Methods of treatment using vaccine compositions

Abstract

The invention relates to methods and compositions for preventing or treating a neuropathology in a subject associated with or induced or caused by a P. gingivalis infection, preventing the deposition of or reducing the level of P. gingivalis gingipain in neuronal tissue, delaying the onset of a P. gingivalis-induced or associated neuropathology, for preventing or slowing the rate of abnormal protein deposition in the neuronal tissue, and / or reducing neuroinflammation, the methods comprising administering an RNA polynucleotide encoding a protein comprising or consisting of: - one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or - the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto, wherein the polynucleotide is capable of being translated in a mammalian cell.

Description

Methods of treatment using vaccine compositionsField of the invention

[0001] The invention relates to methods for preventing and / or treating P. gingivalis-induced or associated neuropathologies (such as dementia and associated neurodegenerative conditions).Related application

[0002] This application claims priority from Australian provisional applicationAU 2024904254, the entire contents of which are hereby incorporated by reference.Background of the invention

[0003] If dental plaque is left to accumulate around the tooth at the gingival (gum) margin this causes gingival inflammation (gingivitis). Chronic gingivitis can allow the emergence of a periodontal pathogen Porphyromonas gingivalis (P. gingivalis) at the base of a periodontal pocket to result in a chronic infection and the development of severe disease. This severe form of periodontal disease is called periodontitis and can lead to tooth loss in an attempt by the immune system to eliminate the infection.

[0004] One in three adults have moderate to severe periodontitis. From epidemiological surveys, periodontitis has been linked to an increased risk of inflammatory diseases including cardiovascular diseases, certain cancers, preterm birth, rheumatoid arthritis and dementia.

[0005] More recent research has linked chronic infection by P. gingivalis with dementia and rheumatoid arthritis. For example, in one study, 96% of Alzheimer’s disease (AD) brain samples showed the presence of P. ginigvalis. Another study shows that chronic oral infection of mice with P. gingivalis resulted in the brain plaques associated with AD in humans and that the P. gingivalis proteases could cleave amyloid precursor and tau proteins to form the plaques and tangles associated with AD.

[0006] A number of virulence factors have been reported to contribute to the pathogenicity of P. gingivalis including; LPS, fimbriae, hemagglutinin, hemolysin and extracellular hydrolytic enzymes (especially the Arg-X and Lys-X specific proteinases), otherwise known as " P. gingivalis gingipains".1006265400

[0007] The magnitude of the public health problem is such that there is a need for a vaccine that provides a strong protective response to P. gingivalis infection and means for providing same.

[0008] There is currently no commercially approved vaccine for use in preventing or reducing the incidence and / or severity of P. gingivalis infection or for treating P. gingivalis infection and disease in subjects.

[0009] There is therefore a need for alternative and / or improved approaches for the design and manufacture of P. gingivalis vaccines, and alternative and / or improved vaccines produced from P. gingivalis.

[0010] There is a need for reagents and methods for preventing and / or treating P. gingivalis - induced or associated neuropathologies such as those that contribute or lead to dementias.

[0011] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the invention

[0012] In a first aspect, the present invention provides a method for preventing or treating a neuropathology in a subject, wherein the neuropathology is associated with or induced or caused by a P. gingivalis infection, the method comprising administering to the subject an RNA polynucleotide encoding a protein comprising or consisting of:- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein the polynucleotide is capable of being translated in a mammalian cell,1006265400thereby preventing or treating P. gingivalis- induced or associated neuropathology in the subject.

[0013] Optionally, the protein encoded by the RNA polynucleotide may further comprise:- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto.

[0014] The neuropathology may comprise a pathology associated with or caused by the presence of, or exacerbated by P. gingivalis gingipain proteins in neuronal (eg brain) tissue. The P. gingivalis gingipain may comprise a Lys- or Arg-gingipain, such as Kgp or Rgp, as further described herein.

[0015] Accordingly, in a second aspect, the present invention also provides a method for preventing the deposition of or reducing the level of P. gingivalis gingipain in neuronal tissue of a subject, the method comprising administering to the subject an RNA polynucleotide encoding a protein comprising or consisting of:- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein the polynucleotide is capable of being translated in a mammalian cell,thereby preventing the deposition of or reducing the level of P. gingivalis gingipain in neuronal tissue of the subject. Optionally, the protein encoded by the RNA polynucleotide may further comprise:- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto.

[0016] The subject in which the level of P. gingivalis gingipain is to be prevented or of which reduction is required may be a subject considered at risk for development of, or a subject having dementia or other neurodegenerative condition.1006265400

[0017] The present invention also provides, in a third aspect, a method for delaying the onset of a P. gingivalis- induced or associated neuropathology in a subject, the method comprising administering to the subject an RNA polynucleotide encoding a protein comprising or consisting of:- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein the polynucleotide is capable of being translated in a mammalian cell,thereby delaying the onset of a P. gingivalis- induced or associated neuropathology in the subject. Optionally, the protein encoded by the RNA polynucleotide may further comprise:- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto.

[0018] The P. g / ng / vaf / s-i nduced or associated neuropathology may comprise or be associated with cognitive decline, a cognitive disorder or a decline or change in motor function. The P. gingivalis- induced or associated neuropathology may comprise a neurodegenerative disease or condition or a pathology resulting in physical or chemical changes to neuronal tissue.

[0019] In any aspect of the invention, the neurodegenerative condition may be characterised by the presence of abnormal protein deposits in the brain, including amyloidopathies, synucleinopathies or tauopathies.

[0020] In any embodiment, the neurodegenerative condition or disorder associated with or caused by or induced by P. gingivalis infection may include Alzheimer’s disease (AD), Lewy-bodies disease (Dementia with Lewy bodies (DLB)), Huntington's disease, Creutzfeldt-Jakob disease (CJD), Gaucher Disease Type 3, or Parkinson's disease. The neurodegenerative disease may be a dementia such as mild cognitive and / or memory impairment, vascular dementia, frontotemporal dementia or other form of dementia not typically associated with deposition of abnormal protein deposits.1006265400

[0021] Preferably, the neurodegenerative condition or disease is Alzheimer’s disease.

[0022] In a fourth aspect, the present invention also provides a method for preventing or slowing the rate of abnormal protein deposition in the neuronal tissue of a subject; preferably wherein the abnormal protein deposition is associated with or caused by P. gingivalis infection, the method comprising administering to the subject an RNA polynucleotide encoding a protein comprising or consisting of:- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein the polynucleotide is capable of being translated in a mammalian cell, thereby preventing or slowing the rate of abnormal protein deposition in the neuronal tissue of the subject. Optionally, the protein encoded by the RNA polynucleotide may further comprise:- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto.

[0023] The abnormal protein deposition may be abnormal deposition of amyloid β, phosphorylated tau protein, or alpha- synuclein.

[0024] Accordingly, in preferred embodiments, the methods of the present invention also relate to methods for preventing or reducing the progression of amyloidopathies, synucleinopathy or tauopathy in a subject.

[0025] Further, the methods of the present invention also relate to methods for preventing or reducing the progression of a neurodegenerative condition selected from: Alzheimer’s disease (AD), Lewy-bodies disease (Dementia with Lewy bodies (DLB)), Huntington's disease, Creutzfeldt-Jakob disease (CJD), Gaucher Disease Type 3, or Parkinson's disease, a dementia such as mild cognitive and / or memory impairment, vascular dementia, frontotemporal dementia or other form of dementia not typically associated with deposition of abnormal protein deposits.1006265400

[0026] In a fifth aspect, the present invention also provides a method for reducing neuroinflammation, preferably neuroinflammation associated with or caused by P. gingivalis infection, the method comprising administering to the subject an RNA polynucleotide encoding a protein comprising or consisting of:- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein the polynucleotide is capable of being translated in a mammalian cell, thereby reducing neuroinflammation in the subject. Optionally, the protein encoded by the RNA polynucleotide may further comprise:- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto.

[0027] In any embodiment of the fifth aspect of the invention, reducing neuroinflammation may comprise reducing the level or amount of any inflammatory marker in the brain of the subject. Optionally, the reducing may comprise reducing the level of one or more of: 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 and others.

[0028] In any embodiment of any aspect of the invention, the RNA polynucleotide induces an immune response to P. gingivalis or to P. gingivalis gingipains in the subject.

[0029] It will be appreciated that in any embodiment, the immune response elicited by administration of a RNA polynucleotide described herein, is preferably antigen-specific. In any embodiment, the compositions, RNA polynucleotide and methods of the invention may be for strengthening an immune response (such as a protective immune response) of a subject to P. gingivalis.1006265400

[0030] In any aspect of the invention, a method of the invention may also comprise administration of one or more of: an antimicrobial compound, an anti-inflammatory agent or further immunogen for inducing an immune response to P. gingivalis or P. gingivalis gingipains.

[0031] The invention also provides use of an RNA polynucleotide encoding a protein comprising or consisting of:- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein the polynucleotide is capable of being translated in a mammalian cell, in the manufacture of a medicament for:- preventing or treating a neuropathology in a subject, preferably wherein the neuropathology is associated with or caused by P. gingivalis infection;- preventing the accumulation of P. gingivalis gingipain in neuronal tissue of a subject;- reducing the level of P. gingivalis gingipain in neuronal tissue of a subject;- delaying the onset of a P. g / ng / vaf / s-associated neuropathology,- preventing or slowing the rate of abnormal protein deposition in the neuronal tissue of a subject;- preventing or reducing progression of an amyloidopathy, synucleinopathy or tauopathy in a subject;- preventing or slowing the rate of progression of a neurodegenerative disease, optionally selected from Alzheimer’s disease (AD), Lewy-bodies disease (Dementia with Lewy bodies (DLB)), Huntington's disease, Creutzfeldt-Jakob disease (CJD), Gaucher Disease Type 3, or Parkinson's disease, a dementia such as mild cognitive and / or memory impairment, vascular dementia,1006265400frontotemporal dementia or other form of dementia not typically associated with deposition of abnormal protein deposits; or- reducing neuroinflammation in a subject, preferably neuroinflammation associated with or caused by P. gingivalis infectionwherein optionally the P. gZngZva / Zs-induced or associated neuropathology comprises cognitive decline, a cognitive disorder, or a pathology resulting in physical or chemical changes to neuronal tissue; or is a neurodegenerative disorder as further herein defined. Optionally, the protein encoded by the RNA polynucleotide may further comprise:- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto.

[0032] The invention also provides an RNA polynucleotide encoding a protein comprising or consisting of:- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein the polynucleotide is capable of being translated in a mammalian cell, for use in:- preventing or treating a neuropathology in a subject, preferably wherein the neuropathology is associated with or caused by P. gingivalis infection;- preventing the accumulation of P. gingivalis gingipain in neuronal tissue of a subject;- reducing the level of P. gingivalis gingipain in neuronal tissue of a subject;- delaying the onset of a P. gingivalis-associated neuropathology,- preventing or slowing the rate of abnormal protein deposition in the neuronal tissue of a subject;1006265400- preventing or reducing progression of an amyloidopathy, synucleinopathy or tauopathy in a subject;- preventing or slowing the rate of progression of a neurodegenerative disease, optionally selected from Alzheimer’s disease (AD), Lewy-bodies disease (Dementia with Lewy bodies (DLB)), Huntington's disease, Creutzfeldt-Jakob disease (CJD), Gaucher Disease Type 3, or Parkinson's disease, a dementia such as mild cognitive and / or memory impairment, vascular dementia, frontotemporal dementia or other form of dementia not typically associated with deposition of abnormal protein deposits; or- reducing neuroinflammation in a subject, preferably neuroinflammation associated with or caused by P. gingivalis infectionwherein optionally the P. g / ng / vaf / s-induced or associated neuropathology comprises cognitive decline, a cognitive disorder, or a pathology resulting in physical or chemical changes to neuronal tissue; or is a neurodegenerative disorder as further herein defined. Optionally, the protein encoded by the RNA polynucleotide may further comprise:- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto.

[0033] In any method, use or RNA molecule for use according to the invention, the subject may be any subject that has or is at risk of having an infection with P. gingivalis, for example a chronic P. gingivalis infection. The subject may be a human. The subject may be a veterinary subject, such as a companion animal that has, or is at risk of having an infection with P. gingivalis.

[0034] In any aspect herein, the protein encoded by the RNA may comprise the aforementioned domains in any order: for example: the one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis may be located N-terminally to the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, and / or the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P. gingivalis', or the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, may be located N-terminally to the one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis and / or the amino1006265400acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P. gingivalis, The domains may be directly joined in the context of the chimeric or fusion protein, or may be joined via a linker region, of one or more amino acid residues, as further defined herein.

[0035] In other words, the protein may comprise or consist of, N to C terminus: active site (K) - ABMs (A); or active site (R) - ABMs (A); or active site (K) - DUF 2436 (D); or active site (R) - DUF2436 (D); or AMBs (A) - active site (K); or ABMs (A)-active site (R); or DUF2436 (D) - active site (K); or DUF2436 (D) - active site (R); or active site (R) -ABMs (A) - active site (K); or active site (R) - ABMs (A) - active site (R); or active site (K) -ABMs (A) - active site (R); or DUF2436 (D) — ABMs (A); or DUF2436 (D) — ABMs (A) - active site (R) or (K); or active site (K) - DUF 2436 (D) - ABMs (A); or active site (K) -DUF 2436 (D) - ABMs (A) - active site (K); or active site (R) - DUF 2436 (D) - ABMs (A) - active site (K); or active site (K) - DUF 2436 (D) - ABMs (A); or active site (R) - DUF 2436 (D) - ABMs (A).

[0036] In any embodiment herein, the RNA polynucleotide is in the form of a messenger RNA (mRNA) molecule. However, it will be appreciated that the RNA polynucleotide may be in any suitable format for being translated in a mammalian cell and enabling synthesis of the protein encoded by the RNA.

[0037] In certain embodiments, the RNA polynucleotide may be composed entirely of ribose-containing nucleotides, or alternatively, may comprise a combination of ribose-containing nucleosides and of 2’-deoxyribose-containing nucleotides.

[0038] In any embodiment, the RNA polynucleotide may be a synthetic RNA molecule.

[0039] In any embodiment, the RNA polynucleotide may be a circular RNA (circRNA) molecule.

[0040] In any embodiment, the RNA polynucleotide may be a complementary RNA (cRNA) molecule.

[0041] In any embodiment, the RNA polynucleotide made be a self-amplifying RA (saRNA) molecule or trans-amplifying (taRNA) molecule.

[0042] Further examples of various RNA molecule forms are described in Fang et al., (2022) Signal Transduction and Targeted Therapy, 7: article 94, incorporated herein by reference.1006265400

[0043] In any embodiment herein, the RNA may further encode an N-terminal signal peptide for enabling secretion of the protein following translation thereof. The N-terminal signal peptide may comprise any amino acid sequence which enables the protein encoded by the RNA to be processed by ribosomes bound to the rough endoplasmic reticulum (ER) of a cell, and thereby results in threading of the protein into the ER. From the ER, the protein is capable of being transported to the plasma membrane and secreted from the mammalian cell. N-terminal secretion peptides are known to the skilled person and are further described herein.

[0044] In preferred embodiments, the RNA may further comprise a 5’ untranslated region (UTR) and a 3’ UTR. The RNA may also comprise a 5’ cap analog, such as 7mG(5')ppp(5')NlmpNp. The RNA may also comprise a polyadenine (polyA) tail. The poly(A) tail may be non-segmented or segmented with a short spacer element.

[0045] The RNA may comprise a chemical modification. Examples of suitable chemical modification include a N1-methylpseudouridine modification or a N1-ethylpseudouridine modification or may comprise any chemical modification described herein.

[0046] Preferably, the polynucleotide has a uridine content of less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20% or less than about 15%. In preferred embodiments, the polynucleotide has a uridine content of between about 15% and about 35%, preferably between about 15% and about 25%.

[0047] In preferred embodiments, the uridines in the polynucleotide are replaced with a chemical modification such as N1-methyl-pseudouridine. Preferably, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uridine nucleosides are replaced with N1-methyl-pseudouridine.

[0048] In any embodiment of the invention, the RNA polynucleotide is in the form of a codon optimised RNA molecule, optionally depleted of uridine nucleosides. In any embodiment, the codon optimisations comprises conversion of codons encoding serine to UCG.1006265400

[0049] Exemplary amino acid sequences of an Arg- or Lys-gingipain of P. gingivalis encoded by an RNA polynucleotide for use according to the present invention are further described herein. Preferably, the amino acid sequence of an active site of an Arg- or Lys-gingipain of P. gingivalis comprises the amino acid sequence of KAS or RAS (the Lysine or Arginine active site histidine sequence), ie a peptide including the active site histidine and surrounding area of the active site.

[0050] In certain embodiments, the amino acid sequence of an active site of an Arg-gingipain of P. gingivalis, (also designated “R” herein), comprises the amino acid sequence of SEQ ID NO: 38, encoded by the RNA sequence as set forth in SEQ ID NO: 50, or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 98%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0051] In certain embodiments, the amino acid sequence of an active site of a Lys-gingipain of P. gingivalis (also designated “K” herein), comprises the amino acid sequence of SEQ ID NO: 8, encoded by the RNA sequence as set forth in SEQ ID NO: 43, or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 98%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0052] Preferably, the amino acid sequence of the active site of an Arg- or Lys-gingipain does not comprise the entire catalytic domain of the gingipain.

[0053] In certain embodiments the chimeric or fusion protein encoded by the RNA polynucleotide comprises i) an amino acid sequence that comprises or consists of an amino acid sequence of the active site of an Arg-gingipain of P. gingivalis, or sequences that are at least 80% identical thereto; and ii) an amino acid sequence that comprises or consists of an amino acid sequence of the active site of a Lys-gingipain of P. gingivalis, or sequences that are at least 80% identical thereto.

[0054] Optionally, the chimeric or fusion protein encoded by the RNA polynucleotide comprises at least two amino acid sequences that comprise or consist of an amino acid sequence of the active site of an Arg- or Lys-gingipain of P. gingivalis, or sequences that1006265400are at least 80% identical thereto. The at least two amino acid sequences may be located contiguously in the chimeric or fusion protein, or may be located in different locations within the chimeric or fusion protein. Optionally, one of the at least two amino acid sequences may be located at the N terminus of the chimeric or fusion protein while the second of the at least two amino acid sequences may be located at the C-terminus of the chimeric or fusion protein. Optionally, one of the at least two amino acid sequences may be located at the N or C terminus of the chimeric or fusion protein while the second of the at least two amino acid sequences may be located within the chimeric or fusion protein (ie not at either N or C termini). Optionally, the at least two amino acid sequences may (both) be located at the N terminus of the chimeric or fusion protein or the at least two amino acid sequences may (both) be located at the C-terminus of the chimeric or fusion protein.

[0055] In any embodiment, the chimeric or fusion protein encoded by the RNA polynucleotide may further comprise:- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto.

[0056] Optionally, the amino acid sequence comprising the amino acid sequence of a DUF2436 domain is located between the amino acid sequence of the active site of the gingipain of P. gingivalis and the amino acid sequence of the one or more adhesin binding motifs (ABMs). (In other words, such that the chimeric or fusion protein comprises, N to C terminus or C to N terminus: active site (K) or (R) - DUF domain (D) - ABMs (A); or active site (K) or (R) - DUF domain (D) - ABMs (A) - active site (K) or (R)).

[0057] In certain embodiments, the amino acid sequence of a DUF2436 domain of an Arg or Lys gingipain of P. gingivalis comprises or consists of the amino acid sequence of SEQ ID NO: 35 or 76, encoded by the RNA sequence as set forth in SEQ ID NO: 51, or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 98%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.1006265400

[0058] In any embodiment, a cysteine residue in the DUF2436 domain may be substituted to a serine or valine residue, preferably to a serine residue (such as shown in SEQ ID NO: 36).

[0059] In any embodiment, the one or more adhesin binding motifs (ABMs) comprise or consist of the amino acid sequence of ABM2 and / or ABM 1 (for example as set forth in SEQ ID NO: 22 and SEQ ID NO: 21, respectively, or comprising the amino acid sequence as set forth in SEQ ID NO: 24 (ABM2+1), or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 98%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto. Such amino acid sequences may be encoded by an RNA comprising the sequence set forth in SEQ ID NOs: 52, 53, 55, herein.

[0060] The ABMs may further comprise ABM3 (eg SEQ ID NO: 73 or encoded by SEQ ID NO: 54).

[0061] The ABMs may be provided in any order, but are preferably in the order ABM2 - ABM1 - ABM3.

[0062] Optionally, the one or more adhesin binding motifs (ABMs) may comprise or consist of the amino acid sequence of ABM2, ABM1 and ABM3 (for example as set forth in SEQ ID NO: 25, 72 or 73 and encoded by an RNA comprising the sequence of SEQ ID NO: 56 or 77 or 78).

[0063] In any embodiment, the one or more adhesin binding motifs may comprise one or more modifications selected from:a) one or more cysteine amino acid substitutions compared to the naturally occurring Arg- or Lys-gingipain sequences in corresponding regions;b) substitution of the proline and / or an asparagine residues in the sequence PxxN corresponding to, or at a position equivalent to, residues 6 to 9 of the sequence of SEQ ID NO: 21 (ABM1);c) substitution of the motif NxFA to SxYQ in the sequence, corresponding to, or at a position equivalent to residues 2 to 5 of the sequence of SEQ ID NO: 21 (ABM1);1006265400d) substitution of the second tyrosine residue, corresponding to or at a position equivalent to residues at position 5 of SEQ ID NO: 22 (ABM2), and of the tryptophan residue, corresponding to or at a position equivalent to residue at position 23 of SEQ ID NO: 21 (ABM1) to alanine residues.

[0064] The one or more cysteine amino acid substitutions may be a substitution to a serine residue or to a valine residue. Preferably, the one or more cysteine substitutions may comprise one or more substitutions to a serine residue.

[0065] In certain embodiments, only one cysteine residue may be substituted. In other embodiments, two or three cysteine residues may be substituted. In particularly preferred embodiments, the cysteine residues are substituted to a combination of valine and serine residues. In other embodiments, all substituted cysteine residues are substituted to serine or all substitute cysteine residues are substituted to valine.

[0066] The motif PxxN (eg PVQN, SEQ ID NO: 106), corresponding to or at a position equivalent to residues 6 to 9 of SEQ ID NO: 21, may comprise a substitution of the proline and asparagine residues.

[0067] The proline amino acid substitution is preferably a substitution to an alanine residue.

[0068] The asparagine amino acid substitution may be a substitution to a proline residue or an alanine residue. Preferably the asparagine residue is substituted to a proline residue. In other embodiments, the asparagine residue is not substituted.

[0069] Preferably, the substitution is from PxxN to AxxP, (eg AVQP, SEQ ID NO: 107) (such as exemplified in the amino acid sequences of SEQ ID NOs: 30 to 32).

[0070] In certain further embodiments, the one or more adhesin binding motifs comprise the amino acid sequence as set forth in any one of SEQ ID NOs: 21 to 25, and comprising:a) one or more cysteine amino acid substitutions compared to the naturally occurring Arg- or Lys-gingipain sequences in corresponding regions, preferably substitution of all cysteine residues; and1006265400b) substitution of the motif PxxN corresponding to, or at a position equivalent to, residues 6 to 9 of the sequence of SEQ ID NO: 21 (ABM1), to AxxP.

[0071] Accordingly, in such embodiments, the one or more adhesin binding motifs comprise or consist the amino acid sequence as set forth in any one of SEQ ID NOs: 26 to 34, or sequences at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 98%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, provided that the sequences comprise the aforementioned substitutions of the cysteine and proline and asparagine residues.

[0072] In particularly preferred embodiments of the invention, the RNA (preferably mRNA) encodes a chimeric or fusion protein comprising or consisting of:a)- one or more amino acid sequences of active site of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto; and- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein preferably the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 39 or any of SEQ ID NOs: 58 to 63; or SEQ ID NO: 81 to 87; orb)- one or more amino acid sequences of active site of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto;- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto, and- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,1006265400wherein preferably the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 4, 12, 16 or 20;more preferably wherein the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 4; orc)- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto, and- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein preferably the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 14; ord)- one or more amino acid sequences of active site of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto; and- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto,wherein preferably the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO: 108; ore)- one or more amino acid sequences of active site of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto;- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto, and- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,1006265400wherein preferably the chimeric or fusion protein comprises the amino acid sequence of SEQ ID NO: 6.

[0073] In certain embodiments a linker region may be included between the amino acid sequence of a DUF2436 domain and the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain. The skilled person will be familiar with suitable linkers which may be used for joining any of the aforementioned domains. In certain embodiments, and as exemplified in the Tables herein, the linker comprises or consists of the sequence EVEDDSP (SEQ ID NO: 109). In certain embodiments, the chimeric or fusion protein encoded by the RNA does not include a linker sequence between the DUF2436 domain and one or more adhesin domains.

[0074] In certain embodiments, the amino acid sequence of an AMB2 domain as defined herein (eg in SEQ ID NO: 22 or SEQ ID NO 24 to 34) may further comprise at its N-terminus, the amino acid sequence of EVEDDSP (SEQ ID NO: 109), which is derived from the native P. gingivalis gingipain polyprotein sequence.

[0075] It will also be appreciated that in some circumstances, N-terminal methionine residues of polypeptides are cleaved following translation of the mRNA into protein. As such, the present disclosure provides basis for the generation of a chimeric or fusion protein from an RNA molecule, wherein the chimeric or fusion protein does not comprise an N terminal methionine residue.

[0076] In other embodiments of the invention, the RNA molecule comprises or consists of a nucleotide sequence encoding a protein comprising or consisting of the amino acid sequence of any one of: SEQ ID NO: 2, SEQ ID NO: 8 or SEQ ID NO: 38.

