recombinants with folded D and A domains and no disulfide crosslinking are superior to mixtures of denatured and native forms produced by dissolving the chimera from inclusion bodies in 8 M urea, with low yields. Figure 6 , 8 and 9).

[0586] These results suggest that the presence of cysteine ​​residues in the antigen may pose problems for solubility and the expression and purification of well-defined commercially developed candidates. Therefore, the possibility of mutating these cysteine ​​residues to serine residues was explored. Mutating four cysteine ​​residues to four serine residues did not prevent the β-chain exchange polymerization of the native ABM(A) domain, indicating that the ABM domain still folds correctly in the “4C” to “4S” mutant. Figure 3 ).

[0587] Furthermore, these results indicate that mutations in the key sequences of the ABM domain that were predicted to allow β-chain flipping at the Pro hinge bend (PVQN > AVQP) eliminated β-chain exchange polymerization. Figure 3 Therefore, the combination of mutating four Cys residues to Ser and mutating the hinge region PVQN (SEQ ID NO: 86) to AVQP (SEQ ID NO: 85) eliminates multimerization under both oxidative and reducing conditions. Figure 3 In this regard, the mutant KDAK1nK-4S-AVQP provided protection against periodontal bone loss and produced good antibody titers against predicted protective epitopes (KAS, DUF, ABM) in animal periodontitis models. Figure 7 ).

[0588] Antibody responses to different segments (K, D, A, and K1) and suspected important epitopes (KAS, ABM2, ABM3, EP1) of chimeras using both protective and non-protective sera did not provide any clear pattern. Although it is clear that these protected chimeric variants tend to show strong (IgG / IgG1) responses to intact *Porphyromonas gingivalis* cells and to known protective epitopes (KAS, DUF, ABM), it appears that protection may not be related to any single epitope, but rather to a combination of epitopes required to ensure adequate protection against periodontal bone loss. However, there is a trend towards one epitope standing out, involving the active site sequence KAS of the chimeric variants that provides protection.

[0589] Although no single epitope explicitly indicates its greater importance, allowing a response to it to be used as a substitute or biomarker for vaccine-induced protection, it is clear that protective chimeric variants tend to produce a favorable antibody response against the protease active site sequence (KAS or K). Antibodies generated against the active site sequence have previously demonstrated neutralizing proteolytic activity against gingival proteases (Kgp and RgpA / B), the major virulence factor of *Porphyromonas gingivalis*. Therefore, in a method to increase antibody titers against the active site and thus enhance protection, the inventors added an additional copy of the active site sequence (K) to KDA and KDAK1n, and both constructs exhibited improved protection in animal models of periodontitis. Figure 8 The soluble, non-polymerized mutant KDAK1nK-4S-AVQP, with two copies of the KAS(K) motif, provides excellent protection against periodontal bone loss induced by *Porphyromonas gingivalis*. Figure 9 ).

[0590] Other soluble non-multimerized mutants, including KDAK-3S-AVQP, KDAK-1V-2S-AVQP, and KDAK-2S-AVQP, also provide good protection against Porphyromonas gingivalis-induced periodontal bone loss. Figures 9 to 13 ).

[0591] Example 4: Developing a model of neuropathology induced by Porphyromonas gingivalis

[0592] The aim of this study was to demonstrate in a mouse model that *Porphyromonas gingivalis* and / or its outer membrane vesicles (OMV) and / or secreted gingival proteases are agents of neuropathology (e.g., pathology similar to Alzheimer's disease), and covered the following tasks: 1. Establish a 6-week oral challenge animal model. Mice were fed 1 x 10^10 CFU of Porphyromonas gingivalis or sham-fed three times a week for 6 weeks. Mid-term checkpoint at 3 weeks.

[0593] 2. Immunohistochemical analysis was performed on the brains of mice sham-fed or fed *Porphyromonas gingivalis* for 3 and 6 weeks to determine the presence of *Porphyromonas gingivalis* and / or OMV and / or secreted gingival proteases, as well as AD-like pathology.

[0594] 3. Analyze periodontal bone loss in control and Porphyromonas gingivalis-infected animals at 6 weeks of age.

[0595] Materials and methods

[0596] Growth of Porphyromonas gingivalis and preparation of a mouse model

[0597] *Porphyromonas gingivalis* strain W50 was obtained from the culture collection of the Collaborative Centre for Oral Health, Faculty of Dentistry, University of Melbourne, Australia. Typically, *Porphyromonas gingivalis* W50 cultures are grown in 200 mL of brain heart infusion (37 g / L BHI; Oxoid, Hampshire, UK) supplemented with 5 mg / L heme and 0.5 g / L cysteine ​​at the MK3 anaerobic workstation (Don Whitley Technologies, Adelaide, Australia) and pre-reduced at 37°C for approximately 30 hours in an anaerobic atmosphere containing 85% N2, 10% CO2, and 5% H2. Each pre-reduced BHI broth was inoculated with a frozen cell pellet of *Porphyromonas gingivalis* W50 collected from 2 mL aliquots of late exponential growth cultures, rotated at 8,000 xg for 5 minutes, the supernatant was discarded, and the pellet was flash-frozen in liquid N2 before storage at -80°C. Following inoculation of frozen cell pellets, cultures were incubated for 16 hours in an anaerobic workstation before harvesting during the late exponential growth phase. The optical density of batches of cultures at 650 nm was monitored using a spectrophotometer (Model 295E, PerkinElmer, Germany). Bacterial purity was typically confirmed by Gram staining. The number of *Porphyromonas gingivalis* W50 cells present in each culture was calculated using the formula y = 1 x 10^9 (OD650 / 3.513) + 1 x 10^8 cfu / mL. Since a dose of 1 x 10^10 cfu was required per mouse, an appropriate volume of culture was harvested by centrifugation (8,000 xg, 15 min, 4°C), and the supernatant was discarded. The cell pellet was resuspended in 2% carboxymethyl cellulose in PBS containing 5 mM cysteine, 25 μL per mouse.

[0598] Animal ethics

[0599] All animal experimental procedures were conducted in strict accordance with the recommendations of the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. The experimental protocols have been approved by the University of Melbourne Ethics Committee for Animal Experimentation.

[0600] Mouse neuropathology and periodontitis models

[0601] Mice (female C57BL / 6; 13 weeks old, 13 mice / group) were orally inoculated with *Porphyromonas gingivalis* W50 three times a week for 6 weeks on the gingival margin of the maxillary molars [1 x 10^10 live *Porphyromonas gingivalis* W50 cells per dose suspended in 25 μL of 2% w / v carboxymethyl cellulose (CMC, Sigma-Aldrich) in PBS containing 5 mM cysteine]. Animal groups were orally inoculated with either *Porphyromonas gingivalis* W50 or 2% carboxymethyl cellulose + 5 mM cysteine ​​in PBS as uninfected controls. After 10 doses (3 weeks), 3 mice from each group were asphyxiated with CO2, bled by cardiac puncture, and perfused with 4% paraformaldehyde before brain removal.

[0602] The brain was dissected into two hemispheres, one of which was fixed in formalin for immunohistochemistry (IHC). After 19 doses, all 10 mice fed with Porphyromonas gingivalis and 5 uninfected control mice were euthanized and treated as described above, with the maxilla also removed to determine alveolar bone loss.

