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KR-20260062938-A - Chimeric protein vaccine

KR20260062938AKR 20260062938 AKR20260062938 AKR 20260062938AKR-20260062938-A

Abstract

The present invention provides a chimeric or fusion protein for inducing an immune response against P. gulae , wherein the protein comprises a first polypeptide and a second polypeptide, and A) The first polypeptide comprises or consists of an amino acid sequence of the active site of Arg- or Lys-gingipain of P. gullae or a sequence that is at least 80% identical thereto; B) The above second polypeptide is The amino acid sequence of the DUF2436 domain of Arg- or Lys-zingipine in P. gullae; and It comprises or consists of the amino acid sequence of the adhesion domain of Arg- or Lys-gingipain of P. gullae.

Inventors

  • 레이놀즈 에릭
  • 슬라케스키 나다
  • 시어스 크리스틴

Assignees

  • 카드무스 애니멀 헬스 리미티드

Dates

Publication Date
20260507
Application Date
20240726
Priority Date
20230726

Claims (20)

  1. A chimeric or fusion protein for inducing an immune response against P. gulae , wherein the protein comprises a first polypeptide and a second polypeptide, and A) The first polypeptide comprises or consists of an amino acid sequence of the active site of an Arg- or Lys-gingipain homologue of P. gullae or a sequence that is at least 80% identical thereto; B) The above second polypeptide is The amino acid sequence of the DUF2436 domain of the Arg- or Lys-zingipane surface complex of P. goulae; and A chimeric or fusion protein comprising or consisting of an amino acid sequence of an adhesion domain of a surface complex of an Arg- or Lys-gingipa homologue of P. goulae, wherein, preferably, the adhesion domain comprises at least the amino acid sequence of SEQ ID NO. 86 and/or SEQ ID NO. 85 or a sequence that is at least 80% identical to the same.
  2. A chimeric or fusion protein according to claim 1, wherein the amino acid sequence of the DUF2436 domain of the gingipain homologue surface complex comprises or is composed of the amino acid sequence presented in SEQ ID NO. 3 or 4 or a sequence that is at least 80% identical to the same.
  3. A chimeric or fusion protein according to claim 1, wherein the amino acid sequence of the DUF2436 domain of the gingipain homologue surface complex comprises or consists of the amino acid sequence presented in SEQ ID NO. 4 or a sequence that is at least 80% identical to the same.
  4. A chimeric or fusion protein according to any one of claims 1 to 3, wherein the amino acid sequence of the adhesion domain of the gingipain homologue surface complex comprises or consists of the amino acid sequence of SEQ ID NO. 88 or 20 or a sequence that is at least 80% identical thereto.
  5. A chimeric or fusion protein according to any one of claims 1 to 4, comprising one or more additional polypeptides comprising or consisting of an amino acid sequence of the active site of an Arg- or Lys-gingipa homologue of P. gullae or a sequence that is at least 80% identical thereto.
  6. In claim 5, a chimeric or fusion protein comprising one or more additional polypeptides, which include or are composed of an active site of an Arg- or Lys-gingipa homologue of P. gullae, located at the N-terminus of the first polypeptide.
  7. In claim 5, a chimeric or fusion protein comprising one or more additional polypeptides, which include or are composed of an active site of an Arg- or Lys-gingipa homologue of P. gullae, located at the C-terminus of the first polypeptide.
  8. In claim 5, a chimeric or fusion protein comprising one or more additional polypeptides, which include or are composed of an active site of an Arg- or Lys-gingipa homologue of P. gullae, located at the N-terminus of the second polypeptide.