[0077] In particularly preferred embodiments of the invention, the RNA molecule comprises or consists of a nucleotide sequence of any one of:a) SEQ ID NO: 48 or 57b) SEQ ID NO: 45, 47 or 49 or SEQ ID NO: 41;c) SEQ ID NO: 46;d) SEQ ID NO: 44;e) SEQ ID NO: 421006265400or sequences at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 98%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0078] In other embodiments of the invention, the RNA comprises or consists of a nucleotide sequence of any one of: SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 50.

[0079] In preferred embodiments, the RNA molecule is provided in the form of a composition, including a pharmaceutical composition comprising an RNA as described herein. Preferably, the composition comprises one or more pharmaceutically acceptable excipients. Optionally, the composition may comprise one or more agents for enabling delivery of the RNA to a mammalian cell, and thereby enabling translation of the RNA in the cell. In any embodiment, the composition may comprise a combination of one or more of the RNA molecules described herein.

[0080] The agent for delivery of the RNA into mammalian cells may be any suitable agent known to the skilled person for delivery of RNA. Such agents may include: cell penetrating peptides, lipid-based formulations.

[0081] Preferably the lipid component comprises a cationic and / or ionisable lipid, a phospholipid, a PEG lipid, and a structural lipid.

[0082] In any embodiment, the ionisable lipid may be substituted or combined with an adjuvant lipidoid for enhancing RNA delivery.

[0083] The further embodiments, the composition also comprises a lipid component. The RNA (e.g., RNA) vaccines for the use of the invention can be formulated using one or more liposomes, lipid vesicle, lipoplexes (such as a lipid-polycation complex), or lipid nanoparticles

[0084] In preferred embodiments, the RNA is formulated in a lipid nanoparticle, liposomes, lipid vesicle, or lipoplexes (such as a lipid-polycation complex).

[0085] In one embodiment, the RNA as described herein is the only polynucleotide species present in the composition or in the liposomes, lipid vesicle, lipoplexes (such as a lipid-polycation complex), or lipid nanoparticles. Preferably, the polynucleotide as described herein is the only active ingredient present in the composition or in the1006265400liposomes, lipid vesicle, lipoplexes (such as a lipid-polycation complex), or lipid nanoparticles.

[0086] In further examples, the composition or liposomes, lipid vesicle, lipoplexes (such as a lipid-polycation complex), or lipid nanoparticles may comprise more than one RNA (polynucleotide) species, as described herein (eg thereby providing RNA molecules encoding more than one chimeric or fusion protein amino acid sequences). Further still, the composition or the liposomes, lipid vesicle, lipoplexes (such as a lipid-polycation complex), or lipid nanoparticles may comprise a single polynucleotide construct containing one or more RNA sequences as described herein (and thereby also encoding more than one chimeric or fusion protein amino acid sequences). Accordingly, the present invention contemplates the provision of compositions, liposomes, lipid vesicle, lipoplexes (such as a lipid-polycation complex), or lipid nanoparticles for delivering combinations of two or more of any of the RNA molecules described herein.

[0087] Lipid nanoparticles are well known in the art and are further described herein. Preferably the lipid nanoparticle comprises a cationic and / or ionisable lipid, a phospholipid, a PEG (or PEGylated) lipid, and a structural lipid.

[0088] In any embodiment, the lipid nanoparticle may comprise:- a cationic and / or ionisable lipid comprising from about 25 % to about 75 mol % of the total lipid present in the nanoparticle;- a sterol (structural lipid) comprising from about 5 mol % to about 60 mol % of the total lipid present in the nanoparticle;- a phospholipid comprising from about 5 mol % to about 50 mol % of the total lipid present in the nanoparticle;- a PEGylated lipid comprising from about 0.5 mol % to 20 mol % of the total lipid present in the nanoparticle.

[0089] In non-limiting examples, the lipid nanoparticle comprises:- an ionisable lipid in the form of [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315),- a sterol in the form of cholesterol,1006265400- a phospholipid in the form of distearoylphosphatidylcholine (DSPC), and- a PEGylated lipid in the form of 2-[(polyethylene glycol)-2000]-N, N- ditetradecylacetamide (ALC-0159).

[0090] Preferably the lipids are present in the lipid nanoparticle at molar lipid ratios (%) of 46.3 ALC-0315: 42.7 cholesterol : 9.4 DSPC : 1.6 ALC-0159, optionally in Tris / sucrose buffer (25 mM Tris pH 7.4, 8.8% sucrose w / v).

[0091] In any embodiment, the RNA polynucleotide may be provided in the form of a nucleic acid construct or vector, comprising a polynucleotide as described herein.

[0092] The vector may be any vector suitable for production of RNA from a DNA template. The vector may additionally comprise 3’IITR and 5’llTRs and polyadenine fragments.

[0093] Examples of such vectors include: IVT RNA vector or similar vectors that comprise T7, T3 and SP6 signals for expression. The vector can be from a plasmid or produced through PCR or Phi29 DNA polymerase (e.g. GenomiPhi™ V2 DNA) or other bacterial constructs.

[0094] The vector may be a self-amplifying RNA replicon, such as, but not limited to a self-amplifying RNA vector from an alphavirus, optionally Venezuelan Equine Encephalitis Virus (VEEV), bipartite VEEV, or variants thereof (including the TC83 mutated variant). Examples of self-amplifying mRNA platforms are known to the skilled person, and are described for example in Maruggi et al., (2017), Vaccines 35: 361-368, incorporated herein by reference.

[0095] 5’ capping of the polynucleotide may be performed using any commercially available capping reagent. Such reagents are known to the skilled person, such as the commercial capping reagent Cap1 from TriLink Biotechnologies Inc. Other capping reagents may be used, including but not limited to Cap 0 and Cap 2.

[0096] As used herein “at least 80% identity” should be taken to provide basis for “at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 98%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity”.1006265400

[0097] As used herein a sequence defined as having “at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 98%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity” to a particular SEQ ID NO, may also be referred to as a “substitutional variant”.

[0098] Throughout this specification, unless the context requires otherwise, the words “comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term “comprising” and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”. Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. By “consisting essentially of” is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of” indicates that the listed elements are required or mandatory, but that other elements (e.g., 1, 2, 3, 4, 5, 6,7, 8,9,10, 11, 12 13, 14, 15, 16, 17,18, 19, 20, or more than 20 additional amino acid residues at the N-terminus or C-terminus of a polypeptide sequence) are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0099] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0100] Figure 1: Schematic showing domain architecture of Kgp polyprotein and chimeric proteins derived therefrom. (KDcAKIn: K = Kgp active site, De = N terminally truncated DUF2436 domain, A = ABM2, ABM1 and ABM3, K1n = C terminally truncated CAD domain; KDAK: K = Kgp active site, D = DUF2436 domain, A = ABM2, ABM1 and ABM3; KDAK-3S-AVQP: K = Kgp active site, D = DUF2436 domain, A = ABM2, ABM1 and ABM3, 3S-AVQP = PVQN (SEQ ID NO: 106) to AVQP (SEQ ID NO:1006265400107) substitution in ABM1 domain and substitution of the cysteine residues in the D and A domains to serine).

[0101] Figure 2: Exemplary structure of mRNA. Cap = 5’ cap for maximising RNA stability; 5’UTR and 3’ UTR = 5’ and 3’ untranslated sequences; SP = signal peptide; Antigen sequence = mRNA sequence encoding protein antigen for expression in cell upon translation of mRNA; Poly(A) tail: polyadenosine tail for providing RNA stability and maximising translation.

[0102] Figure 3: Expression and secretion by HeLa cells of mRNA constructs encoding chimeric proteins. A. HeLa supernatant. Lane 1 = SEAP-KDcAK1n, Lane 2 = KDcAKIn (no secretion peptide), Lane 3 = Mock, Lane 4 = Protein size marker. B. HeLa supernatant Lane 1: SEAP-KDAK, Lane 2 = SEAP-KDAK-3S-AVQP, Lane 3 = Mock, Lane 5 = Protein size marker. C. HeLa supernatant Lane 1 = SEAP-KDAK-3S-AVQP, Lane 2 = KDAK-3S-AVQP (no secretion peptide), Lane 3 = Mock, Lane 4 = Protein size marker. SEAP = SEAP secretion peptide from secreted embryonic alkaline phosphatase. Mock = negative control (mock transfection).

[0103] Figure 4: schematic of study protocol for determining in vivo efficacy of vaccines.

[0104] Figure 5: Antibody responses in the in mouse model of P. gingivalis-induced neuropathology.. A. KDAK-3S-AVQP total IgG titres. B. KDAK-3S-AVQP lgG1 titres. C. KDAK-3S-AVQP lgG2a titres. D. Heat-killed P. gingivalis total IgG titres. E. Heat-killed P. gingivalis lgG1 titres. F. Heat-killed P. gingivalis lgG2a titres. G. RgpA-Kgp complex total IgG titres. H. RgpA-Kgp complex IgG 1 titres. I. RgpA-Kgp complex lgG2a titres. Naive = unvaccinated; infected = no vaccine control; KDAK-3S-AVQP = mRNA encoding KDAK-3S-AVQP protein (SEQ ID NO: 6). m1ψ = N1-methylpseudouridine modified mRNA sequence. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001

[0105] Figure 6: Summary of efficacy of vaccination with m1ψ-KDAK-3S-AVQP in mouse model of P. gingivalis-induced neuropathology. A. percentage of RgpA positive cells. B. percentage of amyloid β plaque positive pixels (area). C. percentage of phospho-tau positive cells. D. percentage of IL-6 positive cells. E. percentage of IL-1β positive cells. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, **** = P < 0.00011006265400

[0106] Figure 7: Expression and secretion of constructs encoding truncated antigens. A. Western blot of lysates at 24 hours after transfection. Left hand panel: using anti-KDAK-3S-AVQP antibody. Right hand panel: using anti-KAS antibody. B. Western blot of supernatants at 24 hours after transfection. Left hand panel: using anti-KAS antibody. Right hand panel: using anti-KDAK-3S-AVQP antibody. A and B: Lanes: L = ladder; 1 = K; 2 = KA; 3 = KD; 4 = DA; 5 = KDAΔABM3; 6 = KDA; 7 = RDA; 8 = KDA-3S-AVQP (positive control); 9 = mock transfection (negative control). All constructs included SEAP secretion peptide. C. Expression of K over time compared to KDAK-3S-AVQP: time-lapse of K expression at 6 hours, 24 hours and 48 hours after transfection. Lanes: L = ladder; 1 = K; 2 @ 6 hours = KDAK-3S-AVQP @ 6 hours; 3 = Mock transfection @ 6 hours; 4 = K; 5 = KDAK-3S-AVQP @ 24 hours; 6 = Mock transfection @ 24 hours; 7 = K @ 48 hours 8 = KDA-3S-AVQP @ 48 hours; 9 = mock transfection @ 48 hours. All constructs included SEAP secretion peptide.

[0107] Figure 8: P. gingivalis induced alveolar bone loss (mm) following vaccination with different mRNA constructs. Naive = uninfected. Infected = no vaccination control. Positive control: alum adjuvanted protein KDAK-3S-AVQP at 200 g. mψ1 = modified mRNA sequence. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

[0108] Figure 9: Antibody responses in mice following alveolar bone loss experiments using mRNA vaccine constructs encoding truncated antigens. A.KDAK-3S-AVQP total IgG titres. B. KDAK-3S-AVQP lgG1 titres. C. KDAK-3S-AVQP lgG2a titres. D. Heat-killed P. gingivalis total IgG titres. E. Heat-killed P. gingivalis lgG1 titres. F. Heat-killed P. gingivalis lgG2a titres. G. Kgpcat total IgG titres. H. Kgpcat IgG 1 titres. I. Kgpcat lgG2a titres. J. KAS2 titres. Naive = unvaccinated; infected = no vaccine control; KDAK-3S-AVQP = mRNA encoding KDAK-3S-AVQP protein (SEQ ID NO: 6). K.Kgp-RgpA complex total IgG titres. L. Kgp-RgpA complex lgG1 titres. M. Kgp-RpgA complex lgG2a titres. m1ψ = N1-methylpseudouridine modified mRNA sequence. KA = mRNA encoding KA protein (SEQ ID NO: 18). KD = mRNA encoding KD protein (SEQ ID NO: 10). DA = mRNA encoding DA protein (SEQ ID NO: 14). KDA21 = mRNA encoding KDAΔAMB3 protein (SEQ ID NO: 16). Protein control: alum-adjuvanted protein KDAK-3S-AVQP at 200pg. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

[0109] Figure 10: Immunogenicity of truncated antigens RDA and K. A. KDAK-3S-AVQP total IgG titres. B. KDAK-3S-AVQP lgG1 titres. C. KDAK-3S-AVQP lgG2a titres.1006265400D. Heat-killed P. gingivalis total IgG titres. E. Heat-killed P. gingivalis lgG1 titres. F. Heat-killed P. gingivalis lgG2a titres. G. Kgpcat total IgG titres. H. Kgpcat lgG1 titres. I. Kgpcat lgG2a titres. J. KAS2 titres. K. RgpA-Kgp complex total IgG titres. L. RgpA-Kgp complex I lgG1 titres M. RgpA-Kgp complex lgG2a titres. Naive = unvaccinated; K = mRNA encoding K protein (SEQ ID NO: 8 ). KD = mRNA encoding KD protein (SEQ ID NO: 10). RDA = mRNA encoding RDA protein (SEQ ID NO: 20). * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.Sequence information