[0603] Immunohistochemistry

[0604] For immunohistochemistry (IHC), formalin-fixed paraffin-embedded (FFPE) mouse brain sections (5 μm thick) were mounted onto Superfrost slides. Sections of FFPE tissue were selected from the hippocampus (including the neocortex) so that each slide contained three brain tissue sections from one mouse. The slides were dewaxed and rehydrated according to standard protocols to prepare for IHC staining. The following primary antibodies were used for IHC: rabbit polyclonal Porphyromonas gingivalis gingival protease R1 (RgpA) antibody (Biorbyt orb243611) or rabbit monoclonal β-amyloid (Aβ) antibody (Ingenieur; catalog number 700254), clone H31L21. Optimal staining conditions were determined using heat-treated antigen retrieval, citrate buffer at pH 6.0, and a 1 / 500 primary antibody dilution.

[0605] According to the manufacturer's instructions, slides were treated using the Mouse and Rabbit Specific HRP / DAB IHC Detection Kit - Micropolymer (ab236466, Abcam, Australia). In short, rehydrated slides were treated with hydrogen peroxide (Abcam, Australia) followed by heat-induced epitope repair (HIER) for 15 minutes with citrate buffer at pH 6.0. The slides were then incubated with a protein blocking reagent (Abcam, Australia) for 10 minutes and then treated overnight at 4°C with specific antibodies against RgpA (1:500) or Aβ (1:500). The next day, after washing three times with PBS, the slides were incubated with goat anti-rabbit IgG secondary antibody (Abcam, Australia) at room temperature for 15 minutes and developed using HRP-conjugated DAB substrate (Abcam, Australia). The treated slides were washed and counterstained with hematoxylin according to standard protocol. The slides were scanned using a Panoramic Scan II digital scanner, a high-throughput bright-field digital slide scanner equipped with an x20 objective lens that generates histological images.

[0606] Finally, the positive staining counts were graded based on digital images of scanned slides obtained using the QuPath open-source digital software v. 0.2.011. In short, the QuPath "Cell Positive Detection" function was used to automatically identify positively stained cells within brain samples. Here, settings were adjusted to detect cells stained with both hematoxylin and DAB. From the default settings of the "Cell Positive Detection" function, "Detection Image" was set to "Total Optical Density," "Requested Pixel Size" was set to "0.2 μm," the "Background Radius" parameter was set to "10 μm," and the "Median Filter Radius" was set to "2 μm." After automation, the cells were then evaluated visually. This analysis method was used for all selected samples. Each slide contained three slices of the brain, and the mean number of positive cells per mouse was calculated. Amyloid plaque formation induced by *Porphyromonas gingivalis* was calculated by subtracting the mean number of positive cells per mouse or mean number of positive cells / mm² / mouse from the mean number of positive cells per mouse in the uninfected control group from the mean number of positive cells per mouse in the *Porphyromonas gingivalis*-infected group. Data were obtained from 34 brain slices per mouse, with 3 brain slices per group. Data are presented as mean ± standard deviation, representing biological variation among mice, and were analyzed using an unpaired t-test.

[0607] Measurement results of alveolar bone loss in the maxilla of mice

[0608] The maxillae with bone loss to be examined were boiled in deionized water (1 minute), mechanically demineralized, and immersed in 2% w / v potassium hydroxide (16 hours, 25°C). The maxillae were washed twice with deionized water (25°C), dried (1 hour, 37°C), and stained with 0.5% w / v methylene blue aqueous solution. Coded digital images of the buccal side of the maxilla were captured using an Olympus DP12 digital camera mounted on a dissecting microscope to assess horizontal bone loss, analyzed using ImageJ imaging software (https: / / imagej.nih.gov / ij / index.html). The maxillae were oriented so that the buccal and lingual molar cusps overlapped. Images were captured in micrometers within frames to allow for normalization of measurements for each image. Horizontal bone loss was defined as loss occurring in a horizontal plane perpendicular to the alveolar ridge, resulting in a decrease in ridge height. The visible area from the cementum-enamel junction (CEJ) to the alveolar ridge (ABC) of each molar was measured using ImageJ version 1.3k imaging software, yielding results in mm. 2Total visible CEJ-ABC area, in mm². *Porphyromonas gingivalis*-induced alveolar bone loss, in mm², was calculated by subtracting the total visible CEJ-ABC area of ​​the uninfected control group from the total visible CEJ-ABC area of ​​the *Porphyromonas gingivalis*-infected group. Alveolar bone loss measurements were determined twice in a randomized and blinded protocol. Data are presented as mean ± standard deviation (in mm²). 2 The results were expressed in units of 1, and the analysis was performed using one-way ANOVA and Dunnett's T3 post-hoc test.

[0609] Results and discussion

[0610] After 10 doses of *Porphyromonas gingivalis* W50 or sham-feeding 10 doses of PBS containing 2% CMC + 5 mM cysteine, 3 mice from each group were euthanized and their brains were removed for immunohistochemistry to detect RgpA in brain slices covering the hippocampus. Figure 18 A) and AD-like pathology in the form of β-amyloid formation ( Figure 18 B).

[0611] Using a dosing regimen of 10 doses of 1x10^10 CFU of Porphyromonas gingivalis for 3 weeks, Porphyromonas gingivalis and / or its OMV significantly infiltrated the brain, as detected by RgpA-specific antibody.

[0612] Compared to sham-fed mice, mice fed *Porphyromonas gingivalis* had an average of 143,526 ± 14,458 RgpA-positive cells per brain, compared to 232 ± 323. Surprisingly, *Porphyromonas gingivalis*-induced β-amyloid protein was also detected in the brains of mice fed *Porphyromonas gingivalis*, with an average of 2,194 ± 786 positive cells.

[0613] Bone loss induced by feeding with Porphyromonas gingivalis was also assessed during the same time period. Figure 18 C shows the *Porphyromonas gingivalis*-induced bone loss (mm) in control (sham-fed; CMC control) mice compared to mice fed *Porphyromonas gingivalis*. These data indicate that *Porphyromonas gingivalis* induces bone loss, accompanied by the aforementioned neurogenic changes.

[0614] After 19 doses of *Porphyromonas gingivalis* W50 or sham-feeding only 19 doses of PBS containing 2% CMC + 5 mM cysteine, the remaining 10 *Porphyromonas gingivalis*-infected mice and 5 mice from the uninfected control group were sacrificed, and their brains were removed for immunohistochemistry to detect RgpA in brain slices covering the hippocampus. Figure 18 D) and β-amyloid protein ( Figure 18The presence of E). After 3 weeks, the mean number of RgpA-positive cells and Porphyromonas gingivalis-induced β-amyloid protein was significantly increased, with 201,441 ± 18,945 cells and 9602 ± 2065 cells per mouse brain, respectively. Figure 18 F).

[0615] Examination of the maxilla revealed that *Porphyromonas gingivalis* induced significant alveolar bone loss over a 6-week period, due to a feeding regimen of 19 doses. Figure 18 C).

[0616] Based on these results, it was determined that in subsequent experiments (such as the prevention model outlined in Example 5), 19 doses of Porphyromonas gingivalis would be used instead of 10 doses, because at 6 weeks, the level of Alzheimer's-like pathology in the form of β-amyloid was increased compared to 3 weeks.

[0617] Example 5: Prophylactic vaccination with chimeric proteins to induce an immune response against Porphyromonas gingivalis.