  9. In claim 5, a chimeric or fusion protein comprising one or more additional polypeptides, which include or are composed of an active site of an Arg- or Lys-gingipa homologue of P. gullae, located at the C-terminus of the second polypeptide.
  10. A chimeric or fusion protein according to any one of claims 1 to 9, comprising at least two additional polypeptides comprising or consisting of an amino acid sequence of the active site of an Arg- or Lys-gingipa homologue of P. gullae or a sequence that is at least 80% identical thereto.
  11. A chimeric or fusion protein according to claim 10, wherein two or more additional polypeptides comprising or consisting of an active site of an Arg- or Lys-gingipa homologue of P. gullae are located at the N-terminus of the second polypeptide, the C-terminus of the second polypeptide, or both the N-terminus and the C-terminus of the second polypeptide.
  12. A chimeric or fusion protein according to any one of claims 5 to 11, wherein the one or more additional polypeptides are connected to the first or second polypeptide of the chimeric or fusion protein through a linker of 50 or fewer amino acids, or are directly connected to the first or second polypeptide.
  13. A chimeric or fusion protein according to any one of claims 1 to 12, wherein the first polypeptide comprises or consists of an amino acid sequence selected from the sequence group identical to SEQ ID NO. 1 or 2 by at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  14. A chimeric or fusion protein according to any one of claims 5 to 13, wherein the one or more additional polypeptides comprise or consist of an amino acid sequence selected from the sequence group identical to SEQ ID NO. 1 or 2, or at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% thereof.
  15. A chimeric or fusion protein according to any one of claims 5 to 14, wherein the first polypeptide and the one or more additional polypeptides comprise or consist of at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical amino acid sequences.
  16. A chimeric or fusion protein according to any one of claims 5 to 14, wherein the first polypeptide and the one or more additional polypeptides have the same amino acid sequence or are composed thereof.
  17. A chimeric or fusion protein according to any one of claims 5 to 15, wherein the first polypeptide comprises or is composed of the amino acid sequence of the active site of a Kgp gingipain homologue (e.g., presented in SEQ ID NO. 1), and the additional polypeptide comprises or is composed of the amino acid sequence of the active site of an Rgp gingipain homologue (e.g., presented in SEQ ID NO. 2).
  18. A chimeric or fusion protein according to any one of claims 5 to 15, wherein the first polypeptide comprises or is composed of the amino acid sequence of the active site of an Rgp gingipain homologue (e.g., presented in SEQ ID NO. 2), and the additional polypeptide comprises or is composed of the amino acid sequence of the active site of a Kgp gingipain homologue (e.g., presented in SEQ ID NO. 1).
  19. A chimeric or fusion protein according to any one of claims 5 to 15, wherein the first polypeptide and the additional polypeptide comprise or consist of the amino acid sequence of the active site of a Kgp gingipain homologue (e.g., as presented in SEQ ID NO. 1).
  20. A chimeric or fusion protein according to any one of claims 5 to 15, wherein the first polypeptide and the additional polypeptide comprise or consist of the amino acid sequence of the active site of an Rgp gingipain homologue (e.g., as presented in SEQ ID NO. 2).