[0110] Table 1: DNA and amino acid sequences| Descriptor | SEQ ID No | Sequencej KDcAK1n (DNA | 1 | AATACCGGAGTCAGCTTTGCAAACTATACAGCGCATGGATCTGAGAC j sequence) | | CGCATGGGCTGATCCACTTCTGACTACTTCTCAACTGAAAGCACTCA j | CTAATAAGGACAAATGGGGAGACAATACGGGTTACCAGTTCTTGTTG | | | GATGCCGATCACAATACATTCGGAAGTGTCATTCCGGCAACCGGTCC j | TCTCTTTACCGGAACAGCTTCTTCCAATCTTTACAGTGCGAACTTCGA j | GTATTTGATCCCGGCCAATGCCGATCCTGTTGTTACTACACAGAATA j | | TTATCGTTACAGGACAGGGTGAAGTTGTAATCCCCGGTGGTGTTTAC j | GACTATTGCATTACGAACCCGGAACCTGCATCCGGAAAGATGTGGAT | | CGCAGGAGATGGAGGCAACCAGCCTGCACGTTATGACGATTTCACA j | TTCGAAGCAGGCAAGAAGTACACCTTCACGATGCGTCGCGCCGGAA j | TGGGAGATGGAACTGATATGGAAGTCGAAGACGATTCACCTGCAAG j | | CTATACCTACACGGTGTATCGTGACGGCACGAAGATCAAGGAAGGT j | CTGACAGCTACGACATTCGAAGAAGACGGTGTAGCTGCAGGCAATC | | ATGAGTATTGCGTGGAAGTTAAGTACACAGCCGGCGTATCTCCGAAG j | GTATGTAAAGACGTTACGGTAGAAGGATCCAATGAATTTGCTCCTGT j | ACAGAACCTGACCGGTAGTTCAGTAGGTCAGAAAGTAACGCTTAAGT j | | GGGATGCACCTAATGGTACCCCGAATCCGAATCCAAATCCGAATCC j | GAATCCGGGAACAACACTTTCCGAATCATTCGAAAATGGTATTCCGG | | CATCTTGGAAGACGATCGATGCAGACGGTGACGGGCATGGCTGGAA j | ACCTGGAAATGCTCCCGGAATCGCTGGCTACAATAGCAATGGTTGTG j | TATATTCAGAGTCATTCGGTCTTGGTGGTATAGGAGTTCTTACCCCT | | | GACAACTATCTGATAACACCGGCATTGGATTTGCCTAACGGAGGT j KDcAK1n (amino acid | 2 | NTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDKWGDNTGYQFLLD j sequence) | | ADHNTFGSVIPATGPLFTGTASSNLYSANFEYLIPANADPVVTTQNIIVTG | | QGEVVIPGGVYDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKK j | YTFTMRRAGMGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTATTFEE j | DGVAAGNHEYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSV | | | GQKVTLKWDAPNGTPNPNPNPNPNPGTTLSESFENGIPASWKTIDADG j | DGHGWKPGNAPGIAGYNSNGCVYSESFGLGGIGVLTPDNYLITPALDLP | NGG1006265400| KDAK (DNA ] 3 |AATACCGGAGTCAGCTTTGCAAACTATACAGCGCATGGATCTGAGAC ^sequence) | | CGCATGGGCTGATCCACTTCTGACTACTTCTCAACTGAAAGCACTCA CTAATAAGGACAAAGCAGAAGGTTCCCGTGAAGTAAAACGGATCGG|AGACGGTCTTTTCGTTACGATCGAACCTGCAAACGATGTACGTGCCA ACGAAGCCAAGGTTGTGCTTGCGGCAGACAACGTATGGGGAGACAA| TACGGGTTACCAGTTCTTGTTGGATGCCGATCACAATACATTCGGAA | GTGTCATTCCGGCAACCGGTCCTCTCTTTACCGGAACAGCTTCTTCC AATCTTTACAGTGCGAACTTCGAGTATTTGATCCCGGCCAATGCCGA| TCCTGTTGTTACTACACAGAATATTATCGTTACAGGACAGGGTGAAG | TTGTAATCCCCGGTGGTGTTTACGACTATTGCATTACGAACCCGGAA | CCTGCATCCGGAAAGATGTGGATCGCAGGAGATGGAGGCAACCAGC | CTGCACGTTATGACGATTTCACATTCGAAGCAGGCAAGAAGTACACC | TTCACGATGCGTCGCGCCGGAATGGGAGATGGAACTGATATGGAAG | TCGAAGACGATTCACCTGCAAGCTATACCTACACGGTGTATCGTGAC GGCACGAAGATCAAGGAAGGTCTGACAGCTACGACATTCGAAGAAG|ACGGTGTAGCTGCAGGCAATCATGAGTATTGCGTGGAAGTTAAGTAC |ACAGCCGGCGTATCTCCGAAGGTATGTAAAGACGTTACGGTAGAAG GATCCAATGAATTTGCTCCTGTACAGAACCTGACCGGTAGTTCAGTA| GGTCAGAAAGTAACGCTTAAGTGGGATGCACCTAATGGTACCCCGA ATCCGAATCCAAATCCGAATCCGAATCCGGGAACAAATACCGGAGTC|AGCTTTGCAAACTATACAGCGCATGGATCTGAGACCGCATGGGCTG |ATCCACTTCTGACTACTTCTCAACTGAAAGCACTCACTAATAAGGACA ^AA| KDAK (amino acid 14 I NTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDKAEGSREVKRIGD ^sequence) | | GLFVTIEPANDVRANEAKWLAADNVWGDNTGYQFLLDADHNTFGSVIP I ATGPLFTGTASSNLYSANFEYLIPANADPWTTQNIIVTGQGEWIPGGV|YDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAG | MGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHE YCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDA| PNGTPNPNPNPNPNPGTNTGVSFANYTAHGSETAWADPLLTTSQLKAL TNKDKj KDAK-3S-AVQP | 5 AATACCGGAGTCAGCTTTGCAAACTATACAGCGCATGGATCTGAGAC | (DNA sequence) | | CGCATGGGCTGATCCACTTCTGACTACTTCTCAACTGAAAGCACTCA | CTAATAAGGACAAAGCAGAAGGTTCCCGTGAAGTAAAACGGATCGG AGACGGTCTTTTCGTTACGATCGAACCTGCAAACGATGTACGTGCCA|ACGAAGCCAAGGTTGTGCTTGCGGCAGACAACGTATGGGGAGACAA | TACGGGTTACCAGTTCTTGTTGGATGCCGATCACAATACATTCGGAA I GTGTCATTCCGGCAACCGGTCCTCTCTTTACCGGAACAGCTTCTTCC|AATCTTTACAGTGCGAACTTCGAGTATTTGATCCCGGCCAATGCCGA [TCCTGTTGTTACTACACAGAATATTATCGTTACAGGACAGGGTGAAG | TTGTAATCCCCGGTGGTGTTTACGACTATAGCATTACGAACCCGGAA | CCTGCATCCGGAAAGATGTGGATCGCAGGAGATGGAGGCAACCAGC CTGCACGTTATGACGATTTCACATTCGAAGCAGGCAAGAAGTACACC| TTCACGATGCGTCGCGCCGGAATGGGAGATGGAACTGATATGGAAG [TCGAAGACGATTCACCTGCAAGCTATACCTACACGGTGTATCGTGAC1006265400| GGCACGAAGATCAAGGAAGGTCTGACAGCTACGACATTCGAAGAAG |ACGGTGTAGCTGCAGGCAATCATGAGTATAGCGTGGAAGTTAAGTAC I ACAGCCGGCGTATCTCCGAAGGTAAGTAAAGACGTTACGGTAGAAG| GATCCAATGAATTTGCTGCTGTACAGCCGCTGACCGGTAGTTCAGTA GGTCAGAAAGTAACGCTTAAGTGGGATGCACCTAATGGTACCCCGA|ATCCGAATCCAAATCCGAATCCGAATCCGGGAACAAATACCGGAGTC |AGCTTTGCAAACTATACAGCGCATGGATCTGAGACCGCATGGGCTG I ATCCACTTCTGACTACTTCTCAACTGAAAGCACTCACTAATAAGGACA AA| KDAK-3S-AVQP 16 | NTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDKAEGSREVKRIGD | (amino acid | I GLFVTIEPANDVRANEAKWLAADNVWGDNTGYQFLLDADHNTFGSVIP ^sequence) | |ATGPLFTGTASSNLYSANFEYLIPANADPWTTQNIIVTGQGEWIPGGV |YDYSITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAG I MGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHE|YSVEVKYTAGVSPKVSKDVTVEGSNEFAAVQPLTGSSVGQKVTLKWDA I PNGTPNPNPNPNPNPGTNTGVSFANYTAHGSETAWADPLLTTSQLKAL| TNKDK| K (DNA sequence) 17 |AATACCGGAGTCAGCTTTGCAAACTATACAGCGCATGGATCTGAGAC | CGCATGGGCTGATCCACTTCTGACTACTTCTCAACTGAAAGCACTCA CTAATAAGGACAAA| K (amino acid 18 NTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDK^sequence) || KD (DNA sequence) | 9 | AATACCGGAGTCAGCTTTGCAAACTATACAGCGCATGGATCTGAGAC | CGCATGGGCTGATCCACTTCTGACTACTTCTCAACTGAAAGCACTCA | CTAATAAGGACAAAGCAGAAGGTTCCCGTGAAGTAAAACGGATCGG |AGACGGTCTTTTCGTTACGATCGAACCTGCAAACGATGTACGTGCCA ACGAAGCCAAGGTTGTGCTTGCGGCAGACAACGTATGGGGAGACAA| TACGGGTTACCAGTTCTTGTTGGATGCCGATCACAATACATTCGGAA GTGTCATTCCGGCAACCGGTCCTCTCTTTACCGGAACAGCTTCTTCC|AATCTTTACAGTGCGAACTTCGAGTATTTGATCCCGGCCAATGCCGA | TCCTGTTGTTACTACACAGAATATTATCGTTACAGGACAGGGTGAAG [TTGTAATCCCCGGTGGTGTTTACGACTATTGCATTACGAACCCGGAA | CCTGCATCCGGAAAGATGTGGATCGCAGGAGATGGAGGCAACCAGC | CTGCACGTTATGACGATTTCACATTCGAAGCAGGCAAGAAGTACACC | TTCACGATGCGTCGCGCCGGAATGGGAGATGGAACTGATATG | KD (amino acid | 10 | NTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDKAEGSREVKRIGD ^sequence) | | GLFVTIEPANDVRANEAKWLAADNVWGDNTGYQFLLDADHNTFGSVIP I ATGPLFTGTASSNLYSANFEYLIPANADPWTTQNIIVTGQGEWIPGGV|YDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAG I MGDGTDM| KDA (DNA sequence) | 11 | AATACCGGAGTCAGCTTTGCAAACTATACAGCGCATGGATCTGAGAC | CGCATGGGCTGATCCACTTCTGACTACTTCTCAACTGAAAGCACTCA | CTAATAAGGACAAAGCAGAAGGTTCCCGTGAAGTAAAACGGATCGG AGACGGTCTTTTCGTTACGATCGAACCTGCAAACGATGTACGTGCCA1006265400ACGAAGCCAAGGTTGTGCTTGCGGCAGACAACGTATGGGGAGACAA TACGGGTTACCAGTTCTTGTTGGATGCCGATCACAATACATTCGGAA GTGTCATTCCGGCAACCGGTCCTCTCTTTACCGGAACAGCTTCTTCC AATCTTTACAGTGCGAACTTCGAGTATTTGATCCCGGCCAATGCCGA TCCTGTTGTTACTACACAGAATATTATCGTTACAGGACAGGGTGAAG TTGTAATCCCCGGTGGTGTTTACGACTATTGCATTACGAACCCGGAA CCTGCATCCGGAAAGATGTGGATCGCAGGAGATGGAGGCAACCAGC CTGCACGTTATGACGATTTCACATTCGAAGCAGGCAAGAAGTACACC TTCACGATGCGTCGCGCCGGAATGGGAGATGGAACTGATATGGAAG TCGAAGACGATTCACCTGCAAGCTATACCTACACGGTGTATCGTGAC GGCACGAAGATCAAGGAAGGTCTGACAGCTACGACATTCGAAGAAG ACGGTGTAGCTGCAGGCAATCATGAGTATTGCGTGGAAGTTAAGTAC ACAGCCGGCGTATCTCCGAAGGTATGTAAAGACGTTACGGTAGAAG GATCCAATGAATTTGCTCCTGTACAGAACCTGACCGGTAGTTCAGTA GGTCAGAAAGTAACGCTTAAGTGGGATGCACCTAATGGTACCCCGA ATCCGAATCCAAATCCGAATCCGAATCCGGGAACA| KDA (amino acid | 12 | NTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDKAEGSREVKRIGD ^sequence) | GLFVTIEPANDVRANEAKVVLAADNVWGDNTGYQFLLDADHNTFGSVIP ATGPLFTGTASSNLYSANFEYLIPANADPVVTTQNIIVTGQGEVVIPGGV YDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAG MGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHE YCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDA PNGTPNPNPNPNPNPGT| DA (DNA sequence) | 13 | GCAGAAGGTTCCCGTGAAGTAAAACGGATCGGAGACGGTCTTTTCG TTACGATCGAACCTGCAAACGATGTACGTGCCAACGAAGCCAAGGTT GTGCTTGCGGCAGACAACGTATGGGGAGACAATACGGGTTACCAGT TCTTGTTGGATGCCGATCACAATACATTCGGAAGTGTCATTCCGGCA ACCGGTCCTCTCTTTACCGGAACAGCTTCTTCCAATCTTTACAGTGC GAACTTCGAGTATTTGATCCCGGCCAATGCCGATCCTGTTGTTACTA CACAGAATATTATCGTTACAGGACAGGGTGAAGTTGTAATCCCCGGT GGTGTTTACGACTATTGCATTACGAACCCGGAACCTGCATCCGGAAA GATGTGGATCGCAGGAGATGGAGGCAACCAGCCTGCACGTTATGAC GATTTCACATTCGAAGCAGGCAAGAAGTACACCTTCACGATGCGTCG CGCCGGAATGGGAGATGGAACTGATATGGAAGTCGAAGACGATTCA CCTGCAAGCTATACCTACACGGTGTATCGTGACGGCACGAAGATCAA GGAAGGTCTGACAGCTACGACATTCGAAGAAGACGGTGTAGCTGCA GGCAATCATGAGTATTGCGTGGAAGTTAAGTACACAGCCGGCGTATC TCCGAAGGTATGTAAAGACGTTACGGTAGAAGGATCCAATGAATTTG CTCCTGTACAGAACCTGACCGGTAGTTCAGTAGGTCAGAAAGTAACG CTTAAGTGGGATGCACCTAATGGTACCCCGAATCCGAATCCAAATCC GAATCCGAATCCGGGAACA| DA (amino acid | 14 | AEGSREVKRIGDGLFVTIEPANDVRANEAKVVLAADNVWGDNTGYQFLL ^sequence) | DADHNTFGSVIPATGPLFTGTASSNLYSANFEYLIPANADPVVTTQNIIVT GQGEWIPGGVYDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGK| KYTFTMRRAGMGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTATTFE1006265400EDGVAAGNHEYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSV GQKVTLKWDAPNGTPNPNPNPNPNPGT| KDAΔABM3 AATACCGGAGTCAGCTTTGCAAACTATACAGCGCATGGATCTGAGAC | (DNA sequence) CGCATGGGCTGATCCACTTCTGACTACTTCTCAACTGAAAGCACTCA CTAATAAGGACAAAGCAGAAGGTTCCCGTGAAGTAAAACGGATCGG AGACGGTCTTTTCGTTACGATCGAACCTGCAAACGATGTACGTGCCA ACGAAGCCAAGGTTGTGCTTGCGGCAGACAACGTATGGGGAGACAA TACGGGTTACCAGTTCTTGTTGGATGCCGATCACAATACATTCGGAA GTGTCATTCCGGCAACCGGTCCTCTCTTTACCGGAACAGCTTCTTCC AATCTTTACAGTGCGAACTTCGAGTATTTGATCCCGGCCAATGCCGA TCCTGTTGTTACTACACAGAATATTATCGTTACAGGACAGGGTGAAG TTGTAATCCCCGGTGGTGTTTACGACTATTGCATTACGAACCCGGAA CCTGCATCCGGAAAGATGTGGATCGCAGGAGATGGAGGCAACCAGC CTGCACGTTATGACGATTTCACATTCGAAGCAGGCAAGAAGTACACC TTCACGATGCGTCGCGCCGGAATGGGAGATGGAACTGATATGGAAG TCGAAGACGATTCACCTGCAAGCTATACCTACACGGTGTATCGTGAC GGCACGAAGATCAAGGAAGGTCTGACAGCTACGACATTCGAAGAAG ACGGTGTAGCTGCAGGCAATCATGAGTATTGCGTGGAAGTTAAGTAC ACAGCCGGCGTATCTCCGAAGGTATGTAAAGACGTTACGGTAGAAG GATCCAATGAATTTGCTCCTGTACAGAACCTGACCGGTAGTTCAGTA GGTCAGAAAGTAACGCTTAAGTGGGATGCACCTAATGGTACC| KDAΔABM3 | 16 | NTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDKAEGSREVKRIGD | (amino acid GLFVTIEPANDVRANEAKWLAADNVWGDNTGYQFLLDADHNTFGSVIP | sequence) ATGPLFTGTASSNLYSANFEYLIPANADPWTTQNIIVTGQGEWIPGGV YDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAG MGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHE YCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDA PNGT| KA (DNA sequence) AATACCGGAGTCAGCTTTGCAAACTATACAGCGCATGGATCTGAGAC CGCATGGGCTGATCCACTTCTGACTACTTCTCAACTGAAAGCACTCA CTAATAAGGACAAAGAAGTCGAAGACGATTCACCTGCAAGCTATACC TACACGGTGTATCGTGACGGCACGAAGATCAAGGAAGGTCTGACAG CTACGACATTCGAAGAAGACGGTGTAGCTGCAGGCAATCATGAGTAT TGCGTGGAAGTTAAGTACACAGCCGGCGTATCTCCGAAGGTATGTAA AGACGTTACGGTAGAAGGATCCAATGAATTTGCTCCTGTACAGAACC TGACCGGTAGTTCAGTAGGTCAGAAAGTAACGCTTAAGTGGGATGCA CCTAATGGTACCCCGAATCCGAATCCAAATCCGAATCCG AATCCGGGAACA| KA (amino acid NTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDKEVEDDSPASYTYT | sequence) VYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGT1006265400| RDA ] 19 |AACGGAGGAATCTCGTTGGCCAACTATACGGGCCACGGTAGCGAAA | (DNA sequence) | | CAGCTTGGGGTACGTCTCACTTCGGCACCACTCATGTGAAGCAGCTT | ACCAACAGCAACCAGGCAGAAGGTTCCCGTGAAGTAAAACGGATCG | GAGACGGTCTTTTCGTTACGATCGAACCTGCAAACGATGTACGTGCC AACGAAGCCAAGGTTGTGCTTGCGGCAGACAACGTATGGGGAGACA|ATACGGGTTACCAGTTCTTGTTGGATGCCGATCACAATACATTCGGA |AGTGTCATTCCGGCAACCGGTCCTCTCTTTACCGGAACAGCTTCTTC CAATCTTTACAGTGCGAACTTCGAGTATTTGATCCCGGCCAATGCCG|ATCCTGTTGTTACTACACAGAATATTATCGTTACAGGACAGGGTGAA GTTGTAATCCCCGGTGGTGTTTACGACTATTGCATTACGAACCCGGA|ACCTGCATCCGGAAAGATGTGGATCGCAGGAGATGGAGGCAACCAG | CCTGCACGTTATGACGATTTCACATTCGAAGCAGGCAAGAAGTACAC CTTCACGATGCGTCGCGCCGGAATGGGAGATGGAACTGATATGGAA| GTCGAAGACGATTCACCTGCAAGCTATACCTACACGGTGTATCGTGA | CGGCACGAAGATCAAGGAAGGTCTGACAGCTACGACATTCGAAGAA | GACGGTGTAGCTGCAGGCAATCATGAGTATTGCGTGGAAGTTAAGTA | CACAGCCGGCGTATCTCCGAAGGTATGTAAAGACGTTACGGTAGAA GGATCCAATGAATTTGCTCCTGTACAGAACCTGACCGGTAGTTCAGT|AGGTCAGAAAGTAACGCTTAAGTGGGATGCACCTAATGGTACCCCG AATCCGAATCCAAATCCGAATCCGAATCCGGGAACAj RDA ] 20 | NGGISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQAEGSREVKRIGD | (amino acid | | GLFVTIEPANDVRANEAKVVLAADNVWGDNTGYQFLLDADHNTFGSVIP ^sequence) | I ATGPLFTGTASSNLYSANFEYLIPANADPVVTTQNIIVTGQGEVVIPGGV |YDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAG I MGDGTDMEVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHE|YCVEVKYTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDA I PNGTPNPNPNPNPNPGT^ABM1 121 | SNEFAPVQNLTGSSVGQKVTLKWDAPNGT| Tryptophan for possible || substitution underlined || amino acid sequence |^ABM2 ^ 22 |ASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKV | amino acid sequence | I CKDVTVEG^ABM3 ]23 | PNPNPNPNPNPGT| amino acid sequence || ABM2+1 | 24 | ASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKV | amino acid sequence | CKDVTVEGS**NEFA**PVQNLTGSSVGQKVTLKWDAPNGT| cysteine residues |^ underlined |1006265400| NEFA motif shown in |j bold; PVQN motif || shown in italics |^ABM2+1+3 ^ 25 ASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKV | amino acid sequence | | CKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGT| cysteine residues |^ underlined || NEFA motif shown in |j bold; PVQN motif || shown in italics |^ABM2+1 ]26 |ASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKV | Cys2 to Ser substitution | | SKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGT ^ (underlined) || amino acid sequence |^ABM2+1 ^ 27 ASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYSVEVKYTAGVSPKV | Cys1 to Ser | | CKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGT| substitution |^ (underlined) || amino acid sequence |^ABM2+1 ]28 |ASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYSVEVKYTAGVSPKV 2 x Cys to Ser | | SKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGT| substitution |^ (underlined) || amino acid sequence |^ABM2+1+3 ^ 29 |ASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYSVEVKYTAGVSPKV 2 x Cys to Ser | | SKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPN | substitution | PGT^ (underlined) || amino acid sequence |^ABM1 PVQN> AVQP ^ 30 SNEFAAVQPLTGSSVGQKVTLKWDAPNGT| amino acid sequence |^ABM2+1 ] 31 ASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKV | PVQN> AVQP | j CKDVTVEGSNEFAAVQPLTGSSVGQKVTLKWDAPNGT| amino acid sequence |1006265400^ABM2+1+3 ] 32 |ASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKV j PVQN> AVQP | I CKDVTVEGSNEFAAVQPLTGSSVGQKVTLKWDAPNGTPNPNPNPNPN | amino acid sequence | PGT^ABM2+1 ^ 33 ASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYSVEVKYTAGVSPKV j PVQN> AVQP | j SKDVTVEGSNEFAAVQPLTGSSVGQKVTLKWDAPNGT2 x Cys to Ser || amino acid sequence |^ABM2+1+3 ^ 34 |ASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYSVEVKYTAGVSPKV j PVQN> AVQP | | SKDVTVEGSNEFAAVQPLTGSSVGQKVTLKWDAPNGTPNPNPNPNPN PGT2 x Cys to Ser || amino acid sequence |j DUF2436 | 35 | AEGSREVKRIGDGLFVTIEPANDVRANEAKVVLAADNVWGDNTGYQFLL | amino acid sequence | | DADHNTFGSVIPATGPLFTGTASSNLYSANFEYLIPANADPVVTTQNIIVT i | GQGEWIPGGVYDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGK KYTFTMRRAGMGDGTDMj DUF2436 Cys to | 36 | AEGSREVKRIGDGLFVTIEPANDVRANEAKVVLAADNVWGDNTGYQFLL i | Serine / Valine | | DADHNTFGSVIPATGPLFTGTASSNLYSANFEYLIPANADPVVTTQNIIVT i | substitution | | GQGEWIPGGVYDY[S / yilTNPEPASGKMWIAGDGGNQPARYDDFTFEA | amino acid sequence | | GKKYTFTMRRAGMGDGTDM| R (Arg gingipain | 37 | AACGGAGGAATCTCGTTGGCCAACTATACGGGCCACGGTAGCGAAA | active site; DNA | | CAGCTTGGGGTACGTCTCACTTCGGCACCACTCATGTGAAGCAGCTT i ^sequence) | | ACCAACAGCAACCAG| R (Arg gingipain | 38 ] NGGISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQ| active site; amino acid |^sequence) || RA ^ 39 | NGGISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQEVEDDSPASYTY | Amino acid sequence | | TVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDVT | VEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGT ^AR ^ 58 | EVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYT i | Amino acid sequence | | AGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNP | NPNPNPNPGTNGGISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQ^AK ^ 59 | EVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYT | amino acid sequence | | AGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNP | NPNPNPNPGTNTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDK1006265400| KAK | 60 | NTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDKEVEDDSPASYTYT | Amino acid sequence | I VYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDVTV | EGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGTNTG | VSFANYTAHGSETAWADPLLTTSQLKALTNKDK| RAK | 61 | NGGISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQEVEDDSPASYTY | Amino acid sequence | | TVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDVT | VEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGTNT | GVSFANYTAHGSETAWADPLLTTSQLKALTNKDKj RAR | 62 | NGGISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQEVEDDSPASYTY | Amino acid sequence | | TVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDVT | VEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGTNG | GISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQj KAR | 63 NTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDKEVEDDSPASYTYT | (amino acid | | VYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDVTV ^sequence) | | EGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGTNGG | ISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQj RA ^ 64 ]AACGGAGGAATCTCGTTGGCCAACTATACGGGCCACGGTAGCGAAA | (DNA sequence) | I CAGCTTGGGGTACGTCTCACTTCGGCACCACTCATGTGAAGCAGCTT |ACCAACAGCAACCAGGAAGTCGAAGACGATTCACCTGCAAGCTATAC [ CTACACGGTGTATCGTGACGGCACGAAGATCAAGGAAGGTCTGACA | GCTACGACATTCGAAGAAGACGGTGTAGCTGCAGGCAATCATGAGT |ATTGCGTGGAAGTTAAGTACACAGCCGGCGTATCTCCGAAGGTATGT AAAGACGTTACGGTAGAAGGATCCAATGAATTTGCTCCTGTACAGAA| CCTGACCGGTAGTTCAGTAGGTCAGAAAGTAACGCTTAAGTGGGAT GCACCTAATGGTACCCCGAATCCGAATCCAAATCCGAATCCGAATCC| GGGAACA^AR | 65 | GAAGTCGAAGACGATTCACCTGCAAGCTATACCTACACGGTGTATCG | (DNA sequence) | | TGACGGCACGAAGATCAAGGAAGGTCTGACAGCTACGACATTCGAA GAAGACGGTGTAGCTGCAGGCAATCATGAGTATTGCGTGGAAGTTA|AGTACACAGCCGGCGTATCTCCGAAGGTATGTAAAGACGTTACGGTA GAAGGATCCAATGAATTTGCTCCTGTACAGAACCTGACCGGTAGTTC|AGTAGGTCAGAAAGTAACGCTTAAGTGGGATGCACCTAATGGTACCC | CGAATCCGAATCCAAATCCGAATCCGAATCCGGGAACAAACGGAGG | AATCTCGTTGGCCAACTATACGGGCCACGGTAGCGAAACAGCTTGG | GGTACGTCTCACTTCGGCACCACTCATGTGAAGCAGCTTACCAACAG CAACCAGj AK | 66 GAAGTCGAAGACGATTCACCTGCAAGCTATACCTACACGGTGTATCG | (DNA sequence) | | TGACGGCACGAAGATCAAGGAAGGTCTGACAGCTACGACATTCGAA | GAAGACGGTGTAGCTGCAGGCAATCATGAGTATTGCGTGGAAGTTA AGTACACAGCCGGCGTATCTCCGAAGGTATGTAAAGACGTTACGGTA| GAAGGATCCAATGAATTTGCTCCTGTACAGAACCTGACCGGTAGTTC AGTAGGTCAGAAAGTAACGCTTAAGTGGGATGCACCTAATGGTACCC| CGAATCCGAATCCAAATCCGAATCCGAATCCGGGAACAAATACCGGA1006265400| GTCAGCTTTGCAAACTATACAGCGCATGGATCTGAGACCGCATGGG | CTGATCCACTTCTGACTACTTCTCAACTGAAAGCACTCACTAATAAGG ACAAAj KAK | 67 AATACCGGAGTCAGCTTTGCAAACTATACAGCGCATGGATCTGAGAC | (DNA sequence) | | CGCATGGGCTGATCCACTTCTGACTACTTCTCAACTGAAAGCACTCA [ CTAATAAGGACAAAGAAGTCGAAGACGATTCACCTGCAAGCTATACC | TACACGGTGTATCGTGACGGCACGAAGATCAAGGAAGGTCTGACAG | CTACGACATTCGAAGAAGACGGTGTAGCTGCAGGCAATCATGAGTAT |TGCGTGGAAGTTAAGTACACAGCCGGCGTATCTCCGAAGGTATGTAA |AGACGTTACGGTAGAAGGATCCAATGAATTTGCTCCTGTACAGAACC |TGACCGGTAGTTCAGTAGGTCAGAAAGTAACGCTTAAGTGGGATGCA | CCTAATGGTACCCCGAATCCGAATCCAAATCCGAATCCGAATCCGGG |AACAAATACCGGAGTCAGCTTTGCAAACTATACAGCGCATGGATCTG I AGACCGCATGGGCTGATCCACTTCTGACTACTTCTCAACTGAAAGCA| CTCACTAATAAGGACAAA| RAK | 68 |AACGGAGGAATCTCGTTGGCCAACTATACGGGCCACGGTAGCGAAA | (DNA sequence) | | CAGCTTGGGGTACGTCTCACTTCGGCACCACTCATGTGAAGCAGCTT |ACCAACAGCAACCAGGAAGTCGAAGACGATTCACCTGCAAGCTATAC | CTACACGGTGTATCGTGACGGCACGAAGATCAAGGAAGGTCTGACA GCTACGACATTCGAAGAAGACGGTGTAGCTGCAGGCAATCATGAGT|ATTGCGTGGAAGTTAAGTACACAGCCGGCGTATCTCCGAAGGTATGT AAAGACGTTACGGTAGAAGGATCCAATGAATTTGCTCCTGTACAGAA| CCTGACCGGTAGTTCAGTAGGTCAGAAAGTAACGCTTAAGTGGGAT | GCACCTAATGGTACCCCGAATCCGAATCCAAATCCGAATCCGAATCC GGGAACAAATACCGGAGTCAGCTTTGCAAACTATACAGCGCATGGAT| CTGAGACCGCATGGGCTGATCCACTTCTGACTACTTCTCAACTGAAA GCACTCACTAATAAGGACAAAj RAR | 69 | AACGGAGGAATCTCGTTGGCCAACTATACGGGCCACGGTAGCGAAA | (DNA sequence) | | CAGCTTGGGGTACGTCTCACTTCGGCACCACTCATGTGAAGCAGCTT |ACCAACAGCAACCAGGAAGTCGAAGACGATTCACCTGCAAGCTATAC [ CTACACGGTGTATCGTGACGGCACGAAGATCAAGGAAGGTCTGACA | GCTACGACATTCGAAGAAGACGGTGTAGCTGCAGGCAATCATGAGT |ATTGCGTGGAAGTTAAGTACACAGCCGGCGTATCTCCGAAGGTATGT |AAAGACGTTACGGTAGAAGGATCCAATGAATTTGCTCCTGTACAGAA | CCTGACCGGTAGTTCAGTAGGTCAGAAAGTAACGCTTAAGTGGGAT GCACCTAATGGTACCCCGAATCCGAATCCAAATCCGAATCCGAATCC| GGGAACAAACGGAGGAATCTCGTTGGCCAACTATACGGGCCACGGT |AGCGAAACAGCTTGGGGTACGTCTCACTTCGGCACCACTCATGTGA I AGCAGCTTACCAACAGCAACCAGj KAR | 70 AATACCGGAGTCAGCTTTGCAAACTATACAGCGCATGGATCTGAGAC | (DNA sequence) | | CGCATGGGCTGATCCACTTCTGACTACTTCTCAACTGAAAGCACTCA [ CTAATAAGGACAAAGAAGTCGAAGACGATTCACCTGCAAGCTATACC | TACACGGTGTATCGTGACGGCACGAAGATCAAGGAAGGTCTGACAG | CTACGACATTCGAAGAAGACGGTGTAGCTGCAGGCAATCATGAGTAT1006265400| TGCGTGGAAGTTAAGTACACAGCCGGCGTATCTCCGAAGGTATGTAA i | AGACGTTACGGTAGAAGGATCCAATGAATTTGCTCCTGTACAGAACC | TGACCGGTAGTTCAGTAGGTCAGAAAGTAACGCTTAAGTGGGATGCA i | CCTAATGGTACCCCGAATCCGAATCCAAATCCGAATCCGAATCCGGG | AACAAACGGAGGAATCTCGTTGGCCAACTATACGGGCCACGGTAGC | GAAACAGCTTGGGGTACGTCTCACTTCGGCACCACTCATGTGAAGC i | AGCTTACCAACAGCAACCAGj KAR | 71 ] NTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDKMEVEDDSPASYT | (amino acid sequence | | YTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDV i | with Methionine at N | | TVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGTN i Herm of A domain) | | GGISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQh 1| A | 72 | EVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYT | AGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNP i ^sequence) | NPNPNPNPGT| A | 73 ] MEVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVK | (amino acid sequence | | YTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTP i | with N terminal Met) | | NPNPNPNPNPGT^A | 74 | GAAGTCGAAGACGATTCACCTGCAAGCTATACCTACACGGTGTATCG i | (DNA sequence) | | TGACGGCACGAAGATCAAGGAAGGTCTGACAGCTACGACATTCGAA | GAAGACGGTGTAGCTGCAGGCAATCATGAGTATTGCGTGGAAGTTA i | AGTACACAGCCGGCGTATCTCCGAAGGTATGTAAAGACGTTACGGTA | GAAGGATCCAATGAATTTGCTCCTGTACAGAACCTGACCGGTAGTTC i | AGTAGGTCAGAAAGTAACGCTTAAGTGGGATGCACCTAATGGTACCC i | CGAATCCGAATCCAAATCCGAATCCGAATCCGGGAACA| A | 75 | ATGGAAGTCGAAGACGATTCACCTGCAAGCTATACCTACACGGTGTA | (alternative DNA| TCGTGACGGCACGAAGATCAAGGAAGGTCTGACAGCTACGACATTC i | sequence with 5’ | | GAAGAAGACGGTGTAGCTGCAGGCAATCATGAGTATTGCGTGGAAG | codon encoding | | TTAAGTACACAGCCGGCGTATCTCCGAAGGTATGTAAAGACGTTACG i ^ methionine) | | GTAGAAGGATCCAATGAATTTGCTCCTGTACAGAACCTGACCGGTAG i | TTCAGTAGGTCAGAAAGTAACGCTTAAGTGGGATGCACCTAATGGTA | CCCCGAATCCGAATCCAAATCCGAATCCGAATCCGGGAACA j DUF2436 ] 76 | AEGSREVKRIGDGLFVTIEPANDVRANEAKWLAADNVWGDNTGYQFLL i | (alternative amino | | DADHNTFGSVIPATGPLFTGTASSNLYSANFEYLIPANADPWTTQNIIVT | acid sequence absent | | GQGEWIPGGVYDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGK i | C terminal | | KYTFTMRRAGMGDGTD^ methionine) |^AR | 79 ATGGAAGTCGAAGACGATTCACCTGCAAGCTATACCTACACGGTGTA | (DNA sequence; 5’ | |TCGTGACGGCACGAAGATCAAGGAAGGTCTGACAGCTACGACATTC | codon encoding | | GAAGAAGACGGTGTAGCTGCAGGCAATCATGAGTATTGCGTGGAAG ^ methionine] | [TTAAGTACACAGCCGGCGTATCTCCGAAGGTATGTAAAGACGTTACG | GTAGAAGGATCCAATGAATTTGCTCCTGTACAGAACCTGACCGGTAG | TTCAGTAGGTCAGAAAGTAACGCTTAAGTGGGATGCACCTAATGGTA I CCCCGAATCCGAATCCAAATCCGAATCCGAATCCGGGAACAAACGG|AGGAATCTCGTTGGCCAACTATACGGGCCACGGTAGCGAAACAGCT1006265400| TGGGGTACGTCTCACTTCGGCACCACTCATGTGAAGCAGCTTACCAA i | CAGCAACCAG§AK § 80 ] ATGGAAGTCGAAGACGATTCACCTGCAAGCTATACCTACACGGTGTA § | (DNA sequence; | § TCGTGACGGCACGAAGATCAAGGAAGGTCTGACAGCTACGACATTC i 15’ codon encoding | | GAAGAAGACGGTGTAGCTGCAGGCAATCATGAGTATTGCGTGGAAG i ^ methionine] | § TTAAGTACACAGCCGGCGTATCTCCGAAGGTATGTAAAGACGTTACG i | GTAGAAGGATCCAATGAATTTGCTCCTGTACAGAACCTGACCGGTAG i | TTCAGTAGGTCAGAAAGTAACGCTTAAGTGGGATGCACCTAATGGTA § § CCCCGAATCCGAATCCAAATCCGAATCCGAATCCGGGAACAAATACC i | GGAGTCAGCTTTGCAAACTATACAGCGCATGGATCTGAGACCGCAT i | GGGCTGATCCACTTCTGACTACTTCTCAACTGAAAGCACTCACTAAT § | AAGGACAAA§ RA ] 81 | NGGISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQMEVEDDSPASYT i | Amino acid sequence | | YTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDV § | (Met at N term of A) | § TVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGT i § AR § 82 § MEVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVK i | Amino acid sequence | | YTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTP i I NPNPNPNPNPGTNGGISLANYTGHGSETAWGTSHFGTTHVKQLTNSN| [N terminal |la I ^ methionine] || KA (amino acid | 83 § NTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDKMEVEDDSPASYT i | sequence; Met at N | | YTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDV i | term of A) | | TVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGT §§ AK ] 84 | MEVEDDSPASYTYTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVK i | (N terminal methionine) | | YTAGVSPKVCKDVTVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTP §§ NPNPNPNPNPGTNTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDK i § KAK | 85 § NTGVSFANYTAHGSETAWADPLLTTSQLKALTNKDKMEVEDDSPASYT i | Amino acid sequence | | YTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDV i | [methionine at | | TVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGTN § | between K and A] | | TGVSFANYTAHGSETAWADPLLTTSQLKALTNKDK§ RAK ] 86 | NGGISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQMEVEDDSPASYT i | Amino acid sequence | | YTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDV §§ TVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGTN i | [methionine at | | TGVSFANYTAHGSETAWADPLLTTSQLKALTNKDK| between R and A] |§ RAR | 87 ] NGGISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQMEVEDDSPASYT § | Amino acid sequence | | YTVYRDGTKIKEGLTATTFEEDGVAAGNHEYCVEVKYTAGVSPKVCKDV i | TVEGSNEFAPVQNLTGSSVGQKVTLKWDAPNGTPNPNPNPNPNPGTN § § GGISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQ§ RD § 108 § NGGISLANYTGHGSETAWGTSHFGTTHVKQLTNSNQAEGSREVKRIGD i | GLFVTIEPANDVRANEAKVVLAADNVWGDNTGYQFLLDADHNTFGSVIP i1006265400| ATGPLFTGTASSNLYSANFEYLIPANADPVVTTQNIIVTGQGEVVIPGGV I | YDYCITNPEPASGKMWIAGDGGNQPARYDDFTFEAGKKYTFTMRRAG i MGDGTDM