[0618] The aim of this series of experiments is to demonstrate that prophylactic vaccination with chimeric protein vaccines as described herein (e.g., KDAK-3S-AVQP, sequence SEQ ID NO: 69) can prevent or delay the development of neuropathologies (e.g., Alzheimer's-like pathologies) in the mouse brain, and covers the following tasks: 1. Administer prophylactic vaccination before Porphyromonas gingivalis challenge. 2. Measure the immunogenicity of the vaccine using ELISA. 3. Quantification of Porphyromonas gingivalis and / or OMV infiltration, Aβ and P-tau neuropathology, and inflammatory cytokines IL-6 and IL-1β in mouse brains using IHC. 4. Use confocal microscopy to determine whether Kgp and Aβ detected in the mouse brain are colocalized. 5. Analysis of periodontal bone loss in all animal groups

[0619] Materials and methods

[0620] Growth of Porphyromonas gingivalis and preparation of a mouse model

[0621] Porphyromonas gingivalis strain W50 was grown as described in Example 4.

[0622] Mouse models of neuropathology similar to Alzheimer's disease and models of periodontitis

[0623] A mouse model of prophylactic vaccination was established using 24 female C57BL / 6 mice (13 weeks old at T=0), which were divided into 4 groups ( Figure 17 ).

[0624] Mice vaccinated received intraperitoneal (IP) and subcutaneous (SC) vaccinations. Each dose of 200 μg recombinant protein KDAK-3S-AVQP (SEQ ID NO: 69) was mixed with alum at a 1:1 (w / w) ratio in histidine buffer (10 mM histidine, 150 mM NaCl, pH 6.5). Figure 17 Mice challenged with *Porphyromonas gingivalis* were intraorally inoculated with *Porphyromonas gingivalis* W50 cells [1 x 10^10 live *Porphyromonas gingivalis* W50 cells per dose suspended in 25 μL of 2% (w / v) CMC containing 5 mM cysteine ​​in PBS]. Using the same protocol, sham-fed mice were orally inoculated with 2% CMC + 5 mM cysteine ​​in PBS as an uninfected control. Figure 17 All mice were euthanized at 12 weeks of age.

[0625] At 12 weeks, all mice were asphyxiated with CO2, bled by cardiac puncture, and then perfused with PBS to remove residual blood before brain removal. The brain was excised and bisected along the midsagittal plane, and the left hemisphere was fixed in 4% paraformaldehyde in PBS for 24 hours and processed to prepare paraffin sections for immunohistochemistry. The right hemisphere was dried and placed in pre-weighed Eppendorf tubes for ICP-MS analysis. The maxilla was also removed to determine alveolar bone loss.

[0626] Subclass antibodies in serum were determined using enzyme-linked immunosorbent assay (ELISA).

[0627] ELISA was performed to evaluate subclass antibodies in serum using heat-inactivated (HK) *Porphyromonas gingivalis* W50 cells, recombinant domain subunits (Kgpcat), or recombinant total protein (KDAK-3S-AVWP) in 0.1 M PBS (pH 7.4) (1 μg / mL) to coat the wells of flat-bottomed polyethylene microplates (4°C, 16 h). After antigen coating, the plates were blocked in 5% (w / v) skim milk powder in PBS. ELISA was developed using 1 / 4000 goat anti-mouse IgG (M8642) and IgG1 (M8770) antibodies (Southern Biotech) and 1 / 4000 horseradish peroxidase-conjugated porcine anti-goat IgG antibody after incubation with mouse serum. Plates were washed three times with 250 μL PBS-T (PBS-0.1% (w / v) Tween 20) between all steps. All ELISAs were developed using ABTS substrates (0.05% (w / v) and 0.02% (w / v) H2O2 in 50 mM citrate). All optical density measurements were performed at 405 nm on a Wallac VICTOR3 1420 multi-tag counter (PerkinElmer).

[0628] Immunohistochemistry

[0629] Immunohistochemistry was performed as described in Example 4. In addition, the following antibodies were used: mouse monoclonal anti-phosphorylated tau (Ser202, Thr205), monoclonal antibody (AT8) (Thermo Fisher Scientific MN1020); rabbit polyclonal anti-IL-6 (Ebola AB6672) and rabbit polyclonal anti-IL-1β (Ebola AB9722). Optimal staining conditions for all primary antibodies were determined to be 1 / 500 using heat-treated antigen retrieval, citrate buffer at pH 6.0, and primary antibody dilutions, except for the anti-phosphorylated tau antibody, which had an optimal dilution of 1 / 40.

[0630] Data are presented as mean ± standard deviation, representing biological variation among mice, and analyzed using one-way ANOVA and Tukey's multiple comparison test. This method was used to quantify positive staining for all primary antibodies except anti-phosphorylated tau. Phosphorylated tau signals were graded based on positive pixel counts of digital images of scanned slides using the QuPath open-source digital software v. 0.2.011 (Bankhead et al. 2017). Briefly, positive pixels within brain samples were automatically identified using QuPath's "Create Thresholder" function. Here, settings were adjusted to detect cells stained with both hematoxylin and DAB. The "Create Thresholder" function was selected from the classification functions, and from the default settings of the "Create Thresholder" function, "Resolution" was set to "High," "Channels" to "DAB," "Pre-Filter" parameter to "Gaussian," and "Smoothing Sigma" to "2.5 μm," while the "Threshold" was manually adjusted to avoid background noise. Positive and negative classes were specified based on the threshold, with "above threshold" designated as "positive." After automation, cells were then evaluated visually. Each slide contained four slices of a brain and was used to calculate the mean positive pixels (%) per mouse. Data were derived from four brain slices per mouse, with three mice in each group. Data are presented as mean ± standard deviation, representing biological variation among mice, and were analyzed using one-way ANOVA followed by Tukey's multiple comparison test.

[0631] Immunofluorescence and confocal microscopy

[0632] FFPE sections of one brain from the *Porphyromonas gingivalis*-fed group (PG1) and one brain from the sham-fed group (CMC3) excited by *Porphyromonas gingivalis* from Milestone 1 at 3 weeks were dewaxed and subjected to antigen retrieval procedures as previously described for IHC. Samples were blocked for 1 hour at room temperature with 10% goat serum in PBS + 1% Tween, followed by two 5-minute washes with PBS. The samples were then blocked using the avidin / biotin blocking system (BioLegend SIG-31126) according to the manufacturer's instructions. After three PBS washes, the samples were incubated overnight at 4°C with the primary antibody mouse biotinylated anti-PG-KAS2 (a self-made antibody targeting the Kgp active site peptide) and rabbit monoclonal anti-β-amyloid (Aβ) antibody clone H31L21 (Ingenium Biotech, Serial No. 700254), each diluted 1 / 500. After three washes with PBS, the samples were subsequently protected from light. Samples were incubated with the fluorescent conjugate streptavidin-Alexa Fluor™ 488 conjugate (Thermo Fisher Scientific S11223) and the goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody-Alexa Fluor™ 647 conjugate (Thermo Fisher Scientific A-21244); both were diluted 1 / 500 in PBS with 1% goat serum and +1% Tween and incubated at room temperature for 1 hour. After three PBS washes, samples were incubated with DAPI (4',6-diamidinyl-2-phenylindole, 1:5000 in PBS) for 5 minutes, followed by three washes with PBS. The slides were mounted with 30–50 μL of ProLong Gold and coverslips, and then imaged for immunofluorescence using a Zeiss LSM800 confocal microscope with 63x objectives / 1.40 Oil DIC M27, 1.3 scanning zoom, 405 nm DAPI, 488 nm and 640 nm channels at the Biomedical Optical Microscopy Platform, The University of Melbourne.