Description

Chimeric protein vaccine The present invention relates to a chimeric polypeptide useful for inducing an immune response against P. gulae , a composition containing the same, and the use thereof for the prevention and treatment of P. gulae-related pathological conditions and diseases. Related applications This application claims priority to Australian provisional application AU 2023902373, the entire contents of which are incorporated herein by reference. The accumulation of dental plaque at the gum line of the teeth causes inflammation of the gums (gingivitis). Chronic gingivitis involves the proliferation of periodontal pathogens, specifically Porphyromonas sp., at the bottom of the periodontal pocket, leading to a chronic infection that can develop into a serious disease. This severe form of periodontal disease is called periodontitis, and it can lead to tooth loss as the immune system attempts to eliminate the infection. Periodontitis is an inflammatory disease of the tooth-supporting tissues in both humans and pets, associated with dysbiotic subgingival plaque, which leads to the destruction of these tissues and loss of tooth attachment. By the age of three, more than 80% of dogs and more than 70% of cats show symptoms of periodontitis. Therefore, the disease burden caused by periodontitis in the pet population is significant. The most common periodontal pathogen in pets, especially dogs, is Porphyromonas gullae (P. gullae). Currently, there are no commercially approved treatments available to prevent or reduce the incidence and/or severity of P. gula infection in companion animals, or to treat P. gula infection and disease. Fig. 1: Expression and solubility ofP. gullae chimeric proteins KDFAK-2S-AVQP and KDA AK-2S-AVQP. (a) Lysate fraction; (b) Small-scale purification test using a Ni-NTA spin column: 1. Purified lysate before column loading; 2. Pass-through; 3. Column wash; 4. Eluted protein fraction. Left panel in both A) and B: KD F AK-2S-AVQP, Right panel: KD A AK-2S-AVQP. Fig. 2: SDS-PAGE analysis of expressed P. gullae antigen and cell lysis fractions. (a) Expression of KD F AK-2S-AVQP and KD A AK-2S-AVQP in LB and TB media for 2 to 3 hours. (b) Lysis under non-reducing conditions. (c) (i & ii) Attempts to solubilize KD F AK-2S-AVQP from the insoluble fractions of non-reducing lysis using reducing buffers (5 mM and 100 mM DTT); (iii) Lysis of KD F AK-2S-AVQP protein under reducing conditions (10 mM DTT). M: Protein standard; TC: Whole cell lysate; Sup: Purified lysate. Fig. 3: Ni-affinity chromatography. (a) Elution profile of KD A AK-2S-AVQP under normal non-reducing conditions and reduction SDS-PAGE of the column fraction. (b) Elution profile of antigen-F under reducing conditions (R) and reduction SDS-PAGE of the column fraction. (c) Elution profile of KD F AK-2S-AVQP under normal non-reducing conditions (NR) and reduction SDS-PAGE of the column fraction. TC: Whole cell lysate; Sp: Purified supernatant before column loading; FT: Passover; W: Column wash. M: Protein standard. Fig. 4: Purification using anion exchange chromatography (AIEX). (a) Elution profile of KD A AK-2S-AVQP under normal non-reducing conditions and reduction SDS-PAGE of the column fraction. (b) Elution profile of KD F AK-2S-AVQP under reducing conditions (R) and reduction SDS-PAGE of the column fraction. (c) Elution profile of KD F AK-2S-AVQP under normal non-reducing conditions (NR) and reduction SDS-PAGE of the column fraction. BL: Before loading; FT: Pass solution; M: Protein standard. Fig. 5: Size exclusion chromatography under non-reducing conditions (NR). (a) Elution profile of KD A AK-2S-AVQP and reduced SDS-PAGE of the column fraction. (b) Elution profile of KD F AK-2S-AVQP purified under reducing conditions (R) prior to this step and reduced SDS-PAGE of the column fraction. (c) Elution profile of KD F AK-2S-AVQP purified under non-reducing conditions (NR) prior to this step and reduced SDS-PAGE of the column fraction. BL: Before loading; M: Protein standard. Fig. 6: PAGE analysis of P. gullae antigen final products. (a) SDS-PAGE; (b) Native PAGE or native gel. A-version: KD A AK-2S-AVQP; FR: KD F AK-2S-AVQP purified under reducing conditions up to the final size exclusion step; F-NR: KD F AK-2S-AVQP purified under non-reducing conditions. R: Reduced; NR: Non-reducing; M: Protein standard. Fig. 7: Mouse periodontitis model: Therapeutic vaccination. Schematic diagram of the experiment schedule Fig. 8: Bone loss caused by P. gullae. Statistical analysis - One-way ANOVA and post-hoc Dunnet's T3. # (p<0.05, compared to native control); ## (p<0.05, compared to infection control). Fig. 9: Anti-P. gullae antibody isotype reactions. Antibody titers of mouse serum (individual) against heat-killed P. gullae whole cells. (a) Total IgG titer; (b) IgG1 subtype titer; (c) IgG2a subtype titer. Fig. 10: IgG antibody response to P. gullae protease complex. Antibody titers of mouse serum (individual) to purified P. gullae RgpA/Kgp pro