[0111] Table 2: exemplary RNA sequences of the invention^Descriptor ^SEQ ID | RNA Sequence[ | No J | ^KDcAK1n | 40 | AAUACCGGAGUCAGCUUUGCAAACUAUACAGCGCAUGGAUCUGAGACCGCAUG | | | GGCUGAUCCACUUCUGACUACUUCUCAACUGAAAGCACUCACUAAUAAGGACAA | | | | AUGGGGAGACAAUACGGGUUACCAGUUCUUGUUGGAUGCCGAUCACAAUACAU | | | | UCGGAAGUGUCAUUCCGGCAACCGGUCCUCUCUUUACCGGAACAGCUUCUUCC | | | | AAUCUUUACAGUGCGAACUUCGAGUAUUUGAUCCCGGCCAAUGCCGAUCCUGU | | | UGUUACUACACAGAAUAUUAUCGUUACAGGACAGGGUGAAGUUGUAAUCCCCG! | I | GUGGUGUUUACGACUAUUGCAUUACGAACCCGGAACCUGCAUCCGGAAAGAUG | | | UGGAUCGCAGGAGAUGGAGGCAACCAGCCUGCACGUUAUGACGAUUUCACAUU | | | | CGAAGCAGGCAAGAAGUACACCUUCACGAUGCGUCGCGCCGGAAUGGGAGAUG | | | | GAACUGAUAUGGAAGUCGAAGACGAUUCACCUGCAAGCUAUACCUACACGGUG | | | | UAUCGUGACGGCACGAAGAUCAAGGAAGGUCUGACAGCUACGACAUUCGAAGA | | ^AGACGGUGUAGCUGCAGGCAAUCAUGAGUAUUGCGUGGAAGUUAAGUACACAG | I | CCGGCGUAUCUCCGAAGGUAUGUAAAGACGUUACGGUAGAAGGAUCCAAUGAA I | | | UUUGCUCCUGUACAGAACCUGACCGGUAGUUCAGUAGGUCAGAAAGUAACGCU I | | | UAAGUGGGAUGCACCUAAUGGUACCCCGAAUCCGAAUCCAAAUCCGAAUCCGA | | | | AUCCGGGAACAACACUUUCCGAAUCAUUCGAAAAUGGUAUUCCGGCAUCUUGG | | | AAGACGAUCGAUGCAGACGGUGACGGGCAUGGCUGGAAACCUGGAAAUGCUCC | | | CGGAAUCGCUGGCUACAAUAGCAAUGGUUGUGUAUAUUCAGAGUCAUUCGGUC | | | UUGGUGGUAUAGGAGUUCUUACCCCUGACAACUAUCUGAUAACACCGGCAUUG | J | GAUUUGGGAUGGAAGGGAGG I ^KDAK | 41 | AAUACCGGAGUCAGCUUUGCAAACUAUACAGCGCAUGGAUCUGAGACCGCAUG | | | GGCUGAUCCACUUCUGACUACUUCUCAACUGAAAGCACUCACUAAUAAGGACAA | | | IAGCAGAAGGUUCCCGUGAAGUAAAACGGAUCGGAGACGGUCUUUUCGUUACGA | I I UCGAACCUGCAAACGAUGUACGUGCCAACGAAGCCAAGGUUGUGCUUGCGGCA | I | GACAACGUAUGGGGAGACAAUACGGGUUACCAGUUCUUGUUGGAUGCCGAUCA | | | bAAUACAUUCGGAAGUGUCAUUCCGGCAACCGGUCCUCUCUUUACCGGAACAGI | | | CUUCUUCCAAUCUUUACAGUGCGAACUUCGAGUAUUUGAUCCCGGCCAAUGCC | | | | GAUCCUGUUGUUACUACACAGAAUAUUAUCGUUACAGGACAGGGUGAAGUUGU | | | IAAUCCCCGGUGGUGUUUACGACUAUUGCAUUACGAACCCGGAACCUGCAUCCG | I I GAAAGAUGUGGAUCGCAGGAGAUGGAGGCAACCAGCCUGCACGUUAUGACGAU | | UCACAUUCGAAGCAGGCAAGAAGUACACCUUCACGAUGCGUCGCGCCGGAAU | | | GGGAGAUGGAACUGAUAUGGAAGUCGAAGACGAUUCACCUGCAAGCUAUACCU | I | IACACGGUGUAUCGUGACGGCACGAAGAUCAAGGAAGGUCUGACAGCUACGACA I | | | UUCGAAGAAGACGGUGUAGCUGCAGGCAAUCAUGAGUAUUGCGUGGAAGUUAA | | | GUACACAGCCGGCGUAUCUCCGAAGGUAUGUAAAGACGUUACGGUAGAAGGAU | | CAAUGAAUUUGCUCCUGUACAGAACCUGACCGGUAGUUCAGUAGGUCAGAAA | | UAACGCUUAAGUGGGAUGCACCUAAUGGUACCCCGAAUCCGAAUCCAAAUCC | | | GAAUCCGAAUCCGGGAACAAAUACCGGAGUCAGCUUUGCAAACUAUACAGCGC | | | IAUGGAUCUGAGACCGCAUGGGCUGAUCCACUUCUGACUACUUCUCAACUGAAA I j j I GCACUCACU AAU AAGGACAAA j ^KDAK-3S- | 42 | AAUACCGGAGUCAGCUUUGCAAACUAUACAGCGCAUGGAUCUGAGACCGCAUG |AVQP | I GGCUGAUCCACUUCUGACUACUUCUCAACUGAAAGCACUCACUAAUAAGGACAA | I GCAGAAGGUUCCCGUGAAGUAAAACGGAUCGGAGACGGUCUUUUCGUUACGA | | | UCGAACCUGCAAACGAUGUACGUGCCAACGAAGCCAAGGUUGUGCUUGCGGCA | | | | GACAACGUAUGGGGAGACAAUACGGGUUACCAGUUCUUGUUGGAUGCCGAUCA | | | | CAAUACAUUCGGAAGUGUCAUUCCGGCAACCGGUCCUCUCUUUACCGGAACAG | | | | CUUCUUCCAAUCUUUACAGUGCGAACUUCGAGUAUUUGAUCCCGGCCAAUGCC | | | GAUCCUGUUGUUACUACACAGAAUAUUAUCGUUACAGGACAGGGUGAAGUUGU | | bAUCCCCGGUGGUGUUUACGACUAUAGCAUUACGAACCCGGAACCUGCAUCCGI | | | GAAAGAUGUGGAUCGCAGGAGAUGGAGGCAACCAGCCUGCACGUUAUGACGAU | || | UUCACAUUCGAAGCAGGCAAGAAGUACACCUUCACGAUGCGUCGCGCCGGAAU |1006265400| | | GGGAGAUGGAACUGAUAUGGAAGUCGAAGACGAUUCACCUGCAAGCUAUACCU | | | | ACACGGUGUAUCGUGACGGCACGAAGAUCAAGGAAGGUCUGACAGCUACGACA | | | | UUCGAAGAAGACGGUGUAGCUGCAGGCAAUCAUGAGUAUAGCGUGGAAGUUAA | | | GUACACAGCCGGCGUAUCUCCGAAGGUAAGUAAAGACGUUACGGUAGAAGGAU | | | CCAAUGAAUUUGCUGCUGUACAGCCGCUGACCGGUAGUUCAGUAGGUCAGAAA | | | GUAACGCUUAAGUGGGAUGCACCUAAUGGUACCCCGAAUCCGAAUCCAAAUCC | | | | GAAUCCGAAUCCGGGAACAAAUACCGGAGUCAGCUUUGCAAACUAUACAGCGC | | | | AUGGAUCUGAGACCGCAUGGGCUGAUCCACUUCUGACUACUUCUCAACUGAAA | j GCACUCACUAAUAAGGACAAA |K | 43 IAAUACCGGAGUCAGCUUUGCAAACUAUACAGCGCAUGGAUCUGAGACCGCAUG | I | GGCUGAUCCACUUCUGACUACUUCUCAACUGAAAGCACUCACUAAUAAGGACAA | | _ |A _|KD | 44 | AAUACCGGAGUCAGCUUUGCAAACUAUACAGCGCAUGGAUCUGAGACCGCAUG | | | GGCUGAUCCACUUCUGACUACUUCUCAACUGAAAGCACUCACUAAUAAGGACAA | | | AGCAGAAGGUUCCCGUGAAGUAAAACGGAUCGGAGACGGUCUUUUCGUUACGA | | | | UCGAACCUGCAAACGAUGUACGUGCCAACGAAGCCAAGGUUGUGCUUGCGGCA | | | GACAACGUAUGGGGAGACAAUACGGGUUACCAGUUCUUGUUGGAUGCCGAUCA | | | CAAUACAUUCGGAAGUGUCAUUCCGGCAACCGGUCCUCUCUUUACCGGAACAG | | | CUUCUUCCAAUCUUUACAGUGCGAACUUCGAGUAUUUGAUCCCGGCCAAUGCC | | | | GAUCCUGUUGUUACUACACAGAAUAUUAUCGUUACAGGACAGGGUGAAGUUGU | | | AAUCCCCGGUGGUGUUUACGACUAUUGCAUUACGAACCCGGAACCUGCAUCCG | | | | GAAAGAUGUGGAUCGCAGGAGAUGGAGGCAACCAGCCUGCACGUUAUGACGAU | | | UUCACAUUCGAAGCAGGCAAGAAGUACACCUUCACGAUGCGUCGCGCCGGAAU | | GGGAGAUGGAACUGAUAUG | |KDA | 45 | AAUACCGGAGUCAGCUUUGCAAACUAUACAGCGCAUGGAUCUGAGACCGCAUG | | | GGCUGAUCCACUUCUGACUACUUCUCAACUGAAAGCACUCACUAAUAAGGACAA | | | AGCAGAAGGUUCCCGUGAAGUAAAACGGAUCGGAGACGGUCUUUUCGUUACGA | | | | UCGAACCUGCAAACGAUGUACGUGCCAACGAAGCCAAGGUUGUGCUUGCGGCA | | | | GACAACGUAUGGGGAGACAAUACGGGUUACCAGUUCUUGUUGGAUGCCGAUCA | | | CAAUACAUUCGGAAGUGUCAUUCCGGCAACCGGUCCUCUCUUUACCGGAACAG | | | CUUCUUCCAAUCUUUACAGUGCGAACUUCGAGUAUUUGAUCCCGGCCAAUGCC | | | | GAUCCUGUUGUUACUACACAGAAUAUUAUCGUUACAGGACAGGGUGAAGUUGU | | | AAUCCCCGGUGGUGUUUACGACUAUUGCAUUACGAACCCGGAACCUGCAUCCG | | | | GAAAGAUGUGGAUCGCAGGAGAUGGAGGCAACCAGCCUGCACGUUAUGACGAU | | | UUCACAUUCGAAGCAGGCAAGAAGUACACCUUCACGAUGCGUCGCGCCGGAAU | | | GGGAGAUGGAACUGAUAUGGAAGUCGAAGACGAUUCACCUGCAAGCUAUACCU | | | ACACGGUGUAUCGUGACGGCACGAAGAUCAAGGAAGGUCUGACAGCUACGACA | | | | UUCGAAGAAGACGGUGUAGCUGCAGGCAAUCAUGAGUAUUGCGUGGAAGUUAA | | | | GUACACAGCCGGCGUAUCUCCGAAGGUAUGUAAAGACGUUACGGUAGAAGGAU | | | | CCAAUGAAUUUGCUCCUGUACAGAACCUGACCGGUAGUUCAGUAGGUCAGAAA | | | GUAACGCUUAAGUGGGAUGCACCUAAUGGUACCCCGAAUCCGAAUCCAAAUCC | | GAAUCCGAAUCCGGGAACA j IDA 146 I GCAGAAGGUUCCCGUGAAGUAAAACGGAUCGGAGACGGUCUUUUCGUUACGAU | | | CGAACCUGCAAACGAUGUACGUGCCAACGAAGCCAAGGUUGUGCUUGCGGCAG | | | ACAACGUAUGGGGAGACAAUACGGGUUACCAGUUCUUGUUGGAUGCCGAUCAC | | | | AAUACAUUCGGAAGUGUCAUUCCGGCAACCGGUCCUCUCUUUACCGGAACAGC | | | UUCUUCCAAUCUUUACAGUGCGAACUUCGAGUAUUUGAUCCCGGCCAAUGCCG | | | AUCCUGUUGUUACUACACAGAAUAUUAUCGUUACAGGACAGGGUGAAGUUGUA | | AUCCCCGGUGGUGUUUACGACUAUUGCAUUACGAACCCGGAACCUGCAUCCGG | | | |AAAGAUGUGGAUCGCAGGAGAUGGAGGCAACCAGCCUGCACGUUAUGACGAUU I I I I UCACAUUCGAAGCAGGCAAGAAGUACACCUUCACGAUGCGUCGCGCCGGAAUG | | | | GGAGAUGGAACUGAUAUGGAAGUCGAAGACGAUUCACCUGCAAGCUAUACCUA | | | CACGGUGUAUCGUGACGGCACGAAGAUCAAGGAAGGUCUGACAGCUACGACAU | | | UCGAAGAAGACGGUGUAGCUGCAGGCAAUCAUGAGUAUUGCGUGGAAGUUAAG | | | UACACAGCCGGCGUAUCUCCGAAGGUAUGUAAAGACGUUACGGUAGAAGGAUC | | | | CAAUGAAUUUGCUCCUGUACAGAACCUGACCGGUAGUUCAGUAGGUCAGAAAG | | | | UAACGCUUAAGUGGGAUGCACCUAAUGGUACCCCGAAUCCGAAUCCAAAUCCG | j AAUCCGA^ | |KDAΔABM3 | 47 IAAUACCGGAGUCAGCUUUGCAAACUAUACAGCGCAUGGAUCUGAGACCGCAUG | I I GGCUGAUCCACUUCUGACUACUUCUCAACUGAAAGCACUCACUAAUAAGGACAA | I AGCAGAAGGUUCCCGUGAAGUAAAACGGAUCGGAGACGGUCUUUUCGUUACGA | | | | UCGAACCUGCAAACGAUGUACGUGCCAACGAAGCCAAGGUUGUGCUUGCGGCA | || I GACAACGUAUGGGGAGACAAUACGGGUUACCAGUUCUUGUUGGAUGCCGAUCA |1006265400| | | CAAUACAUUCGGAAGUGUCAUUCCGGCAACCGGUCCUCUCUUUACCGGAACAG | | | | CUUCUUCCAAUCUUUACAGUGCGAACUUCGAGUAUUUGAUCCCGGCCAAUGCC | | | | GAUCCUGUUGUUACUACACAGAAUAUUAUCGUUACAGGACAGGGUGAAGUUGU | | AAUCCCCGGUGGUGUUUACGACUAUUGCAUUACGAACCCGGAACCUGCAUCCG | | | GAAAGAUGUGGAUCGCAGGAGAUGGAGGCAACCAGCCUGCACGUUAUGACGAU | | | UUCACAUUCGAAGCAGGCAAGAAGUACACCUUCACGAUGCGUCGCGCCGGAAU | | | | GGGAGAUGGAACUGAUAUGGAAGUCGAAGACGAUUCACCUGCAAGCUAUACCU | | | | ACACGGUGUAUCGUGACGGCACGAAGAUCAAGGAAGGUCUGACAGCUACGACA | | | | UUCGAAGAAGACGGUGUAGCUGCAGGCAAUCAUGAGUAUUGCGUGGAAGUUAA | | | GUACACAGCCGGCGUAUCUCCGAAGGUAUGUAAAGACGUUACGGUAGAAGGAU | I | CCAAUGAAUUUGCUCCUGUACAGAACCUGACCGGUAGUUCAGUAGGUCAGAAA | | UAACGCUUAAGUGGGAUGCACCUAAUGGUACC §KA | 48 | AAUACCGGAGUCAGCUUUGCAAACUAUACAGCGCAUGGAUCUGAGACCGCAUG § § | GGCUGAUCCACUUCUGACUACUUCUCAACUGAAAGCACUCACUAAUAAGGACAA § | | AGAAGUCGAAGACGAUUCACCUGCAAGCUAUACCUACACGGUGUAUCGUGACG § J | GCACGAAGAUCAAGGAAGGUCUGACAGCUACGACAUUCGAAGAAGACGGUGUA § § | GCUGCAGGCAAUCAUGAGUAUUGCGUGGAAGUUAAGUACACAGCCGGCGUAUC J | | | UCCGAAGGUAUGUAAAGACGUUACGGUAGAAGGAUCCAAUGAAUUUGCUCCUG § § | UACAGAACCUGACCGGUAGUUCAGUAGGUCAGAAAGUAACGCUUAAGUGGGAU § | GCACCUAAUGGUACCCCGAAUCCGAAUCCAAAUCCGAAUCCGAAUCCGGGAAC § I I |Al | RDA 149 | AACGGAGGAAUCUCGUUGGCCAACUAUACGGGCCACGGUAGCGAAACAGCUUG] I | | GGGUACGUCUCACUUCGGCACCACUCAUGUGAAGCAGCUUACCAACAGCAACC | | | | AGGCAGAAGGUUCCCGUGAAGUAAAACGGAUCGGAGACGGUCUUUUCGUUACG | | AUCGAACCUGCAAACGAUGUACGUGCCAACGAAGCCAAGGUUGUGCUUGCGGC | | AGACAACGUAUGGGGAGACAAUACGGGUUACCAGUUCUUGUUGGAUGCCGAUC | | | | ACAAUACAUUCGGAAGUGUCAUUCCGGCAACCGGUCCUCUCUUUACCGGAACA | | | | GCUUCUUCCAAUCUUUACAGUGCGAACUUCGAGUAUUUGAUCCCGGCCAAUGC | | | | CGAUCCUGUUGUUACUACACAGAAUAUUAUCGUUACAGGACAGGGUGAAGUUG | | | UAAUCCCCGGUGGUGUUUACGACUAUUGCAUUACGAACCCGGAACCUGCAUCC | | | GGAAAGAUGUGGAUCGCAGGAGAUGGAGGCAACCAGCCUGCACGUUAUGACGA | | | UUUCACAUUCGAAGCAGGCAAGAAGUACACCUUCACGAUGCGUCGCGCCGGAA | | | | UGGGAGAUGGAACUGAUAUGGAAGUCGAAGACGAUUCACCUGCAAGCUAUACC | | | | UACACGGUGUAUCGUGACGGCACGAAGAUCAAGGAAGGUCUGACAGCUACGAC | | | AUUCGAAGAAGACGGUGUAGCUGCAGGCAAUCAUGAGUAUUGCGUGGAAGUUA | | | AGUACACAGCCGGCGUAUCUCCGAAGGUAUGUAAAGACGUUACGGUAGAAGGA | I | UCCAAUGAAUUUGCUCCUGUACAGAACCUGACCGGUAGUUCAGUAGGUCAGAA | | | AGUAACGCUUAAGUGGGAUGCACCUAAUGGUACCCCGAAUCCGAAUCCAAAUC | J | CGAAUCCGAAUCCGGGAACA| R | 50 | AACGGAGGAAUCUCGUUGGCCAACUAUACGGGCCACGGUAGCGAAACAGCUUG | | | | GGGUACGUCUCACUUCGGCACCACUCAUGUGAAGCAGCUUACCAACAGCAACC [ [ | AG j |DUF2436 ]51 ] GCAGAAGGUUCCCGUGAAGUAAAACGGAUCGGAGACGGUCUUUUCGUUACGAU | | | CGAACCUGCAAACGAUGUACGUGCCAACGAAGCCAAGGUUGUGCUUGCGGCAG | | | ^ACAACGUAUGGGGAGACAAUACGGGUUACCAGUUCUUGUUGGAUGCCGAUCAC | I | IAAUACAUUCGGAAGUGUCAUUCCGGCAACCGGUCCUCUCUUUACCGGAACAGC I | | | UUCUUCCAAUCUUUACAGUGCGAACUUCGAGUAUUUGAUCCCGGCCAAUGCCG | | | AUCCUGUUGUUACUACACAGAAUAUUAUCGUUACAGGACAGGGUGAAGUUGUA | I ^AUCCCCGGUGGUGUUUACGACUAUUGCAUUACGAACCCGGAACCUGCAUCCGG | | ^AAAGAUGUGGAUCGCAGGAGAUGGAGGCAACCAGCCUGCACGUUAUGACGAUU | | | | UCACAUUCGAAGCAGGCAAGAAGUACACCUUCACGAUGCGUCGCGCCGGAAUG | J | GGAGAUGGAACUGAUAUG|ABM1 | 52 ] UCCAAUGAAUUUGCUCCUGUACAGAACCUGACCGGUAGUUCAGUAGGUCAGAA | | | | AGUAACGCUUAAGUGGGAUGCACCUAAUGGUACC1006265400IABM2 I 53 | GCAAGCUAUACCUACACGGUGUAUCGUGACGGCACGAAGAUCAAGGAAGGUCU | I I | GACAGCUACGACAUUCGAAGAAGACGGUGUAGCUGCAGGCAAUCAUGAGUAUU | I I | GCGUGGAAGUUAAGUACACAGCCGGCGUAUCUCCGAAGGUAUGUAAAGACGUU | | | | ACGGUAGAAGGA^ABM3 | 54 | CCGAAUCCGAAUCCAAAUCCGAAUCCGAAUCCGGGAACA § | ABM 2+1 | 55 ] GCAAGCUAUACCUACACGGUGUAUCGUGACGGCACGAAGAUCAAGGAAGGUCU | | | GACAGCUACGACAUUCGAAGAAGACGGUGUAGCUGCAGGCAAUCAUGAGUAUU | | | | GCGUGGAAGUUAAGUACACAGCCGGCGUAUCUCCGAAGGUAUGUAAAGACGUU § | | | ACGGUAGAAGGAUCCAAUGAAUUUGCUCCUGUACAGAACCUGACCGGUAGUUC | | | | AGUAGGUCAGAAAGUAACGCUUAAGUGGGAUGCACCUAAUGGUACC| ABM 2+1+3 | 56 | GCAAGCUAUACCUACACGGUGUAUCGUGACGGCACGAAGAUCAAGGAAGGUCU | I I | GACAGCUACGACAUUCGAAGAAGACGGUGUAGCUGCAGGCAAUCAUGAGUAUU | I I | GCGUGGAAGUUAAGUACACAGCCGGCGUAUCUCCGAAGGUAUGUAAAGACGUU | | I | ACGGUAGAAGGAUCCAAUGAAUUUGCUCCUGUACAGAACCUGACCGGUAGUUC | I I IAGUAGGUCAGAAAGUAACGCUUAAGUGGGAUGCACCUAAUGGUACCCCGAAUC ICGAAUCCAAAUCCGAAUCCGAAUCCGGGAACA j §RA | 57 IAACGGAGGAAUCUCGUUGGCCAACUAUACGGGCCACGGUAGCGAAACAGCUUG I | | | GGGUACGUCUCACUUCGGCACCACUCAUGUGAAGCAGCUUACCAACAGCAACC | | | | AGGAAGUCGAAGACGAUUCACCUGCAAGCUAUACCUACACGGUGUAUCGUGAC | | | | GGCACGAAGAUCAAGGAAGGUCUGACAGCUACGACAUUCGAAGAAGACGGUGU | | | AGCUGCAGGCAAUCAUGAGUAUUGCGUGGAAGUUAAGUACACAGCCGGCGUAU | | | | CUCCGAAGGUAUGUAAAGACGUUACGGUAGAAGGAUCCAAUGAAUUUGCUCCU | I I | GUACAGAACCUGACCGGUAGUUCAGUAGGUCAGAAAGUAACGCUUAAGUGGGA | I I | UGCACCUAAUGGUACCCCGAAUCCGAAUCCAAAUCCGAAUCCGAAUCCGGGAA | J j CA| A | 77 | GAAGUCGAAGACGAUUCACCUGCAAGCUAUACCUACACGGUGUAUCGUGACGG | | | | CACGAAGAUCAAGGAAGGUCUGACAGCUACGACAUUCGAAGAAGACGGUGUAG | | | | CUGCAGGCAAUCAUGAGUAUUGCGUGGAAGUUAAGUACACAGCCGGCGUAUCU | | | | CCGAAGGUAUGUAAAGACGUUACGGUAGAAGGAUCCAAUGAAUUUGCUCCUGU | | | | ACAGAACCUGACCGGUAGUUCAGUAGGUCAGAAAGUAACGCUUAAGUGGGAUG | | | | CACCUAAUGGUACCCCGAAUCCGAAUCCAAAUCCGAAUCCGAAUCCGGGAACA | A (with 5’ AUG ^ 78 |[AUG]GAAGUCGAAGACGAUUCACCUGCAAGCUAUACCUACACGGUGUAUCGUG | codon) I IACGGCACGAAGAUCAAGGAAGGUCUGACAGCUACGACAUUCGAAGAAGACGGU | | | | GUAGCUGCAGGCAAUCAUGAGUAUUGCGUGGAAGUUAAGUACACAGCCGGCGU | | | | AUCUCCGAAGGUAUGUAAAGACGUUACGGUAGAAGGAUCCAAUGAAUUUGCUC | | | | CUGUACAGAACCUGACCGGUAGUUCAGUAGGUCAGAAAGUAACGCUUAAGUGG I | | | GAUGCACCUAAUGGUACCCCGAAUCCGAAUCCAAAUCCGAAUCCGAAUCCGGG | | | ^AACA |Detailed description of the embodiments

[0112] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0113] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and1006265400equivalents, which may be included within the scope of the present invention as defined by the claims.