[0633] To quantify the colocalization between fluorescently labeled Kgp and β-amyloid probes, Pearson's correlation coefficient and Manders' correlation coefficient (M1 and M2) (Schindelin et al. 2012) were determined from corresponding confocal images using FIJI software and the JACoP plugin (Bolte and Cordelières, 2006). Automatically adjusted thresholds for two distinct channels were obtained using a method without Costes randomization. In each JACoP run, the automatic thresholds were adjusted, with channels 1 and 2 representing β-amyloid (red) and Kgp (green), respectively. All images were analyzed using the same script, with the thresholds for each individual channel adjusted each time. Pearson's and Manders' correlation coefficients (M1 and M2) were extracted from the JaCoP analysis for data representation.

[0634] Measurement results of alveolar bone loss in the maxilla of mice

[0635] As described in Example 4, assess alveolar bone loss.

[0636] Results and discussion

[0637] A preventive vaccination model for Porphyromonas gingivalis-induced neuropathology and periodontal disease was successfully established by collecting serum and tissue samples from four mouse groups used for analysis: a control group vaccinated only, a control group uninfected (sham-fed), a control group infected with Porphyromonas gingivalis, and a group vaccinated and then infected with Porphyromonas gingivalis.

[0638] Serum analysis using ELISA showed that vaccination with three doses of KDAK-3S-AVQP was highly effective in stimulating an IgG1 antibody response. Figure 19 Evaluation of antibody titers against the antigen KDAK-3S-AVQP, heat-inactivated whole cells of *Porphyromonas gingivalis* (HKPg), and Kgpcat from four mouse groups showed that mice repeatedly fed *Porphyromonas gingivalis* could produce an antibody response, but this response was not as strong as the immune response induced by vaccination. The immune response in mice vaccinated only was similar to that in mice vaccinated and challenged with *Porphyromonas gingivalis* for 6 weeks, indicating that challenge with *Porphyromonas gingivalis* did not enhance the antibody response. High antibody titers against KDAK-3S-AVQP were observed in both vaccinated mice and mice vaccinated prior to *Porphyromonas gingivalis* infection, with titers exceeding 200,000 (…). Figure 19 ).

[0639] Figure 20 This study demonstrated the efficacy of the chimeric protein KDAK-3S-AVQP in preventing bone loss—confirming the vaccine's efficacy before examining changes in neurogenic / inflammatory markers.

[0640] Immunohistochemistry was then used to detect and quantify a specific antigen, RgpA gingival protease, in brain tissue from three mice in each group. Figure 21 ), and biomarkers commonly associated with pathologies similar to Alzheimer's disease, such as Aβ ( Figure 22 ) and P-tau ( Figure 23 ), and the inflammatory cytokine IL-6 ( Figure 24 ) and IL-1β ( Figure 25 ).

[0641] Vaccination with KDAK-3S-AVQP significantly reduced the infiltration of *Porphyromonas gingivalis* and / or OMV into the brains of mice, with a mean reduction of 61.2% ± 12.9% of RgpA-positive cells per mouse brain compared to 39% ± 3.9% in mice vaccinated with and fed *Porphyromonas gingivalis*. Figure 21 Given the acute and extreme nature of Porphyromonas gingivalis stimulation, the reduction in the infiltration of Porphyromonas gingivalis and / or OMV and / or secreted gingival proteases into the brains of vaccinated and stimulated mice is impressive.

[0642] β-amyloid protein was detected in the brains of mice from all groups, which is expected, as the production of Aβ occurs naturally during aging. Figure 22 Alzheimer's disease pathology is associated with abnormally high levels of Aβ accumulation. Vaccination with KDAK-3S-AVQP significantly reduced the amount of Aβ produced, with 19.07% ± 1.48% Aβ in the brains of mice vaccinated with and fed with *Porphyromonas gingivalis* compared to 12.75% ± 2.08% Aβ. Figure 22 ).

[0643] Figure 28 This study demonstrated the co-localization of Kgp gingival protease and β-amyloid in mice orally infected with *Porphyromonas gingivalis*. The second biomarker of Alzheimer's disease pathology, phosphorylated tau protein (P-tau), was also significantly reduced in mice vaccinated with KDAK-3S-AVQP prior to *Porphyromonas gingivalis* challenge, compared to 21.4% ± 2.8% P-tau, at 9.65% ± 1.45% P-tau. Figure 23 ).

[0644] Compared with mice fed with *Porphyromonas gingivalis*, mice vaccinated with and fed with *Porphyromonas gingivalis* also showed a significant decrease in the inflammatory biomarker IL-6, with IL-6 levels of 11.8% ± 0.12% compared to 19.4% ± 4.3%. Figure 24 Another biomarker, inflammatory IL-1β, also showed a significant decreasing trend, with statistical significance approaching significance (p = 0.05), indicating that using more animals and improving statistical power would achieve significance. Figure 25 ).

[0645] Figure 26 The results show the summary of efficacy of KDAK-3S-AVQP vaccination in reducing biomarkers of Porphyromonas gingivalis-induced neuropathology.

[0646] Confocal microscopy and fluorescent probes detecting Kgp gingival protease and Aβ were used to determine whether Aβ colocalized with *Porphyromonas gingivalis* and / or its OMV in mouse brains (data not shown). In image analysis, the Pearson coefficient was r = 0.854, and the overlap coefficient was r = 0.835, which increased to r = 0.925 when thresholds of thrA = 39 and thrB = 34 were applied; this indicates that the signals are colocalized. Figure 28 ).

[0647] Vaccination with KDAK-3S-AVQP also reduced alveolar bone loss in mice stimulated with 19 doses (6 weeks) of Porphyromonas gingivalis to naturally occurring levels; this significantly reduced alveolar bone loss compared to unvaccinated mice stimulated with Porphyromonas gingivalis.

[0648] A second group of *Porphyromonas gingivalis* challenge experiments was conducted, this time shortening the challenge period to 3 weeks instead of 6 weeks, followed by a 3-week recovery period, with 10 animals used in each group. The results were consistent with those of the first group (not shown).

[0649] Example 6: A model for treating Porphyromonas gingivalis-induced or related neuropathology

[0650] To explore the therapeutic efficacy of the chimeric protein KDAK-3S-AVQP (as described elsewhere in this article) in a model of Porphyromonas gingivalis-induced neuropathology, a series of preliminary experiments were conducted.

[0651] Mice were fed *Porphyromonas gingivalis* according to the methods described in Examples 4 and 5 of this paper. The establishment of *Porphyromonas gingivalis*-induced neuropathology was assessed by staining β-amyloid protein (similar to the methods described in Examples 4 and 5).

[0652] After establishing Porphyromonas gingivalis infection, mice were administered 200 μg of KDAK-3S-AVQP with alum as an adjuvant.

[0653] like Figure 27 As shown in Figure A, β-amyloid levels were detectable in naïve mice (i.e., mice not infected with *Porphyromonas gingivalis*), but significantly higher levels were observed after *Porphyromonas gingivalis* infection. β-amyloid levels were significantly reduced in the brains of mice vaccinated with KDAK-3S-AVQP after *Porphyromonas gingivalis* infection.

[0654] Figure 27 B showed that the reduction in β-amyloid protein in mice treated with KDAK-3S-AVQP was also associated with a reduction in bone loss, further demonstrating the therapeutic efficacy of KDAK-3S-AVQP protein in this experiment.

Claims

1. A method for preventing or treating neuropathology in a subject, wherein the neuropathology is associated with or caused by a Porphyromonas gingivalis (P. gingivalis) infection, the method comprising administering to the subject a therapeutically effective amount of a chimeric or fusion protein comprising a first polypeptide and a second polypeptide, wherein: the first polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1; and the second polypeptide comprises an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

2. P. gingivalis ​ A) the first polypeptide comprises or consists of an amino acid sequence of the active site of an Arg-gingivase or Lys-gingivase of P. gingivalis or a sequence at least 80% identical thereto; and, B) the second polypeptide comprises or consists of an adhesin domain of an Arg-gingivase or Lys-gingivase of P. gingivalis; wherein the second polypeptide comprises a sequence of one or more adhesin binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain of P. gingivalis gingivases and the cleaved adhesin domain (CAD); and wherein the second polypeptide: a) does not comprise a sequence of a cleaved adhesin domain (CAD) or a portion thereof, preferably a sequence of a CAD having the sequence set forth in SEQ ID NO: 12 or 13 or a sequence at least 80% identical thereto; and / or, b) comprises an amino acid sequence corresponding substantially to the full length of the DUF2436 domain of an Arg-gingivase or Lys-gingivase, preferably an amino acid sequence as set forth in SEQ ID NO: 23, or a sequence at least 80% identical thereto; and / or, c) comprises one or more amino acid substitutions selected from: one or more cysteine amino acid substitutions in the adhesin domain compared to the naturally occurring Arg-gingivase or Lys-gingivase sequence in the corresponding region; and / or, one or more amino acid motif substitutions selected from: i) a substitution of a proline and / or asparagine residue in the sequence PxxN corresponding to or at a position equivalent to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1); ii) a substitution of a motif NxFA in the sequence corresponding to or at a position equivalent to residues 2 to 5 of the sequence of SEQ ID NO: 14 or 19 (ABM1) to SxYQ; iii) a substitution of a second tyrosine residue corresponding to or at a position equivalent to residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue corresponding to or at a position equivalent to residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) to an alanine residue; optionally wherein the chimeric or fusion protein comprises one or more additional polypeptides comprising or consisting of an amino acid sequence of the active site of an Arg-gingivase or Lys-gingivase of P. gingivalis or a sequence at least 80% identical thereto; thereby preventing or treating P. gingivalis-induced neuropathology in the subject.

2. A method for reducing the level of P. gingivalis gingipain in a neural tissue of a subject, the method comprising administering to the subject a therapeutically effective amount of a chimeric or fusion protein comprising a first polypeptide and a second polypeptide, wherein: A) the first polypeptide comprises or consists of an amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis or a sequence at least 80% identical thereto; and, B) the second polypeptide comprises or consists of an amino acid sequence of the adhesin domain of Arg- or Lys-gingipain of P. gingivalis; wherein the second polypeptide comprises the sequence of one or more adhesin binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain and the cleaved adhesin domain (CAD) of P. gingivalis gingipain; and wherein the second polypeptide: a) does not comprise: the sequence of a cleaved adhesin domain (CAD) or a portion thereof, preferably the sequence of a CAD having the sequence set forth in SEQ ID NO: 12 or 13 or a sequence at least 80% identical thereto; and / or, b) comprises an amino acid sequence corresponding substantially to the full length of the DUF2436 domain of Arg- or Lys-gingipain, preferably the amino acid sequence set forth in SEQ ID NO: 23, or a sequence at least 80% identical thereto; and / or, c) comprises one or more amino acid substitutions selected from: one or more cysteine amino acid substitutions in the adhesin domain compared to the naturally occurring Arg- or Lys-gingipain sequence in the corresponding region; and / or, one or more amino acid motif substitutions selected from: i) substitution of a proline and / or asparagine residue in the sequence PxxN corresponding to or at a position equivalent to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1); ii) substitution of the motif NxFA in the sequence corresponding to or at a position equivalent to residues 2 to 5 of the sequence of SEQ ID NO: 14 or 19 (ABM1) to SxYQ; iii) substitution of a second tyrosine residue corresponding to or at a position equivalent to residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue corresponding to or at a position equivalent to residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) to alanine residues; optionally wherein the chimeric or fusion protein comprises one or more additional polypeptides comprising or consisting of an amino acid sequence of the active site of Arg- or Lys-gingipain of P. gingivalis or a sequence at least 80% identical thereto; thereby reducing the level of P. gingivalis gingipains in the neural tissue of the subject.

3. A method for delaying the onset of P. gingivalis-associated or induced neuropathology in a subject, the method comprising administering to the subject a therapeutically effective amount of a chimeric or fusion protein comprising a first polypeptide and a second polypeptide, wherein: A) the first polypeptide comprises or consists of an amino acid sequence of the active site of Arg gingipain or Lys gingipain of P. gingivalis or a sequence at least 80% identical thereto; and, B) the second polypeptide comprises or consists of an amino acid sequence of the adhesin domain of Arg gingipain or Lys gingipain of P. gingivalis; wherein the second polypeptide comprises the sequence of one or more adhesin binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain and the cleaved adhesin domain (CAD) of P. gingivalis gingipains; and wherein the second polypeptide: a) does not comprise: the sequence of a cleaved adhesin domain (CAD) or a portion thereof, preferably the sequence of a CAD having the sequence set forth in SEQ ID NO: 12 or 13 or a sequence at least 80% identical thereto; and / or, b) comprises an amino acid sequence corresponding substantially to the full length of the DUF2436 domain of Arg gingipain or Lys gingipain, preferably the amino acid sequence set forth in SEQ ID NO: 23, or a sequence at least 80% identical thereto; and / or, c) comprises one or more amino acid substitutions selected from: one or more cysteine amino acid substitutions in the adhesin domain compared to the naturally occurring Arg gingipain or Lys gingipain sequence in the corresponding region; and / or, one or more amino acid motif substitutions selected from: i) substitution of a proline and / or asparagine residue in the sequence PxxN corresponding to or located at a position equivalent to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1); ii) substitution of the motif NxFA in the sequence corresponding to or located at a position equivalent to residues 2 to 5 of the sequence of SEQ ID NO: 14 or 19 (ABM1) to SxYQ; iii) substitution of a second tyrosine residue corresponding to or located at a position equivalent to residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue corresponding to or located at a position equivalent to residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) to alanine residues; optionally, wherein the chimeric or fusion protein comprises one or more additional polypeptides comprising or consisting of an amino acid sequence of the active site of Arg-gingivase or Lys-gingivase of P. gingivalis or a sequence at least 80% identical thereto; thereby delaying the onset of P. gingivalis-related or induced neuropathology in the subject.

4. A method for preventing or slowing the rate of P. gingivalis-induced or related neuropathology in a subject, the method comprising administering to the subject a therapeutically effective amount of a chimeric or fusion protein comprising a first polypeptide and a second polypeptide, wherein: A) the first polypeptide comprises or consists of an amino acid sequence of the active site of Arg-gingivase or Lys-gingivase of P. gingivalis or a sequence at least 80% identical thereto; and, B) the second polypeptide comprises or consists of an amino acid sequence of the adhesin domain of Arg-gingivase or Lys-gingivase of P. gingivalis; wherein the second polypeptide comprises a sequence of one or more adhesin binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain and the cleaved adhesin domain (CAD) of P. gingivalis gingivases; and wherein the second polypeptide: a) does not comprise a sequence of a cleaved adhesin domain (CAD) or a portion thereof, preferably a sequence of a CAD having the sequence set forth in SEQ ID NO: 12 or 13 or a sequence at least 80% identical thereto; and / or, b) comprises an amino acid sequence corresponding substantially to the full length of the DUF2436 domain of Arg-gingivase or Lys-gingivase, preferably an amino acid sequence as set forth in SEQ ID NO: 23, or a sequence at least 80% identical thereto; and / or, c) comprises one or more amino acid substitutions selected from: one or more cysteine amino acid substitutions in the adhesin domain compared to the naturally occurring Arg-gingivase or Lys-gingivase sequence in the corresponding region; and / or, one or more amino acid motif substitutions selected from: i) substitution of a proline and / or asparagine residue in the sequence PxxN corresponding to or located at a position equivalent to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1); ii) substitution of the motif NxFA in the sequence corresponding to or located at a position equivalent to residues 2 to 5 of the sequence of SEQ ID NO: 14 or 19 (ABM1) to SxYQ; and / or, iii) substitution of a glutamine residue in the sequence corresponding to or located at a position equivalent to residue 10 of the sequence of SEQ ID NO: 14 or 19 (ABM1) to a glutamic acid residue. iii) a substitution of a second tyrosine residue corresponding to or located at a position equivalent to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue corresponding to or located at a position equivalent to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) to an alanine residue; optionally wherein the chimeric or fusion protein comprises one or more additional polypeptides comprising or consisting of an amino acid sequence of the active site of Arg-gingipain or Lys-gingipain of P. gingivalis or a sequence at least 80% identical thereto; thereby preventing or slowing the rate of abnormal protein deposition in neural tissue of the subject.