[0114] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0115] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0116] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0117] In work leading to the present invention, the inventors investigated various chimeric or fusion proteins for use in inducing immune responses to P. gingivalis and methods for large-scale production of such chimeras for use as vaccine candidates.

[0118] One such candidate fusion protein (termed KDcAK1n as further described herein), was found to elicit a robust immune response to P. gingivalis, but suffered from large-scale manufacturing and production issues.

[0119] The inventors initially considered whether the manufacturing issues for this protein could be overcome when the protein was provided to subjects when encoded by an RNA vaccine. Unexpectedly, the inventors found that an RNA vaccine encoding KDcAK1n did not express well and therefore KDcAK1n was not a preferred candidate for use in an RNA vaccine.

[0120] Surprisingly, the inventors found that robust immune responses were obtained when providing an RNA encoding alternative chimeric proteins containing various domains derived from P. gingivalis Arg- or Lys-gingipain proteins.

[0121] The inventors therefore designed new RNA vaccines encoding protein antigens, or chimeric or fusion proteins comprising protein antigens, for use in inducing an immune response to P. gingivalis, and methods and uses comprising the same. Having1006265400determined that the RNA vaccines were useful for inducing an immune response to P. gingivalis, the inventors then sought to determine whether the RNA vaccines could be useful for treating, preventing or reducing the progression of P. g / ng / vaf / s-induced or associated neuropathologies (such as formation of amyloid plaque and / or phosphorylation of tau proteins in the brain, which are known to be associated with the onset of neurodegenerative diseases including dementias, Alzheimer’s disease and related pathologies).

[0122] There are extensive preclinical, epidemiological, clinical, and other data to support the oral pathogen P. gingivalis as playing a role in Alzheimer’s disease (AD) progression in a susceptible subpopulation of AD sufferers with active periodontal disease. The presence of a variety of P. gingivalis biomolecules including nucleic acids, lipopolysaccharide (LPS) and the surface proteases (gingipains), has been determined in multiple regions of human AD brains, including the hippocampus, cortical grey matter, the basal forebrain, and hypothalamic regions. These biomolecules have been colocalised with neurons, tau tangles and intracellular Ap pathology in the brain of AD patients, and cerebral loads of gingipains significantly correlated with AD diagnosis, tauopathy and ubiquitin pathology. Higher serum anti-P. gingivalis immunoglobulin G (IgG) titres have been reported as a risk factor for AD development, increased AD incidence and impaired delayed memory and calculation. A recent study of 20 AD patients and 20 patients with dementia of other forms detected elevated levels of anti-P. gingivalis IgG antibodies in cerebrospinal fluid for both groups.Gingipains

[0123] The pathogenicity of P. gingivalis is attributed to a number of surface-associated virulence factors that include cysteine proteinases (gingipains), fimbriae, haem-binding proteins, and outer membrane transport proteins amongst others. In particular, the extracellular Arg- and Lys-specific proteinases ‘gingipains’ (RgpA / B and Kgp) of P. gingivalis have been implicated as major virulence factors that are critical for colonisation, penetration into host tissue, dysregulation of the immune response, dysbiosis and disease.

[0124] The gingipains, in particular the Lys-specific proteinase Kgp are essential for P. gingivalis to induce alveolar bone resorption in the mouse periodontitis model. The gingipains have also been found in gingival tissue at sites of severe periodontitis at high1006265400concentrations proximal to the subgingival plaque and at lower concentrations at distal sites deeper into the gingival tissue. Lys-specific and Arg-specific proteinases have been shown to degrade a variety of host proteins in vitro, e.g., fibrinogen, fibronectin, and laminin. Plasma host defence and regulatory proteinase inhibitors α-trypsin, α2-macroglobulin, anti-chymotrypsin, antithrombin III and antiplasmin are also degraded by Lys- and Arg- proteinases from P. gingivalis. This has led to the development of a cogent mechanism to explain the keystone role played by P. gingivalis in the development of chronic periodontitis.

[0125] The RgpA, RgpB and Kgp genes all encode an N-terminal signal peptide of ~22 amino acids in length, an unusually long propeptide of ~200 amino acids in length, and a catalytic domain of ~480 amino acids. C-terminal to the catalytic domain is a large hemagglutinin-adhesin (HA) domain which is comprised of adhesin binding domains (ABMs, of which 5 distinct sequences have been described), a “Domain of Unknown Function” (termed DUF2436 which is defined as conserved Pfam Domain of Unknown Function; IPR018832) and C-terminal adhesin domains or cleaved adhesin domains (or CADs). The particular arrangement of the ABMs, DUF and CADs varies between naturally occurring Kgp and RgpA / B.

[0126] The architecture of the domains in the Kgp polyprotein is illustrated in Figure 1. For example, Kgp comprises (N terminus to C terminus): a catalytic domain, a first ABM (ABM1), DUF2436, a domain comprising ABM2, ABM1, ABM3, two CAD domains (termed K1 and K2), a further domain comprising ABM2 and ABM 1, a further CAD domain (termed K3), ABM2, and a C-terminal domain.

[0127] As used herein, reference to ABMs 1, 2 and 3 will be understood to generally refer to the ABMs found in the order ABM2, ABM1 and ABM3 in the sequence immediately C terminal to DUF2436 of Kgp, as depicted in Figure 1.

[0128] The catalytic domains of RgpB and RgpA share a high-degree of sequence homology. However, RgpB lacks the HA domains and is located in a monomeric form on the outer membrane. Some of the HA domains have been alternatively described as C-terminal adhesin domains or cleaved adhesin domains (CADs) and some are DUF (“Domain of Unknown Function”) 2436 domains (conserved Pfam Domain of Unknown Function; IPR018832).1006265400

[0129] The RgpA and Kgp precursor proteins are cleaved into multiple domains that remain non-covalently associated forming large outer membrane protein complexes. In vivo, Arg- and Lys-specific proteinases are therefore found in a cell-associated complex of non-covalently associated proteinases and adhesins. One such complex has been designated the RgpA-Kgp proteinase-adhesin complex (previously referred to as the PrtR-PrtK proteinase-adhesin complex). The complex is composed of a 45kDa Argspecific calcium-stabilised cysteine proteinase and seven sequence-related adhesin domains,

[0130] As used herein a Lys-gingipain catalytic domain may also be referred to as a KAS domain or PAS domain. As used herein an Arg-gingipain catalytic domain may also be referred to as a RAS domain or PAS domain. Typically, the catalytic domain of the Lys-gingipain or Arg-gingipains is located in the N-terminal ~480 amino acid region of the protein. The active site within the catalytic domain is typically located at amino acid residues 426-446 (for RgpA) and 432-453 (for Kgp).

[0131] As used herein an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis will be understood to typically refer to the region of an Arg- or Lys-gingipain that is C-terminal to the catalytic or active site domain. The adhesin domain (also referred to as the HA domain) typically comprise a Domain of Unknown Function (DUF) domain (especially DUF 2436 conserved Pfam Domain of Unknown Function; IPR018832) and several adhesin binding motifs (ABM) domains and a cleaved adhesin domain (CAD).Treatment of P. gingivalis induced or associated neuropathologies

[0132] The present invention provides methods and compositions for treating or preventing, or delaying the onset, of neuropathologies associated with or caused by or induced by P. gingivalis infection.

[0133] Further the invention provides methods for reducing the level of P. gingivalis gingipain (such as RgpA and / or KgpA) in neuronal tissue (eg brain tissue) of a subject.

[0134] The invention also relates to methods for reducing neuroinflammation in a subject, wherein the neuroinflammation may be caused or associated with P. gingivalis infection.1006265400

[0135] The P. g / ng / vaf / s-induced or associated neuropathology may comprise cognitive decline, a neurocognitive disorder, or a pathology resulting in physical or chemical changes to neuronal tissue; or is a neurodegenerative disorder as further herein defined.

[0136] In any embodiment, the chemical change may comprise inflammation in neuronal tissue.

[0137] In any aspect, the neurodegenerative condition may be characterised by the presence of abnormal protein deposits in the brain, including amyloidopathies, synucleinopathies or tauopathies. The neurodegenerative condition or disorder may include Alzheimer’s disease (AD), Lewy-bodies disease (Dementia with Lewy bodies (DLB)), Huntington's disease, Creutzfeldt-Jakob disease (CJD), Gaucher Disease Type 3, or Parkinson's disease. The neurodegenerative disease may be vascular dementia, frontotemporal dementia or other form of dementia not typically associated with deposition of abnormal protein deposits.

[0138] As used herein, cognitive disorder, or neurocognitive disorder (NCD) refers to a mental health and / or neurological disorder that primarily affects cognitive abilities including learning, memory, perception, and / or problem solving. Neurocognitive disorders include delirium and mild and major neurocognitive disorder (previously known as dementia). They are defined by deficits in cognitive ability that are acquired (as opposed to developmental), typically represent decline, and may have an underlying brain pathology. The DSM-5 defines six key domains of cognitive function: executive function, learning and memory, perceptual-motor function, language, complex attention, and social cognition.

[0139] Neurocognitive disorders are diagnosed as mild and major based on the severity of their symptoms. The skilled person will be familiar with standard approaches for identifying subjects at risk of dementia, or related neurocognitive disorder, or diagnosing a subject with a neurodegenerative disease, including any of those described herein.

[0140] Similarly, the skilled person will be familiar with standard methods for identifying whether a patient has an intellectual disability, e.g., associated with a neurocognitive1006265400disorder as described herein. Such methods may include various behavioural and cognitive tests.

[0141] In the context of the present invention, a further test for determination of likelihood or risk of P. gingivalis induced or associated neuropathology, may be comprised of diagnosis of P. gingivalis infection (or periodontal disease indicative of P. gingivalis infection). Diagnosis of periodontal disease is well known in the art. Diagnosis of P. gingivalis infection may be accomplished by testing for the presence of orally detectable levels of P. gingivalis in the saliva of the subject. Such tests are well known to the skilled person and may include various methods using the polymerase chain reaction (PCR), such as qPCR, RT-PCR and ddPCR to detect P. gingivalis DNA and / or RNA in the saliva, tongue surface, tooth pockets (including subgingival pockets) or elsewhere in the mouth. Other tests may include lateral flow and antigen tests utilising antibodies, such as described by O’Brien-Simpson et al, 2017, incorporated herein by reference. Examples of PCR / qPCR based methods are described in WO 2020 / 069397, also incorporated herein by reference. In still a further example of a salivary test that is known, the BANA Test, is commercially available for dental applications to test for proteases from P. gingivalis and other oral bacteria. The BANA test is a small plastic card to which is attached two separate reagent matrices, seen as strips on the card. The lower white reagent matrix is impregnated with N-benzoyl-DL-arginine-B-naphthylamide (BANA). Subgingival plaque samples are applied to the lower matrix, and then distilled water is applied to the upper matrix. Then the lower matrix is folded back to make contact with the upper matrix. The upper buffer reagent matrix contains a chromogenic diazo reagent which reacts with one of the hydrolytic products of the enzyme reaction forming a blue color. The reaction occurs when the plastic strip is inserted into an incubator set at 35 degrees C for 5 minutes. The BANA substrate detects at least three different oral bacteria however and is not specific to P. gingivalis. The BANA test could be used to identify people at risk for P. g / ng / vaf / s-induced or associated neuropathology or eligible for treatment according to the present invention. Alternatively, the BANA substrate can be substituted in similar formats or in a liquid assay with an RgpA, RgpB and / or Kgp specific substrate. Reagents that bind to active gingipains are known in the art, including in WO 2017 / 083433, by way of non-limiting example.1006265400

[0142] In further embodiments, the determination of a likelihood of a P. gingivalis-induced or associated neurodegenerative or neurocognitive condition may include a prior history of P. gingivalis infection or periodontal disease.

[0143] The National Institute of Neurological and Communicative 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 Alzheimer's Criteria for diagnosis in 1984, extensively updated in 2007.

[0144] The present invention also finds utility in improving cognitive function, or delaying a decline in cognitive function in subjects having, or suspected of having dementia with Lewy bodies. Dementia with Lewy bodies (DLB) is a dementia that has the primary symptoms of visual hallucinations and " Parkinsonism". Parkinsonism is the symptoms of Parkinson's disease, which includes tremor, rigid muscles, and a face without emotion. The visual hallucinations in DLB are generally very vivid hallucinations of people or animals and they often occur when someone is about to fall asleep or just waking up. Other prominent symptoms include problems with attention, organization, problem solving and planning (executive function), and difficulty with visual-spatial function.

[0145] Although imaging studies do not necessarily make the diagnosis of DLB, some signs are particularly common. A person with DLB often shows occipital hypoperfusion on SPECT scan or occipital hypometabolism on a PET scan. Generally, a diagnosis of DLB is straightforward and unless it is complicated, a brain scan is not always necessary.

[0146] In preferred embodiments of the invention, the impairment of cognitive function is caused by, or attributed to, Alzheimer's disease. In another embodiment, the impairment of cognitive function is caused by, or attributed to, mild cognitive impairment (MCI). In a further embodiment, the impairment of cognitive function is caused by, or attributed to, dementia.

[0147] As used herein, the terms “treatment of conditions associated with decline or loss of cognitive ability” or “to counteract cognitive decline” or “treatment of a cognitive disorder” or “delaying the progression of a cognitive disorder” or “improving the1006265400cognitive function” or “counteracting the decline of the cognitive function” used throughout this specification shall mean promoting cognitive function (affecting impaired cognitive function in the subject so that it more closely resembles the function of an aged-matched normal, unimpaired subject, including affecting states in which cognitive function is reduced compared to a normal subject) and preserving cognitive function (affecting normal or impaired cognitive function such that it does not decline or does not fall below that observed in the subject upon first presentation or diagnosis, e.g. to the extent of expected decline in the absence of treatment).

[0148] As used herein, the terms "treatment" or "treating" of a subject includes the application or administration of a composition of the invention to a subject (or application or administration of a compound of the invention to a cell or tissue from a subject) with the purpose of delaying, slowing, stabilizing, curing, regressing, healing, alleviating, relieving, altering, remedying, less worsening, ameliorating, improving, or affecting the disease or condition, the symptom of the disease or condition, or the risk of (or susceptibility to) the disease or condition. The term "treating" refers to any indication of success in the treatment or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement; remission; lessening of the rate of worsening; lessening severity of the disease; stabilization, diminishing of symptoms or making the injury, pathology or condition more tolerable to the subject; slowing in the rate of degeneration or decline; making the final point of degeneration less debilitating; or improving a subject's physical or mental well-being.

[0149] As used herein, "preventing" or "prevention" is intended to refer to at least the reduction of likelihood of the risk of (or susceptibility to) acquiring a disease or disorder (i.e., causing at least one of the clinical symptoms of the disease not to develop in a subject that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease). Biological and physiological parameters for identifying such subjects are provided herein and are also well known by physicians. As such, it will be appreciated that the methods of the invention include methods for preventing development of any of the conditions (eg cognitive impairment) or pathologies described herein.

[0150] The methods of the present invention extend equally to both human and / or veterinary medicine. As used herein, the term “subject” shall be taken to mean any animal including humans, for example a mammal. Exemplary subjects include but are not limited1006265400to humans and non-human primates. For example, the subject may be a human. In further examples, the subject may be a veterinary subject, such as a companion animal (cat, dog, guinea pig, and the like).

[0151] As used herein, the terms “subject”, “individual” and “patient” may be used interchangeably. The vaccine compositions for the use of the invention can be administered to subjects felt to be in greatest need thereof.

[0152] In any embodiment, the methods of the invention may comprise administering an RNA polynucleotide or composition as described herein to a subject who is considered to be at risk of or who is considered to have an aging-related neuronal condition, such as dementia, or a neurodegenerative condition (such as Alzheimer’s disease, or any other neurodegenerative disease as disclosed herein). In such embodiments, the methods may be useful for preventing the further progression of the neuronal condition or neurodegenerative condition (such as by reducing or preventing the accumulation of gingipain proteins and or other abnormal protein deposits in neuronal tissue).

[0153] Just as the skilled person will be familiar with methods for identifying a subject requiring treatment according to the present invention, the skilled person will also be able to determine the success of treatment. Success of treatment with an RNA polynucleotide as described herein can be determined using standard techniques known to the skilled person. For example, in the context of determining whether a treatment with an RNA polynucleotide described herein, has resulted in successful treatment of a neuropathology (such as cognitive dysfunction or symptoms of neurodegenerative disorders), the skilled person can make use of standard cognition tests for determining whether there is an improvement in cognition or one or more other signs of P. g / ng / vaf / s-induced neuropathology.

[0154] In some embodiments, an “improvement” may refer to a reduction of score on the scale or subscale of the Neuropsychiatric Inventory-Nursing Home Version (NPI-NH) or the Alzheimer's Disease Cooperative Study-Clinical Global Impression of Change (ADSC-CGIC). For example, an improvement refers to a reduction of a patient's total NPI-NH score from a score of 50 to a score of 40. In some embodiments, the improvement may optionally refer to one or more patients.1006265400

[0155] The terms “improvement,” “improved” and “improves” as used herein with respect to the clinical setting refer to a clinically relevant effect being achieved greater 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% when compared to baseline after a specified period of time. In some embodiments, the improvement refers to improved efficacious effect in a single patient after the administration of an RNA polynucleotide or composition as described herein as compared to baseline (i.e., prior to the administration of the protein). In other embodiments, the improvement refers to the demonstration of efficacy by a greater percentage of patients demonstrating an efficacious effect after a specified period of time as compared to placebo or lack of treatment. In various embodiments, the percentage of patients demonstrating an efficacious effect is increased by greater 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% when compared to placebo or lack of treatment. In some embodiments, the specified period of time is about two weeks, four weeks or six weeks. In one embodiment, the specified period of time is six weeks.

[0156] The terms “reduction,” “reduced” and “reduces” as used herein with respect to the clinical setting refer to a clinically relevant effect being achieved less 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% when compared to baseline after a specified period of time or when compared to placebo or lack of treatment. In some embodiments, the specified period of time is about two weeks, four weeks or six weeks. In one embodiment, the specified period of time is six weeks.

[0157] In any embodiment, the methods of the invention also comprise treating a subject as herein described with one or more adjunct therapies. In some examples, the adjunct therapy comprises a treatment to reduce the oral load of P. gingivalis of the subject. For example, in certain embodiments the adjunct therapy may comprise scaling and root planing (SRP) to remove P. gingivalis oral biofilm from the subject, antibiotic treatment to reduce bacterial load / infection, or anti-inflammatory agents to reduce swelling and inflammation. The adjunct therapy may also comprise a therapy or treatment for a neuropathology, such as a treatment for dementia, neurodegeneration, cognitive decline, abnormal protein deposits in the brain, including amyloidopathies,1006265400synucleinopathies or tauopathies. In any embodiment, the adjunct treatment may comprise a therapy or treatment for Alzheimer’s disease (AD), Lewy-bodies disease (Dementia with Lewy bodies (DLB)), Huntington's disease, Creutzfeldt-Jakob disease (CJD), Gaucher Disease Type 3, or Parkinson's disease.Methods of administration

[0158] The methods of the present invention typically comprise administering a polynucleotide as described herein, preferably formulated in a vaccine composition (eg lipid nanoparticle) as described herein, to a subject in need thereof.

[0159] The term "vaccine composition" used herein is defined as a composition used to elicit an immune response against an antigen (immunogen) encoded by the RNA in the composition in order to protect or treat an organism against disease.

[0160] As used herein, the terms “immunostimulating composition”, “vaccine composition” and “immunogenic composition” may generally be used interchangeably.

[0161] In some embodiments, a polynucleotide RNA (e.g., mRNA) vaccine is administered to a subject by intradermal or intramuscular injection, subcutaneous, intravenous, or intranasal route, or any other suitable route for delivery of an RNA-based vaccine.

[0162] A polynucleotide (e.g., mRNA) vaccine of the present disclosure is administered to a subject in an effective amount (eg an amount effective to induce an immune response and / or to treat or prevent a disease or condition as herein described).

[0163] In some embodiments, the polynucleotide (e.g., mRNA) vaccine is formulated in an effective amount to produce an antigen specific immune response in a subject.

[0164] Dosages of the vaccine composition described herein can vary between wide limits, depending upon the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.

[0165] This dosage can be repeated as often as appropriate. For example, an initial dose of the vaccine may be administered and then a booster administered at a later date.1006265400

[0166] In preferred embodiments, vaccines of the invention (e.g., LNP-encapsulated mRNA vaccines) produce prophylactically- and / or therapeutically-efficacious levels, concentrations and / or titres of antigen-specific antibodies in the blood or serum of a vaccinated subject.

[0167] In one embodiment of the invention, the subject receiving treatment has normal cognitive function (i.e., has not yet displayed signs of cognitive impairment or decline), which is improved following administration of an RNA polynucleotide according to the presently described methods.

[0168] In a further embodiment the subject exhibits cognitive impairment associated with dementia or any other neuropathology as described herein, but demonstrates improved cognitive function following treatment.Nucleic acids

[0169] The term “nucleic acid,” in its broadest sense, includes any compound and / or substance that comprise a polymer of nucleotides. These polymers are often referred to as polynucleotides. Typically, the polynucleotides of the invention are in the form of an RNA molecule, preferably an mRNA. As used herein, the term “messenger RNA” (RNA) refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo. The skilled artisan will appreciate that, except where otherwise noted, polynucleotide sequences set forth in the instant application will recite “T”s in a representative DNA sequence but where the sequence represents RNA (e.g., RNA), the “T”s would be substituted for “U”s. Thus, any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., RNA) sequence encoded by the DNA, where each “T” of the DNA sequence is substituted with “II.”

[0170] The basic components of an RNA molecule include at least a coding region, a 5'UTR, a 3'UTR, a 5' cap and a poly-A tail. Polynucleotides of the present disclosure may function as RNA but can be distinguished from wild-type RNA in their functional and / or structural design features, which serve to overcome existing problems of effective polypeptide expression using nucleic-acid based therapeutics.1006265400

[0171] A “5' untranslated region” (5'UTR) refers to a region of an RNA that is directly upstream (i.e., 5') from the start codon (i.e., the first codon of an RNA transcript translated by a ribosome) that does not encode a polypeptide.

[0172] A “3' untranslated region” (3'IITR) refers to a region of an RNA that is directly downstream (i.e., 3') from the stop codon (i.e., the codon of an RNA transcript that signals a termination of translation) that does not encode a polypeptide.

[0173] An “open reading frame” is a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a polypeptide.

[0174] A “polyA tail” is a region of RNA (typically mRNA) that is downstream, e.g., directly downstream (i.e., 3'), from the 3' UTR that contains multiple, sometimes consecutive adenosine monophosphates. A polyA tail may contain 10 to 300 adenosine monophosphates. For example, a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a polyA tail contains 50 to 250 adenosine monophosphates. In some embodiments, a segmented polyA tail may be used (typically segments of consecutive adenosine monophosphates separated via a short spacer region between segments). In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus and translation.

[0175] In some embodiments, a polynucleotide includes 200 to 3,000 nucleotides. For example, a polynucleotide may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides.

[0176] The present invention also contemplates the use of one or more structural and / or chemical modifications or alterations which impart useful properties to the polynucleotide including, in some embodiments, the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced. As such, modified RNA molecules of the present invention may also be termed “mmRNA.” As used herein, a “structural” feature or modification is one in which two or more linked nucleotides1006265400are inserted, deleted, duplicated, inverted or randomized in a polynucleotide, primary construct or mRNA without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”. The same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”. Here, the dinucleotide “CO” has been inserted, resulting in a structural modification to the polynucleotide.

[0177] The RNA molecules of the invention may also comprise a 5’ terminal cap. In some embodiments, the 5' terminal cap is 7mG(5')ppp(5')NlmpNp although it will be appreciated that any number of different 5’ terminal caps commonly used in the art may be employed.