5. A method for preventing or slowing the rate of abnormal protein deposition in neural tissue of a subject, preferably wherein the abnormal protein deposition is associated with or caused by a P. gingivalis infection, the method comprising administering to the subject a therapeutically effective amount of a chimeric or fusion protein comprising a first polypeptide and a second polypeptide, wherein: A) the first polypeptide comprises or consists of an amino acid sequence of the active site of Arg-gingipain or Lys-gingipain of P. gingivalis or a sequence at least 80% identical thereto; and, B) the second polypeptide comprises or consists of an amino acid sequence of the adhesin domain of Arg-gingipain or Lys-gingipain of P. gingivalis; wherein the second polypeptide comprises a sequence of one or more adhesin binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain and the cleaved adhesin domain (CAD) of P. gingivalis gingipain; and wherein the second polypeptide: a) does not comprise a sequence of a cleaved adhesin domain (CAD) or a portion thereof, preferably a sequence of a CAD having the sequence set forth in SEQ ID NO: 12 or 13 or a sequence at least 80% identical thereto; and / or, b) comprises an amino acid sequence corresponding substantially to the full length of the DUF2436 domain of Arg-gingipain or Lys-gingipain, preferably an amino acid sequence as set forth in SEQ ID NO: 23, or a sequence at least 80% identical thereto; and / or, c) comprises one or more amino acid substitutions selected from: one or more cysteine amino acid substitutions in the adhesin domain compared to the naturally occurring Arg-gingipain or Lys-gingipain sequence in the corresponding region; and / or, one or more amino acid motif substitutions selected from: i) a substitution of proline and / or asparagine residues in the sequence PxxN corresponding to or located at positions equivalent to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1); and / or, ii) a substitution of a tyrosine residue corresponding to or located at a position equivalent to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) to an alanine residue; and / or, iii) a substitution of a second tyrosine residue corresponding to or located at a position equivalent to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue corresponding to or located at a position equivalent to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) to an alanine residue. ii) a substitution of the motif NxFA to SxYQ in the sequence corresponding to or located at a position equivalent to residues 2 to 5 of the sequence of SEQ ID NO: 14 or 19 (ABM1); iii) a substitution of the second tyrosine residue corresponding to or located at a position equivalent to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and the tryptophan residue corresponding to or located at a position equivalent to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) to an alanine residue; optionally wherein the chimeric or fusion protein comprises one or more additional polypeptides comprising or consisting of an amino acid sequence of the active site of Arg-gingipain or Lys-gingipain of P. gingivalis or a sequence at least 80% identical thereto; thereby preventing or slowing the rate of abnormal protein deposition in neural tissue of the subject.

6. The method of claim 5, wherein the abnormal protein deposition comprises abnormal deposition of P. gingivalis gingipain, amyloid beta, phosphorylated tau protein, or alpha-synuclein.

7. The method of any one of claims 1 to 6, wherein the P. gingivalis-induced neuropathology or the abnormal protein deposition comprises or is associated with cognitive decline or a cognitive disorder.

8. The method of any one of claims 1 to 6, wherein the P. gingivalis-induced neuropathology or the abnormal protein deposition comprises or is associated with a neurodegenerative disease or condition or a pathology that causes a physical or chemical change in neural tissue.

9. The method of claim 8, wherein the neurodegenerative condition is characterized by the presence of abnormal protein deposits in the brain, including amyloidosis, synucleinopathy, or tauopathy.

10. The method of claim 8 or 9, wherein the neurodegenerative condition or disorder is selected from Alzheimer's disease (AD), Lewy-bodies disease (DLB), Huntington's disease, Creutzfeldt-Jakob disease (CJD), Gaucher Disease Type 3, or Parkinson's disease, vascular dementia, frontotemporal dementia, or other forms of dementia not typically associated with deposition of abnormal protein deposits.

11. The method of claim 8, wherein the neurodegenerative condition or disease is Alzheimer's disease.

12. The method of any one of claims 1 to 4, wherein the neuropathology comprises or is caused by the presence of P. gingivalis gingipain protein in neural (e.g., brain) tissue.

13. A method for reducing neuroinflammation, preferably neuroinflammation associated with or caused by a P. gingivalis infection, the method comprising administering to a subject a therapeutically effective amount of a chimeric or fusion protein comprising a first polypeptide and a second polypeptide, wherein: A) the first polypeptide comprises or consists of an amino acid sequence of the active site of an Arg-gingipain or Lys-gingipain of P. gingivalis or a sequence at least 80% identical thereto; and, B) the second polypeptide comprises or consists of an amino acid sequence of the adhesin domain of an Arg-gingipain or Lys-gingipain of P. gingivalis; wherein the second polypeptide comprises a sequence of one or more adhesin binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain and the cleaved adhesin domain (CAD) of P. gingivalis gingipain; and wherein the second polypeptide: a) does not comprise a sequence of a cleaved adhesin domain (CAD) or a portion thereof, preferably a sequence of a CAD having the sequence set forth in SEQ ID NO: 12 or 13 or a sequence at least 80% identical thereto; and / or, b) comprises an amino acid sequence corresponding substantially to the full length of the DUF2436 domain of Arg-gingipain or Lys-gingipain, preferably an amino acid sequence as set forth in SEQ ID NO: 23, or a sequence at least 80% identical thereto; and / or, c) comprises one or more amino acid substitutions selected from: one or more cysteine amino acid substitutions in the adhesin domain compared to the naturally occurring Arg-gingipain or Lys-gingipain sequence in the corresponding region; and / or, one or more amino acid motif substitutions selected from: i) a substitution of a proline and / or asparagine residue in the sequence PxxN corresponding to or at a position equivalent to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1); ii) a substitution of a motif NxFA in the sequence corresponding to or at a position equivalent to residues 2 to 5 of the sequence of SEQ ID NO: 14 or 19 (ABM1) to SxYQ; iii) a substitution of a second tyrosine residue corresponding to or at a position equivalent to residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue corresponding to or at a position equivalent to residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) to an alanine residue; optionally, wherein the chimeric or fusion protein comprises one or more additional polypeptides comprising or consisting of an amino acid sequence of the active site of an Arg-gingipain or Lys-gingipain of P. gingivalis or a sequence at least 80% identical thereto, ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ thereby reducing neuroinflammation in the subject.

14. The method of claim 13, wherein reducing neuroinflammation comprises reducing the level or amount of an inflammatory marker in the brain of the subject.

15. The method of claim 13 or 14, wherein the reducing neuroinflammation comprises reducing the level of one or more of IL-6, IL-1 beta, C-reactive protein (CRP), TNF-alpha and its receptors TNFR-I and TNFR-II, VCAM-I, d-dimer, and sirtuin signaling, YKL-40, IL-7, IL-8, IL-15, IL-12, IP-10, ICAM-1, Flt-1, monocyte chemoattractant protein 1, nitric oxide (NO), COX-2, GM-CSF, and the like.

16. The method of any one of the preceding claims, wherein the chimeric or fusion protein induces an immune response in the subject against P. gingivalis or against a P. gingivalis gingipain.

17. The method of any one of the preceding claims, wherein more than one chimeric or fusion protein as defined in any one of the preceding claims is administered.

18. The method of any one of the preceding claims, wherein the method further comprises administering one or more of an antimicrobial compound, an anti-inflammatory agent, and an adjuvant or an additional immunogen to induce an immune response against P. gingivalis or a P. gingivalis gingipain.

19. Use of a chimeric or fusion protein for the manufacture of a medicament for: • preventing or treating neuropathology in a subject, preferably wherein the neuropathology is associated with or caused by a P. gingivalis infection; • preventing accumulation of P. gingivalis gingipain in neural tissue of a subject; • reducing the level of P. gingivalis gingipain in neural tissue of a subject; • delaying the onset of P. gingivalis -associated neuropathology; • preventing or slowing the rate of abnormal protein deposition in neural tissue of a subject; • preventing or reducing the progression of an amyloidosis, synucleinopathy, or tauopathy in a subject; • reducing neuroinflammation in a subject, preferably P. gingivalis -associated or caused by a P. gingivalis infection; wherein the chimeric or fusion protein comprises a first polypeptide and a second polypeptide, wherein: A) the first polypeptide comprises or consists of an amino acid sequence of the active site of Arg gingipain or Lys gingipain of P. gingivalis or a sequence at least 80% identical thereto; and, B) the second polypeptide comprises or consists of an amino acid sequence of the adhesin domain of Arg gingipain or Lys gingipain of P. gingivalis; wherein the second polypeptide comprises the sequence of one or more adhesin binding motifs (ABMs), preferably wherein the ABMs correspond to some or all of the ABMs between the DUF2436 domain of P. gingivalis gingipain and the cleaved adhesin domain (CAD). and wherein said second polypeptide: a) does not comprise: a sequence of a cleaved adhesin domain (CAD) or a portion thereof, preferably a sequence of a CAD having the sequence set forth in SEQ ID NO: 12 or 13 or a sequence at least 80% identical thereto; and / or, b) comprises an amino acid sequence corresponding substantially to the full length of a DUF2436 domain of Arg-gingipasin or Lys-gingipasin, preferably an amino acid sequence as set forth in SEQ ID NO: 23, or a sequence at least 80% identical thereto; and / or, c) comprises one or more amino acid substitutions selected from: one or more cysteine amino acid substitutions in said adhesin domain compared to the naturally occurring Arg-gingipasin or Lys-gingipasin sequence in the corresponding region; and / or, one or more amino acid motif substitutions selected from: i) a substitution of a proline and / or asparagine residue in the sequence PxxN corresponding to or at a position equivalent to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1); ii) a substitution of a motif NxFA in the sequence corresponding to or at a position equivalent to residues 2 to 5 of the sequence of SEQ ID NO: 14 or 19 (ABM1) to SxYQ; iii) a substitution of a second tyrosine residue corresponding to or at a position equivalent to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue corresponding to or at a position equivalent to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) to an alanine residue; optionally wherein said chimeric or fusion protein comprises one or more additional polypeptides comprising or consisting of an amino acid sequence of the active site of Arg-gingipasin or Lys-gingipasin of P. gingivalis or a sequence at least 80% identical thereto.

20. The use of claim 19, wherein said P. gingivalis-related neuropathology comprises cognitive decline, cognitive impairment, or a pathology that causes a physical or chemical change in neurogenic tissue; or is a neurodegenerative disorder.

21. The method or use according to any one of the preceding claims, wherein the first polypeptide comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 1 to 11, or a functional equivalent thereof comprising a sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, preferably wherein said first polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

8.

22. The method or use of any one of the preceding claims, wherein said first polypeptide and second polypeptide are directly linked or joined by a linker or polypeptide sequence.

23. The method or use of any one of the preceding claims, wherein said second polypeptide: a) a sequence which does not comprise a cleaved adhesin domain (CAD), preferably having the sequence set forth in SEQ ID NO: 12 or 13, or a sequence which is at least 80% identical thereto.

24. The method or use of any one of claims 1 to 22, wherein the chimeric or fusion protein comprises a first polypeptide and a second polypeptide, wherein the second polypeptide: a) comprises an amino acid sequence which corresponds substantially to the full length of a DUF2436 domain of Arg gingipain or Lys gingipain, preferably as set forth in SEQ ID NO: 23, or a sequence which is at least 80% identical thereto.

25. The method or use of any one of claims 1 to 22, wherein the second polypeptide: b) comprises an amino acid sequence which corresponds substantially to the full length of a DUF2436 domain of Arg gingipain or Lys gingipain, preferably as set forth in SEQ ID NO: 23, or a sequence which is at least 80% identical thereto.

26. The method or use of any one of claims 1 to 22, wherein the second polypeptide comprises one or more amino acid substitutions selected from: a) one or more cysteine amino acid substitutions in the adhesin domain compared to the naturally occurring Arg gingipain or Lys gingipain sequence in the corresponding region; and / or, b) one or more amino acid motif substitutions selected from: i) substitution of a proline and / or asparagine residue in the sequence PxxN corresponding to or at a position equivalent to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1); ii) substitution of the motif NxFA in the sequence corresponding to or at a position equivalent to residues 2 to 5 of the sequence of SEQ ID NO: 14 or 19 (ABM1) to SxYQ; iii) substitution of a second tyrosine residue corresponding to or at a position equivalent to residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue corresponding to or at a position equivalent to residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) to an alanine residue.

27. The method or use of any one of claims 1 to 22, wherein the second polypeptide comprises one or more amino acid substitutions selected from: a) one or more cysteine amino acid substitutions in the adhesin domain compared to the naturally occurring Arg gingipain or Lys gingipain sequence in the corresponding region; and / or, b) one or more amino acid motif substitutions selected from: i) substitution of a proline and / or asparagine residue in the sequence PxxN corresponding to or at a position equivalent to residues 6 to 9 of the sequence of SEQ ID NO: 14 or 19 (ABM1); iii) a substitution of a second tyrosine residue corresponding to or located at a position equivalent to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue corresponding to or located at a position equivalent to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) to an alanine residue.