[0178] In some embodiments, the RNA molecule comprises at least one chemical modification. The terms “chemical modification” and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribonucleosides in at least one of their position, pattern, percent or population. Generally, these terms do not refer to the ribonucleotide modifications in naturally occurring 5'-terminal RNA cap moieties. With respect to a polypeptide, the term “modification” refers to a modification relative to the canonical set 20 amino acids.

[0179] Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise various (more than one) different modifications. In some embodiments, a particular region of a polynucleotide contains one, two or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, a modified RNA polynucleotide (e.g., a modified RNA polynucleotide), introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified polynucleotide. In some embodiments, a modified RNA polynucleotide (e.g., a modified RNA polynucleotide), introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response).

[0180] Modifications of polynucleotides include, without limitation, those described herein. Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) may1006265400comprise modifications that are naturally-occurring, non-naturally-occurring or the polynucleotide may comprise a combination of naturally-occurring and non-naturally-occurring modifications. Polynucleotides may include any useful modification, for example, of a sugar, a nucleobase, or an internucleoside linkage (e.g., to a linking phosphate, to a phosphodiester linkage or to the phosphodiester backbone).

[0181] Polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the polynucleotides to achieve desired functions or properties. The modifications may be present on internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminus of a chain or anywhere else in the chain. Any of the regions of a polynucleotide may be chemically modified.

[0182] The present disclosure provides for modified nucleosides and nucleotides of a polynucleotide (e.g., RNA polynucleotides, such as mRNA polynucleotides). A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Polynucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodioester linkages, in which case the polynucleotides would comprise regions of nucleotides.

[0183] Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base / sugar or linker may be incorporated into polynucleotides of the present disclosure.1006265400

[0184] The at least one chemical modification may be selected from pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine and 2'-O-methyl uridine. In some embodiments, the chemical modification is in the 5-position of the uracil. In some embodiments, the chemical modification is a N1-methylpseudouridine. In some embodiments, the chemical modification is a N1 -ethylpseudouridine. In some embodiments, polynucleotides include a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases.

[0185] In some embodiments, polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a polynucleotide can be uniformly modified with 5-methyl-cytidine (m5C), meaning that all cytosine residues in the RNA sequence are replaced with 5-methyl-cytidine (m5C). Similarly, a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

[0186] Exemplary nucleobases and nucleosides having a modified cytosine include N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2-thio-5-methyl-cytidine.

[0187] In some embodiments, a modified nucleobase is a modified uridine. Exemplary nucleobases and nucleosides having a modified uridine include 5-cyano uridine, and 4'-thio uridine.

[0188] In some embodiments, a modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A).

[0189] In some embodiments, a modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-1006265400inosine (mil), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (mIG), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine.

[0190] The polynucleotides of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a polynucleotide of the disclosure, or in a given predetermined sequence region thereof (e.g., in the mRNA including or excluding the polyA tail). In some embodiments, all nucleotides X in a polynucleotide of the present disclosure (or in a given sequence region thereof) are modified nucleotides, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

[0191] The polynucleotide may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). Any remaining percentage is accounted for by the presence of unmodified A, G, U, or C.

[0192] The polynucleotides may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the polynucleotides may contain a modified pyrimidine such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the polynucleotide1006265400is replaced with a modified uracil (e.g., a 5-substituted uracil). The modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures), In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the polynucleotide is replaced with a modified cytosine (e.g., a 5-substituted cytosine). The modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).

[0193] In some embodiments, the RNA (e.g., RNA) vaccines comprise a 5'UTR element, an optionally codon optimized open reading frame, and a 3'UTR element, a poly(A) sequence and / or a polyadenylation signal wherein the RNA is not chemically modified.

[0194] Polynucleotides of the present disclosure, in some embodiments, are codon optimized. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase RNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g. glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and RNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art — non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and / or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.

[0195] In some embodiments, a codon optimized sequence shares less than 95% sequence identity, less than 90% sequence identity, less than 85% sequence identity, less than 80% sequence identity, or less than 75% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type RNA sequence1006265400encoding a polypeptide or protein of interest (e.g., an antigenic protein or antigenic polypeptide)).

[0196] In some embodiments, a codon-optimized sequence shares between 65% and 85% (e.g., between about 67% and about 85%, or between about 67% and about 80%) sequence identity to a naturally-occurring sequence or a wild-type sequence (e.g., a naturally-occurring or wild-type RNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)). In some embodiments, a codon-optimized sequence shares between 65% and 75%, or about 80% sequence identity to a naturally-occurring sequence or wild-type sequence (e.g., a naturally-occurring or wildtype RNA sequence encoding a polypeptide or protein of interest (e.g., an antigenic protein or polypeptide)).

[0197] In some embodiments a codon-optimized RNA (e.g., mRNA) may, for instance, be one in which the levels of G / C are enhanced. The G / C-content of nucleic acid molecules may influence the stability of the RNA. RNA having an increased amount of guanine (G) and / or cytosine (C) residues may be functionally more stable than nucleic acids containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. WO02 / 098443 discloses a pharmaceutical composition containing an RNA stabilized by sequence modifications in the translated region. Due to the degeneracy of the genetic code, the modifications work by substituting existing codons for those that promote greater RNA stability without changing the resulting amino acid. The approach is limited to coding regions of the RNA.

[0198] Naturally-occurring eukaryotic RNA molecules have been found to contain stabilizing elements, including, but not limited to untranslated regions (UTR) at their 5'-end (5'UTR) and / or at their 3'-end (3'UTR), in addition to other structural features, such as a 5'-cap structure or a 3'-poly(A) tail. Both the 5'UTR and the 3'UTR are typically transcribed from the genomic DNA and are elements of the premature RNA. Characteristic structural features of mature RNA, such as the 5'-cap and the 3'-poly(A) tail are usually added to the transcribed (premature) RNA during RNA processing. The 3'-poly(A) tail is typically a stretch of adenine nucleotides added to the 3'-end of the transcribed RNA. It can comprise up to about 400 adenine nucleotides. In some embodiments the length of the 3'-poly(A) tail may be an essential element with respect to the stability of the individual RNA.1006265400

[0199] In some embodiments the RNA (e.g., mRNA) vaccine may include one or more stabilizing elements. Stabilizing elements may include for instance a histone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa protein has been identified. It is associated with the histone stem-loop at the 3'-end of the histone messages in both the nucleus and the cytoplasm. Its expression level is regulated by the cell cycle; it peaks during the S-phase, when histone RNA levels are also elevated. The protein has been shown to be essential for efficient 3'-end processing of histone pre-RNA by the U7 snRNP. SLBP continues to be associated with the stem-loop after processing, and then stimulates the translation of mature histone RNAs into histone proteins in the cytoplasm. The RNA binding domain of SLBP is conserved through metazoa and protozoa; its binding to the histone stem-loop depends on the structure of the loop. The minimum binding site includes at least three nucleotides 5' and two nucleotides 3' relative to the stem-loop.

[0200] In some embodiments, the RNA (e.g., mRNA) vaccines include a coding region, at least one histone stem-loop, and optionally, a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal generally should enhance the expression level of the encoded protein. The encoded protein, in some embodiments, is not a histone protein, a reporter protein (e.g. Luciferase, GFP, EGFP, p-Galactosidase, EGFP), or a marker or selection protein (e.g. alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT)).

[0201] In some embodiments, the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop, even though both represent alternative mechanisms in nature, acts synergistically to increase the protein expression beyond the level observed with either of the individual elements. It has been found that the synergistic effect of the combination of poly(A) and at least one histone stem-loop does not depend on the order of the elements or the length of the poly(A) sequence.

[0202] In some embodiments, the RNA (e.g., mRNA) vaccine does not comprise a histone downstream element (HDE). “Histone downstream element” (HDE) includes a purine-rich polynucleotide stretch of approximately 15 to 20 nucleotides 3' of naturally occurring stem-loops, representing the binding site for the U7 snRNA, which is involved in processing of histone pre-RNA into mature histone RNA. Ideally, the inventive nucleic acid does not include an intron.1006265400

[0203] In some embodiments, the RNA (e.g., mRNA) vaccine may or may not contain an enhancer and / or promoter sequence, which may be modified or unmodified or which may be activated or inactivated. In some embodiments, the histone stem-loop is generally derived from histone genes, and includes an intramolecular base pairing of two neighbored partially or entirely reverse complementary sequences separated by a spacer, including (e.g., consisting of) a short sequence, which forms the loop of the structure. The unpaired loop region is typically unable to base pair with either of the stem loop elements. It occurs more often in RNA, as is a key component of many RNA secondary structures, but may be present in single-stranded DNA as well. Stability of the stem-loop structure generally depends on the length, number of mismatches or bulges, and base composition of the paired region. In some embodiments, wobble base pairing (non- Watson-Crick base pairing) may result. In some embodiments, the at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides.

[0204] In other embodiments the RNA (e.g., mRNA) vaccine may have one or more All-rich sequences removed. These sequences, sometimes referred to as AU RES are destabilizing sequences found in the 3'UTR. The AURES may be removed from the RNA (e.g., mRNA) vaccines. Alternatively the AURES may remain in the RNA (e.g., mRNA) vaccine.

[0205] In still further embodiments, the RNA of the invention (eg mRNA) may comprise a ribosome skipping sequence, such as a 2A skipping sequence. The use of such sequences in RNA coding sequences are known to the skilled person and may enable the expression of multiple proteins or peptides from a single mRNA. Accordingly, in any embodiment, an mRNA of the invention may encode two or more of the domains K, D, A (including AAAMB3) or R as defined elsewhere herein and also defined in Table 1, or may encode two or more of the proteins exemplified in Table 1 as being proteins that can be encoded by an RNA sequence of the invention. In certain non-limiting examples, an mRNA of the invention could encode one or more of a KA chimeric protein, a DA chimeric protein, an RA chimeric protein, an AR chimeric protein, an AK chimeric protein, an AD chimeric protein, a KDA chimeric protein, an RDA chimeric protein, a DAR chimeric protein, a DAK chimeric protein or combinations thereof.

[0206] Non-limiting examples of 2A peptide sequence for use to introduce ribosome skipping include the T2A or T2A-like sequences derived from Thosea asigna virus and from Porcine teschovirus-1 2A.1006265400Polypeptides

[0207] It will be appreciated that the polynucleotides for the use of the invention encode a chimeric or fusion protein. The protein encoded by the RNA molecules of the invention may also be termed an “antigenic polypeptide” or simply “antigen”.

[0208] As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances the polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked. Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[0209] As used herein, a chimeric or fusion protein refers to a polypeptide that comprises amino acid sequences that are not arranged in the same spatial configuration as occurs in nature. For example, and in the context of the present invention, the chimeric or fusion protein encoded by the polynucleotides of the invention, comprises portions of an Arg or Lys- gingipain from P. gingivalis, which are in a different spatial arrangement to full length gingipain.Linkers

[0210] In the context of the present invention, the RNA preferably encodes chimeric or fusion proteins comprising various domains (as defined herein), derived from a P. gingivalis gingipain. The domains may be directly joined within the chimeric or fusion protein, or the chimeric or fusion protein may comprise linkers for joining the domains.1006265400

[0211] Suitable linkers for joining amino acid sequences are well known to persons of skill in the art. Preferably, the linker is non-immunogenic. Typically, the linker is comprised of amino acids, and may therefore be termed a peptide linker.

[0212] A linker is usually a peptide having a length of up to 20 amino acids, although may be longer. The term “linked to” or “fused to” refers to a covalent bond, e.g., a peptide bond, formed between two moieties. Accordingly, in the context of the present invention the linker may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 or more amino acids. For example, the chimeric or fusion proteins encoded by the RNAs of the invention, may comprise a linker between the amino acid sequence of a P. gingivalis gingipain active site, and the amino acid sequence of the adhesin domain of a P. gingivalis gingipain. Such linkers have the advantage that they can make it more likely that the different polypeptides of the fusion protein fold independently and behave as expected. Suitable linkers may be up to 50 amino acids in length, although less than 20, less than 15 or less than five amino acids is preferred. The linker may function to bring the domains into a closer spatial arrangement than normally observed in a P. gingivalis trypsin-like enzyme. Alternatively, it may space domains apart.

[0213] Suitable linkers for use in protein constructs, including those with minimal impact on solubility are known in the art. The linker may be any linker known in the art to the skilled person and may be a flexible linker (such as those comprising repeats of glycine and serine residues), a rigid linker (such as those comprising glutamic acid and lysine residues, flanking alanine repeats) and / or a cleavable linker (such as sequences that are susceptible by protease cleavage). Examples of such linkers are known to the skilled person and are described for example, in Chen et al., (2013) Advanced Drug Delivery Reviews, 65: 1357-1369.

[0214] Useful linkers include glycine-serine (GlySer) linkers, which are well-known in the art and comprise glycine and serine units combined in various orders. Examples include, but are not limited to, (GS), (GSGGS)n (SEQ ID NO: 88), (GGGS)n (SEQ ID NO: 89) and (GGGGS)n (SEQ ID NO: 90), where n is an integer of at least one, typically an integer between 1 and about 10, for example, between 1 and about 8, between 1 and about 6, or between 1 and about 5.

[0215] In some embodiments, the peptide linker may include the amino acids glycine and serine in various lengths and combinations. In some aspects, the peptide linker can1006265400include the sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS, SEQ ID NO: 89) or Gly-Gly-Gly-Gly-Ser (GGGGS, SEQ ID NO: 90) and variations or repeats thereof. In some aspects, the peptide linker can include the amino acid sequence GGGGS (a linker of 6 amino acids in length, SEQ ID NO: 90) or even longer. The linker may be a series of repeating glycine and serine residues (GS) of different lengths, i.e., (GS)n where n is any number from 1 to 15 or more. For example, the linker may be (GS)3 (i.e., GSGSGS, SEQ ID NO: 91) or longer (GS)11 or longer. It will be appreciated that n can be any number including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more. Fusion proteins having linkers of such length are included within the scope of the present invention. Similarly, the linker may be a series of repeating glycine residues separated by serine residues. For example (GGGGS)3 (i.e., the linker may comprise the amino acid sequence GGGGSGGGGSGGGGS, (G4S)3, SEQ ID NO: 92) and variations thereof.

[0216] In one embodiment, the peptide linker can include the amino acid sequence GGGGS (a linker of 6 amino acids in length) or even longer. The linker may a series of repeating glycine and serine residues (GS) of different lengths, i.e., (GS)n where n is any number from 1 to 15 or more. For example, the linker may be (GS)3 (i.e., GSGSGS, SEQ ID NO: 91) or longer (GS)11 or longer. It will be appreciated that n can be any number including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or more.

[0217] Other useful linkers include DSSG (SEQ ID NO: 93), DSSGAS (SEQ ID NO: 94), KLDSSG (SEQ ID NO: 95) and variations thereof. Examples of other suitable linkers are described in Chen et al., (2013) Advanced Drug Delivery Reviews, 65: 1357-1369.

[0218] The term “polypeptide variant” refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and / or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence. Ordinarily, variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous) to a native or reference sequence.

[0219] The present invention contemplates several types of compositions which encode polypeptides, including variants and derivatives. These include substitutional, insertional, deletion and covalent variants and derivatives. The term “derivative” is used1006265400synonymously with the term “variant” but generally refers to a molecule that has been modified and / or changed in any way relative to a reference molecule or starting molecule.

[0220] As such, RNAs of the invention encoding polypeptides containing substitutions, insertions and / or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide sequences disclosed herein, are included within the scope of this invention. For example, sequence tags or amino acids, such as one or more lysines, can be added to the peptide sequences of the invention (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble, or linked to a solid support.

[0221] “Substitutional variants” when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. Substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more (e.g., 3, 4 or 5) amino acids have been substituted in the same molecule. In certain embodiments, the substitutions may be conservative amino acid substitutions.

[0222] As used herein the term “conservative amino acid substitution” refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine,1006265400methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and / or a polar residue for a non-polar residue.

[0223] Amino acid deletions or insertions can also be made relative to the native sequence of the P. gingivalis protein. Thus, for example, amino acids which do not have a substantial effect on the activity of the polypeptide, or at least which do not eliminate such activity, can be deleted.

[0224] As used herein when referring to polypeptides the term “domain” refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).

[0225] In any embodiment herein, the skilled person may make modifications to an RNA molecule of the invention so that it includes codons encoding additional amino acid residues derived from the naturally occurring domain sequences of the gingipain. For example, where an RNA of the invention encodes a chimeric protein (such as RA, KA, KDA, KDAK and the like), and wherein the sequences of K, R, D and A are as herein defined, it will be within the purview of the skilled person to include additional codons encoding additional amino acids at the N or C termini of each domain, for example, in order to further stabilise the encoded protein. Typically, the additional amino acids correspond to naturally occurring gingipain sequence. In one non-limiting example, a methionine may be encoded at the N terminal region of the A domain. It will be appreciated that the RNA may encode 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more additional amino acid residues. In another example, the D domain may comprise a C terminal methionine residue (or alternatively this residue may be omitted from the sequence of the D domain).

[0226] As used herein the terms “termini” or “terminus” when referring to polypeptides or polynucleotides refers to an extremity of a polypeptide or polynucleotide respectively. Such extremity is not limited only to the first or final site of the polypeptide or polynucleotide but may include additional amino acids or nucleotides in the terminal regions. Polypeptide-based molecules may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by1006265400non-covalent forces (multimers, oligomers). These proteins have multiple N- and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.

[0227] As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides of interest. For example, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical) of a reference protein having a length of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or longer than 100 amino acids. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 (contiguous) amino acids that are 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% identical to any of the sequences described herein can be utilized in accordance with the disclosure. In some embodiments, a polypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided herein or referenced herein. In another example, any protein that includes a stretch of 20, 30, 40, 50, or 100 amino acids that are greater than 80%, 90%, 95%, or 100% identical to any of the sequences described herein, wherein the protein has a stretch of 5, 10, 15, 20, 25, or 30 amino acids that are less than 80%, 75%, 70%, 65% to 60% identical to any of the sequences described herein can be utilized in accordance with the disclosure.

[0228] Polypeptide or polynucleotide molecules of the present disclosure may share a certain degree of sequence similarity or identity with recited SEQ ID NOs. The term “identity,” as known in the art, refers to a relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between two sequences as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (e.g., “algorithms”). Identity of related peptides can be readily calculated by known methods. “% identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid1006265400sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for the alignment are well known in the art. Identity depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation. Generally, variants of a particular polynucleotide or polypeptide have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, et al. (1997).” Gapped BLAST and PSI-BLAST: a new generation of protein database search programs,” Nucleic Acids Res. 25:3389-3402). Another popular local alignment technique is based on the Smith-Waterman algorithm (Smith, T. F. & Waterman, M. S. (1981) “Identification of common molecular subsequences.” J. Mol. Biol. 147:195-197). A general global alignment technique based on dynamic programming is the Needleman-Wunsch algorithm (Needleman, S. B. & Wunsch, C. D. (1970) “A general method applicable to the search for similarities in the amino acid sequences of two proteins.” J. Mol. Biol. 48:443-453). More recently, a Fast Optimal Global Sequence Alignment Algorithm (FOGSAA) was developed that purportedly produces global alignment of nucleotide and protein sequences faster than other optimal global alignment methods, including the Needleman-Wunsch algorithm.Signal peptides

[0229] The polypeptides encoded by the polynucleotides of the invention typically comprise N-terminal signal peptides. Signal peptides, comprising the N-terminal 15-60 amino acids of proteins, are typically needed for the translocation across the membrane on the secretory pathway and, thus, universally control the entry of most proteins both in eukaryotes and prokaryotes to the secretory pathway. Signal peptides generally include three regions: an N-terminal region of differing length, which usually comprises positively charged amino acids; a hydrophobic region; and a short carboxy-terminal peptide region. In eukaryotes, the signal peptide of a nascent precursor protein (pre-protein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing. ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by an1006265400ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor. A signal peptide may also facilitate the targeting of the protein to the cell membrane. The signal peptide, however, is not responsible for the final destination of the mature protein. Secretory proteins devoid of additional address tags in their sequence are by default secreted to the external environment. During recent years, a more advanced view of signal peptides has evolved, showing that the functions and immunodominance of certain signal peptides are much more versatile than previously anticipated.

[0230] In any embodiment, the N-terminal secretion signal peptide may comprise any amino acid sequence which enables the chimeric or fusion protein to be processed by ribosomes bound to the rough endoplasmic reticulum (ER) of a cell, and thereby results in threading of the chimeric or fusion protein into the ER.

[0231] Preferably the N-terminal secretion signal peptide is any peptide that enables secretion of the encoded protein by the cell in which the RNA is expressed or translated.

[0232] In some embodiments, the signal peptide fused to the antigenic polypeptide is an artificial signal peptide. In some embodiments, an artificial signal peptide fused to the antigenic polypeptide encoded by the RNA (e.g., mRNA) vaccine is obtained from an immunoglobulin protein, e.g., an IgE signal peptide or an IgG signal peptide. In some embodiments, a signal peptide fused to the antigenic polypeptide encoded by a RNA (e.g., mRNA) vaccine is an Ig heavy chain epsilon-1 signal peptide (IgE HC SP) having the sequence of: MDWTWILFLVAAATRVHS (SEQ ID NO: 96).

[0233] In some embodiments, a signal peptide fused to the antigenic polypeptide encoded by the RNA (e.g., mRNA) vaccine is an IgGk chain V-lll region HAH signal peptide (IgGk SP) having the sequence of METPAQLLFLLLLWLPDTTG (SEQ ID NO: 97). In some embodiments, the signal peptide is selected from: Japanese encephalitis PRM signal sequence (MLGSNSGQRVVFTILLLLVAPAYS, SEQ ID NO: 98), VSVg protein signal sequence (MKCLLYLAFLFIGVNCA, SEQ ID NO: 99) and Japanese encephalitis JEV signal sequence (MWLVSLAIVTACAGA, SEQ ID NO: 100).

[0234] Further examples of suitable signal peptides include sequences derived from tPA (tissue plasminogen activator): MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 101) variants, such as: tPA (VSA): MDAMKRGLCCVLLLCGAVFVSA (SEQ ID NO: 102),1006265400tPA (VSAR): MDAMKRGLCCVLLLCGAVFVSAR (SEQ ID NO: 103), tPA (VSP): MDAMKRGLCCVLLLCGAVFVSP (SEQ ID NO: 104), tPA (VSPS): MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 101).

[0235] In certain embodiments, the amino acid sequence of the signal peptide comprises the sequence of SEAP (secreted embryonic alkaline phosphatase): MLLLLLLLGLRLQLSLG[A] (SEQ ID NO: 105), such that the expressed RNA product comprises the sequence MLLLLLLLGLRLQLSLG[A] (SEQ ID NO: 105) N terminal to the sequences defined herein including in Table 1.

[0236] In preferred embodiments the invention, the signal peptide comprises the sequence as set forth in any of these examples, of a sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

[0237] The examples disclosed herein are not meant to be limiting and any signal peptide that is known in the art to facilitate targeting of a protein to ER for processing and / or targeting of a protein to the cell membrane may be used in accordance with the present disclosure.

[0238] A signal peptide may have a length of 15-60 amino acids. For example, a signal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, a signal peptide has a length of 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15-20 amino acids.

[0239] A signal peptide is typically cleaved from the nascent polypeptide at the cleavage junction during ER processing. The mature antigenic polypeptide produces by an RNA vaccine of the present disclosure typically does not comprise a signal peptide.

[0240] It will be well within the purview of the skilled person to design a polypeptide encoded by an RNA for the use of the invention, to facilitate expression and translation thereof in vivo. For example, in certain non-limiting examples, the RNA of the invention1006265400may include sequence encoding an N terminal methionine, and / or other residues (such as alanine) for enabling expression, secretion and / or cleavage of the signal peptide.

[0241] Accordingly, in any embodiment, the RNA for the use of the invention may encode one, two, three, four or more N terminal amino acids to the sequences defined herein in Table 1. For example, an RNA encoding an amino acid sequence as set forth in any of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 38 or 39 may encode one or more additional N terminal amino acids, optionally an N terminal alanine residue and / or optionally an N terminal methionine residue.

[0242] In further examples, an RNA encoding an amino acid sequence of a D domain (eg in SEQ ID NOs: 35 and 36) may comprise the C terminal methionine residue (as shown in these sequence) or the C terminal methionine residue may be omitted from the D domain sequence.Compositions and lipid nanoparticles

[0243] The present invention contemplates the provision of a polynucleotide (preferably an RNA) encoding a chimeric or fusion protein for various methods of treatment, preferably formulated in a lipid nanoparticle. Accordingly, the present invention also provides a lipid nanoparticle comprising a polynucleotide as described herein. It will be appreciated that in any embodiment, the nanoparticles of the invention may also be described as “vaccine” compositions or “immune stimulating” compositions.

[0244] In some embodiments, the RNA for the use of the invention is formulated in a lipid-polycation complex, referred to as a cationic lipid nanoparticle. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and / or polyarginine. In some embodiments, the RNA may be formulated in a lipid nanoparticle that includes a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

[0245] Lipid nanoparticle formulations typically comprise a lipid, in particular, an ionizable cationic lipid, and further comprise a non-cationic lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid.1006265400

[0246] In some embodiments, a cationic lipid is an ionizable cationic lipid and the noncationic lipid is a neutral lipid, and the sterol is a cholesterol.

[0247] In some embodiments, lipid nanoparticle formulations consist essentially of a lipid mixture in molar ratios of 20-70% cationic lipid: 5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid. In some embodiments, lipid nanoparticle formulations consist essentially of a lipid mixture in a molar ratio of 20-60% cationic lipid: 5-25% neutral lipid: 25-55% cholesterol: 0.5-15% PEG-modified lipid.

[0248] A lipid nanoparticle formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. In one example by Semple et al. (Nature Biotech. 201028:172-176), the lipid nanoparticle formulation is composed of 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA.

[0249] In some embodiments, the lipid nanoparticle comprises a molar ratio of about 20-60% cationic lipid, 0.5-15% PEG-modified lipid, 25-55% sterol, and 25% non-cationic lipid. In some embodiments, the cationic lipid is an ionizable cationic lipid and the noncationic lipid is a neutral lipid, and the sterol is a cholesterol.

[0250] In some embodiments, lipid nanoparticle formulations may comprise 35 to 45% cationic lipid, 40% to 50% cationic lipid, 50% to 60% cationic lipid and / or 55% to 65% cationic lipid.

[0251] In some embodiments, the ratio of lipid to RNA (e.g., mRNA) in lipid nanoparticles may be 5:1 to 20:1, 10:1 to 25:1, 15:1 to 30:1 and / or at least 30:1.

[0252] In some embodiments, the ratio of PEG in the lipid nanoparticle formulations may be increased or decreased and / or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and / or biodistribution of the lipid nanoparticle formulations.

[0253] The amino alcohol cationic lipid may be a lipid described in and / or made by the methods described in U. S. Patent Publication No. US20130150625, herein incorporated by reference in its entirety.1006265400

[0254] The cationic lipid may be any one of N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), 1,2-dioeoyloxy-3-(dimethylamino)propane (DODAP), 1,2-dioleyloxy-N, N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N, N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTAP), 3-(N — (N', N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl)-N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N, N-dimethyl-1-propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3.beta.-oxy)-3'-oxapentoxy)-3-dimethy-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N, N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N, N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N, N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 4-Hydroxybutyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), 8-[(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino]-octanoic acid, 1 -octylnonyl ester (SM-102) and mixtures thereof.

[0255] Optionally, the cationic lipid may be 4-Hydroxybutyl)azanediyl)bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315).

[0256] The phospholipid moiety may be selected from the group consisting of: phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.

[0257] The phospholipid may have a fatty acid moiety selected from the non-limiting group consisting of: lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoicacid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid.

[0258] The phosopholipid may be lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides,1006265400dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

[0259] The phospholipid may be distearoylphosphatidylcholine (DSPC).

[0260] The phospholipid may comprise from about 5 mol % to about 20 mol %, from about 5 mol % to about 15 mol %, from about 5 mol % to about 10 mol %, from about 10 mol % to about 20 mol %, from about 15 mol % to about 20 mol % of the total lipid present in the particle.

[0261] The structural lipid may be selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof.

[0262] The structural lipid may be cholesterol. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or a combination thereof. Further, the structural lipid may be squalene, squalene or combination thereof.

[0263] The structural lipid may include lipids containing geranyl acetate, farnesyl acetate or geranyl-geranyl, or ether, ester, or other derivatives.

[0264] The structural lipid may comprise from about 30 mol % to about 50 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 30 mol % to about 35 mol %, from about 35 mol % to about 50 mol %, from about 40 mol % to about 50 mol %, or from about 45 mol % to about 50 mol % of the total lipid present in the particle.1006265400

[0265] Examples of lipid nanoparticle compositions and methods of making same are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety).

[0266] In any embodiment, the PEGylated lipid may comprise about 0.05 mol %, about 0.1 mol %, about 0.15 mol %, about 0.2 mol %, about 0.25 mol %, about 0.3 mol %, about 0.35 mol %, about 0.4 mol %, about 0.45 mol %, about 0.5% mol, about 0.6% mol, about 0.7% mol, about 0.8% mol, about 1 % mol, about 1.2% mol, about 1,4 % mol, about 1.6% mol, about 1.8 % mol, or about 2 % mol or more of the total lipid present in the particle.

[0267] The PEGylated lipid may be selected from the group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof.

[0268] In some embodiments, the pharmaceutical compositions of the RNA (e.g., mRNA) vaccines may include at least one of the PEGylated lipids described in International Publication No. WO2012099755, the contents of which are herein incorporated by reference in their entirety.

[0269] The PEGylated lipid may have a PEG component that has a molecular weight between about 100 Da and about 100,000 Dam between about 100 Da and about 100,000 Da, between about 1000 Da and 9,000 Da, between about 1000 Da and 8,000 Da, between about 1000 Da and 7,000 Da, between about 1000 Da and 6,000 Da, between about 1000 Da and 5,000 Da, between about 1000 Da and 4,000 Da, between about 1000 Da and 3,000 Da, or between about 1000 Da and 2,000 Da.

[0270] The PEGylated lipid may be ALC-0159 (2-[(polyethylene glycol)-2000]-N, N-ditetradecylacetamide). ALC-0159 is a PEG / lipid conjugate (i.e. PEGylated lipid), specifically, it is the N, N-dimyristylamide of 2-hydroxyacetic acid, O-pegylated to a PEG chain mass of about 2 kilodaltons (corresponding to about 45-46 ethylene oxide units per molecule of N, N-dimyristyl hydroxyacetamide). It is a non-ionic surfactant by its nature.

[0271] Non-limiting examples of lipid nanoparticle compositions and methods of making them are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28:172-1006265400176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety).

[0272] In some embodiments, the RNA vaccine composition of the invention may comprise a polynucleotide described herein, formulated in a lipid nanoparticle comprising ALC — 0315 ([(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate)) Cholesterol, DSPC and ALC-0159 (2-[(polyethylene glycol)-2000]-N, N-ditetradecylacetamide), the buffer Tris-sucrose and water for injection.

[0273] As a non-limiting example, the composition comprises: 0.6 mg / mL of drug substance (e.g., polynucleotides encoding a chimeric or fusion protein described herein and comprising components of a P. gingivalis gingipain polyprotein complex), 8.58 mg / mL ofALC-0315, 3.99 mg / mL of cholesterol, 1.80 mg / mL of DSPC, 0.95 mg / mL of ALC-0159 (2-[(polyethylene glycol)-2000]-N, N-ditetradecylacetamide), 3.03 mg / mL of Tris (trishydroxymethyl)aminomethoane), 88 mg / mL of sucrose in water, with a typical volume for injection of 50 pL.

[0274] In alternative embodiments, the RNA vaccine composition of the invention may comprise the four lipids DLin-MC3-DMA, Cholesterol, DSPC and DMG-PEG 2000 at a ratio of 50:39.8:10:0.2 (mol ratio).

[0275] In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 10-500 nm, 20-400 nm, 30-300 nm, 40-200 nm. In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has a mean diameter of 50-150 nm, 50-200 nm, 80-100 nm or 80-200 nm.

[0276] In some embodiments the RNA (e.g., RNA) vaccine may be associated with a cationic or polycationic compounds, including protamine, nucleoline, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), polyarginine, basic polypeptides, cell penetrating peptides (CPPs).) and of one or more hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole), etc.

[0277] In other embodiments the RNA (e.g., RNA) vaccine is not associated with a cationic or polycationic compounds.1006265400

[0278] Other examples of suitable lipid nanoparticle formulations are provided in US 10,702,600, the contents of which are hereby incorporated by reference.

[0279] The lipid nanoparticles described herein may be made in a sterile environment.

[0280] The nanoparticle formulations may comprise a phosphate conjugate. The phosphate conjugate may increase in vivo circulation times and / or increase the targeted delivery of the nanoparticle. As a non-limiting example, the phosphate conjugates may include a compound of any one of the formulas described in International Application No. WO2013033438, the contents of which are herein incorporated by reference in its entirety.

[0281] The nanoparticle formulation may comprise a polymer conjugate. The polymer conjugate may be a water soluble conjugate. The polymer conjugate may have a structure as described in U. S. Patent Application No. 20130059360, the contents of which are herein incorporated by reference in its entirety. In

[0282] The nanoparticle formulations may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a RNA (e.g., mRNA) vaccine. As a non-limiting example, the carbohydrate carrier may include, but is not limited to, an anhydride-modified phytoglycogen or glycogen-type material, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin. (See e.g., International Publication No. WO2012109121; the contents of which are herein incorporated by reference in their entirety).

[0283] Nanoparticle formulations of the present disclosure may be coated with a surfactant or polymer in order to improve the delivery of the particle. In some embodiments, the nanoparticle may be coated with a hydrophilic coating such as, but not limited to, PEG coatings and / or coatings that have a neutral surface charge. As a nonlimiting example nanoparticles comprising a hydrophilic coating and methods of making such nanoparticles are described in U. S. Patent Publication No. US20130183244, the contents of which are herein incorporated by reference in their entirety.

[0284] In some embodiments, the lipid nanoparticles of the present disclosure may be hydrophilic polymer particles. Non-limiting examples of hydrophilic polymer particles and methods of making hydrophilic polymer particles are described in U. S. Patent Publication1006265400No. US20130210991, the contents of which are herein incorporated by reference in their entirety.

[0285] In some embodiments, the lipid nanoparticles of the present disclosure may be hydrophobic polymer particles.

[0286] In any embodiment, a lipid-based formulation including any LNP disclosed herein may further comprise one or more adjuvants. For example, in any embodiment, an ionisable lipid present in the nanoparticle formulation may be substituted or combined with an adjuvant lipidoid for enhancing RNA delivery. Examples of such approaches are described in the prior art, such as in Han et al., (2023) Nature Nanotechnology, https: / / doi.org / 10.1038 / s41565-023-01404-4, and Salleh et al., (2022), Peer J, 10:e13083; incorporated herein by reference.

[0287] serum dilution required to achieve a 50% reduction in the number of plaques.

[0288] In some embodiments, the disclosure features a pharmaceutical composition comprising a nanoparticle composition according to the preceding embodiments and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be as described herein, and may also include one or more agents for facilitating storage of the composition at low temperatures. For example, the pharmaceutical composition may be refrigerated or frozen for storage and / or shipment (e.g., being stored at a temperature of 4° C or lower, such as a temperature between about -150° C. and about 0° C. or between about -80° C and about -20° C (e.g., about -5° C, -10° C, -15° C, -20° C, -25° C, -30° C, -40° C, -50° C, -60° C, -70° C, -80° C, -90° C, -130° C or -150° C.). For example, the pharmaceutical composition is a solution that is refrigerated for storage and / or shipment at, for example, about -20° C, -30° C, -40° C, -50° C, -60° C, -70° C, or -80° C. Accordingly, it will be appreciated that the compositions described herein may further comprise one or more cryoprotectants or cryopreservatives. Optionally the cryopreservative or cryoprotectant may comprise a sugar such as sucrose, glucose or related sugar-based cryoprotectant.Liposomes and Lipoplexes, and Lipid Nanoparticles

[0289] The RNA (e.g., mRNA) vaccines of the disclosure can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In some embodiments, pharmaceutical compositions of RNA (e.g., mRNA) vaccines include liposomes.1006265400

[0290] In some embodiments, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from 1,2-dioleyloxy-N, N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by reference in its entirety) and liposomes which may deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

[0291] In some embodiments, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from the synthesis of stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 23:1002-1007; Zimmermann et al., Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 201028:172-176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132; U. S. Patent Publication No US20130122104; all of which are incorporated herein in their entireties).

[0292] In some embodiments, liposome formulations may comprise from about 25.0% cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol to about 45.0% cholesterol, from about 35.0% cholesterol to about 50.0% cholesterol and / or from about 48.5% cholesterol to about 60% cholesterol. In some embodiments, formulations may comprise a percentage of cholesterol selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0% and 43.5%. In some embodiments, formulations may comprise from about 5.0% to about 10.0% DSPC and / or from about 7.0% to about 15.0% DSPC.

[0293] In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated in a lipid vesicle, which may have crosslinks between functionalized lipid bilayers.

[0294] In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated in a lipid-polycation complex. The formation of the lipid-polycation complex may be accomplished by methods known in the art and / or as described in U. S. Pub. No.100626540020120178702, herein incorporated by reference in its entirety. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and / or polyarginine. In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated in a lipid-polycation complex, which may further include a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

[0295] Further examples of suitable formulations for mRNA delivery are described in Guevara et al., (2020), Frontiers in Chemistry, 8:589959; Zhang et al., (2019), Frontiers in Immunology, 10:594; and Liu et al., (2022), Polymers, 14: 4195; incorporated by reference herein in their entirety.Kits

[0296] In another embodiment there is provided a kit or article of manufacture including one or more RNAs as described herein for use according to the invention in any method as described above.

[0297] In yet another aspect, the present invention provides a kit of parts comprising a vaccine composition for use according to the methods of the invention and one or more adjuvants for separate, subsequent or simultaneous administration to a subject.

[0298] In other embodiments there is provided a kit for use in a therapeutic or prophylactic application mentioned above, the kit including:- a container holding an RNA polynucleotide composition as described herein;- a label or package insert with instructions for use.

[0299] In any embodiment the kit may contain one or more further active principles or ingredients for eliciting an immune response to P. gingivalis in a subject.

[0300] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0301] The following examples are presented in order to more fully illustrate some embodiments of the invention. They should, in no way be construed, however, as1006265400limiting the broad scope of the invention. One skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.Examples

[0302] Example 1: Materials and methods

[0303] Production of mRNA-LNPs

[0304] The mRNAs were in vitro transcribed using T7 in vitro transcription kit (NEB) according to manufacturer’s instructions, from linearised DNA templates encoding 5’ and 3’ UTRs, signal peptide, the candidate sequence and a 125 nucleotide poly(A) tail and were capped co-transcriptionally using Clean Cap mRNA capping technology (TriLink Biotech). Where clearly stated, the UTP was replaced by N1-methyl pseudouridine (N1-methyl pseudo-UTP, ml^P) during mRNA production. DNA was removed using DNAse I (NEB), and double-stranded RNA (dsRNA) was removed using cellulose binding as previously described previously [Baiersddrfer et al. (2019) Mol Ther Nucleic Acids. Apr 15;15:26-35] and formulated in lipid nanoparticles (LNPs) of the following lipid composition: ALC-0315, Cholesterol, DSPC, and ALC-0159 at molar lipid ratios (%) of 46.3:42.7:9.4:1.6, in Tris / sucrose buffer (25 mM Tris pH 7.4, 8.8% sucrose w / v).

[0305] Expression of mRNA vaccine candidates in cell culture in vitro

[0306] HeLa cells were cultured in culture media DM EM, high glucose, GlutaMAX™ Supplement, pyruvate (ThermoFisher, CAT#10569010) with 10% FBS at 37°C, 5% CO2, according to standard protocols. mRNA candidates (prior to LNP formulation), were formulated with Lipofectamine MessengerMAX (IThermoFisher) and used to transfect cells according to manufacturer’s instructions. 1.8 pg of an mRNA was used to transfect 35,000 cells / well on a six well plate. For supernatant collection, cells were pelleted by centrifuging at 14,000 g for 15 mins. Supernatants were transferred to a fresh tube and frozen at -20°C until Western Blotting. For whole cell lysate collection, cells were rinsed in each well with ~2 mL DPBS. The liquid was removed and 250 pL of RIPA lysis buffer with protease inhibitors (ThermoFisher) was added to each well, and gently swirled to mix for 10s. Using a cell scraper the lysates were transferred into tubes1006265400and centrifuged at 4°C at 16,000 g for 15 mins to pellet debris. The clarified lysates were collected into new tubes and stored at -20°C until Western Blotting.

[0307] Western Blot

[0308] Samples were mixed with 4X Laemmli sample buffer (10%v / v) supplemented with 2-mercaptoethanol and 100mM DTT, centrifuged at 13,000 g speed for 30 seconds and incubated at room temperature for 20 min, then denatured at 95°C for 10 min. Samples were loaded onto Any kD™ Mini-PROTEAN® TGX™ Precast Protein Gels (BioRad, ca# 4569036) and proteins were separated by SDS-PAGE with Tris / Glycine / SDS running buffer (BioRad, cat# 1610732) at 150V for 45 minutes.Precision Plus Protein™ WesternC™ Blotting Standard was used as a protein marker (BioRad, cat# 161-0376). Proteins were transferred onto a PDV membrane (Life Technologies, cat# LC2002) that had been pre-activated in methanol for 30 s and washed in transfer buffer (10x Tris / Glycine Buffer for Western Blots and Native Gels (BioRad, cat# 1610734), using Trans-Blot® Turbo™ Transfer System (BioRad) with the standard protocol: 25V and 1.0A for 30 min.

[0309] PVDF membranes with proteins transferred onto them were blocked for 2 hours in blocking buffer (5% skim milk powder in PBS-T) on a rocker and probed with in-house primary antibodies for binding to KAS2 (1:2500 in blocking buffer) or KDAK-3S-AVQP (1:3500 in blocking buffer) overnight at 4°C on a rocker. Membranes were washed 5 times with PBS-T and incubated with goat anti mouse HRP (1:2500 in blocking buffer, supplemented with Precision Protein StrepTactin-HRP Conjugate BioRad, cat# 1610381 at 1:10,000) at room temperature for an hour on a rocker, washed 5 times with PBS-T, developed using Clarity™ Western ECL Substrate (BioRad, cat# 170-5060) and imaged using ChemiDoc™ Touch Imaging System using chemiluminescence detection.

[0310] Culture of bacteria for mouse model of periodontitis

[0311] P. gingivalis strain; W50 (serotype C); was obtained from the culture collection of the Oral Health Cooperative Research Centre, The Melbourne Dental School, University of Melbourne, Australia. P. gingivalis W50 was grown on Horse Blood Agar (HBA) (20 g / L HBA; Oxoid Ltd., Hampshire, UK) supplemented with 10% v / v lysed horse blood (37°C) in an anaerobic N2 atmosphere containing 5% CO2 in a MK31006265400Anaerobic Workstation (Don Whitley Scientific Ltd., Adelaide, Australia). Colonies were inoculated into starter culture comprised of 20 mL sterilised brain heart infusion (37 g / L BHI; Oxoid Ltd., Hampshire, UK) medium supplemented with 5 mg / L hemin and 0.5 mg / L cysteine and incubated anaerobically (24 h, 37 °C). Absorbance of batch cultures were monitored at OD650nm using a spectrophotometer (model 295E, Perkin-Elmer, Germany). Bacterial cells were harvested during late exponential growth by centrifugation (7,000 g, 20 min, 4 °C). Bacterial purity was routinely confirmed by Gram stain [Slots (1982). In: Host-Parasite Interaction in Periodontal Disease, Genco, R. J. and Merganhagan, S. E. (eds). Washington D. C.: American Society for Microbiology, pp.27-45.].

[0312] Preparation of heat-killed bacteria

[0313] P. gingivalis W50 culture was harvested (6,500 g, 4 °C), washed once with phosphate buffered saline (PBS) (0.01 M Na2HPO4, 1.5 mM KH2PO4 and 0.15 M NaCI, pH 7.4) then pelleted by centrifugation (7,000 g, 20 min 4°C). Bacterial cells were resuspended in PBS and heated to 65°C for 15 minutes. 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 a cell density of 2 x 1010cells / mL, and protein concentration determined using Biorad Protein Assay Dye Reagent Concentrate (Life Science, NSW, Australia).

[0314] Mouse model of P. ainaivalis-induced Alzheimer’s disease

[0315] Mice (female C57BL6) were intra-orally inoculated with P. gingivalis [1 x 1010viable P. gingivalis W50 cells per dose suspended in 20 pL PG buffer [50 mM Tris-HCL, 150 mM NaCI, 10 mM MgSO4 and 14.3 mM mercaptoethanol, pH 7.4 containing 2% (w / v) carboxymethylcellulose]. The inoculum begun on day 42 and was repeated three times a week for 3 weeks. The inocula were prepared anaerobically and then immediately applied to the gingival margin of the maxillary molar teeth. The number of viable bacteria in each inoculum was verified by CFU counts on blood agar.

[0316] Groups of animals consisted of: P. gingivalis W50 orally inoculated (infected control); a non-bacterial inoculated control (naive) and vaccinated group. On Day 84, mice were killed and bled by cardiac puncture. Mice were transcardially perfused with1006265400ice cold PBS at a flow rate of 2 mL per minute using 20 mL of PBS. Following perfusion, brains were removed, sectioned longitudinally and the right hemisphere stored in 4% (v / v) PFA at 4°C for 48 hours, and subsequently transferred into PBS containing 0.01% (w / v) sodium azide and stored at 4°C until sectioning.

[0317] Determination of subclass antibody in sera using ELISA

[0318] ELISAs were performed to evaluate subclass antibody in sera as described in Pathirana et al. (2007). Infect Immun 75: 1436-1442) using a solution of either heat killed W50 cells (10 pg / mL), domain subunits or epitopes (1 pg / mL) in 0.1 M PBS (pH 7.4) to coat wells (16 h, 4 °C) of fl at- bottom polyvinyl microtitre plates (Microtiter;Dynatech Laboratories, McLean, VA, US). In these experiments the following antibody dilutions were used; a dilution of 1 / 4000 dilution of goat anti-mouse; IgG (M8642), I gG 1 (M8770), lgG2a (M4434) antibodies (Sigma, New South Wales, Australia). A 1 / 4000 dilution of a horseradish peroxidase-conjugated swine anti-goat IgG antibody (M5420; Sigma, New South Wales, Australia) was used to develop ELISA experiment. For the epitope ELISAs biotinylated peptides were bound to pre-blocked streptavidin coated flat bottom plates (Pierce; Thermo-Fisher) at 10 pg / mL. Following incubation with sera, the ELISA was developed with 1 / 4000 goat anti-mouse IgG and 1 / 4000 horseradish peroxidase-conjugated swine anti-goat IgG antibody. In all ELISA experiments washing was performed between steps using three washes of 220pL of PBS-Tween 20 (0.1% v / v). All optical density measurements were conducted on a Wallac VICTORS 1420 Multilabel counter (Perkin Elmer) at 405nm and data were analysed using a one-way ANOVA or Kruskal-Wallis test.

[0319] Brain sectioning and analysis

[0320] For histological analysis, the right hemisphere of the brain was coronally sectioned from the hippocampal region at approximately Bregma (-170) formalin-fixed, paraffin-embedded (FFPE). 7 pm thick sections were cut and mounted onto Superfrost slides.

[0321] Slides were dewaxed and rehydrated, using the Mouse and Rabbit Specific HRP / DAB IHC Detection Kit - Micro-polymer (ab236466, Abeam, Australia) according to manufacturer's instructions. Briefly, rehydrated slides were treated with hydrogen peroxide, which was followed by heat-induced epitope-retrieval (HI ER) with citrate1006265400buffer pH 6.0 for 15 min. The sections were then incubated with the protein-blocking reagent for 10 min and treated with specific primary antibodies at 4°C overnight. After being washed with PBS three times the next day, the sections were incubated with goat anti-rabbit IgG secondary antibody at RT for 15 min and developed using HRP-conjugated DAB substrate. Treated sections were washed and counter stained with hematoxylin according to standard protocols and covered with coverslips. Slides were scanned using the 3DHISTECH-Pannoramic Scanll Digital Scanner, a high throughput brightfield digital slide scanner fitted with a x20 objective that generates histological images.

[0322] IHC was performed using the following primary antibodies: P. gingivalis Gingipain R1 (RgpA) rabbit polyclonal antibody (Biorbyt Catalogue number: orb243611), rabbit beta amyloid (Ap) antibody clone H31L21 (Invitrogen; #700254), mouse monoclonal anti-Phospho-Tau (Ser202, Thr205) (clone AT8) (Thermo Fisher, MN1020), rabbit polyclonal anti-IL-6 (Abeam, ab6672), and rabbit polyclonal anti-IL-ip (Abeam, ab9722). Optimal staining conditions were determined by performing heat-treated antigen retrieval using citrate buffer at pH 6.0, followed by hydrogen peroxide treatment.

[0323] Detected AD-like pathology and brain neuroinflammation were quantified using QuPath digital software and compared between mouse groups to determine the effect of vaccination on the development of P. g / ng / vaf / s-i nduced AD-like pathology and neuroinflammation. For the evaluation of P-Tau, IL-1β, and IL-6 immunoreactivity, the stained sections were graded based on the count of positively stained cells. Regions including the hippocampus, cerebral cortex, and midbrain were identified and annotated using software tools. Automation was employed to identify cells with DAB (3,3'-diaminobenzidine substrate) positive staining within the brain region of interest using the "cell positive detection" function by setting image intensity and nucleus size thresholds. Following automation, the cells underwent visual assessment. Data is expressed as the number of positive cells within the area (mm2) for the region of interest of the brain tissue.

[0324] Example 2: Expression of mRNA constructs encoding chimeric proteins

[0325] The inventors obtained mRNA constructs encoding chimeric proteins containing postulated antigenic domains from the Kgp gingipain protein. The mRNA constructs comprised the general architecture as shown in Figure 2 and the chimeric proteins1006265400(antigens) encoded by the mRNA expression constructs comprised the architectures designated KDcAKIn, KDAK and KDAK-3S-AVQP as shown in Figure 1 (amino acid sequences as set forth in SEQ ID NOs: 2, 4 and 6, respectively, mRNA sequences as set forth in SEQ ID NOs: 40, 41 and 42, respectively).

[0326] The mRNAs were tested for in vitro antigen expression and secretion in 293T (not shown) or HeLa cells and using various secretion peptides including tPA-derived signal peptides and SEAP signal peptide. Cells were transfected with the mRNA constructs using lipofectants as outlined in Example 1. After 48 hours, supernatants were collected and subjected to Western Blot analysis to test for secreted polypeptide.