28. The method or use according to any one of claims 1 to 22, wherein the second polypeptide: a) does not comprise: a sequence of a cleaved adhesin domain (CAD) or a portion thereof, preferably a sequence of a CAD having the amino acid sequence set forth in SEQ ID NO: 12 or 13 or a sequence at least 80% identical thereto; and, b) comprises an amino acid sequence corresponding substantially to the full length of a DUF2436 domain of Arg-gingipain or Lys-gingipain, preferably the amino acid sequence set forth in SEQ ID NO: 23 or a sequence at least 80% identical thereto; and, comprises one or more amino acid substitutions: c) one or more cysteine amino acid substitutions in the amino acid sequence of the DUF 2436 domain and ABM compared to the naturally occurring Arg-gingipain or Lys-gingipain sequence in the corresponding region; and / or (preferably and), d) one or more amino acid motif substitutions: i) a substitution of a proline and / or asparagine residue in the sequence PxxN corresponding to or located at a position equivalent to residues 6 to 9 of SEQ ID NO: 14 or 19 (ABM1); ii) a substitution of a motif NxFA in the sequence corresponding to or located at residues 2 to 5 of SEQ ID NO: 14 or 19 (ABM1) to SxYQ; iii) a substitution of a second tyrosine residue corresponding to or located at a position equivalent to the residue at position 5 of SEQ ID NO: 15 or 20 (ABM2) and a tryptophan residue corresponding to or located at a position equivalent to the residue at position 23 of SEQ ID NO: 14 or 19 (ABM1) to an alanine residue. the amino acid sequence of the active site of Arg-gingipain or Lys-gingipain of P. gingivalis or a sequence at least 80% identical thereto.

29. The method or use of any one of the preceding claims, wherein the chimeric or fusion protein comprises one or more additional polypeptides comprising or consisting of:

30. The method of use according to claim 29, wherein the one or more additional polypeptides are located N-terminal to the first polypeptide, C-terminal to the first polypeptide, or N-terminal to the second polypeptide or C-terminal to the second polypeptide. ​ 31. The method or use according to any one of claims, wherein the one or more additional polypeptides comprises or consists of an amino acid sequence selected from or composed of the group consisting of: SEQ ID NO: 1 to 11, or a functional equivalent thereof comprising a sequence at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto, preferably wherein the first polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

8.

32. The method or use of any one of the preceding claims, wherein the first polypeptide and the further polypeptide are derived from the active site of a heterologous gingipain (e.g., a gingipain from different strains of P. gingivalis).

33. The method or use of any one of the preceding claims, wherein the first polypeptide and the further polypeptide have amino acid sequences derived from different gingipains (e.g., wherein one of the polypeptides has an amino acid sequence from the active site of Kgp and the other polypeptide has an amino acid sequence from the active site of Rgp; or alternatively, wherein one of the polypeptides has an amino acid sequence from the active site of RgpA and the other polypeptide has an amino acid sequence from the active site of RgpB).

34. The method of any one of the preceding claims 24 to 33, wherein the amino acid sequence corresponding substantially to the full length of the DUF2436 domain, or a functional equivalent thereof comprising a sequence at least 80% identical thereto, refers to a sequence comprising 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 gingipain or Lys gingipain.

35. The method of claim 34, wherein the amino acid sequence of the DUF2436 domain of Arg gingipain or Lys gingipain is the sequence set forth in SEQ ID NO: 23, or a functional equivalent thereof comprising a sequence at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

36. The method of any one of claims 34 or 35, wherein the second polypeptide comprises or consists of the sequence set forth in SEQ ID NO: 34, or a functional equivalent thereof comprising a sequence at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

37. The method of any one of claims 34 or 35, wherein the second polypeptide comprises the sequence set forth in SEQ ID NO: 76, or a functional equivalent thereof comprising a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

38. The method of any one of claims 34 to 37, wherein the chimeric or fusion protein comprises or consists of the amino acid sequence set forth in any one of SEQ ID NO: 28, 50, 51, 52, 53, or 54, 65, 66, or a functional equivalent thereof comprising a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

39. The method or use of claims 26 to 38, wherein the one or more cysteine amino acid substitution is a substitution to a serine residue or to a valine residue.

40. The method or use of any one of claims 26 to 39, wherein the one or more cysteine substitution comprises one or more substitution to a serine residue.

41. The method or use of any one of claims 26 to 40, wherein the cysteine residue in the DUF2436 domain is substituted to a serine or valine, preferably serine.

42. The method or use of any one of claims 26 to 40, wherein the cysteine residue in the DUF2436 domain is not substituted, and preferably one or more cysteine residues in the remainder of the adhesin domain are substituted.

43. The method or use of any one of claims 26-41, wherein the adhesin domain comprises or consists of: the sequence set forth in SEQ ID NO: 23 or a sequence at least 80% identical thereto, wherein the cysteine residue at position 115 is substituted to a serine or valine residue.

44. The method or use of any one of claims 26-41, wherein the adhesin domain comprises or consists of: the sequence set forth in SEQ ID NO: 34 or SEQ ID NO: 76 or a sequence at least 80% identical thereto, wherein one or more cysteine residues are substituted to a serine or valine residue.

45. The method or use of any one of claims 26 to 44, wherein the second polypeptide: a) comprises one or more substitution of cysteine to serine amino acids compared to the naturally occurring adhesin domain sequence; and, b) comprises a substitution of proline and / or asparagine in the sequence PxxN corresponding to or at a position equivalent to residues at positions 6 to 9 of the sequence of SEQ ID NO: 14 or 19.

46. The method or use of claim 45, wherein the adhesin domain comprises the sequence set forth in SEQ ID NO: 18 or 22 or a functional equivalent thereof comprising a sequence at least 80% identical thereto, and wherein one or both cysteine residues in SEQ ID NO: 18 or 22 are substituted for a serine residue, and wherein the proline and / or asparagine residues in the sequence PxxN at positions 63 to 66 of SEQ ID NO: 18 or SEQ ID NO: 22, or equivalent thereto, are substituted.

47. The method or use of claim 46, wherein the proline residue is substituted for an alanine residue, and / or the asparagine residue is substituted for a proline or alanine residue, preferably wherein the proline is substituted for an alanine and the asparagine is substituted for a proline, such that the sequence at positions 63 to 66 of SEQ ID NO: 18 or SEQ ID NO: 22 is AxxP (e.g. AVQP, SEQ ID NO: 85).

48. The method or use of claim 47, wherein one, two or three cysteine residues are substituted for a serine residue, and wherein the proline and asparagine residues in the sequence PxxN at positions 235 to 238, or equivalent thereto, are substituted.

49. The method or use of any one of claims 26 to 48, wherein the second polypeptide comprises or consists of the sequence as set forth in any one of SEQ ID NOs: 35 to 49, or a functional equivalent thereof comprising a sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, preferably wherein the second polypeptide comprises or consists of the amino acid sequence of SEQ ID NO:

49.

50. The method or use of any one of claims 26 to 48, wherein the chimeric or fusion protein comprises or consists of the amino acid sequence as set forth in any one of SEQ ID NOs: 55, 56 or 57, or a functional equivalent thereof comprising a sequence 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto.

51. The method or use of any one of claims 26 to 48, wherein the chimeric or fusion protein comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 62 to 63, or a functional equivalent thereof comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

52. The method or use of any one of claims 26 to 48, wherein the chimeric or fusion protein comprises or consists of a sequence as set forth in any one of SEQ ID NOs: 58, 59, 60, 61, and 69, 70 to 75, or a functional equivalent thereof comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

53. The method or use of any one of claims 1 to 21, wherein the chimeric or fusion protein comprises or consists of a sequence as set forth in SEQ ID NO: 69, or a functional equivalent thereof comprising at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical thereto.

54. The method or use of any one of claims 1 to 35, wherein the DUF2436 domain and the ABM domain derived from Arg-gingivain or Lys-gingivain are directly linked or joined by a linker or by a polypeptide sequence.

55. The method or use of claim 54, wherein the DUF2436 domain and the ABM domain (having representative amino acid sequences of SEQ ID NOs: 23 to 26 and 18, 29 to 33, respectively) can be linked by a peptide sequence having the amino acid sequence EVEDDSP (SEQ ID NO: 90).

56. The method or use of any one of the preceding claims, wherein the subject has or is at risk of having P. gingivalis infection.

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