[0327] Figure 3 shows Western Blots demonstrating expression and secretion by HeLa cells of mRNA constructs encoding KDcAKIn, KDAK-3S-AVQP and KDAK. Various signal peptides were tested. The results using the SEAP secretion peptide are shown.

[0328] The results indicate that constructs encoding KDcAKIn did not express well, whereas constructs encoding KDAK and KDAK-3S-AVQP expressed abundant amounts of protein which was secreted to the supernatant of the HeLa cells.

[0329] Example 3: in vivo efficacy and immunogenicity of an mRNA vaccine candidate

[0330] The mRNA vaccine candidate encoding KDAK-3S-AVQP (formulated in LNPs) was assessed in the mouse model of P. gZngv / va / Zs-induced Alzheimer’s disease (Figure 4). The mRNA was ml ip-modified and was administered at a dose of 30 pg mRNA.

[0331] The results, shown in Figure 5, indicate that the mRNA vaccine candidate encoding KDAK-3S-AVQP provided robust protection against P. gZngZva / Zs-induced brain pathologies resembling Alzheimer’s disease.

[0332] The mRNA vaccine candidate protected from brain infiltration by gingipains (RgpA) caused by oral P. gingivalis infection.

[0333] The mRNA vaccine candidate protected from amyloid accumulation in the brain caused by oral P. gingivalis infection.

[0334] The mRNA vaccine candidate protected from Tau phosphorylation in the brain caused by oral P. gingivalis infection.1006265400

[0335] The mRNA vaccine candidate protected from inflammation in the brain caused by oral P. gingivalis infection. This was evident from the decreased levels of IL-6 and IL1-P compared to infected mice that were not vaccinated.

[0336] Antisera were used to probe against the absorbed antigens: recombinant protein KDAK-3S-AVQP heat killed P. gingivalis strain W50 (HKPg) and RgpA-Kgp complex. The results are shown in Figure 6.

[0337] Antibody responses are expressed as the ELISA titre obtained minus triple the background level, with each titre representing the mean ± s.d. of the 10 individual mice. The mRNA vaccine candidate induced strong total IgG, I gG 1 and lgG2 responses against KDAK-3S-AVQP.

[0338] The mRNA vaccine candidate induced strong total IgG and lgG1 titres against HKPg.

[0339] The mRNA vaccine candidate induced strong total IgG and lgG1 titres against RgpA-Kgp complex.

[0340] Example 4: expression and secretion of truncated antigens encoded by alternative mRNA constructs

[0341] Various truncated mRNA constructs were assessed, each expressing truncated variations of the protein antigens encoded by the mRNAs tested in the above examples. All mRNAs were ml ip-modified.

[0342] The mRNA constructs tested encoded:- K (also referred to herein as KAS or KAS2 active site domain; amino acid sequence SEQ ID NO: 8)- KD (amino acid sequence: SEQ ID NO: 10)- KA (amino acid sequence SEQ ID NO: 18)- DA (amino acid sequence SEQ ID NO: 14)- KDA (amino acid sequence SEQ ID NO: 12)- KDAAAMBS (ie wherein the adhesin domain comprises ABMs 2 and 1 but not ABM3;amino acid sequence SEQ ID NO: 16)- RDA (replacing Arginine-dependent gingipain active site KAS, with active site from a Lysine-dependent gingipain RAS) (amino acid sequence: SEQ ID NO: 20)1006265400- positive control: KDAK-3S-AVQP.

[0343] The mRNAs encoding the target antigens were tested for in vitro expression and secretion in HeLa cells by Western Blot. Figure 7 shows the results of these studies. SEAP signal peptide was encoded at the N-terminus of each antigen.

[0344] All constructs expressed well except the construct encoding K (ml^P-K) which was poorly expressed (potentially due to protein degradation following expression). The m1 -K construct was initially not detected in either the lysate or the supernatant in the 24 hour transfected samples (Figure 7A). An additional time course analysis of 6 - 48 hours, plus an increased loading (x 3 volume) of ml^P-K samples revealed that it was poorly expressed and rapidly degraded (Figure 7B). All other constructs: ml^P-KD, ml^P-KA, ml^P-DA, ml^P-KDA, m14J-KDA21 showed good expression and secretion (Figure 7A).

[0345] The RDA antigen which was predominantly found in the whole cell lysate rather than supernatant (suggesting poor secretion).

[0346] Example 5: in vivo efficacy and immunogenicity of truncated constructs

[0347] mRNAs (formulated in LNPs) encoding KDA, KDAAABMS, DA, KA and KD were assessed in this next study and compared to KDAK-3S-AVQP mRNA vaccine. All mRNAs were ml ip-modified and were administered at a dose of 30 pg mRNA.

[0348] The results, shown in Figure 8, indicate that the mRNA vaccine encoding KA, DA, KDAAABMS and KDA provided robust protection against P. g / ng / vaf / s-induced alveolar bone loss.

[0349] Vaccines containing ml^P-KDAK-SS-AVQP, m14J-KDA21 protected against bone loss in the animal model, similarly to the alum-adjuvanted recombinant protein KDAK-3S-AVQP. ml^P-DA and ml^P-KA provided partial protection (Figure 8), suggesting the importance of A and K domains for protection.

[0350] Antisera were used to probe against the absorbed antigens: recombinant protein KDAK-3S-AVQP heat killed P. gingivalis strain W50 (HKPg) and Kgpcat corresponding to sequences encoded by the tested mRNA vaccine candidates.1006265400

[0351] Antibody responses are expressed as the ELISA titre obtained minus triple the background level, with each titre representing the mean ± s.d. of the 10 individual mice. All tested constructs induced strong total IgG, lgG1 and lgG2 responses against KDAK-3S-AVQP (Figure 9).

[0352] All tested constructs induced strong total IgG and lgG1 titres against HKPg. Only ml^-P-DA and m14J-KDA21 induced lgG2a responses.

[0353] All constructs except ml^P-DA (as it doesn’t contain the K domain that is part of the Kgpcat) induced IgG (primarily lgG1) responses against Kgpcat, but the response of ml^P-KDAK-SS-AVQP was weaker and did not show statistical significance for lgG1. Only m14J-KDA21 and, ml^P-KDAK induced lgG2 titres.

[0354] All constructs except m14J-KDA21 (as it doesn’t contain the KAS2 epitope) induced antibodies against the KAS2 peptide.

[0355] Example 6: further assessment of immunogenicity of truncated constructs

[0356] A further series of immunogenicity assessments were performed using constructs: ml^P-K 30 pg and ml^P-RDA 30 pg.

[0357] Mice were intramuscularly immunised with the mRNA-LNPs encoding the target antigens. Serum antibody subclass responses of immunised mice were examined by ELISA. Antisera were used to probe against the absorbed antigens: KDAK-3S-AVQP polypeptide, heat killed P. gingivalis strain W50 (HKPg), and Kgpcat.

[0358] Antibody responses are expressed as the ELISA titre obtained minus triple the background level, with each titre representing the mean ± s.d. of the 5 individual mice (Figure 10). Only ml^-P-RDA induced strong responses to KDAK-3S-AVQP inducing strong total IgG, IgG 1 and lgG2 titres. It also induced significant total IgG primarily lgG1 but not lgG2, against HKPg and significant lgG1 titres against Kgpcat. It also induced antibody titres against the KAS2 and ABM3 peptides. ml^P-K showed no immunogenicity, likely due to poor expression / stability as described above.

[0359] The results in Examples 5 and 6 indicate that the mRNAs encoding KDA, RDA, KDAAABMS, DA, KDA21, KA and KD perform similarly to the KDAK-3S-AVQP mRNA vaccine in terms of generating an immune response to P. gingivalis and protecting against1006265400P. gingivalis- induced alveolar bone loss in this model. It is therefore expected that mRNA vaccines encoding KDA, KDAAABMS, DA, KA and KD would perform similarly to the KDAK-3S-AVQP mRNA vaccine in providing protection against P. gingivalis- induced brain pathologies resembling Alzheimer’s disease (such as such as brain infiltration by gingipains (RgpA), amyloid accumulation in the brain, Tau phosphorylation in the brain and inflammation in the brain).

[0360] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.1006265400

Claims

CLAIMS1. A method for preventing or treating a neuropathology in a subject, wherein the neuropathology is associated with or induced or caused by a P. gingivalis infection, the method comprising administering to the subject an RNA polynucleotide encoding a protein comprising or consisting of:- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein the polynucleotide is capable of being translated in a mammalian cell, thereby preventing or treating P. gingivalis- induced or associated neuropathology in the subject.

2. A method for preventing the deposition of or reducing the level of P. gingivalis gingipain in neuronal tissue of a subject, the method comprising administering to the subject an RNA polynucleotide encoding a protein comprising or consisting of:- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein the polynucleotide is capable of being translated in a mammalian cell, thereby preventing the deposition of or reducing the level of P. gingivalis gingipain in neuronal tissue of the subject.

3. A method for delaying the onset of a P. gingivalis-induced or associated neuropathology in a subject, the method comprising administering to the subject an RNA polynucleotide encoding a protein comprising or consisting of:1006265400- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein the polynucleotide is capable of being translated in a mammalian cell, thereby delaying the onset of a P. gingivalis-induced or associated neuropathology in the subject.

4. A method for preventing or slowing the rate of a P. gingivalis-induced or associated neuropathology in a subject, the method comprising administering to the subject an RNA polynucleotide encoding a protein comprising or consisting of:- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein the polynucleotide is capable of being translated in a mammalian cell, thereby preventing or slowing the rate of a P. gingivalis-induced or associated neuropathology the subject.

5. A method for preventing or slowing the rate of abnormal protein deposition in the neuronal tissue of a subject; preferably wherein the abnormal protein deposition is associated with or caused by P. gingivalis infection, the method comprising administering to the subject an RNA polynucleotide encoding a protein comprising or consisting of:- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or1006265400- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein the polynucleotide is capable of being translated in a mammalian cell,thereby preventing or slowing the rate of abnormal protein deposition in the neuronal tissue of the subject.

6. The method of claim 5, wherein the abnormal protein deposition comprises abnormal deposition of P. gingivalis gingipain, amyloid β, 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 resulting in physical or chemical changes to neuronal tissue.

9. The method of claim 8, wherein the neurodegenerative condition is characterised by the presence of abnormal protein deposits in the brain, including amyloidopathies, synucleinopathies or tauopathies.

10. The method of claim 8 or 9, wherein the neurodegenerative condition or disorder is selected from: Alzheimer’s disease (AD), Lewy-bodies disease (Dementia with Lewy bodies (DLB)), Huntington's disease, Creutzfeldt-Jakob disease (CJD), Gaucher Disease Type 3, or Parkinson's disease, vascular dementia, frontotemporal dementia or other form 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 a pathology associated with or caused by the presence of P. gingivalis gingipain proteins in neuronal (eg brain) tissue.100626540013. A method for reducing neuroinflammation, preferably neuroinflammation associated with or caused by P. gingivalis infection, the method comprising administering to the subject an RNA polynucleotide encoding a protein comprising or consisting of:- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein the polynucleotide is capable of being translated in a mammalian cell,thereby reducing neuroinflammation, preferably neuroinflammation associated with or caused by P. gingivalis infection, 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-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 and others16. The method of any one of the preceding claims, wherein the RNA polynucleotide induces an immune response to P. gingivalis or to P. gingivalis gingipains in the subject.

17. The method of any one of the preceding claims, wherein the method further comprises administration of one or more of: an antimicrobial compound, an antiinflammatory agent, and adjuvant or further immunogen for inducing an immune response to P. gingivalis or P. gingivalis gingipains.

18. Use of an RNA polynucleotide encoding a protein comprising or consisting of:- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and / or1006265400- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,wherein the polynucleotide is capable of being translated in a mammalian cell, in the manufacture of a medicament for:- preventing or treating a neuropathology in a subject, preferably wherein the neuropathology is associated with or caused by P. gingivalis infection;- preventing the accumulation of P. gingivalis gingipain in neuronal tissue of a subject;- reducing the level of P. gingivalis gingipain in neuronal tissue of a subject;- delaying the onset of a P. gingivalis-associated neuropathology,- preventing or slowing the rate of abnormal protein deposition in the neuronal tissue of a subject;- preventing or reducing progression of an amyloidopathy, synucleinopathy or tauopathy in a subject;- preventing or slowing the rate of progression of a neurodegenerative disease, optionally selected from Alzheimer’s disease (AD), Lewy-bodies disease (Dementia with Lewy bodies (DLB)), Huntington's disease, Creutzfeldt-Jakob disease (CJD), Gaucher Disease Type 3, or Parkinson's disease, a dementia such as mild cognitive and / or memory impairment, vascular dementia, frontotemporal dementia or other form of dementia not typically associated with deposition of abnormal protein deposits; or- reducing neuroinflammation in a subject, preferably neuroinflammation associated with or caused by P. gingivalis infection,19. The use of claim 18, wherein the P. gingivalis-associated neuropathology comprises cognitive decline, a cognitive disorder, or a pathology resulting in physical or chemical changes to neuronal tissue; or is a neurodegenerative disorder.100626540020. The method or use of any one of the preceding claims, wherein the subject has or is at risk of having an infection with P. gingivalis.

21. The method or use of any one of the preceding claims, wherein the protein encoded by the RNA comprises or consists of:one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto.

22. The method or use of claim 21, wherein the protein encoded by the RNA polynucleotide further comprises:- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto.

23. The method or use of any one of the preceding claims, wherein the protein encoded by the RNA polynucleotide is a chimeric or fusion protein comprising or consisting of:- one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto.

24. The method or use of any one of the preceding claims, wherein the RNA encodes an amino acid sequence of an active site of an Arg-gingipain of P. gingivalis comprising the amino acid sequence of SEQ ID NO: 38 (eg encoded by the RNA sequence as set forth in SEQ ID NO: 50), or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 98%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.100626540025. The method or use of any one of the preceding claims, wherein the RNA encodes an amino acid sequence of an active site of a Lys-gingipain of P. gingivalis comprising the amino acid sequence of SEQ ID NO: 8, (eg encoded by the RNA sequence as set forth in SEQ ID NO: 43), or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 98%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

26. The method or use of any one of the preceding claims, wherein the RNA encodes a chimeric or fusion protein that comprises:i) an amino acid sequence that comprises or consists of an amino acid sequence of the active site of an Arg-gingipain of P. gingivalis, or sequences that are at least 80% identical thereto; andii) an amino acid sequence that comprises or consists of an amino acid sequence of the active site of a Lys-gingipain of P. gingivalis, or sequences that are at least 80% identical thereto.

27. The method or use of any one of the preceding claims, wherein the RNA encodes a chimeric or fusion protein that comprises at least two amino acid sequences that comprise or consist of an amino acid sequence of the active site of an Arg- or Lys-gingipain of P. gingivalis, or sequences that are at least 80% identical thereto.

28. The method or use of any one of claim 27, wherein the at least two amino acid sequences are located contiguously in the chimeric or fusion protein.

29. The method or use of any one of claim 27, wherein one of the at least two amino acid sequences is located at the N terminus of the chimeric or fusion protein and the second of the at least two amino acid sequences is located at the C-terminus of the chimeric or fusion protein.

30. The method or use of any one of claim 27, wherein one of the at least two amino acid sequences is located at the N or C terminus of the chimeric or fusion protein and the second of the at least two amino acid sequences is located within the chimeric or fusion protein.100626540031. The method or use of any one of claim 27, wherein the at least two amino acid sequences are (both) located at the N terminus of the chimeric or fusion protein or the at least two amino acid sequences are (both) located at the C-terminus of the chimeric or fusion protein.

32. The method or use of any one of claims 23 to 31, wherein the chimeric or fusion protein encoded by the RNA polynucleotide further comprises:- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto.

33. The method or use of claim 32, wherein the RNA encodes:one or more amino acid sequences of an active site of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; and the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto; andwherein the amino acid sequence of a DUF2436 domain is located between an amino acid sequence of an active site of the gingipain of P. gingivalis and the amino acid sequence of the or more adhesin binding motifs (ABMs).

34. The method or use of claim 32 or 33, wherein RNA encodes an amino acid sequence of a DUF2436 domain of an Arg or Lys gingipain of P. gingivalis comprising or consisting of the amino acid sequence of SEQ ID NO: 35, (eg encoded by the RNA sequence as set forth in SEQ ID NO: 50), or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 98%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

35. The method or use of any one of claims 32 to 34, wherein the RNA encodes a substitution of a cysteine residue in the DUF2436 domain, such as a substitution to a serine or valine residue, preferably wherein the RNA encodes an amino acid sequence of a DUF2436 domain comprising a cysteine to serine substitution (such as shown in SEQ ID NO: 36).100626540036. The method or use of any one of the preceding claims, wherein the RNA encodes one or more adhesin binding motifs (ABMs) comprising or consisting of the amino acid sequence of ABM2 and ABM1.

37. The method or use of claim 36, wherein the RNA encodes the amino acid sequence of a polypeptide comprising ABM2 and ABM1 as set forth in SEQ ID NO: 22 and SEQ ID NO: 21, respectively, or comprising the amino acid sequence as set forth in SEQ ID NO: 24 (ABM2+1), or a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 98%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto.

38. The method or use of claim 37, wherein the RNA comprises the nucleotide sequence as set forth in any one of SEQ ID NOs: 52 to 55.

39. The method or use of any one of claims 36 to 38, wherein the RNA encodes one or more adhesin binding motifs (ABMs) comprising or consisting of the amino acid sequence of ABM2, ABM1 and ABM3.

40. The method or use of claim 39, wherein the RNA encodes the amino acid sequence as set forth in SEQ ID NO: 25 (eg encoded by an RNA comprising the sequence of SEQ ID NO: 56).

41. The method or use of claim 36 to 40, wherein the RNA encodes one or more adhesin binding motifs comprising one or more modifications selected from:a) one or more cysteine amino acid substitutions compared to the naturally occurring Arg- or Lys-gingipain sequences in corresponding regions;b) substitution of the proline and / or an asparagine residues in the sequence PxxN corresponding to, or at a position equivalent to, residues 6 to 9 of the sequence of SEQ ID NO: 21 (ABM1);c) substitution of the motif NxFA to SxYQ in the sequence, corresponding to, or at a position equivalent to residues 2 to 5 of the sequence of SEQ ID NO: 21 (ABM1); d) substitution of the second tyrosine residue, corresponding to or at a position equivalent to residues at position 5 of SEQ ID NO: 22 (ABM2), and of the tryptophan1006265400residue, corresponding to or at a position equivalent to residue at position 23 of SEQ ID NO: 21 (ABM1) to alanine residues.

42. The method or use of claim 41, wherein the RNA encodes one or more adhesin binding motifs comprising a substitution of one or more cysteine residues to a serine residue or to a valine residue.

43. The method or use of claim 42, wherein the RNA encodes one or more adhesin binding motifs that comprise substitution of all cysteine residues to serine or valine residues.

44. The method or use of any one of claims 41 to 43, wherein the RNA encodes one or more adhesin binding motifs that comprise a proline and / or asparagine substitution of the motif PxxN (eg PVQN, SEQ ID NO: 106), corresponding to or at a position equivalent to residues 6 to 9 of SEQ ID NO: 21.

45. The method or use of claim 44, wherein the proline amino acid substitution is a substitution to an alanine residue.

46. The method or use of claim 44 or 45, wherein the asparagine amino acid substitution is a substitution to a proline residue or an alanine residue.

47. The method or use of any one of claims 41 to 46, wherein the RNA encodes one or more adhesin binding motifs comprising a substitution from PxxN to AxxP, (eg AVQP, SEQ ID NO: 107) in the region encoding the ABM1 (such as exemplified in the amino acid sequences of SEQ ID NOs: 30 to 32).

48. The method or use of any one of claims 41 to 47, wherein the RNA encodes one or more adhesin binding motifs that comprise the amino acid sequence as set forth in any one of SEQ ID NOs: 21 to 25, and comprising:a) one or more cysteine amino acid substitutions compared to the naturally occurring Arg- or Lys-gingipain sequences in corresponding regions, preferably substitution of all cysteine residues; andb) substitution of the motif PxxN corresponding to, or at a position equivalent to, residues 6 to 9 of the sequence of SEQ ID NO: 21 (ABM1), to AxxP.100626540049. The method or use of claim 48, wherein the RNA encodes one or more adhesin binding motifs comprising or consisting of the amino acid sequence as set forth in any one of SEQ ID NOs: 26 to 34, or sequences at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 98%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical thereto, provided that the sequences comprise the aforementioned substitutions of the cysteine and proline and asparagine residues.

50. The method or use of any one of claims 1 to 20, wherein the protein encoded by the RNA polynucleotide comprises or consists of:- one or more amino acid sequences of active site of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto; and- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto,51. The method or use of claim 50, wherein the RNA encodes a chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 18 or 83 or SEQ ID NO: 39 or 81, or SEQ ID NO: 58 to 63, or SEQ ID NO: 82 to 87.

52. The method or use of any one of claims 1 to 20, wherein the protein encoded by the RNA polynucleotide comprises or consists of:- one or more amino acid sequences of active site of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto;- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto, and- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto.100626540053. The method or use of claim 52, wherein the RNA encodes a chimeric or fusion protein comprising or consisting of the amino acid sequence of the amino acid sequence of SEQ ID NO: 12, 16 or 20.

54. The method or use of claim 52, wherein the RNA encodes a chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 4.

55. The method or use of any one of claims 1 to 20, wherein the protein encoded by the RNA polynucleotide comprises or consists of:- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto, and- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto.

56. The method or use of claim 55, wherein the RNA encodes a chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 14.

57. The method or use of any one of claims 1 to 20, wherein the protein encoded by the RNA polynucleotide comprises or consists of:- one or more amino acid sequences of active site of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto; and- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto.

58. The method or use of claim 57, wherein the RNA encodes a chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 10 or 108.

59. The method or use of any one of claims 1 to 20, wherein the protein encoded by the RNA polynucleotide comprises or consists of:- one or more amino acid sequences of active site of an Arg- or Lys-gingipain of P.gingivalis, or a sequence that is at least 80% identical thereto;1006265400- the amino acid sequence of a DUF2436 domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto, and- the amino acid sequence of one or more adhesin binding motifs (ABMs) of an adhesin domain of an Arg- or Lys-gingipain of P. gingivalis, or a sequence that is at least 80% identical thereto.

60. The method or use of claim 59, wherein the RNA encodes a chimeric or fusion protein comprising or consisting of the amino acid sequence of SEQ ID NO: 6.

61. The method or use of any one of claims 1 to 20, wherein the RNA polynucleotide comprises or consists of a nucleotide sequence encoding a protein comprising or consisting of the amino acid sequence of any one of: SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 38.

62. The method or use of any one of claims 1 to 20, wherein the RNA comprises or consists of a nucleotide sequence of any one of:a) SEQ ID NO: 48, 57,b) SEQ ID NO: 45, 47 or 49 or SEQ ID NO: 41;c) SEQ ID NO: 46;d) SEQ ID NO: 44;e) SEQ ID NO: 42.

63. The method or use of any one of claims 1 to 20, wherein the RNA comprises or consists of a nucleotide sequence of any one of: SEQ ID NO: 40, SEQ ID NO: 43 or SEQ ID NO: 50.

64. The method or use of any one of the preceding claims, wherein the RNA is an mRNA.

65. The method or use of any one of the preceding claims wherein the RNA further encodes an N-terminal signal peptide for enabling secretion of the protein following translation thereof.100626540066. The method or use of any one of the preceding claims, wherein the RNA further comprises a 5’ untranslated region (UTR) and / or a 3’ UTR.

67. The method or use of any one of the preceding claims, wherein the RNA also comprises a 5’ cap analog, such as 7mG(5')ppp(5')NlmpNp.

68. The method or use of any one of the preceding claims wherein the RNA also comprises a polyadenine (polyA) tail.

69. The method or use of any one of the preceding claims, wherein the RNA comprises a chemical modification, preferably wherein the chemical modification is a 1-methylpseudouridine modification ora 1 -ethylpseudouridine modification.

70. The method or use of any one of the preceding claims wherein the RNA has a uridine content of less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20% or less than about 15%.

71. The method or use of any one of the preceding claims, wherein the uridines in the RNA are replaced with a chemical modification such as N-methyl-pseudouridine.

72. The method or use of claim 71, wherein at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uridine nucleosides are replaced with N-methyl-pseudouridine.

73. The method or use of any one of the preceding claims wherein the RNA is in the form of a codon optimised RNA molecule.

74. The method or use of any one of the preceding claims, wherein the RNA is provided in the form of a lipid nanoparticle composition.

75. The method or use of, wherein the lipid nanoparticle composition comprises: - a cationic and / or ionisable lipid comprising from about 25 % to about 75 mol % of the total lipid present in the nanoparticle;- a sterol (structural lipid) comprising from about 5 mol % to about 60 mol % of the total lipid present in the nanoparticle;1006265400- a phospholipid comprising from about 5 mol % to about 50 mol % of the total lipid present in the nanoparticle;- a PEGylated lipid comprising from about 0.5 mol % to 20 mol % of the total lipid present in the nanoparticle.

76. The method or use of claim 74 or 75, wherein the lipid nanoparticle composition comprises:- an ionisable lipid in the form of [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315),- a sterol in the form of cholesterol,- a phospholipid in the form of distearoylphosphatidylcholine (DSPC), and- a PEGylated lipid in the form of 2-[(polyethylene glycol)-2000]-N, N- ditetradecylacetamide (ALC-0159).

77. The method or use of claim 76, wherein the lipids are present in the lipid nanoparticle at molar lipid ratios (%) of 46.3 ALC-0315: 42.7 cholesterol : 9.4 DSPC : 1.6 ALC-0159, optionally in Tris / sucrose buffer (25 mM Tris pH 7.4, 8.8% sucrose w / v).1006265400

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